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Regulation of Cardiac Lipoprotein Lipase During Hypertension and Diabetes
Regulation of Cardiac Lipoprotein Lipase During Hypertension and Diabetes
AND DIABETES
by
Nandakumar Sambandam
DOCTOR OF PHILOSOPHY
in
September 1999
DE-6 (2/88)
ii
ABSTRACT
Hypertension and diabetes often co-exist in humans and the cardiac dysfunction greatly
exceeds that observed with either condition alone. Induction of streptozotocin (STZ) diabetes in
the spontaneously hypertensive rat (SHR) produces a more extensive cardiomyopathy relative to
normotensive rats. Both diabetes and hypertension are associated with early metabolic
alterations that could underlie the pathogenesis of cardiac dysfunction. In diabetes, the heart is
exclusively dependent on free fatty acid (FFA) oxidation for its energy production. On the other
hand, during hypertension, the heart has been shown to utilize glucose predominantly.
Interestingly, when diabetes was induced, SHR hearts showed a remarkable shift towards F F A
oxidation that strongly correlated with severe cardiac dysfunction observed in SHR-diabetic
hearts. To date, the underlying mechanism(s) for these metabolic lesions is not understood.
free fatty acid (FFA) to the heart and therefore could play a potential role in cardiac metabolism.
We hypothesized that changes in cardiac metabolism that occur during hypertension and diabetes
suggest that hypertension and hypertriglyceridemia co-exist, and may be causally related to each
muscle and adipose tissues in hypertensive patients, Dahl-hypertensive rats and in stroke-prone
SHR rats. We investigated the effect of hypertension on cardiac L P L in two hypertensive animal
models: a) the Spontaneously hypertensive (SHR) rat that has a genetic propensity to develop
hypertension, and b) the fructose hypertensive rat which is an acquired model of hypertension.
Hearts from spontaneously hypertensive (SHR) rats were examined before or after the
development of severe hypertension in SHR rats. Age matched Wistar Kyoto (WKY) rats were
iii
used as normotensive controls. With the development of hypertension in SHR rats, there was a
activity. Neither insulin action nor cell-associated enzyme activity could explain this low L P L
2+
activity in coronary blood vessels. However, acute vasodilation with nifedipine (a Ca influx
male Wistar rats. Acute fructose treatment did not alter cardiac heparin-releasable L P L activity,
whereas a significant decrease in L P L activity was seen in the chronic treated group.
activity. Similar to the SHR study, acute vasodilation by in vitro perfusion of coronary
the chronic group to control levels. These studies demonstrate that hypertension may play a
significant role in regulating cardiac L P L activity and that the decrease in enzyme could be a
The diabetic heart has elevated levels of FFA and T G , being supplied from various
sources. The relative contribution of L P L to this supply of F F A during diabetes is not clear. We
previously demonstrated that moderate diabetes (induced by 55 mg/kg STZ) augments heparin
releasable L P L and that this augmentation is possibly due to an increased translocation of the
enzyme from its site of synthesis (i.e. cardiomyocytes). To determine the precise location of the
augmented L P L , a modified Langendorff retrograde perfusion was used to isolate the enzyme at
the coronary lumen from that in the interstitial effluent. In response to heparin, a 4-fold increase
in L P L activity and protein mass was observed in the coronary perfusate after 2 weeks of STZ-
diabetes. Release of LPL activity into the interstitial fluid of control hearts was slow but
iv
progressive, whereas in diabetic hearts, peak enzyme activity was observed within 1 to 2 min
sections confirmed that the augmented L P L in diabetic hearts was mainly localized at the
examined rat hearts at various times after the onset of hyperglycemia. A n increased heparin-
releasable L P L activity in diabetic rats was demonstrated shortly (6 to 24 hours) after STZ
was also increased after an overnight fast. These studies indicate that the intravascular heparin-
releasable fraction of cardiac LPL activity is acutely regulated by short-term changes in insulin
rather than glucose. Thus, during short periods (hours) of hypoinsulinemia, increased L P L
activity at the capillary endothelium can increase the delivery of FFAs to the heart.
To study the effect of this enlarged LPL pool on triglyceride (TG) rich lipoproteins, we
examined the metabolism of very low-density lipoprotein (VLDL) perfused through control and
diabetic hearts. Diabetic rats had elevated lipoprotein T G levels compared to control. However,
fasting for 16 hours abolished this difference. When the plasma lipoprotein fraction of density
< 1.006 g/ml from fasted-control and -diabetic rats (and thus containing mainly V L D L ) were
incubated in vitro with purified bovine or rat LPL, diabetic V L D L was hydrolyzed as efficiently
as V L D L obtained from control animals. Moreover, V L D L s derived from diabetic rats were
plasma lipolytic activity was comparable between control and diabetic animals. [ H]VLDL
3
obtained from control rats was metabolized at a significantly faster rate by perfused diabetic
hearts than by control rat hearts. This increased V L D L - T G hydrolysis was essentially abolished
by prior perfusion of the diabetic heart with heparin, implicating L P L in this process. These
findings suggest that the enlarged L P L pool in the diabetic heart is present at a functionally
rats. The decreased L P L at its functional location could possibly restrict the supply of F F A
(derived from circulating TGs) to the cardiac tissue leading to an increased glucose oxidation.
On the other hand, cardiac L P L is enhanced within hours after induction of diabetes, inducing a
F F A oxidation in the diabetic heart. Interestingly, previous studies in our lab indicate that
activity and significantly increased functional L P L in the heart. This may serve to increase the
delivery of F F A to the heart and the resultant metabolic changes may lead to the severe
TABLE OF CONTENTS
Title page i
Abstract ii
Table of Contents vi
Table Legends x
Figure Legends xi
List of Abbreviations xvii
Acknowledgments xx
Dedication xxi
1. INTRODUCTION 1
1.1. Lipoprotein lipase 1
1.2. LPL Molecular Biology 2
1.2.1 L P L gene 2
1.2.2 L P L structure 3
1.3. Synthesis, Activation and Translocation 8
1.3.1 L P L synthesis and activation 8
1.3.2 L P L secretion 12
1.3.3 L P L translocation 13
1.4. LPL in the Heart 18
1.5. LPL Regulation 20
1.5.1 Transcriptional regulation 20
1.5.2 Po st-transcriptional regulation 21
1.5.3 Post-translational regulation 23
1.5.3.1 Glycosylation 23
1.5.3.2 Binding sites 24
1.5.3.3 LPL turnover by intracellular degradation/recycling 26
1.5.3.4 LPL regulation by substrates 27
1.6. LPL Regulation by Various Physiological Factors 28
1.6.1. Fasting and Feeding 28
1.6.2. Hormonal regulation of L P L 30
1.6.2.1. Growth hormone 30
1.6.2.2. Thyroid hormone 31
1.6.2.3. Catecholamines 32
1.6.2.4. Other Factors 33
1.7. L P L Regulation by Pathological Conditions 34
1.7.1 L P L in Hypertension 36
1.7.2 L P L in Diabetes 38
1.7.2.1. Diabetes Mellitus: An Overview 38
1.7.2.2. LPL regulation during diabetes 41
RATIONALE, HYPOTHESIS AND OBJECTIVES 48
2.1 Hypertension and Diabetes 48
2.2 Metabolic lesions during Hypertension and Diabetes 49
2.3 Possible role of LPL in the development of cardiac dysfunction 50
2.4 Objective 1 51
2.5 Objective 2 52
METHODS 54
3.1 Hypertension Study 54
3.1.1. Experimental animals 54
3.1.1.1. Spontaneously Hypertensive Rats 54
3.1.1.2. Fructose Hypertensive Rats 55
3.1.2. Measurement of blood pressure 55
3.1.3. Isolated whole heart perfusion 56
3.1.4. Preparation of cardiac myocytes 58
3.1.5. L P L assay 59
3.1.5.1. LPL assay in the medium 59
3.1.5.2. Cellular LPL assay 59
3.1.5.3. Post heparin plasma lipolytic activity 60
3.1.6. Triglyceride secretion rate 60
3.1.7. Triglyceride clearance rate 61
3.2. Diabetes Study 62
3.2.1. Cardiac L P L regulation during diabetes 62
3.2.1.1 Experimental animals 62
3.2.1.1.1. Induction of diabetes 62
3.2.1.1.2. Insulin reduction 63
3.2.1.2. Modified Langendorjfperfusion 64
3.2.1.3. Preparation of cardiac myocytes 65
3.2.1.4. Heart tissue homogenization 65
3.2.1.5. Enzyme-linked immunosorbant assay for LPL mass 66
3.2.1.6. Immunolocalization of LPL 67
3.2.1.7. Effect offood restriction on LPL activity 68
3.2.2. Hydrolysis of V L D L in the diabetic heart 68
3.2.2.1. Separation of lipoproteins and lipid profile 68
3.2.2.2. VLDL purification and characterization 69
3.2.2.3. Analysis of apolipoproteins of VLDL using gradient gel
electrophoresis 70
3.2.2.4. In vitro lipolysis of VLDL by bLPL and rLPL 70
3.2.2.5. In vivo radiolabeling of VLDL 71
3.2.2.6. 3
H-VLDL perfusion of the isolated beating heart 72
3.3. Plasma measurements 74
3.4. Materials 74
3.5. Statistical analysis 75
RESULTS 76
4.1. Hypertension study 76
4.1.1. L P L regulation in spontaneously hypertensive rat hearts 76
4.1.1.1. General characteristics and plasma parameters 76
4.1.1.2. Coronary endothelial LPL 76
4.1.1.3. Cardiac myocyte LPL 11
4.1.1.4. Insulin and LPL activity 78
4.1.1.5. Acute effects of vasodilators 78
4.1.1.6. Secretion and clearance of TGs and plasma lipolytic activity 19
4.1.2. L P L regulation in fructose hypertensive rats 95
4.1.2.1. General characteristics 95
4.1.2.2. Coronary endothelial LPL 95
4.1.2.3. Myocyte surface and intracellular LPL activity 96
4.1.2.4. Effects of insulin and vasodilators perfusion on endothelial LPL
activity 96
4.2 Diabetes Study 111
4.2.1. L P L regulation in the diabetic heart 111
4.2.1.1. General characteristics of diabetic rats 111
4.2.1.2. Modified Langendorffperfusion 111
4.2.1.3. LPL activity in whole heart homogenate 112
4.2.1.4. Immunolocalization LPL 112
4.2.2. Acute L P L regulation by hypoinsulinemia 113
4.2.2.1. Insulin depletion study 113
4.2.2.2. Insulin withdrawal study 113
4.2.2.3. Fasting 114
4.2.3. Hydrolysis o f V L D L in the diabetic heart 137
4.2.3.1. Plasma lipid profile and apolipoprotein analysis 137
4.2.3.2. Post-heparin plasma lipolytic activity 137
4.2.3.3. Lipolysis of VLDL by bovine or rat LPL 13 8
4.2.3.4. 3
H- VLDL clearance by isolated rat hearts 139
5. DISCUSSION 158
5.1 Cardiac LPL regulation during hypertension 158
5.1.1. L P L regulation in spontaneously hypertensive rat heart 158
5.1.2. L P L regulation in fructose hypertensive rat hearts 161
5.2 LPL during diabetes 164
5.2.1. L P L regulation in the diabetic rat heart 164
5.2.2. Hydrolysis of V L D L in the diabetic rat heart 168
6. SUMMARY AND CONCLUSIONS 172
7. FUTURE DIRECTIONS 176
8. REFERENCES 178
TABLES
Tables Page No
STZ injection.
from hearts isolated from 2-week D55 rats and age matched
controls
5 Fed and fasted plasma parameters of control and diabetic rats 141
XI
FIGURE LEGENDS
Figures Page No
cardiac myocytes
hearts from W K Y and SHR rats at 7-8 (A), 11-12 (B) and 15-16 (C)
weeks of age.
age.
period for 7-8 weeks (A), and 15-17 weeks (B) old SHR and W K Y rats
is shown.
10 Post heparin plasma lipase activity of 15-17 week old SHR and W K Y 93
rats.
11 Plasma insulin (A), triglyceride (B), and blood pressure (C) of acute (2 99
animals that were withdrawn from fructose treatment for 2 weeks after
chronic treatment.
14 Effect of increasing concentration of insulin (100, 200 and 500 ng/ml) 105
in coronary perfusate (A) and interstitial fluid (B) from C O N and D55
rat hearts
capillaries.
STZ.
20 The chronological changes in plasma insulin (left panel) and glucose 123
22 Effect of insulin treatment in STZ diabetic rats. Animals were made 127
severely diabetic with 100 mg/kg STZ (D100), and then treated
for 7 days. Body weight (A) and fluid intake (B) were measured before
injecting insulin.
23 Plasma glucose (A) and insulin (B) levels of control (CON) and 129
treatment.
METHODS.
lipoprotein fraction (< 1.006 8) separated from the fed serum of control
released/ml/min.
(increasing concentrations viz., 10, 20, 50, 100 mU) and (B) 20 m U of
XV
31 The FPLC elution profile of LPL and HL from heparin sepharose 150
32 In vitro lipolysis of CON and D55 VLDL-TGs (0.3 mM) by (A) CON 152
LPL and by (B) D55 LPL. LPL was purified from post-heparin plasma
chromatography.
using VLDL samples from three animals from CON and D55 groups.
isolated-perfused hearts from CON and D55 rats. Hearts were perfused
LIST OF ABBREVIATIONS
ER Endoplasmic reticulum
FFA Free fatty acids
FSE-2 Fat specific element-2
GH Growth hormone
GlcNAc N-acetyl glucose
Gly Glycine
Gp Glycoprotein
GPAGE Gradient polyacrylamide gel electrophoresis
GRE Glucocorticoid responsive element
HDL High density lipoprotein
His Histidine
HL Hepatic lipase
hLPL Human L P L
hrp 116 Heparin sensitive protrein
HS-oligos Heparan sulfate oligosaccharides
HSPG Heparan sulfate proteoglycans
i.p. Intraperitoneal
i.v. Intravenous
IRS Insulin responsive sequence
KDa Kilodalton
LDL Low density lipoprotein
Leu Leucine
LPDS Lipoprotein deficient serum
Lys Lysine
MEF Myocyte enhancer factor
Mr Relative molecular weight
mRNA Messenger ribonucleic acid
NCSS Number Cruncher Statistical System
Oct-1 Octamer-1
OTF Octamer transcription factor
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PL Pancreatic lipase
RAP Receptor associated protein
RIA Radioimmunoassay
rLPL Rat LPL
s.c. Sub-cutaneous
SDS Sodium dodecyl sulfate
Ser Serine
SNAP Soluble NSF attachment protein
SNARE SNAP-receptors
STZ Streptozotocin
TG Triglyceride
TGCR Triglyceride clearance rate
TGSR Triglyceride secretion rate
Thr Threonine
TRE Thyroid responsive element
Tip Tryptophan
t-SNARE target membrane-SNARE
VDRE Vitamin D3 responsive element
VLDL Very low density lipoprotein
v-SNARE vesicular-SNARE
WAT White adipose tissues
XX
ACKNOWLEDGMENTS
DEDICATION
To my Mom, Dad and my sister for their love and support in all walks of life
To my wife Sumathi and son Santhosh for their love and understanding
1. I N T R O D U C T I O N
Triacylglycerols (TG) constitute 90% of the dietary fat of an average adult food in the
Western society (Enerback and Gimble 1993). After being absorbed, lipids are packaged and
and very low-density lipoproteins (VLDL, secreted by the liver). The T G component of these
lipoproteins must be enzymatically hydrolyzed to release free fatty acids (FFA) which are then
re-esterified and stored as intracellular TGs or utilized as an energy source (Enerback and Gimble
1993). In adult extrahepatic tissues, lipoprotein lipase (LPL) plays a primary role in the
L P L [EC 3.1.1.34] was initially identified as a "clearing factor" by Hahn (1943) since it
cleared severe alimentary lipemia that occur due to irregular eating habbits in dogs. Heparin
injection to these dogs cleared milky plasma which was later found to be due to the lipolytic
action of an enzyme called lipoprotein lipase (Anfinsen et al, 1952; Korn 1955). Subsequent
studies have established that LPL is present in several tissues and is located close to the capillary
wall (Hamosh and Hamosh 1983). Thus, LPL, present on the luminal surface of the capillary
and releases FFA, which are subsequently utilized by tissues for various metabolic tasks. In this
way, L P L plays a rate limiting role in the clearance of TG-rich lipoproteins from the circulation
L P L activity and mRNA have been detected in a wide variety of tissues such as white and
brown adipose, adult heart, skeletal muscle, lungs, lactating mammary glands, kidney, spleen,
2
thoracic aorta, ovary, small intestine, testes, hippocampus of brain, and neonatal liver (Braun and
Severson 1992). Adult liver has very little L P L activity and no LPL-mRNA. In white adipose
tissue (WAT), L P L provides FFAs which are then re-esterified as T G for storage. In brown
an energy source in skeletal and cardiac muscles. In lactating mammary glands, L P L facilitates
milk formation. The physiological role for L P L in some tissues, like brain, is not clear (Braun
1.2. L P L - m o l e c u l a r biology
1.2.1. LPL-gene
The L P L gene has been mapped to the short arm of chromosome 8 (8p22) and spans
Exons 1 -9, which code for the protein sequence are highly conserved among various species and
exon 10, the largest of all, shows the maximum species variability (Enerback and Gimble 1993).
Exon 1 encodes the 5' untranslated region (UTR) and the signal peptide, exon 2 codes for N -
linked glycosylation site (Asn-Xaa-Ser or Thr, where Xaa is any amino acid but proline), exon 4
for the interfacial lipid binding region, exon 5 for catalytic site (consensus sequence Gly-Xaa-
Ser-Xaa-Gly), exon 6 for a putative heparin binding site, exon 8 for a second N-linked
glycosylation site and exon 10 codes for 3' U T R (Braun and Severson 1992). 5'-upstream region
is highly conserved up to ~210 bp in various species including humans and rats. Several cis-
acting elements have been identified within the 5'-flanking region. The notable control elements
include: octamer-1 (Oct-1), fat specific element (FSE-2), glucocorticoid responsive elements
(GRE), thyroid responsive element (TRE), A P I , AP2, S p l , LP a/p\ and more recently, FSE-2
like, G A T A , insulin responsive sequence (IRS), vitamin D3 responsive element (VDRE), and
3
myocyte enhancer factor (MEF-2) (Bey et al 1998). It is proposed that differences in these cis-
acting sites may be responsible for the differential regulation of LPL across the species.
LPL is a glycoprotein, which in its active form occurs as a non-covalent homodimer. The
molecular weight (Mr) of LPL, as predicted from mRNA, is 50,394 assuming removal of the 17-
27 amino acid signal peptide. Mature LPL is therefore a 448 amino acid long polypeptide
(Auwerx et al 1992). Sedimentation equilibrium measurements give a Mr of 41,700 for the LPL
(Olivecrona et al 1985). The species difference in molecular weights was primarily attributed to
LPL has a significant homology with other lipases like hepatic lipase (HL) and pancreatic
lipase (PL). Although the crystallographic structure for LPL is not yet deduced, it is proposed to
be similar to that of human PL (Hide et al 1992). The LPL molecule has an amino (N)-terminal
spanning amino acids 1-312, and a carboxy (C)-terminal from 313-448 (Murthy et al 1996). N-
terminal region has several active sites and C-domain has lipid and heparin binding sites. LPL
monomer has 8 putative functional sites: (1) a catalytic triad, (2) an oxyanion hole, (3) a 'lid' or
'flap' structure, (4) a f$-5 loop, (5) an apolipoprotein CII binding site, (6) an interfacial/lipid
binding site, (7) a heparin binding site and (8) a non-covalent dimer formation site (Braun and
Human LPL has 8 highly conserved serine (Ser) residues out of which Ser 132 is the
crucial residue required for enzyme catalysis (Faustinella et al 1991). Ser 132 is part of the
consensus sequence Gly-Xaa-Ser-Xaa-Gly that is present in all serine proteinases and in human
4
PL. It is also proposed that these highly conserved Ser residues could play a potential role in
conserving three-dimensional structure of LPL similar to PL. Recently, the catalytic triad was
identified as Serl32-Aspl56-His241 and is well conserved in all three lipases of human and of
all other species studied so far (Hide et al 1992). The oxyanion hole and p-5 loop along with the
'lid' structure probably control access of the substrate to the active site. The oxyanion hole is
formed by Trp55 and Leul33, which are next to Serl32 of the catalytic triad. The ' l i d ' or the
'flap' structure is a mobile surface loop that covers the active site and by repositioning, it permits
the substrate to have access to the catalytic site. The lid is flanked by two cysteine residues
(Cys216 and Cys239) which form disulfide bridges in the LPL molecule and stabilizes the lid
within the protein during activation. Mutation analysis reveals that the charge and periodicity in
the proximal and distal segments of the lid is crucial for L P L activity while apical residues (up to
8 amino acids length) of the loop contribute very little to LPL activity (Henderson et al 1993). It
is proposed that these differences in the open (active) conformation of the lid structures in LPL
and P L may account for their different substrate specificity. The (3-5 loop, another mobile loop
that occupies His53 to Trp64, is also involved in rendering the active site more accessible to the
substrates by bringing the oxyanion hole into a catalytically competent position (Murthy et al
, terminal domain. This site interacts with the dipeptide (Lys-Gly, Glu-Glu) present in the C-
terminal region of apoCII. The heparin binding sites on LPL interacts with cell surface heparan
sulfate proteoglycans (HSPG). The hypothetical consensus sequences situated in the N-terminal
residue and X denotes a neutral residue. Additionally, positively charged clusters like Lysl47,
Lysl48, Argl51 may also be involved in heparin binding activity. Recently, Sendak and
5
Bensadoun (1998), using mutational analysis, demonstrated that the distal carboxy-terminal
domain comprising a cluster of positively charged Lys321, Arg405, Arg407, Lys409, Lys416
residues constitute the major heparin-binding domain. The disulfide bridges between Cys264-
Cys275 and Cys278-Cys283 in the L P L molecule may stabilize these regions upon heparin
binding (Murthy et al 1996). Therefore, it appears that each L P L molecule has several heparin
binding sites.
particularly in the last 56 amino acids (Lookene and Bengtsson-Olivecrona 1993). In a recent
study, Lookene et al (1997) expressed several mutants by replacing tryptophan (Tip) residues
390, 393 and 394 with alanine (Ala) and determined that these residues are important for the
productive orientation of L P L at the lipid/water interface. In the same study, the authors
proposed that Tip residues in the N-terminal region (55 and 114) might be involved in subunit
interaction and dimer formation. Previato et al (1994) showed that the C-terminal domain, in
addition to its lipid and heparin binding function, may also play an important role in the
conformational changes in the LPL molecule such that the lid structure opens up and exposes the
hydrophobic residues of the lid to undergo interfacial activation. This activation results in the
formation of a cleft to which the F A chains (snl or 3) of T G probably bind and the glycerol
backbone of the T G occupies the oxyanion hole. The catalytic triad then executes the hydrolysis
of T G (Figure 1). The sn-2 fatty acyl chain probably interacts with the lipidic matrix and
independent lipoprotein uptake requires L P L in its dimeric form. Moreover, heparin and lipid
binding sites between amino acids 390-421 are necessary for LPL-mediated lipoprotein uptake
(Krappetal 1995).
7
triacylglycerol (TAG) by LPL (below). (These models are adapted with permission from
Oxyanion hole
NH NH
$5-loop
8
1.3. Synthesis, activation a n d translocation
(Previato et al 1991), Ob-17 preadipocytes, and Chinese hamster ovarian (CHO) cell cultures
(Ben-Zeev et al 1992) were some of the cell lines used to delineate biosynthetic pathways for
reticulum (ER) and activated somewhere between the ER and Golgi apparatus. The active
enzyme is a dimer with asparagine (Asn) linked glycosylation. Glycosylation of the N-terminal
domain at the Asn residue of LPL is required for its catalytic activity. There are two Asn
residues to which oligosaccharide moieties are added to the L P L molecule. Asn 43 (N-terminal
domain) and 359 (C-terminal domain) in guinea pigs, Asn 44 and 361 in cow and Asn 45 in
chicken LPL, are glycosylated. Asn 43 in is the most important glycosylation site of human L P L
(Auwerx et al 1992). In the rat, two Asn (43 and 359) glycosylation sites are conserved as in
the C-terminal domain is not required for the expression of fully active LPL. This was also
reinforced by Busca et al. (1995) who showed that replacing Asn 43 with A l a (Asn43->Ala43)
using site directed mutagenesis caused retention of non-glycosylated human L P L (hLPL) in ER.
Neither L P L activity nor protein was found in the medium of cells expressing mutant hLPL and
all detectable protein was present exclusively in the ER. In addition, this accumulation of non N -
glycosylated L P L also caused morphological changes in ER and prevented the transport of other
proteins like B°' amino acid transporter (rBAT) and glucose transporter (glut-4) to plasma
+
Severson 1992). This glycoprotein with mannose rich glycosyl residues in the ER is endo-(3-N-
series of modifications: 1) terminal glucose residues are removed by glucosidase I and II in ER,
2) one mannose residue is then removed by a-mannosidase giving rise to high mannose peptide
[Man -(GlcNAc) -protein] which is then transferred to cis-Golgi, a receiving center of the Golgi
8 2
complex. Although the exact location of Golgi processing enzymes is not clear, three mannose
medial-Golgi, GlcNAc is added and two more mannose residues are removed by mannosidase II
protein is further modified by a series of transferases, which add GlcNAc, galactose and sialic
acid residues to give rise to a endo-H resistant glycoprotein (Figure 2, Braun and Severson 1992).
Vannier and Alihaud (1989) proposed that LPL exists as an inactive monomer within the
ER and the activation of LPL occurs only after translocation to the cis/medial-Golgi. However,
required for full expression of enzyme activity. Various approaches like brefeldin A treatment
(to disassemble the cis/medial-Golgi complex), incubation at 16°C (to prevent the movement of
proteins from ER to Golgi), and tagging with KDEL (a specific ER retention signal) caused
retention of fully active LPL in its high mannose form within the ER, blocking secretion into the
medium. Also, it was shown that mannose trimming was not necessary for the activation and
activity and secretion occurred when glucosidase inhibitors like castanospermin and N-methyl-
oligosaccharide processing or translocation to the cis-Golgi is not required for full expression of
lipolytic activity (Ben-Zeev et al 1992). Liu et al (1993), utilizing metabolic labeling of perfused
guinea pig hearts, demonstrated that L P L is assembled into dimers in the E R and that the
using the cld/cld mouse model showed that glycosylation and dimerization alone do not produce
active L P L but transport of L P L out of the ER is essential for the secretion of active enzyme.
L P L accumulates in the trans-Golgi in its fully active form with a half time of approximately one
1
•Fully matured
Secretory
active LPL dimer
Vesicles
Lysosomal
1" degradation
Temporary binding to
cell surface HSPGs
Interstitial
space Translocation to luminal surface
Vascular
lumen
Figure 2. Schematic diagram of L P L synthesis, activation and secretion from cardiac myocytes.
12
1.3.2. LPL secretion
LPL, like other proteins, reaches the plasma membrane from the ER through a vesicular
transport system. In general, vesicles are sculpted from the ER with the help of ER coat proteins
like COP I/COP II (Rothman and Wieland 1996). These coat proteins are then removed to allow
the fusion of the vesicle to the target membranes like Golgi and plasma membranes. The specific
proteins called SNAP receptors (SNARE) bring about the docking of the vesicles to the target
the target membranes) to dock the vesicle to appropriate target membranes. NSF (N-
ethylmaleimide-sensitive fusion protein) and SNAP (soluble NSF attachment protein) then bind
to the S N A R E complex and initiate an energy dependent fusion process. NSF mediated A T P
hydrolysis produces energy required in this process. The target specific vesicular transport is
dictated by the intrinsic signal from the protein being transported. A protein may have multiple
sorting signals, each determining the fate of that protein at successive stages and jointly
controlling its journey (Rothman and Wieland 1996). In the L P L molecule, a signal peptide of
17-27 amino acids and oligosaccharides were proposed to be involved in the directional transport
of the protein (Eckel 1989, Braun and Severson 1992), but the details of the transport process
remain to be elucidated.
constitutive and regulated mechanisms (Braun and Severson 1992). The constitutive release of
L P L occurs spontaneously and the rate of release matches the rate of synthesis. The regulated
release occurs in response to a secretogogue (e.g. heparin). For regulated release to occur, the
enzyme has to be packaged and stored in secretory vesicles until it is released. A l l cell types that
produce L P L exhibit constitutive release while some cells exhibit both constitutive and regulated
13
release (for example Ob-17, 3T3-L1 adipocytes, cardiac myocytes and mesenchymal cells).
Heparin is a major secretogogue, acts by releasing L P L from its cell surface binding sites and
therby stimulates secretion of additional enzyme from within the cells (Eckel 1989). Release of
L P L from cells is also possibly controlled by metabolic demand (Olivecrona and Bengtsson-
Olivecrona 1993). Addition of increasing concentrations of oleic acid prevented the release of
LPL, after being secreted, binds to parenchymal cells transiently before it is translocated
to the endothelial surface in the vascular lumen. Translocation of L P L from its site of synthesis
(myocytes/adipocytes) to the endothelial cell surface in the vascular lumen is not clearly
understood. The subendothelial basement membranes are proposed to sequester and stabilize the
the subendothelial space is composed of collagens (type I, III, and IV), fibronectin, laminin and
proteoglycans of which L P L binds mainly to the heparan sulfate proteoglycans (HSPG). It was
first proposed that L P L could move along bridges of HSPG molecules that connect parenchymal
HSPG at the basolateral surface of the endothelium was shown to be necessary for an efficient
transport to the apical surface (Saxena et al 1991). The removal of HSPGs by heparinase
HSPG and L P L within the transport vesicles was not required for the secretion of active L P L as
14
shown in HSPG deficient Chinese hamster ovarian (CHO) cells (Berryman and Bensadoun
1995). Nevertheless, L P L produced by wild type CHO cells (which express HSPG) was found to
be more stable than L P L produced by the mutant cells. Stins et al (1992) further proved that the
movement of L P L across the endothelial cells was polarized towards the apical (also known as
luminal) surface. Transport of L P L was therefore appreciably greater from the basolateral (also
known as abluminal) to the apical surface than in the opposite direction. Recently, fragments of
HSPG, heparan sulfate (HS) oligosaccharides have been shown to act like extracellular
chaperones for L P L and enable the transport of the enzyme in its active form across the
endothelial cells (Sivaram et al 1997). Endothelial cells release a heparanase-like enzyme during
the lypolytic encounter of L P L with V L D L in the co-cultures of endothelial cells and adipocytes.
This heparanase-like enzyme then cleaves surface bound HSPGs to release HS-oligosaccharides
them to the apical surface of endothelial cells and probably prevent L P L degradation in
interstitial fluid. Moreover, HS-oligosaccharides have also been shown to enhance transport of
L P L across endothelial cells (Sivaram et al 1997). HS-oligosaccharides only partly saturate the
heparin binding domains on the L P L molecule and, therefore do not affect further binding of L P L
to cell surface HSPGs (Sivaram et al 1997). A l l of these in vitro results were complemented by
the fact that feeding animals also increases the activity of L P L as well that of heparanase-like
Movement of L P L across endothelial cells has been suggested to occur via a poorly
macromolecules like L P L at the basolateral side of endothelial cells, vesicular transport across
the cell, and exocytosis at the luminal surface. Much of the understanding comes from basic
cellular transport processes that are applicable to various macromolecules in general. Endothelial
15
cells lining the arterioles, capillaries and venules of glomeruli and peritubules of kidney and
capillaries associated with intestinal mucosa and secretory glands are fenestrated. However,
endothelial cells lining the microvessels of the heart are not fenestrated but continuous.
Therefore, the transport of macromolecules across the endothelial barrier has to be either by fluid
phase or transcytotic mechanisms (Michel 1998). Several studies suggest that caveolae could act
as ferries for macromolecules between luminal and abluminal surfaces of the endothelial cells.
supporting a vesicular transport system across the endothelial cells (Oh et al 1998). A
endocytosis, and SNARE-SNAP-NSF complex mediated exocytosis are indicated in this caveolar
transport across endothelium (Niles and Malik 1999). Additionally, involvement of actin
filaments has been suggested in the caveolar transport process since cytochalasin B prevented
transport of these vesicles (Niles and Malik 1999). In this regard, Martinho et al (1996)
demonstrated that binding of L P L to HSPG triggers the aggregation and distribution of HSPGs
along the actin cytoskeleton. In the absence of such ligand binding, HSPGs appear distributed
irregularly on the fibroblast cell surface, without any apparent co-localization with the actin
cytoskeleton. Whether this organization of HSPG along the actin cytoskeleton is involved in the
transcytosis of L P L from the abluminal to the luminal surface is still not known. Earlier studies
in the heart demonstrated that antimitotic agents like colchicine and vinblastin inhibited the
antimitotic drug, cyclophosphamide, also had similar inhibitory effects on the secretion of L P L in
rabbit hearts (Lespine et al 1997). Recently, Ewart et al (1999) demonstrated that the actin
16
cytoskeleton is involved in the transport of L P L from within the cardiac myocytes. Because the
microvascular endothelium of the heart is known to be enriched with caveolae (Michel 1998), it
Abluminal surface
«mc rafter
Luminal Surface
HSPG
• LPL dimer
J
Vesicular S N A R E (v-SNARE)
Target S N A R E (t-SNARE)
N-ethylmaleimide
o
sensitive fusion protein (NSF)
Soluble NSF
attachment protein (SNAP)
The transcytotic process has been depicted to involve four stages (1. Endocytosis, 2. Actin
alignment, 3. Vesicular traffic along the actin cytoskeleton, and 4. Vesicle fusion and
exocytosis).
18
1.4. L P L in the H e a r t
In the heart, L P L gene expression and protein synthesis begins only after birth and
reaches a maximum level within 3 weeks of age. The fetal heart does not express L P L (Hamosh
and Hamosh 1983). Synthesis, processing and translocation of L P L in the heart occurs by
mechanisms similar to those of adipose tissues and has been mainly studied in experimental
animals like mouse, guinea pigs, rats, and rabbits. In the adult mouse heart, L P L is synthesized
myocardium, in situ hybridization for LPL mRNA revealed that L P L was primarily synthesized
by cardiac myocytes (O'Brien et al 1994). In the rat heart, cholera toxin induced L P L m R N A
expression primarily in interstitial cells (Stein et al 1991) although cardiac myocytes also
exhibited a diffuse but a definite localization of L P L mRNA. Since the LPL gene has muscle
specific motifs and calcium- and cAMP-responsive elements, it is likely that under normal
conditions, cardiac myocytes and possibly some vascular pericytes (which comprise a portion of
interstitium in the heart) could be the primary sites of L P L synthesis (O'Brien et al 1994).
myocardium delineated that 78% of total L P L was present in the cardiac myocytes, 3-6% in the
interstitial space, and 18% in the capillary endothelium (Blanchette-Mackie et al 1989). Within
the myocytes, L P L was found localized within the sarcoplasmic reticulum, Golgi complex and
with the plasma membrane and the basal surface of the capillary endothelium. L P L in the
endothelium was seen inside the cells, within the vesicles and along the luminal surface of the
perfused heart was immunocytochemically localized in the vascular lumen (Pedersen et al 1983).
expression was detected (Camps et al 1990). Further, treatment with protein synthesis inhibitors
like cycloheximide, or with transport inhibitors like colchicine reduced the L P L activity in the
vascular lumen (Chajek et al 1975, Liu and Olivecrona 1992). Therefore, it was concluded that
endothelial cells do not synthesize L P L and the enzyme must have been synthesized in the
cardiac myocytes and translocated to the endothelial surface in the vascular lumen. Pulse-chase
studies demonstrated that it takes approximately 30 minutes for the newly synthesized L P L to be
translocated to the luminal surface of vascular endothelium (Liu and Olivecrona 1991).
When perfused with heparin via a Langendorff retrograde perfusion, isolated hearts
release LPL. This heparin-releasable LPL was shown to occur in three phases over a 60 minute
period in guinea pig hearts (Liu and Olivecrona 1992, Rodrigues et al 1997): 1) a rapid phase,
occuring within seconds after heparin perfusion, probably from the endothelial luminal surface;
2) a shoulder of release, occuring between 2-30 minutes, probably from within and from the
abluminal surface of endothelial cells; and 3) a slow steady release of LPL, probably explained
by release from the site of synthesis. The rapid, heparin releasable, vascular-bound L P L fraction
is also called 'functional' LPL since it is involved in the hydrolysis of circulating TGRLs. It is
this heparin-releasable fraction of L P L that is more sensitive to physiological (e.g. fasting and
feeding, Braun and Severson 1992), and pathological (e.g. diabetes, Tavangar et al 1992) changes
and this can correspondingly affect exogenous FFA supply to the heart.
20
1.5. L P L Regulation
stages by various physiological conditions like fasting, feeding, cold exposure, lactation etc., and
by various pathological conditions like diabetes, hypertension, obesity etc. For example, L P L
synthesis can be observed in the fetal liver but the adult liver does not synthesize LPL. On the
contrary, heart LPL activity is very low in the fetus but reaches maximum levels within three
weeks after birth (Hamosh and Hamosh 1983). Several transcriptional, post-transcriptional and
A greater understanding of transcriptional regulation of LPL has been possible due to the
availability of cDNA for L P L from various species including human, mouse, rat, baboon, bovine,
chicken and guinea pig (Enerback and Gimble 1993). Most of the transcriptional regulation of
mature adipocytes, several genes are activated, earliest among them being the L P L gene
(Enerback and Gimble 1993). The L P L gene has several cis-acting regulatory sequences in its
-46 (relative to the transcriptional start site) is one such site which binds to trans-acting factors
like OTF-1 (octamer transcription factor). Using transient transfection studies, deletion of the
Oct-1 site has been shown to decrease the transcription of L P L gene. The specificity of Oct-1
and 2 protein binding to this octamer sequence is determined by the D N A sequence adjacent to
the Oct-1 site (Previato et al 1991, Currie and Eckel 1992). N F - Y , another protein which
specifically binds to the C A A T motif at position -65, is also involved in the transcriptional
21
regulation of L P L (Currie and Eckel 1992). The presence of positive (-368 to -35, increases LPL
expression) and negative (-724 to -565, suppresses L P L expression) regulatory sequences in the
5'flanking region have been indicated to play a role in the tissue specific regulation of L P L
(Previato et al 1991). Similarly, LP-cx/p\ Ap-1 complex, and C/EBP proteins are all involved in
the transcriptional regulation of L P L (Enerback and Gimble 1993). Differences that exist in
these regulatory sequences and in the expression of trans-acting factors have been postulated to
The involvement of the 3'untranslated region (3'UTR) region in the tissue specific
regulation of L P L is increasingly evident from recent studies. L P L cDNA from various species
including human, bovine and mouse exhibits two polyadenylation signals (corresponding to the
human sequence at nucleotides 3155 and 3550) in its 3'-UTR and these sites are highly
conserved in all species examined so far (Ranganathan et al 1995). Adipose tissue in humans
expresses two L P L mRNA species (3.2 and 3.6 kb) probably due to a random choice of one of
the two polyadenylation sites (Ranganathan et al 1995). However, skeletal and heart muscles
express only the 3.6 kb mRNA form probably due to a non-random choice. In addition to
humans, several other species express multiple mRNAs for LPL. However, in rat adipose and
muscle tissues only the second polyadenylation site is utilized to express the 3.6 kb L P L m R N A
(Ranganathan et al 1995). Therefore, in addition to the 5'-flanking region, the 3'UTR can be
speculated to play a role in the tissue and species specific regulation of L P L expression.
Two mRNA species have so far been identified for L P L ; one is 3.2 kb and the other 3.6
kb in length. When transfected in CHO cells, the longer mRNA form more efficiently translated
to L P L protein that had higher specific activity than the 3.2 kb m R N A transcript (Ranganathan et
22
al 1995). Steady state mRNA levels depend on message stability as well as gene transcription.
Stability and translatability of mRNA determine the amount and rate of protein translation. The
polyadenylation tail confers stability to mRNA. Although the exact mechanism by which the 3.6
kb mRNA translates more efficiently is not known, it is proposed that in general, changes in the
secondary structure of mRNA may allow better interactions of certain trans-acting binding
proteins. The 3'-UTR may also be involved in mRNA stability and in its translational efficiency
(Ranganathan et al 1995). Hormones like insulin and epinephrine may alter the translational
Turnover of L P L mRNA is usually very slow under normal conditions (~40 hours). This
slow turnover of mRNA means a continuous production of active L P L even when there is no
concentration at a level that is absolutely necessary to fulfill the physiological demand (Mitchell
et al 1992). The rate of L P L protein translation from its mRNA is commonly measured by S - 3 5
synthesis can either be dependent on or independent of mRNA levels. For example, insulin
glucose further increased L P L synthesis in these cells without altering mRNA levels (Ong and
Kern 1989). In another instance, the diurnal variation (both increase and decrease) in L P L mass
and activity was not accompanied by corresponding changes in L P L mRNA levels indicating a
posttranslational regulation (Bergo et al 1996a). However, certain conditions like treatment with
TNF-a, and cold/warm adaptations may markedly alter LPL mRNA production and degradation
multiple levels such as transcriptional, pretranslational and posttranslational levels was observed
in Djungarian hamsters' brown adipose tissue in response to cold adaptation. In early phases of
23
cold adaptation posttranslational mechanisms are activated whereas, prolonged cold exposure
al 1996).
turnover of LPL protein, dimerization to an active form and also through control of LPL binding
sites on the endothelium. The LPL activity can also be regulated by physiological substrates such
1.5.3.1. Glycosylation
This carbohydrate is in the form of N-glycosylated sugars on the LPL protein. Core
glycosylation and glucose trimming occurs in ER and further processing takes place in the Golgi
led to the production of inactive monomeric LPL that had low affinity for heparin. However,
Golgi processing) produced active, dimeric LPL that had high affinity for heparin. Therefore, it
was concluded that glycosylation and subsequent removal of distal glucose residues was
necessary but processing by the Golgi mannosidase I was not required for the production of
active and dimeric LPL that had high affinity for heparin binding (Masuno and Okuda 1994, Park
etal 1995).
24
1.5.3.2. Binding Sites
L P L , after secretion binds to HSPG binding sites that are present both on parenchymal
and endothelial cell surfaces (Cheng et al 1981, Shimada et al 1981, Wang-Iverson and Brown
1982, Williams et al 1983, Cisar et al 1989, Chajek-Shaul et al 1989,). Recently, the active
This active fragment may bind to peptide region(s) containing basic amino acid residues of L P L
L P L secretion from the parenchymal cells. The exact mechanism by which this stimulatory
effect of heparin occurs is not yet known. Binding of L P L to HSPG also stabilizes L P L activity
(Berryman and Bensadoun 1995). Unbound L P L present in the medium loses its activity much
faster than the bound form. In vivo, L P L released into the circulation from its endothelial
binding sites is subsequently taken up by the liver and degraded (Chevreuil et al 1993).
Intravenous injection of heparin results in a release of large amount of functional L P L from all
extrahepatic binding sites into the circulation. Heparin delays but does not abolish the hepatic
its HSPG binding sites by physiological factors like antithrombin III, a-thrombin and its inactive
better accessibility of the enzyme for its substrate (e.g. V L D L , CHYL) and proposed to be an
additional mechanism by which HSPG binding could regulate L P L action (de Man et al 1997).
25
Glycosylphosphatidylinositol (GPI) is another binding site identified on the cell surface
for LPL. GPI binding sites can be selectively cleaved by phsophatidylinositol (PI) specific
3T3-L1 adipocytes with insulin resulted in a PLC mediated release of LPL from GPI binding
sites on the surface (Eckel et al 1978, Spooner et al 1979, and Chan et al 1988). In addition to
insulin, agents like glimepiride (a sulfonylurea) also induced release of LPL anchored to GPI
from rat adipocytes via activation of GPI specific phospholipase (GPI-PL). Glucose free
medium completely abolished the stimulatory effect of insulin as well as glimepiride on GPI-PL
and therefore on L P L release (Muller et al 1994). This set of data indicates that increased
glucose transport rather than insulin stimulates GPI-PL and release of LPL from rat adipocytes.
Bruin et al (1994) have questioned the above possibility by showing that the C-terminal domain
of LPL does not have an addition signal for GPI anchoring. C-ethanolamine, a component of
l4
the GPI anchor, was not incorporated into LPL molecules by metabolic labeling, suggesting that
Other binding sites for L P L include low density lipoprotein receptor related protein
(LRP), a glycoprotein (gp330), a heparin releasable protein (hrp-116), and a novel 85-kDa
glycoprotein (Wolle et al 1995). Physiological importance of these non-HSPG LPL binding sites
is not yet known. Similarly, another putative binding of LPL with unknown physiology has been
shown to occur with oc2-macroglobulin (0C2-M) in the plasma. a2-M is a broad spectrum protease
inhibitor known to bind various basic proteins like nerve growth factor, basic fibroblast growth
factor, cationic aspartate aminotransferase, myelin basic protein and eosinophil cationic protein.
LPL, being a basic protein, also binds to CC2-M in a non-covalent manner via its heparin binding
domain and thus loses its ability to bind to cell surface HSPGs. Whether this binding of LPL to
0C2-M plays any physiological role is not yet known (Vilella et al 1994, Neilsen et al 1997).
26
1.5.3.3. LPL turnover by intracellular degradation/recycling:
by which L P L activity is rapidly regulated during conditions like feeding and fasting (Lee et al
1998). About 80% of newly synthesized L P L are degraded before being secreted. This turnover
by intracellular degradation in adipocytes is a very rapid process with a ti/2 of about 40 minutes.
tunicamycin inhibitable, ER compartment (Braun and Severson 1992). Surface bound L P L also
undergoes degradation after being internalized. Adipocytes degrade only a fraction (<28%) of
total surface bound L P L and this process involves internalization of the enzyme and a subsequent
hepatocytes (Obunike et al 1996, Hoogewerf et al 1991, and Sehayek et al 1995). Berryman and
Bensadoun (1995) demonstrated that HSPG binding sites are essential for the internalization and
degradation of the enzyme by cultured CHO-K1 cells. The degree of sulfation of HSPGs can
on the adipocytes decreased the maximum binding of L P L to the cell surface. This decreased
binding of L P L to HSPGs also resulted in less degradation of the enzyme as a result of decreased
the surface bound L P L by heparin. Release of surface bound L P L by heparin stimulates secretion
of the enzyme from within cells. In adipocytes, this secretory process diverts the newly
intracellular degradation. In cardiac myocytes, release of surface bound L P L does not alter the
in cardiac myocytes is slow (Liu and Olivecrona 1992). In addition to HSPGs, receptor-
27
associated protein-sensitive LRP binding sites also mediate this L P L turnover process, however,
Unlike adipocytes and cardiac myocytes, endothelial cells degrade L P L to a very small
extent. Using in vitro cell cultures, endothelial cells were in fact shown to recycle the surface
bound L P L in its active dimeric form (Saxena et al 1990). Involvement of HSPG binding sites is
once again emphasized. The probable recycling mechanism involves internalization of HSPG-
favors HSPG-LPL binding, thereby enabling the complex to be released back into the medium or
inserted onto the cell surface. However, the following issues have yet to be clarified to
L P L during the recycling process? If it is undegraded HSPG, then is the HSPG-LPL complex
their interaction with LPL. For example, binding of apolipoprotein CII (apoCII) present on the
TGRLs, has been shown to inhibit L P L activity (Jong et al 1997a). L P L activity is also regulated
by the substrates via a feed-back mechanism. For example, F F A released from the TGs by the
Olivecrona and Olivecrona 1980). However, albumin can prevent this feed-back inhibition due
to its higher affinity for FFAs (Bengtsson-Olivecrona and Olivecrona 1980). Another
28
mechanism by which L P L activity can be inhibited by its substrates is by displacement of L P L
from its binding sites (Saxena and Goldberg 1990). L P L bound to endothelial cells can be
displaced from its binding sites by TGRLs and oleic acid. In vitro experiments demonstrate that
FFA binding interferes with the LPL-heparin interaction. In addition, F F A also affects L P L
binding to its activator apolipoprotein CII (Saxena and Goldberg 1990). It is proposed that when
more F F A than the tissue can utilize is released by L P L , F F A would probably bind to L P L and
displace it from its binding sites, thereby regulating the enzyme activity via a negative feed-back
mechanism. The above results generated by cell culture experiments were questioned due to the
fact that no L P L was released when FFAs were perfused through isolated beating hearts from rats
(Rodrigues et al 1992). Alternatively, incubation of cardiac myocytes with oleic acid resulted in
a decreased L P L activity, not due to release of the enzyme from its binding sites but due to
1.6. L P L r e g u l a t i o n b y v a r i o u s p h y s i o l o g i c a l factors
Fasting increases L P L activity in muscles (skeletal and cardiac) but decreases the enzyme
activity in adipose tissues in different animal models such as rats, guinea pigs and hamsters.
Feeding has opposite effects in these tissues (Robinson 1960, Salaman and Robinson 1966,
Robinson and Jennings 1965, Pedersen and Schotz 1980). Regulation at post-transcriptional and
post-translational levels has been suggested. Decrease in rat adipose L P L due to short term
fasting was restored within 4 hours of refeeding. However, long term fasting (36 hr, 60 hr, and
3-5 days) took several days of refeeding before L P L levels were restored (Bergo et al 1996a). It
was shown that short-term fasting, similar to diurnal rhythms did not affect L P L mRNA levels
(Bergo 1996a) and was in fact due to an increase in the proportion of inactive form of L P L in
29
adipose tissues (Bergo 1996b). In addition, the short term fasting period was also shown to
increase degradation of newly synthesized L P L and markedly decrease secretion of L P L into the
medium (Lee et al 1998). Thus, the regulation during short term fasting was at a post-
translational level. Prolonged fasting (>24 hours) on the other hand, resulted in a decreased level
of m R N A and thus required transcriptional regulation for the restoration of L P L activity in rat
adipose tissues (Bergo et al 1996a). Recent studies have further confirmed that this differential
level of L P L regulation in rat adipose tissue is associated with the duration of fasting (Lee et al
1998).
Fasting and feeding respectively increased and decreased heparin releasable fraction of
heart L P L in rats. There was no change in L P L activity in soleus or extensor digitorum longus
(EDL) in response to short term fasting or feeding. However, L P L activity and mRNA in skeletal
muscle increased only after a 6-day fast. It was concluded that heart L P L is more sensitive to
fasting/feeding than is the skeletal muscle (Ong et al 1994, Ladu et al 1991). The increase in
H R - L P L in the heart is also attributed to increased uptake of L P L from the blood (Olivecrona et
125
al 1995). Although plasma L P L level was not measured in this study, uptake of exogenous I-
L P L was shown to be increased in the heart during fasting. In the mouse myocardium,
methionine (which incorporates into newly synthesized L P L molecule) and chased with non-
radioactive methionine, guinea pig hearts exhibited no difference in the rate of transport of newly
synthesized [ S]-LPL during fed or fasted states (Liu and Olivecrona 1992). The above studies
35
in general support that changes in heart L P L during fasting and feeding are controlled at the
LPL decreases in humans similar to animal models during fasting, but skeletal muscle exhibits
either no change or increase in LPL activity depending on the duration of fasting (Enerback and
Gimble 1993). LPL regulation in obese subjects is different in that LPL activity decreased both
in adipose tissue as well as in skeletal muscle as a result of fasting. Weight reduction in obese
individuals caused adipose LPL to remain elevated during fasting suggesting that the metabolic
set point is primed for continued weight gain in these individuals (Schwartz and Brunzell 1981,
Eckel and Yost 1987). These alterations in LPL activity are however not accompanied by any
catecholamines, prolactin, and steroids regulate LPL activity in a tissue specific manner. The
effects of insulin on LPL will be discussed under the "diabetes and LPL" section.
Growth hormone (GH) induces LPL gene transcription, increases mRNA stability and
receptor in rat liver cells increased the ability of the cells to augment LPL expression in response
to GH. GH-mediated LPL regulation in adipocytes has been shown to occur at the level of gene
(Barcellini-Couget et al 1993). This c-fos mediated regulation is however inhibited by the rise in
31
intracellular C a 2+
indicating that the inability of pre-adipocytes to mobilize C a 2+
in response to
G H increases L P L activity in the heart and skeletal muscles with a parallel increase in post-
heparin plasma. These effects are not due to changes in L P L mRNA and also are not mediated
adipose and muscle tissues (Ong et al 1994). Hypothyroidism stimulates L P L activity in brown
and white adipose tissues and as well as in cardiac and skeletal muscles. These changes in L P L
activity are not due to changes in mRNA levels and thus the regulation is at a post-transcriptional
level (Enerback and Gimble 1993). It appears that an unknown translation repressor factor that
binds to 3'UTR and inhibits L P L translation in normal adipocytes might be decreased under
hypothyroid conditions (Kern et al 1996). This decrease in repressive trans-acting factor could
recent study, it was found that the increase in muscle L P L activity mainly occurs in heparin
releasable fraction in hypothyroid rats (Ong et al 1994). The exact mechanism of this
posttranslational regulation is not clear and suggested to be due to nutritional status of the
animals. It is to be noted that hypothyroidism also obliterated the inverse relationship between
Catecholamines decrease L P L activity in white adipose tissue (WAT, stores lipids) but
increase the activity in brown adipose tissues (BAT, involved in thermogenesis). The effect of
and Gimble 1993). Surprisingly, sympathetic denervation did not prevent the cold induced
increase of L P L activity (Klingenspor et al 1996). Although the reason for this is not clear, it is
possible that circulating catecholamines in the plasma of cold-exposed hamsters could have
caused the increase in B A T - L P L . In mice, cold exposure and in vivo noradrenaline treatment
caused a similar increase in L P L mRNA and enzyme activity in B A T (Kuusela et al 1997a). This
L P L activity.
either increased or not changed by isoproterenol treatment (Stam and Hulsmann 1984, Friedman
cAMP second messenger system. Although, increases in cAMP (by cholera toxin) produced an
increase in total L P L activity in heart tissue homogenates, isolated cardiac myocytes did not
blockers like propranolol did not decrease activity or mRNA level of L P L in rats (Gouni-
Berthold et al 1997). However, Friedman et al (1992) showed that P-agonists and cAMP-analogs
increased L P L mRNA and activity in cultured cardiac mesenchymal cells. A selective p2-
adrenergic agonist like clenbuterol decreased heart LPL in rats (Belhasen and Deshaies 1992). In
33
humans, catecholamines did not have an effect on adipose L P L activity but stimulated skeletal
muscle L P L activity and specific mRNA levels (Eckel et al 1996). Therefore, the inconsistencies
in the literature could be partly due to non-specific activation or inhibition of the adrenergic
system, differences in cell types in which L P L was measured and species differences.
In the fed state in rats, steroids such as glucocorticoids stimulate L P L activity in muscle
but decrease the activity in adipose tissues in rats (Enerback and Gimble 1993). Recently, it was
shown that the glucocorticoid, dexamethasone, had no effect in isolated cardiac myocytes but
stimulated both the H R - L P L and cellular L P L activity in combination with insulin (Ewart et al
1997). Therefore, the in vivo effects of glucocorticoids under fed conditions may be due to the
fed state and prevented the decline in L P L activity during re-feeding (Jansen et al 1980,
Borensztajn and Rubenstein 1973). This increase in cardiac L P L activity mainly occurs in the
H R - L P L fraction (Stam and Hulsmann 1984) and this change in H R - L P L occurs within 3
minutes of glucagon in the perfused heart (Simpson 1979). This rapid action of glucagon is
suggested to modify the enzyme activity at a posttranslational level. Adipose L P L activity was
not affected by glucagon either during re-feeding or in the fed state (Borensztajn and Rubenstein
1973). It is proposed that these in vitro effects of glucagon, catecholamines and steroids could
possibly contribute to the alterations of LPL in a tissue specific manner during stress, and other
pathophysiological conditions.
34
Prolactin, a pituitary hormone that is responsible for lactation during pregnancy, also
induces L P L enzyme activity and pre-adipocyte differentiation. In vivo, during the immediate 48
hour pre-partum, L P L enzyme activity rises 100 fold in mammary tissue and remained high
throughout the suckling period (Enerback and Gimble 1993). Adipose L P L decreases during this
period possibly to divert T G to the lactating breast (Eckel 1989). Although the mechanism of
this prolactin mediated L P L regulation is not clearly understood, recent evidence on cultured
activity in response to prolactin incubation (Hang and Rillema 1997). Whether this increase in
mediating immune and inflammatory responses. One of the mechanisms by which cytokines
regulate lipid metabolism is via regulating L P L activity (Memon et al 1995). Several pro-
inflammatory cytokines like tumor necrosis factor (TNF-oc), interleukins 1 and 6, interferon y
inhibiting L P L synthesis and therefore control L P L action at translational level of the enzyme
(Enerback and Gimble 1993, Oshumi et al 1994). The effect of these cytokines on the muscle
tissues is variable. The above cytokines also suppressed L P L activity either additively or
(Tengku-Mohammad et al 1998).
autosomal recessive disorder. Some of the symptoms of this disease include recurrent abdominal
pain, pancreatitis, hepatosplenomegaly, eruptive xanthomas, and lipemia retinalis and low levels
of HDL. This disease is also sometimes associated with a deficiency in apoCII, the activator of
LPL. Restriction of dietary fat and the resultant decrease in plasma chylomicrons tend to
dietary obesity, is clearly associated with increased L P L activity in adipose tissues. This
increased L P L activity in adipose tissue is responsible for increased adipose cell size, mass and
resultant obesity. In obese patients, adipose L P L activity is maintained at a higher level even
during a weight loss program, the responsiveness of adipose L P L to insulin and glucose increases
markedly in obese patients. This probably enhances the lipid filling capacity of adipose tissues
and therefore leads to a return of obesity (Eckel 1989). The role of L P L in the pathogenesis of
deficiency leading to low levels of serum H D L can increase the risk of atherogenesis. In
addition, recent evidence suggests that L P L could directly play a role in internalizing atherogenic
remnants and retains them on the blood vessels to be internalized later (Williams et al 1992a).
The products of lipolysis of TGRLs (VLDL and C H Y L ) by L P L like FFAs, lysolecithins, and
36
lysophosphatidylcholine are involved in endothelial damage, one of the initial steps in the
also shown to be up-regulated during the process of atherogenesis and this is proposed to play an
important role in the formation of foam cells. Collectively, all these studies emphasize the
1.7.1. L P L in Hypertension
systolic blood pressure of > 140 mmHg and a diastolic blood pressure of >90 mmHg, after several
repeated measurements (Chalmers and Zanchetti 1996). Hypertension is classified broadly into
essential or primary hypertension (the etiology of which is still unknown) and secondary
is strongly correlated to defects in carbohydrate and lipid metabolism (Reaven 1991a, 1991b,
Bonner 1994). The connection between hypertension and metabolic disorders appears to be
primarily due to insulin resistance and hyperinsulinemia (Pollare et al 1990, Rao 1993, Sowers et
al 1994, Kahn and Song 1995). Patients with essential hypertension have been observed to
display a resistance to insulin-induced glucose disposal (Ferrannini et al 1987, Reaven 1990) and
a resultant hyperinsulinemia (Welborn et al 1966). Another school of thought supports the fact
that altered vascular reactivity of resistance vessels for insulin during insulin resistance results in
vasoconstriction and therefore hypertension (Laakso et al 1990 & 1992, Lembo et al 1993 &
1995). In addition, increased activity of the sympathetic nervous system (Rowe et al 1981,
Landsberg and Drieger 1989) and Na /water retention (DeFronzo et al 1975) during the
+
It has been suggested that the coexistence of hypertension and hypertriglyceridemia is not
incidental, and these two conditions may be causally related to each other (Reaven 1988, Reaven
1991a&b, Ferrannini and Natali 1991, Kotchen et al 1991, Reaven and Chang 1991). Although
the mechanisms are not obvious, there are studies to support the notion that hypertriglyceridemia
in these circumstances could be the result of defective clearance of VLDL and CHYL. Indeed, a
hypertensive patients and rats (Pasanisi et al 1988, Mackintosh et al 1991, Lind and Lithell 1993)
LPL. Vascular hypertrophy, and rarefaction of blood vessels as a result of increased sympathetic
tone could account for the poor peripheral perfusion (Lind and Lithell 1993). Interestingly,
post-heparin plasma (Pasanisi et al 1988, Lind and Lithell 1993, Marotta et al 1995). LPL
activity is also decreased in skeletal muscle and adipose tissue in hypertensive patients
1993, Lind and Lithell 1993) and in stroke prone SHR (Ogawa et al 1991). Of particular interest,
a large body of evidence reveals a genetic correlation between LPL and hypertension. For
example, in a group of Taiwanese type 2 diabetic patients, a definite linkage for systolic blood
pressure was located to a region at or near the LPL locus on the short arm of chromosome 8p22
Mondon et al (1993) showed that total L P L activity in heart tissue homogenates was decreased in
m R N A in the heart progressively decreased with the development of hypertrophy, and a parallel
decrease in V L D L receptor levels was also observed in these hearts (Masuzaki et al 1996).
activity was not measured, it is not known whether the decreased m R N A resulted in a
corresponding decrease in L P L activity. Additionally, none of the above studies have measured
As defined by the recent expert committee on the diagnosis and classification of diabetes
mellitus (Report of the expert committee 1997), "diabetes mellitus is defined as a group of
insulin action, or both." The acute clinical manifestations include polydipsia, polyuria, weight
loss, polyphagia, and fatigue. Uncontrolled hyperglycemia may progress to life threatening
diabetic ketoacidosis and coma. The chronic or secondary complications of diabetes include
Diabetes is classified based on clinical conditions and etiology into two major types: (1) type 1
insulin action and an insufficient compensatory insulin secretory response. Type 1 patients often
markers of the disease. Type 2 diabetics are usually asymptomatic and often diagnosed by
fasting hyperglycemia and by oral glucose tolerance tests (Report of the expert committee 1997).
Type 1 diabetes is further divided into immune-mediated diabetes and idiopathic diabetes.
Several autoantibodies including islet cell autoantibodies (ICAs), insulin autoantibody (IAAs),
autoantibodies (IA-2 and IA-2(3) have been identified in the serum. About 85-90% of the
individuals with fasting hyperglycemia were detected with one or more of these autoantibodies
(Report of the expert committee 1997). The genetic component of this type 1 diabetes is strongly
associated with major histocompatibility complex (MHC) molecules which in humans are known
as human leukocyte-associated antigens (HLA). Recent report suggest that genes like D Q A and
B with the influence of DRB are involved in type 1 diabetes. DR/DQ alleles of H L A can either
predispose or protect an individual to or from diabetes (Report of the expert committee 1997). In
addition to genetic predisposition, environmental factors may also play a role in the pathogenesis
of type 1 diabetes. Idiopathic diabetes, on the other hand has no known etiology; evidence of
autoimmunity, nor any H L A associations or strong familial inheritance is encountered with these
ketoacidosis and an absolute requirement for insulin replacement therapy (Report of the expert
committee 1997).
have insulin resistance and usually have relative insulin deficiency (Report of the expert
40
committee 1997). The specific etiology of this disease is not known, neither autoimmune
destruction nor any other causes mentioned in type 1 contribute to the pathogenesis. Obesity and
/or abdominal body fat are strongly correlated to type 2 diabetes. Although the genetics of type 2
diabetes is more complex, a strong genetic predisposition is observed in type 2, more so than is
the type 1 diabetes. The risk of developing type 2 diabetes increases with age, sedentary life style
and obesity. The development of hyperglycemia is very gradual and slow and often with no
noticeable symptoms of diabetes in the early stages. Ketoacidosis seldom occurs in type 2 and
the individuals may not require insulin at least in initial stages and often throughout their
lifetime. Insulin resistance is often associated with obesity and therefore can be improved by
weight reduction and through pharmacological interventions. Diet and exercise is nevertheless
the first line of therapy for type 2 diabetes, followed by oral anti-diabetic drugs, and insulin
Experimental models of diabetes include those induced chemically (by agents like
streptozotocin (STZ), and alloxan), as well as genetic models (e.g. non-obese diabetic (NOD)
mouse, Zucker diabetic fatty (ZDF) rats, BioBreeding (BB) diabetic rats, etc.). STZ is more
commonly used to induce diabetes in laboratory animals due to its greater selectivity for
pancreatic (3-cells, longer half-life in the body (15 min) and a lesser mortality rate of diabetic
Streptomyces achromogenes. The glucose moiety with a highly reactive nitrosourea side chain is
that closely resembles Glut-2 is said to be involved in STZ-|3-cell selectivity, the exact
mechanism(s) is not known. At the intracellular level, STZ is believed to cause several effects:
(1) N A D +
depletion due to activation of poly ADP-ribose synthetase as a cell repair tool in
41
response to D N A damage by reactive methyl carbonium ions generated by STZ; (2) free radical
generation and a resultant oxidative stress by STZ; and (3) nitric oxide generation from STZ, all
possibly contributing to its cytotoxicity. Recently, it was hypothesized that STZ might generate
peroxynitrite through the production of superoxide. The peroxynitrite would then dissociate into
NO2 and hydroxy radicals which are involved in D N A damage and apoptosis of p-cells
mechanisms including: (1) high affinity sites for STZ on P-cell membranes, (2) unique thiol
groups on the P-cell membrane that make these cells more susceptible to oxidative interactions,
(3) less free-radical scavenging ability of P-cells, and (4) a low ratio of N A D / D N A in islets
+
STZ on other cells cannot be excluded. The severity of p-cell destruction and diabetic condition
is dependent on the dose of STZ used. In rats, for example, a 25 mg/kg STZ dose resulted in no
change in serum glucose, insulin levels or pancreatic insulin content. At 65 mg/kg, STZ
produces a moderate and a stable diabetes within 48 hours which is characterized by overt
hyperglycemia, hyperlipidemia, loss of weight gain and a 50% decrease in serum insulin levels.
Pancreatic insulin content drops down to less than 5% of control. High dose of STZ, in the range
100 mg/kg, produces absolute insulin deficiency due to more than 95% P-cell destruction and a
severe hyperglycemia. These animals die of diabetic ketoacidosis i f not supplemented with
L P L regulation during diabetes has been well documented and is known to occur in a
tissue specific manner. The following sections review tissue specific L P L regulation in adipose
tissue, skeletal muscle, cardiac muscle and post-heparin plasma, during diabetes.
42
Adipose LPL: Adipose L P L activity has been shown to be unequivocally decreased both
at the functional level and at the site of synthesis during diabetes (O'Looney and Vahouny 1987).
This decrease in adipose L P L was partly due to decreased irrrmunoreactive L P L protein and
steady state mRNA levels but primarily as a result of decreased catalytic activity (Tavangar et al
1992). The decrease in L P L activity was reversed by insulin treatment. In vitro studies have
demonstrated a direct effect of insulin on adipose L P L activity (Braun and Severson 1992).
Insulin increased cellular LPL activity, rate of LPL synthesis, L P L m R N A levels and H R - L P L
activity on the cell surface of adipocytes and thus regulates adipose L P L activity at
further complemented by several in vivo experiments which demonstrate that increases in plasma
(Enerback and Gimble 1993). In STZ-diabetic rats and non-diabetic controls, high caloric intake
increased plasma insulin levels with a parallel increase in adipose L P L activity in both groups
m R N A levels. The decreased adipose L P L in diabetic patients (both type 1 and type 2) was
L P L activity was due to both an increased rate of L P L synthesis and a decreased degradation of
the enzyme. Since there was no change in LPL-mRNA levels due to insulin treatment, the
The insulin resistance that occurs in obesity blunts the adipose L P L stimulation in
response to insulin (Eckel et al 1995). This response was even more diminished in type 2
diabetics (Eckel et al 1995). Weight reduction in obesity or exercise in type 2 diabetics increased
suggesting that the stimulatory effect of insulin on adipose L P L depends on insulin sensitivity.
43
The greater the insulin sensitivity, the better the response of adipose L P L and a threshold level of
insulin sensitivity controls the enzyme response in adipose tissue (Eckel et al 1995). In this
regard, it was also shown that the more severe the insulin resistance, the lower is the adipose LPL
mRNA level (Maheux et al 1997). This was attributed to the inability of insulin either to
insulin resistant individuals (Maheux et al 1997). Contrary to all of the above evidence, insulin
resistance and hyperinsulinemia induced by chronic high-fat feeding actually exaggerated the
insulin response in adipose and muscle tissues (Boivin et al 1994). This discrepancy is probably
due to differences in the severity and duration of high fat induced insulin resistance and
hyperinsulinemia.
Skeletal muscle LPL: Skeletal muscle L P L activity during type 1 diabetes was found to
be either decreased, not changed, or increased, depending on type of skeletal muscle and the
degree and duration of diabetes studied (O'Looney and Vahouny 1987). Under normal
during insulin resistance, as seen in obesity, skeletal muscle L P L is increased by insulin (Eckel et
al 1995). When type 2 diabetes was present together with obesity, the severe insulin resistance
that occurs did not increase the LPL activity in the skeletal muscle (Eckel et al 1995). Indeed, in
type 2 diabetic patients infusion of insulin/glucose led to a blunted response on skeletal muscle
L P L activity suggesting that neither plasma insulin nor glucose play an important role in the
diabetics (Kashiwazaki et al 1998). Albuminuria was associated with defects in HSPGs in extra-
cellular matrix (ECM) and therefore type 1 diabetic patients with albuminuria were suggested to
differ although both groups had lower L P L activity as compared to healthy individuals. The
elevated plasma T G levels were correlated to this decreased skeletal muscle. L P L activity in both
animal models. In spite of a plethora of literature on diabetes and its effect on cardiac LPL,
results are inconclusive due to high variability. For example, studies on cardiac L P L regulation
determined in whole heart homogenate) were elevated during diabetes (Kessler 1963, Nomura et
al 1984). Insulin treatment reversed the diabetic conditions as well as fractional and total L P L
activities in the heart to normal levels. Tavangar et al (1992) demonstrated that the elevated
protein but with decreased LPL-mRNA levels. Therefore, the elevated level of L P L activity was
not due to enhanced translation of the protein but was probably due to the depressed rate of L P L
turnover (degradation) in the diabetic heart. Contrary to these studies, isolated hearts from
normalized by in vivo as well as in vitro insulin treatments. Further, the effects of insulin could
be inhibited by a protein synthesis inhibitor, cycloheximide (O'Looney et al 1983). The latter set
of experiments supports the idea that the decrease in functional L P L in the diabetic heart could
be due to a decreased synthesis of the enzyme and that insulin can directly stimulate L P L
synthesis in the heart. It is to be noted that the in vitro effects of insulin either in perfused hearts
or isolated cardiac myocyte culture could not be reproduced by several subsequent studies (Braun
and Severson 1991, 1992, Rodrigues et al 1992b, Ewart et al 1997, Ewart et al 1999).
45
Nevertheless, in vivo insulin administration did stimulate L P L activity in the diabetic heart,
which led to a conclusion that insulin requires a second factor to regulate cardiac L P L activity.
In this regard, Ewart et al (1997, 1999) using cardiac myocytes from control and diabetic rats
demonstrated that incubation of cells with insulin and dexamethasone (a glucocorticoid) together
stimulated L P L activity. Neither insulin nor glucocorticoid by themselves had any effect on
myocyte LPL. Therefore, it appears that insulin regulates heart L P L in vivo in the presence of
another factor (like glucocorticoid). Liu and Severson (1994) further confirmed that functional
functional) activities were depressed in diabetic rats. This decrease in cardiac L P L was neither
accompanied by changes in steady state L P L mRNA levels and stability nor by a decrease in
relative rates of L P L synthesis and turnover (degradation) (Carroll et al 1995). Also, the diabetic
heart did not exhibit any changes in LPL-specific HSPG binding sites (Liu and Severson 1996).
Evidence supporting the idea that there is no change in heart L P L activity during diabetes
has also been noted in the literature. L P L activity in the hearts of STZ-diabetic rats was not
affected either by calorie intake or by the diabetic state per se (Inadera et al 1992). In summary,
diabetes produced variable regulation of L P L in the heart. The disparity in the literature can be
partly explained by differences in chemical agent used to induce diabetes (alloxan/STZ), dose of
STZ, duration of diabetes, differences in animal models used (species and strain differences), and
the techniques used to measure L P L activity (heart tissue homogenate versus isolated heart
perfusion, acetone-ether extracts versus unextracted tissues, etc.) (O'Looney and Vahouny 1987).
46
Post-heparin plasma lipolytic activity (PHPLA): Diabetes is usually accompanied by
hypertriglyceridemia in addition to elevated glucose levels. High plasma T G levels could be due
Hirano et al 1991, Mamo et al 1992, Staprans et al 1992, Duerden and Gibbons 1993, Garg 1994,
Yoshino et al 1996). The decreased clearance of T G R L from blood could partly be due to
contributed by various tissues like, skeletal and cardiac muscles, adipose tissues, etc. P H P L A
was determined to be low in diabetic patients (Taskinen 1987, O'Looney and Vahouny 1987) and
STZ-diabetic hamsters (Ebara et al 1994). Treatment of diabetes with insulin resulted in the
and the compensatory hyperinsulinemia (often associated with type 2 diabetes) can also decrease
plasma L P L activity and cause postprandial lipemia (Jeppesen et al 1995). Maheux et al (1997)
further demonstrated that the degree of insulin resistance was directly correlated to the degree of
decrease in plasma L P L (activity and mass), and to the degree of elevations in plasma T G
concentrations. In fact, numerous studies have shown a genetic correlation between diabetic
1993). Pvu II polymorphism of the L P L gene is also significantly associated with type 2 diabetes
and coronary artery disease (Wang et al 1996). Heterozygous familial lipoprotein lipase
(Julien et al 1997). A novel compound NO-1886, by elevating L P L activity without affecting the
hypoinsulinemic and hyperglycemic states of diabetes, decreased plasma T G levels and increased
reports also demonstrated an increase or no change in plasma lipase activity in diabetic patients
and in experimental animal models (Groop et al 1996, Ebara et al 1994, Hirano et al 1991).
type of diabetes studied, basal insulin or T G levels in the diabetic conditions, and duration of
insulin treatment and withdrawal in diabetic patients (O'Looney and Vahouny 1987).
Decreased clearance of TGRLs from the circulation could also be due to factors other
than defective L P L . A dissociation between L P L activity and plasma T G level was observed
during diabetes (Chen et al 1980) suggesting that factors other than L P L could contribute towards
structure and composition resulting in a poorer interaction with L P L and this was implicated as
O'Looney et al 1985). Major apolipoproteins that are packaged along with the lipids in TGRLs
are apoBlOO, B48, apoEl-5, apoAl-4 and apoCl-3 (Breslow 1989). Among these
apolipoproteins, apoCII is a co-factor necessary for the full expression of L P L activity. ApoCIII
and apoE, on the other hand, inhibit L P L activity. Changes in ratios of apoCII/apoCIII and
apoCs / apoEs have been shown to alter lipolysis of TGRLs by L P L (Bar-On et al 1984, Levy et
2.1. H y p e r t e n s i o n a n d Diabetes
Hypertension and diabetes are two major risk factors implicated in cardiovascular
diseases (CVD) which are collectively the number one killer of western and Asian populations.
1996). Epidemiological studies report that at least 50% of hypertensive patients die of
impairment in coronary flow reserve and chronic myocardial ischemia result in eventual
transformation to a dilated, failing ventricle (Nicholls 1996). Therefore, the presence of left
heart failure (Nicholls 1996). In spontaneously hypertensive rats a similar development of left
ventricular hypertrophy was observed in early stages of hypertension (Anversa et al 1984) which
Diabetes is another potent and independent risk factor for cardiovascular diseases in
humans as well as in experimental animal models (Butler 1998, Dhalla et al 1998). The diabetic
heart suffers a specific muscle disease called diabetic cardiomyopathy that is due to insulin
deficiency but is seen in the absence of coronary artery disease, myocardial infarction, cardiac
autonomic neuropathy, and micro and macroangiopathy (Fein and Sonnenblick 1985, McNeill
and Tahiliani 1986). The cardiomyopathy is characterized by reduced heart rate, depressed left
ventricular developed pressure, and decreased rates of contraction and relaxation. These
2+
sarcoplasmic reticulum and sarcolemmal membrane-bound enzyme systems (Ca -pumps,
49
Na /Ca -exchanger, Na /K -ATPase activity), Ca -ATPase of myofibrils, myosin light chain
+ 2+ + + 2+
(MLC) and MLC-kinase activity, various organelle membrane disturbances probably as a result
overload and eventual dysfunction of the cardiac muscle (Dhalla et al 1998). At least 50% of
type 1 and type 2 diabetics develop hypertension (Bilo and Gans 1998). The incidence of
cardiovascular diseases and mortality increases, at least by two fold, when diabetes and
hypertension exist together (Rodrigues and McNeill 1986, Bilo and Gans 1998). In humans as
well as in experimental animals, hypertension was shown to cause extensive structural damage to
the diabetic heart and a greater depression in cardiac function when compared to non-
Glucose and F F A are major substrates that the heart utilizes to produce energy, F F A
normally contribute at least 70 % in generating cardiac energy in the form of ATP. Evidence is
emerging to suggest a correlation between metabolic lesions and the development of cardiac
dysfunction during hypertension and diabetes (Christe and Rodgers 1994, 1995, Rodrigues et al
1995). In hearts from SHRs, glucose oxidation was shown to be markedly elevated compared to
hearts from Sprague Dawley rats, yielding a 4-5 fold increase in the ratio of Glucose/FFA
oxidation rates (Christe and Rodgers 1994). This shift in metabolism possibly supports an
efficient energy production in the stable, hypertrophied heart. It was also hypothesized that FFAs
are diverted to meet an increased demand on structure building during the development of left
ventricular hypertrophy (Christe and Rodgers 1994). Therefore, it was proposed that changes in
50
cardiac metabolism probably favor the development of ventricular hypertrophy in the
In the diabetic heart, substantial evidence indicates that early metabolic derangements in
the myocardium could underlie the pathogenesis of diabetic cardiomyopathy (Rodrigues and
glucose entry and FFA mediated inhibition of glucose oxidation is probably the major metabolic
lesion that occurs during diabetes (Randle et al 1963). Interestingly, when diabetes was induced
in SHR, there was a remarkable shift towards F F A oxidation in the SHR-diabetic heart (Christe
and Rodgers 1995). This drastic shift from an increased glucose oxidation to an increased F F A
oxidation positively correlated with a much greater functional depression and cardiomyopathy in
McNeill 1986). To date, the underlying mechanism(s) for these metabolic lesions are not
completely understood.
2.3. P o s s i b l e r o l e o f L P L i n the d e v e l o p m e n t o f c a r d i a c d y s f u n c t i o n
Although, F F A are the preferred energy substrate, the heart has a limited potential to
synthesize them (van der Vusse et al 1992). Hence, FFAs are supplied to cardiac cells from
several sources: through lipolysis of endogenous cardiac triglyceride (TG), or from exogenous
sources in the blood (as free acid bound to albumin or as TGRL). F F A from circulating T G R L is
released by the lipolytic action of endothelium-bound LPL in the coronary vasculature and taken
up by the heart for numerous metabolic and structural tasks. L P L plays a rate limiting role in this
hydrolytic process. Increase and decrease in L P L activity at the coronary lumen could be
contribution of L P L towards cardiac lipid metabolism is not known. Isolated heart perfusion
51
studies revealed that most of the FFA released by LPL was directly taken up by the cardiac tissue
muscles resulted in premature death of mice due to skeletal and cardiac myopathy secondary to
soleus muscle, similar to cardiac muscle, utilizes FFA extensively for its energy production and
expresses 10 fold higher L P L when compared to extensor digitorum longus (EDL, type II fibers
or fast twitch fibers). Indeed, E D L fibers are not dependent on FFA oxidation but on glycolytic
and anerobic oxidation (Ong et al 1994). These observations strongly support the notion that
circulating TGs. During diabetes, F F A oxidation is increased in the heart whereas, during
these metabolic alterations observed during hypertension and diabetes. We hypothesize that
changes in cardiac L P L could be responsible for the alterations in cardiac metabolism that were
2.4. OBJECTIVE 1
(Pollare et al 1991, Marotta et al 1995) and Dahl sensitive hypertensive rats (Mondon et al 1993).
In the heart, total L P L activity, as measured in tissue homogenates, was decreased in Dahl salt
However, in the above study L P L protein or activity was not measured. Changes in L P L
synthesis or activity can occur independent of mRNA levels due to involvement of post-
52
translational regulation (Ong and Kern 1989, Bergo et al 1996a). Therefore, it is physiologically
hypertensive heart.
animal models: 1) spontaneously hypertensive (SHR) rats which have the genetic propensity to
develop hypertension and 2) fructose hypertensive rats which acquire hypertension as a result of
fructose feeding. We attempted to study the enzyme activity in functional (luminal) and non-
2.5 OBJECTIVE 2
The diabetic heart has elevated levels of FFA and TG. Various sources like enhanced
adipose tissue derived FFA, increased intracellular T G synthesis and lipolysis of T G stores, could
contribute to this higher FFA level, ensuring fuel supply to the glucose-deprived diabetic heart
(Murthy et al 1983, Kenno and Severson 1985, Chattopadhyay et al 1990). Several studies have
consistently proven that adipose L P L is decreased during diabetes and that the decrease is
directly related to the insulin deficient state (Robinson 1960, O'Looney and Vahouny 1987,
1997). In contrast, studies on cardiac L P L regulation are inconsistent and unclear. L P L activity
and immunoreactive protein have been shown to be either unchanged, increased, or decreased in
the diabetic heart (Rodrigues et al 1997). Factors such as the strain of rats, dose of STZ and
duration of diabetes, and the techniques that were used to measure L P L could have partly
53
contributed to these disparate results. In fact, unlike in severe diabetes, heparin perfusion
released a 2-3 fold greater amount of L P L in the perfusates of the isolated hearts from the
moderately diabetic animals (Rodrigues et al 1997). In addition, we also observed that heparin
phase of release) from the control heart into the medium. Since heparin can traverse the
endothelial barrier (Lovich and Edelman 1995), we believe that the conventional Langendorff-
perfused heart would release L P L not only from the coronary lumen (which probably constitutes
the fast phase) but also from the abluminal, interstitial spaces and the myocyte surface
release (Rodrigues et al 1997). Therefore, we asked two questions: (1) Does the augmented L P L
in the diabetic heart actually represent the functional pool of enzyme at the coronary lumen,
exclusive of the abluminal, interstitial and myocyte pools? This question is of particular
importance because the presence of the enzyme at this location would permit F F A supply to the
diabetic heart in the absence of glucose utilization. (2) Can changes in L P L activity be acutely
carries clinical relevance since during diabetes, poor compliance with insulin treatment causes
C H Y L . These changes make the lipoproteins poorer substrates for LPL. Thus, it was not clear
whether increased L P L at the coronary lumen in the diabetic heart would correspondingly
3.1. H y p e r t e n s i o n study
hypertension, are inbred rats derived from the Wistar Kyoto ( W K Y ) strain which in turn
descended from the Wistar strain (Trippodo and Frohlich 1981). SHRs have been very well
characterized and shown to express similarities to clinical hypertension (of primary or essential
hypertension type) with respect to the degree of blood pressure, hemodynamics, vascular
reactivity, neurohumoral, and renal factors (Trippodo and Frohlich 1981). Like human patients,
SHRs develop left ventricular hypertrophy and heart failure (Bing et al 1995). The W K Y rat is
A l l animals in the study were cared for in accordance with the principles promulgated by
the Canadian Council on Animal Care and The University of British Columbia. Male SHR and
W K Y rats were obtained at 6 weeks of age (Charles River, Montreal, Que.). The rats were
maintained under a 12 h light (7:00-19:00)-dark cycle. Rats were housed two to three per cage
and supplied with a standard laboratory chow diet (Lab Diet #5001, PMI Feeds, Richmond, V A )
and water ad libitum. As blood pressure is significantly higher only after 9-10 weeks of age in
SHR rats, W K Y and SHR rats were killed before (7-8 weeks of age, representing a pre-
hypertensive stage), after the development of severe hypertension (11-12 weeks of age), and after
a prolonged period of hypertension (15-16 weeks of age), and L P L activity was measured in the
heart.
55
3.1.1.2. Fructose Hypertensive Rats
(Dai and McNeill 1995, Dall'Aglio et al 1995, Zavaroni et al 1980, Hirano et al 1989). Adult
male Wistar rats (180-200 g) were obtained from the University of British Columbia Animal
Care Unit (Vancouver, BC). The rats were maintained under a 12 h light (7:00-19:00)-dark
cycle, and supplied with a standard laboratory chow diet (PMI Feeds, Richmond, V A ) , and water
ad libitum. Rats were randomly divided into control and fructose-treated groups. In the acute
study, treated rats were given a 10% (w/v) fructose solution (p-D- (-)-Fructose, ICN, Aurora,
Ohio) in drinking water for 2-weeks. In some rats, fructose treatment was continued for 4 to 6
and 6 week fructose-treated animals, data from these rats were pooled, and this group was then
defined as the chronic treated group. At the end of the acute or chronic treatment period, rats
were killed, and the hearts were removed for measurement of heparin-releasable L P L activity.
To determine i f the effects of fructose were reversible, fructose treatment was stopped after a 6-
week period. These rats (fructose-withdrawn) were subsequently maintained for an additional 2
weeks, after which they were killed and L P L activity was determined. Age matched controls
Measurements of systolic blood pressure were done in conscious rats by the indirect tail
cuff method without external pre-heating (Bunag 1973). Animals were placed in a rat holder in a
chamber (Model 306, IITC Inc., Woodland Hills, CA) maintained at 29 °C. The tail cuff with
pressure sensor was attached to the base of the tail and connected to a cuff pump (Model 20-NW,
IITC Inc.). A semiautomatic BP analyzer and an analog-to-digital recorder (Model 179, IITC
56
Inc.) were connected to the tail cuff to amplify and record the signal from the pressure sensor.
With this method, the photoelectric sensor detects the reappearance of pulsation (on gradual
deflation of the cuff). Three readings out of five were averaged to determine BP. Animals were
always preconditioned to the experimental procedure before conducting the actual measurements.
Our readings were similar (within 5 mm Hg) to those obtained by direct cannulation of
Pharmaceuticals, Cambridge, Ont.), the thoracic cavities were opened and hearts were removed.
Rats were not injected with heparin prior to killing, as it displaces L P L bound to HSPGs on the
capillary endothelium. Therefore, it was necessary to remove and cannulate the heart as quickly
as possible to avoid clotting of blood in the coronary arteries. Immediately upon excision, the
beating heart was immersed in ice-cold (4°C) calcium-free Joklik minimal essential medium (pH
of the aorta, the hearts were perfused retrogradely by the non-recirculating Langendorff technique
for 5 min (or until the perfusate was clear of blood) (Rodrigues et al 1992a, 1992b). This period
is necessary to remove proteases released by tissue damaged during the dissection that would
reduce L P L activity subsequently measured in the hydrolysis assay. The perfusion fluid was
kept at 37 °C with a temperature-controlled circulating water bath. The rate of coronary flow (7-
measure the release of L P L activity into the medium, the perfusion solution was changed to
1977). This concentration of heparin was previously shown to maximally release cardiac L P L
from its binding sites. The coronary effluent was collected for 10 seconds in timed fractions and
Vasoconstriction has been suggested to affect L P L activity (Lind and Lithell 1993).
Nifedipine, a C a 2 +
influx blocker, is known to dilate blood vessels and decrease blood pressure
in SHR rats (Rinaldi et al 1987, Tschudi et al 1994). Moreover, nifedipine can increase coronary
blood flow, and these effects persist over 30 minutes (Ogawa et al 1981). Hence, nifedipine (3
mg/kg in 40% ethanol) was injected into the tail veins of 15-16 week old SHR rats. Thirty
minutes following nifedipine injection, the rats were killed, hearts were removed and perfused
with buffer containing heparin, and the perfusate was collected and analyzed for L P L activity. In
another experiment, hearts from 15-16 week old SHR and W K Y rats were perfused in vitro with
recirculating Joklik buffer containing nifedipine (100 nM) and 1% B S A for 10 minutes. This
was followed by perfusion with heparin, and fractions of perfusate were collected and analyzed
for L P L activity. CGS-21680, an A -purinergic receptor agonist has also been shown to dilate
2
coronary blood vessels with insignificant effects on cardiac function (Vials and Burnstock 1993).
Hence hearts from hypertensive SHR (15-16 weeks) rats were similarly perfused with 10 u M
CGS-21680 (RBI, M A , USA) for 10 min followed by a 10 min perfusion with buffer containing
heparin to release endothelial bound LPL. During perfusion with nifedipine or CGS-21680, the
coronary flow rate was kept constant at 7-8 ml/min. Similar protocols for in vitro experiments in
the 4-6 weeks fructose treated group were followed to investigate the effects of coronary
Perfusion of the heart with buffer containing heparin predominantly releases extracellular,
endothelial-bound LPL. However, heparin non-releasable (cellular) LPL activity can still be
measured in the myocytes. To measure this fraction, calcium-tolerant myocytes were made from
hearts were removed from anesthetized rats and digested by perfusing Joklik buffer containing
collagenase (228 U/ml), 0.5% BSA, and 50 mM CaCh retrogradely through the heart. Myocytes
method of isolation yields a highly enriched population of calcium-tolerant myocytes that are rod
shaped with clear cross striations in the presence of 1 mM Ca . Intolerant cells are intact, but
2+
hypercontract into vesiculated spheres. Yield of myocytes (cell number) was determined
85%) was assessed as the percentage of elongated cells with clear cross striations that excluded
Cardiac myocytes from WKY and SHR rats were suspended in Joklik-minimum essential
medium to a cell density of 0.4 x 10 cells/ml and incubated at 37°C under an atmosphere of
6
95% 02-5% CO2. To release surface-bound LPL activity, heparin (5 U/ml) was added to the
myocyte culture. Aliquots of cell suspension (1 ml) were removed (cell preparation was shaken
before sampling to obtain a homogenously suspended sample) at specified intervals, and the
medium was separated from cells by centrifugation (3,000 x g for 10 sec) in an Eppendorf
microcentrifuge. The supernatant and corresponding cell pellets were stored in -70°C until
emulsion. The standard assay conditions included 0.6 m M glycerol tri[9,10- H]oleate (1 3
inactivated chicken serum (containing the L P L activator, apolipoprotein CII), and 100 pl of either
myocyte medium or heart perfusate in a total volume of 400 pl. The release of [ H]oleate was
3
measured after incubation for 30 min at 30°C. The released [ H]oleate in the reaction mixture
3
1.41:1.25:1.0 and 100 pl of oleic acid) and 100 pl of 0.1 M NaOH (Ramirez et al 1985). After
vortex mixing and centrifugation (TY JS-4.2 rotor, 2,500 x g for 30 minutes) using a Beckman J-
6B centrifuge, the radioactive sodium [ H]oleate in a sample (0.5 ml) of the upper phase was
3
the reaction rate was linear with respect to time and volume of medium assayed. The validity of
the L P L assay has been previously established (Ramirez et al 1985). Results were expressed as
nanomoles of oleate released per hour per ml (coronary perfusate) or 10 cells (myocyte medium
6
or cells).
a 5 second interval, at 4°C) the cell pellets (0.4 x 10 cells) after resuspending them in 0.2 ml of
6
50 m M ammonia buffer (pH 8.0) containing 0.125% (v/v) Triton X-100. After sonication, the
60
volume was adjusted to 1 ml using sucrose buffer [0.25 M sucrose, 1 m M E D T A , 1 m M
dithiothreitol, 10 m M HEPES, [pH 7.4] (Carroll et al 1995). The assay for cell sonicate L P L
activity was done essentially as described above except that 20 ul of cell sonicate was used and
Plasma L P L activity in the basal (non-fasted) state and in response to a heparin injection
was determined in control rats and those fed fructose for 6 weeks. Heparin (0.5 U/g) was
injected into the tail vein of lightly anaesthetized (20 mg/kg sodium pentobarbital i.p.) rats and
blood samples were collected at 5 minutes. Plasma was separated and stored at -70 °C until
assayed for L P L activity (Galan et al 1994). Plasma lipase activity was determined by first
measuring total lipase (hepatic + LPL) activity in 5 pl of plasma sample. Hepatic lipase activity
was measured by incubating plasma with 1 M NaCl (at room temperature for 10 minutes before
exposing to substrate) and conducting the assay in the absence of apolipoprotein CII, to suppress
L P L activity (Galan et al 1994). Plasma LPL activity was calculated as the difference between
total and hepatic lipase activity and is expressed in milliunits (mU), which corresponds to 1 nmol
determined in SHR and W K Y rats by a previously described method (Kazumi et al 1986). Triton
WR1339, when injected intravenously, inhibits T G lipolysis in the circulation. Therefore, newly
synthesized TGs accumulate in the plasma and the rate of accumulation is an indirect measure of
TGSR. We used 15-16 week old SHR and W K Y rats to determine the TGSR. Rats were fasted
61
for 5 hours (900-1400 hrs) to minimize chylomicron contribution in the TGSR determination.
After light anesthesia, rats were injected (i.v.) with Triton WR1339 (25% w/v solution in normal
saline togive a dose of 600 mg/kg body weight). Blood samples were collected at 0, 20, 40, and
60 minutes after the injection. Serum was separated and the T G concentration was measured
using a Boehringer Mannheim diagnostic kit . When T G concentration is plotted against time,
slope of the linear line that is generated gives the TGSR of the animals.
heparin gives the measure of plasma lipolytic activity in vivo. When heparin is injected i.v., it
displaces L P L and H L from all tissue-binding sites and increases the plasma lipolytic activity
clearance of endogenous T G from the circulation. Anesthetized SHR and W K Y rats (15-16
weeks of age) were injected with heparin (i.v., 0.5 U/g body weight), blood samples were
separated from the above blood samples. A n aliquot of 0 and 5 minute plasma samples were also
measured for post heparin plasma lipolytic activity (for P H P L A measurement, refer section
3.1.5.3.).
62
3.2. Diabetes study
Adult male Wistar (280-290 g) rats were obtained from University British Columbia
animal care unit (Vancouver, BC). The rats were maintained and cared for as mentioned earlier.
Selective P-cell death and the ensuing diabetic state can be produced after a single
severity of diabetes is produced by 25-100 mg/kg STZ (Rodrigues et al 1999). After an i.v.,
injection of 55 mg/kg STZ, stable hyperglycemia develops within 24-48 hrs and remains 2-3
times higher than normal in concert with an =50% reduction in plasma insulin levels. Although
these animals are insulin deficient, they do not require insulin supplementation for survival and
do not develop ketoacidosis. On the other hand, a 100 mg/kg dose of STZ causes intense p-cell
necrosis, remarkable elevation of serum glucose within 24 hrs, reduced plasma insulin to ~2%-
5% of control, and a 98% loss of pancreatic insulin stores. Without administration of exogenous
insulin, death in most of these animals occurs within 7-10 days. Rats were randomly divided into
nondiabetic (CON) and diabetic (D55 and D100) groups. Halothane-anesthetized rats were
injected with STZ (55 [D55] or 100 [D100] mg/kg IV, Sigma Chemical Co.) or an equivalent
volume (1 mL/kg) of saline. Glycosuria was determined 24 hours after STZ injection, and
hyperglycemia was tested at 48 hours with a glucometer. A l l STZ-treated rats displayed both
To evaluate the effect of a chronic reduction in plasma insulin on cardiac LPL, D55 rats
were kept for 2 weeks after the STZ injection, at which time they were killed and the hearts
protocols were followed. In the first protocol, rats were injected with 100 mg/kg STZ. In
preliminary experiments, we determined that after this dose of STZ, there is a triphasic pattern of
changes in blood glucose and insulin levels in the 24-hour period after injection. A n initial brief
degranulation, and an enormous release of insulin (Junod et al 1969). Blood glucose then rises to
a hyperglycemic value of 13 mmol/L within 12-16 hours. Rats were monitored individually, and
at the point of hyperglycemia, or 3 or 6 hours after hyperglycemia, animals were killed for the
determination of cardiac L P L activity. One potential drawback with this approach is the varied
metabolic changes that occur before progression to stable hyperglycemia. Hence, with our
second protocol, rats were made severely diabetic with 100 mg/kg STZ. One day after diabetes
induction, the animals were treated subcutaneously with intermediate-acting insulin (D100+1,
Iletin N P H , Beef and Pork; Lilly) once daily. The insulin injection was given at 1000 A M with
the dose adjusted daily to achieve normoglycemia. Treatment was continued for 7 days. This
time was necessary to ascertain the optimal insulin dose (-18-20 U/kg) required to maintain
euglycemia for 24 hours. After the animals' diabetes had been well controlled, insulin injection
was stopped, and plasma glucose was monitored. After the last insulin injection, plasma glucose
levels increased after 24 hours. A plasma glucose concentration of 13 mmol/L was considered a
hyperglycemic value, and when this level was reached, diabetic animals were kept for either 6 or
24 hours before they were killed. Using this method, we were able to achieve a fixed duration of
Langendorff retrograde perfusion technique was used to separate the coronary from the
interstitial effluent (DeDeckere and Hoor 1977, Jansen et al 1980, de Lannoy et al 1997). Rats
were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the thoracic cavity was
opened. The left anterior vena cava was ligated below the azygous vein followed by ligation of
the right anterior vena cava. The hearts were then carefully excised with the aorta, inferior vena
cava, and lungs still attached. Rats were not injected with heparin before being killed because
necessary to cannulate the heart quickly to avoid clotting in the coronary arteries. Immediately
on excision, the beating heart was immersed in cold (4°C) Krebs-Ringer-HEPES buffer (pH 7.4).
The concentrations of solutes in the buffer were (in mmol/L) 1 CaCl , 118 NaCl, 4.96 KC1, 1.19
2
K H P 0 , 1.19 M g S 0 7 H 0 , 24 HEPES, and 10 glucose. After the aorta was cannulated and
2 4 4 2
tied below the innominate artery, the hearts were perfused by the retrograde noncirculating
Langendorff technique. The perfusion fluid was continuously gassed with 95% 0 /5% C 0 in a
2 2
circulating water bath. A peristaltic pump controlled the rate of coronary flow (7-8 mL/min).
The right and left branches of the pulmonary artery were cut before they entered the lungs, and
the 2 branches were then trimmed off at their junction. Afterward, the inferior vena cava and
branches of the right and left pulmonary veins were ligated, the lungs were removed, and the
pulmonary artery was cannulated and tied. At this time, most of the perfusate (=98-99%) starts
flowing through the pulmonary cannula whereas a small amount of fluid (=1-2%) drips down to
the apex of the heart. The pulmonary effluent represents the coronary perfusate whereas the fluid
collected at the apex represents interstitial transudate (DeDeckere and Hoor 1977, Jansen et al
65
1980, de Lannoy et al 1997). To measure the release of L P L activity or protein into the medium,
the perfusion solution was changed to Krebs-Ringer HEPES buffer containing 1% B S A and
heparin (5 U/ml). This concentration of heparin can maximally release cardiac L P L from its
binding sites, an action mediated by the interaction of negative charges on heparin with positively
charged amino residues on the enzyme (Olivecrona 1993). The coronary and interstitial effluents
were collected separately in timed fractions, and frozen until assayed for L P L activity.
Insulin treatment: In some experiments, 2-week D55 rats were treated with a rapid-acting
insulin (Iletin, 15 U , i.v.) 90 minutes before the rats were euthanized, and cardiac heparin-
Calcium tolerant cardiac myocytes were isolated from C O N and D100 rats by the
previously described method (see section 3.1.4.). Cardiac myocytes were isolated to measure
whether acute insulin reduction after withdrawal of insulin treatment in D100 + I group could
also modulate enzyme activity. Insulin treatment reversed a number of effects of STZ-diabetes,
including hyperglycemia, food and fluid intake and body weight gain, etc, to control levels.
When insulin was withdrawn, the rats became hyperglycemic within 24 hours. The animals were
Hearts from C O N and D55 rats were removed and cleared of blood by flushing them
through the aorta with 10 ml of Krebs-Ringer-HEPES buffer (pH 7.4, 37°C). Atria and other
tissues were removed and the ventricles were freeze-clamped in liquid nitrogen. The tissues
were stored in -70°C until L P L activity was measured. For the L P L measurement, frozen tissues
66
were weighed and ground using a mortar and pestle that were pre-cooled in liquid nitrogen. To
the powdered tissue, 20 ml of 50 mM ammonia buffer (pH 8.0) containing 0.125% (v/v) Triton
X-100 and 10 U/mL of heparin was added and homogenized further using a Polytron (PT MR
3100, Kinematica, AG, Switzerland) homogenizer (2x30 seconds with a 10 seconds gap in
between) under ice-cold conditions. After sonication, the volume was adjusted to give 25 mg/ml
3000xg and the supernatant was separated. A 25 pl aliqout of the supernatant was exposed to
sonicated [ H]-triolein emulsion and the LPL assay was performed in triplicate as before (for
3
Changes in the amount of LPL activity do not always represent changes in quantity of
immunoassayed LPL protein (Olivecrona et al 1987). To measure LPL mass, coronary perfusate
(=24 ml) from CON and D55 hearts was collected between 1 and 3 minutes (flow rate at 7-8
ml/min) after heparin perfusion. LPL protein was measured by a previously described sandwich
ELISA method (Anderson et al 1997). Briefly, samples were lyophilized and resuspended in 0.2
1) were coated with 100 pl of anti-LPL antibody (15 pg/ml in 0.05 M carbonate buffer, pH 9.6)
overnight at 4°C. After washing, 100 pl aliquots of samples diluted in phosphate-buffered saline
(PBS) containing 0.05% (wt/vol) Tween-20, 1 mg/ml heparin, 0.4% (wt/vol) BSA, 1 mmol/L
wells. The plates were then sealed and incubated overnight at 4°C. Purified bovine milk LPL
67
was used as standard for the ELISA. After extensive washing, affinity-purified biotin-labeled
anti-LPL antibody was applied to the wells followed by a second overnight incubation at 4°C.
The wells were then incubated with peroxidase-labeled streptavidin in PBS, 1% (wt/vol) fatty
acid-free B S A for 2 hours at 25°C. The plates were washed thoroughly and incubated with o-
phenylenediamine (0.8 mg/ml in 0.15 M citrate buffer, pH 5), and color development was
measured at 495 nm using a Bio-Rad microplate reader. L P L concentration in coronary fluid was
Immediately upon excision, C O N and D55 rat hearts were perfused by the retrograde non-
circulating Langendorff technique with Krebs-Ringer-HEPES buffer for 3 minutes to clear the
heart of blood. Perfusion buffer was then changed to fixative (neutral phosphate-buffered 10%
formalin solution, room temperature) for 2 minutes. After perfusion, hearts were stored in 10%
formalin for 24 h followed by paraffin processing through graded ethanol and xylene. The
blocks were then embedded in Paraplast, sectioned at 3 um and mounted on positively charged
glass slides. For immunostaining, sections were deparaffinized, rehydrated, and treated with 5%
(vol/vol) heat inactivated rabbit serum in Tris-buffered saline (TBS, 0.15 moles/1 NaCl pH 7.4)
to block any nonspecific background. Sections were incubated with the affinity-purified
polyclonal antibody against LPL (1:100 dilution in TBS containing 1% (w/v) BSA) overnight at
room temperature in a humid chamber. The primary antibody was then washed in TBS and
further incubated for 1 hr at room temperature with the secondary biotinylated rabbit anti-chicken
complex (ABC Kit, Vector Inc). After being rinsed, sections were stained with 0.3% (w/v) 3,3'
68
diaminobenzidine hydrochloride/H202 followed by staining with 0.1% (w/v) Nuclear Fast Red in
5% (w/v) aqueous aluminum sulfate. After a final rinse in running tap water, sections were
dehydrated in ethanol, cleared in xylene, and mounted in a resinous mounting medium and
photographed. Sections from C O N and D55 hearts were treated in an identical manner during all
absence of staining was observed when the primary antibody was omitted or replaced by
activity, C O N rats were fasted for 16-hrs (6 PM-10 A M ) and L P L activity was measured in
coronary and interstitial compartments. During fasting, food was withdrawn from the animals
Rats were deeply anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the chest cavity
was opened, and blood was withdrawn from the inferior vena cava using a 10 ml syringe with a
20 gauge needle. The blood was transferred to glass test tubes and centrifuged at 3000x g for 20
minutes in J6B-Beckman centrifuge at 4°C to separate clear serum. A pooled serum sample was
then used for the isolation of major lipoproteins. Lipoproteins like V L D L , L D L , and H D L were
separated from the serum by a one-step density gradient ultracentrifugation method (Redgrave et
(Beckmann), 1.02 g of NaBr (0.34 g/ml) was added to achieve a density of about 1.25 g/mL
69
(Hatch and Lees 1968, Wasan et al. 1999) and kept at 4° C for 2 hours for pre-cooling. 2.8 ml of
pre-cooled buffers of different densities (1.21, 1.063, or 1.006 g/ml) were layered carefully in the
order of decreasing densities. Tubes were kept immersed in ice until they were placed into
Beckman SW 41 titanium rotor buckets. Tubes with buckets were balanced and sealed with
screw caps. Ultracentrifugation at 288,000 x g at 15° C for 18 hours was carried out in L8-80
Beckman Ultracentrifuge. Tubes were removed from the centrifuge with care so as not to disturb
the gradient layers. V L D L / C H Y L float on top (< 1.006 g/ml), while L D L separates at 1.019
g/ml, H D L at 1.21 g/ml and lipoprotein deficient serum (LPDS) stays at the bottom. The
lipoprotein layers were removed using a glass pasteur pipette and the volume of each layer was
measured. Different fractions were then measured for TG, cholesterol and protein contents. T G
and cholesterol were measured using respective diagnostic kits, and protein was measured by the
Bradford method.
Isolation of V L D L with less amount of C H Y L was achieved by using the serum obtained
from fasted animals. Animals were fasted for about 18 hours (1930 to next day 1330). As a
result of fasting for this duration, rats produce more V L D L and less C H Y L (Rajaram et al. 1980).
E D T A was not added to blood samples that were used for isolation of V L D L for in vitro lipolysis
Ca ZT
that is necessary for L P L activity and for heart function. However, E D T A (4.0 m M , pH 7.6)
was added to blood samples that were used for V L D L to analyze apolipoproteins. To 4.0 ml pre-
cooled serum samples in Beckman Ultraclear® centrifuge tubes, 7.5 ml of 1.006 g/ml buffer
(11.4 g NaCl, 0.2 g disodium EDTA, 1 m M NaOH in 1003 ml double distilled water) was added.
The tubes were secured in SW-41 titanium rotor buckets and ultracentrifuged at 288,000 x g for
70
18 hours and at 15°C. V L D L that floats on top of the 1.006 g/ml solution was separated carefully
using a glass pasteur pipette to avoid dilution. From each tube approximately 1.0-1.5 ml of
V L D L fraction was collected and pooled. Using a similar procedure, the lipoprotein fraction
(<1.006 g/ml) was isolated from plasma of fed animals. In the fed state the plasma is known to
V L D L obtained by the above method was characterized for TG, cholesterol and protein
contents as mentioned earlier. A gradient polyacrylamide gel (Ready gel, 4-20%) electrophoresis
was performed to identify major apolipoproteins -ApoB (Mr 220,000), ApoAI (Mr 46,000),
ApoE (Mr 31,000-35,000), and ApoCII (Mr 14,000). V L D L samples (non-delipidized) from
C O N and D55 rats were solublized and reduced in SDS sample buffer (5% SDS, 20% glycerol,
1% (3-mercaptoethanol, 0.5% bromophenol blue in 0.5M Tris, pH 6.8) and boiled for 4 min at 95
°C. A broad range protein molecular weight standard (BioRad) was diluted 1:20 with sample
dilution buffer, digested for 4 minutes at 95°C and 10 pL of this digested molecular weight
standard was loaded into the wells. 25 pL of V L D L sample (adjusted for protein concentration
to 3.2 mg/mL) was loaded and electrophoresis was run at constant current of 40 mAmps/gel.
The gels were stained with Coomassie Brilliant blue R250 for 90 minutes and destained
3.2.2.4. In vitro lipolysis of VLDL by bovine LPL (bLPL) and rat LPL (rLPL)
(Saheki et al. 1993, Fisher et al. 1995, de Man et al. 1997). V L D L was isolated from C O N and
71
D55 rat sera by the methods described earlier. T G concentrations in C O N - and D55-VLDL were
adjusted to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, or 0.35, m M and exposed to bLPL (0.2 pg protein,
V L D L and bLPL stock solutions were diluted with Tris buffer (pH 8.1) containing 0.5%
essentially fatty acid free B S A (Saheki et al. 1993, Fisher et al. 1995, and de Man et al. 1997).
At the end of a 30 minute incubation, the reaction was stopped by adding pre-cooled 0.3 M
Na2P04 buffer (pH 6.9) and immersing the tubes in ice. From this reaction mixture, a 50 uL
aliquot was pipetted in triplicate to measure F F A release due to lipolysis, using an N E F A - C kit.
The results were corrected for the background using an unreacted blank with corresponding
bLPL (Fisher et al. 1995). The release of F F A by bLPL was found to be linear up to 30 minutes
under the conditions specified. Some of the assays were performed with rLPL purified from
C O N and D55 post-heparin plasma. The purification of rLPL was carried out by heparin
column and a fast protein liquid chromatography (FPLC; Pharmacia, Uppsala, Sweden). The
purified rLPL was then adjusted to 20 mU/ml in both C O N and D55 samples and exposed to 0.3
m M of C O N - and D55 V L D L under the above mentioned conditions. Some in vitro lipolysis
experiments with TGRLs obtained from plasma of fed rats against bLPL were also performed.
V L D L was labeled with ^H-oleic acid based on a previously described method (Rajaram
et al. 1980). H-oleic acid was complexed to B S A as follows: 20 ul (=100 uCi) of H-oleic acid
3 3
was mixed with 200 uL of 10% NaHC03 until the solution cleared, 25% B S A in distilled water
was then added to produce albumin-fatty acid complex, and heated for 3 minutes at 60°C (Nestel
72
and Barter 1971). ^pj-oleic acid-BSA complex was then injected intravenously to deeply
anesthetized non-diabetic Wistar rats (CON) that were fasted for 18 hours (between 1930 and
1330). Forty minutes after injection of the ^H-oleic acid, rats were euthanized and blood was
withdrawn from the inferior vena cava as described previously. Serum was then subjected to
confirmed by thin layer chromatographic analysis on chloroform/methanol extract (2:1 v/v) using
silica gel plates (K5F, Whatman) and hexane/diethyl ether/methanol/acetic acid (90:20:3:2 v/v)
solvent system. The spot corresponding to standard triolein was scraped off and counted for the
V L D L . More than 97% of the VLDL-lipoprotein radioactivity was present in the T G moiety.
Rate of V L D L - T G clearance in C O N and D55 hearts was studied by perfusing the hearts
1983). Wistar rats used in this study were induced with diabetes using STZ (55 mg/kg, i.v.) and
kept for 2 weeks before sacrifice. The perfusion buffer contained 0.3 m M of V L D L - T G , 0.5%
BSA, 1.0 m M CaCl2 in 20 ml of Krebs-Henseleit buffer (pH 7.4). The perfusion buffer was
continuously oxygenated with 02:C02 (95:5%). The perfusion apparatus was a specially
designed, closed recirculating system with a double jacketed reservoir, heart chamber (to keep
the heart warm at 37°C), coil condenser to warm the buffer to 37°C, a bubble trap and an
injection port to collect the sample. The total perfusate volume of the apparatus (including the
tubing) was less than 30 mL. This miniature apparatus has enabled us to use small perfusion
volumes (15-20 ml) and diminished the loss of V L D L due to sticking to the surfaces (average
loss was 20% as determined by circulating the buffer containing V L D L through the apparatus
73
without the heart for 90 minutes). After the heart was secured through the aorta, Krebs-Ringer-
mode to clear the blood from capillaries. The perfusion was then switched to recirculating mode
and buffer containing V L D L - T G (0.3 mmoles/1) and B S A (0.5% w/v), and perfused for 90
minutes at the rate of 5.0 to 5.5 ml /min. The flow rate was kept constant with help of a
perfusion pump (Masterflex®, 7519-00, Cole-Parmer Instruments Co., IL, USA). The heart rate
was counted mannually at frequent intervals throughout the perfusion period. Samples were
collected through the injection port at 0 (just before the perfusate reaches the heart), 15, 30, 45,
60, and 90 minutes, placed on the ice and immediately extracted for TG. T G extraction was
performed according to a rapid extraction procedure described by Bligh and Dyer (1959). To a
400 pl aliquot of sample, 500 pl of chloroform, 1.0 ml of methanol followed by another 500 ul of
chloroform and 400 pl of distilled water was added with vortexing after each addition. Thus the
3300xg for 30 minutes of the extraction mixture resulted in separation of two layers: chloroform
layer at the bottom and an aqueous alcoholic layer at the top with an insoluble protein interface.
A n aliquot of 200 ul (in duplicate) of the chloroform layer which contains the T G moiety of
V L D L was added to A C S scintillation fluid and was counted for radioactivity using a liquid
In some of the perfusion studies, hearts were perfused with heparin (5 U/ml) containing
Krebs-Ringer-HEPES buffer (pH 7.4, 37°C) for 5 minutes to deplete functional LPL prior to
V L D L - T G clearance by the perfused heart. After heparin, hearts were perfused with buffer
(containing no B S A and V L D L ) to clear the heparin before commencing the perfusion with
VLDL.
74
3.3. P l a s m a measurements
After a 5 hour fasting period (0900-1400), blood samples from the tail vein were
collected in heparinized glass capillary tubes. Blood samples were immediately centrifuged
(3300 x g for 10 min at 4 °C) and plasma was collected and stored at -20 °C until assayed.
Plasma glucose, triglyceride, cholesterol and FFA levels were measured with kits (Boehringer
Mannheim Canada, Laval, Que.). Plasma insulin was measured using a rat insulin RIA kit.
3.4. Materials
Joklik minimal essential medium was obtained from Gibco Canada (Burlington, ON,
Canada), Iletin; Regular Beef and Pork insulin, Eli Lilly Canada (Toronto, Ont., Canada),
[ H]triolein was purchased from Amersham Canada (Oakville, ON, Canada), [ H]-oleic acid
3 3
from NEN Life Sciences Products (Boston, MA, USA), heparin sodium injection (Hapalean;
1000 U.S.P. U/ml) from Organon Teknika (Toronto, ON, Canada). CGS-21680, Research
purified chicken polyclonal antibodies against LPL purifiedfrombovine milk were a generous
gift from Dr. D.L. Severson (University of Calgary, AB, Canada), rabbit anti-chicken IgG;
Chemicon Corp, TG, Choi diagnostic kitsfromBoehringer Mannheim, Que., Canada, Bradford
protein assay kit from BioRad Laboratories, ON, Canada. Ready gel (4-20%); BioRad
Laboratories, CA, USA), NEFA-C kit (WAKO chemicals GmbH, Neuss, Germany), HiTrap®
column (Pharmacia, Sweden), K5F-TLC plates (Whatman Inc., NJ, USA), triolein standard
(Sigma Chemicals Co, St. Louis, MO, USA), Insulin RIA kit (Linco Research Inc., MO, USA).
75
3.5. Statistical analysis
A l l data are reported as mean ± standard error of mean (SEM) unless otherwise stated.
Unpaired Student's t test was used to determine differences between group means. Changes in
whole heart L P L activity over time in response to heparin was analyzed by multivariate A N O V A
followed by the Newman-Keul's test using the Number Cruncher Statistical System (NCSS®,
Kaysville, Utah). The level of statistical significance was set at p < 0.05.
76
4. R E S U L T S
4.1. H y p e r t e n s i o n study
Table 1 shows the chronological changes in general characteristics and plasma parameters
of SHR and W K Y rats. There were no marked differences between groups in body weight until
week 15, after which body weight in W K Y rats became significantly greater than SHR. At 7-8
weeks, the blood pressure of SHR rats was not significantly different from W K Y controls.
However, SHR blood pressure gradually increased over time reaching -200 mmHg at 11-12
weeks of age. Therefore, SHR represent a chronically hypertensive group by 15-16 weeks of age.
The blood pressure in 11-12 and 15-16 weeks of age groups was 74 mmHg higher in SHR than
in W K Y rats. We did not observe any difference in the heart weight/body weight ratio between
W K Y and SHR rats at 15-16 weeks of age ( W K Y 3.6±0.2; SHR 3.7±0.2, mg/g).
A l l plasma parameters were measured after a 5 hour fast (0900-1400). There was no
significant differences in plasma glucose or insulin levels between W K Y and SHR rats at any age
studied. Plasma T G and total cholesterol were significantly lower in SHR rats when compared to
W K Y animals at 7-8 and 15-16 weeks. However, plasma FFA concentrations in 15-16 week old
Retrograde perfusion of the isolated whole heart with heparin resulted in a release of LPL
into the coronary perfusate (Figure 4). The heparin-induced LPL discharge was rapid, and peak
77
activity that appeared within 2 minutes was suggested to represent L P L located at or near the
endothelial cell surface. On continuous perfusion with heparin, there was a progressive decline
rat hearts was comparable to values obtained from W K Y animals (Figure 4A, inset). However,
with increasing age and blood pressure, a decline in peak and total heparin-releasable L P L
activity was observed in SHR rats relative to W K Y animals (Figure 4B and 4C, insets).
of synthesis, cardiac myocytes from SHR and W K Y rats were isolated at different ages, and
surface bound and intracellular L P L activities were measured. There was no difference in
myocyte viability (% live cells) between the W K Y and SHR rats at 7-8 ( W K Y 79±3; SHR 81±1,
n=9 in each group) or 15-16 ( W K Y 83+1, n=7; SHR 81±2, n=8) weeks of age. Similarly,
myocyte yield (total number of cells x l O ) at 7-8 ( W K Y 1L+1.5; SHR 9.210.6) or 15-16 ( W K Y
6
9.3±1; SHR 8.8±0.9) weeks of age was similar in W K Y and SHR rats. No difference in total
cellular L P L activity was observed in SHR and W K Y rats at 7-8 weeks of age (Figure 5A, left
panel). However, at 15-16 weeks, myocytes from SHR rats had higher total cellular L P L activity
than cells from W K Y rats (Figure 5B, left panel). The increase in cellular L P L activity was not
accompanied by a parallel rise in the secretion of enzyme. At both 7-8 (Figure 5A, right panel)
and 15-16 weeks (Figure 5B, right panel) of age, there was no difference in heparin-induced
Based on reports that: a) insulin can release L P L from its binding sites on the surface of
cultured 3T3-L1 adipocyte cells (Chan et al 1988), and b) the presence of hyperinsulinemia in
SHR rats (Reaven and Chang 1991, Verma et al 1994), we hypothesized that the decrease in
endothelial L P L activity in SHR rats could be a result of elevated insulin levels in these animals.
However, our current results indicate that 5-hour fasted plasma insulin levels in SHR and W K Y
rats were similar at all time points measured (Table 1). Moreover, retrograde perfusion of
isolated rat hearts from 15-16 week old W K Y and SHR rats with insulin failed to release L P L
Nifedipine treatment in vivo effectively reduced the blood pressure of 15-16 week old
SHR rats (108±6 mm Hg, n=5) as compared to pre-treatment values (199±9 mm Hg, n=5) within
30 minutes. This effect lasted over 2 hours, at which point blood pressure returned to pre-
treatment values. Nifedipine had no effect on the blood pressure of 15-16 week old W K Y rats.
15-16 week old W K Y and SHR rats. Perfusion of isolated hearts from W K Y rats with nifedipine
did not change heparin-releasable LPL activity (Figure 7A). However, nifedipine administered
either in vitro for 10 min or in vivo for 30 min prior to removal of the heart increased peak
heparin-releasable L P L activity in SHR rat hearts (Figure 7B). A similar increase in heparin-
releasable L P L activity was observed in 15-16 week old SHR rat hearts on prior perfusion for 10
min with CGS-21680, a coronary vasodilator, in vitro (Figure 8). In vivo treatment of SHR rats
with CGS-21680 could not be performed due to its extremely short duration of action.
79
Nifedipine and CGS-21680 by themselves had no effect in releasing cardiac endothelial L P L
4.1.1.6. Secretion and clearance of circulating TGs and plasma lipolytic activity
In this study, SHR of all age groups were found to have lower plasma T G levels when
compared to age matched W K Y controls (Table 1). Therefore, we measured T G clearance rates
(TGCR) in 7-8 week and 15-17 week age groups. As shown in Figure 9, at 7-8 weeks the T G C R
of SHR is not different from W K Y . At 15-17 weeks, the T G C R of SHR appear to be less when
compared to W K Y . However, post-heparin plasma L P L activity in 15-17 week SHR rats was not
different from W K Y rats (Figure 10). Preliminary experiments suggest that T G secretion rate in
SHR is less compared to W K Y at 15-17 weeks of age ( W K Y = 1.43 mg/ml/min, SHR = 1.09
V
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81
W K Y and SHR rats at 7-8 (A), 11-12 (B) and 15-16 (C) weeks of age. Rats were
perfused with Joklik as described in the methods. At the time indicated by the arrow,
heparin (5 U/ml) was added to the buffer, and L P L activity measured in the coronary
perfusate which was collected for 10 seconds, at the indicated times. The insets
represents area under the curve for heparin-releasable LPL activity in perfused hearts
from W K Y and SHR rats. Results are the mean + S E M from the number of
500 -
2700 - i
400 -
5^1800
O
300 -
< 900 H
200 -
100 -
0 -
I T
0 2 4 8 10
B. 11-12 weeks
700 - i
- • - SHR(n-5)
- 0 - WKY(n=5)
600 -
500 -
2700
400 -
^1800 H
Mil
O
300 - < 900 H
200 -
100 -
0 -
"I 1 1 1
0 4 6 8 10
C. 15-16 weeks
700 - . - • - SHR(n=6)
- 0 - WKY(n=3)
600 -
500 -
2700 - i *
400 - T
••
^ 1800 -
O
300 -
< 900 -
200 -
o -
100 - \
0 -
T" ~l
0 2 4 6 8 10
Figure 5. Effect of hypertension on L P L activity in cardiac myocytes from SHR and W K Y rats
at 7-8 (A) and 15-16 (B) weeks of age. Myocytes were prepared as described in the
pellet and determination of cellular L P L activity (left panel). Heparin (5 U/ml) was
then added to the incubation medium of the homogenized cells at the time indicated
by the arrow and the release of L P L activity into the incubation medium determined
at the indicated times of incubation (right panel). Results are the mean ± S E M from
calculated by a Student's t-test; *,p < 0.05 relative to W K Y for cellular activity at
15-16 weeks.
A. 7-8 weeks
CELLS MEDIUM
3000 H h 600
2500
2000 h 400
1500 -
1000 - 200
SHR(n=9)
500 - G- WKY(n=9)
0 WKY SHR
0
0 0 10 20 30 40 50 60 o
B. 15-16 weeks
CELLS MEDIUM
o
3000 - o
h 600 a
CO
0 S
2500 -
2000 - 400
1500 -
1000 - h 200
SHR (n=8)
500
WKY (n=7)
0 WKY SHR
0
0 10 20 30 50 60
Incubation time (min) Figure
85
Figure 6. Lack of an acute effect of in vitro insulin on the release of L P L from isolated
perfused hearts of 15-16 week old W K Y and SHR rats. Various concentrations of
insulin were added to the perfusion fluid, and samples of coronary perfusate collected
700 -i SHR
WKY
600
500 H
Ins 100 ng/ml Ins 200 ng/ml Ins 500 ng/ml
2 400 H
300 H
200 H
100 H
j-e
oH
"T ~T~
0 10 20 30
Time (min)
Figure 6
87
Figure 7. The effect of nifedipine in vitro and in vivo on heparin releasable LPL activity in
perfused hearts from A) W K Y and B) SHR rats at 15-16 weeks of age. For the in
vitro experiments, hearts from 15-16 week old W K Y and SHR rats were removed
and perfused with recirculating Joklik buffer containing nifedipine (100 nM) for 10
minutes. This was followed by perfusion with heparin, with fractions of perfusate
collected and analyzed for LPL activity as described previously. For the in vivo
study, nifedipine (3 mg/kg) was injected into the tail vein. 30 minutes following
nifedipine injection, the rats were killed, the hearts removed and perfused with
heparin and the perfusate collected and analyzed for LPL activity. Results are the
Figure 7
89
Figure 8. Effect of in vitro treatment with CGS-21680 on heparin releasable LPL activity in
perfused hearts from SHR rats at 15-16 weeks of age. Hearts were perfused for 10
fractions were collected at the indicated times and LPL activity measured. Results
are the mean ± SEM; multivariate ANOVA followed by Newman-Keul's test was
used to determine the probablity value, *,p < 0.05 relative to untreated SHR.
S H R (15-16 weeks)
Figure
91
Figure 9. Plasma triglyceride (TG) clearance over a 60 minute following heparin injection (see
Methods section for details) for 7-8 weeks (A), and 15-17 weeks (B) 15-16 weeks
old SHR and W K Y and SHR nifedipine (SHRN) treated rats are shown. Heparin
(0.5 U/g body weight) was injected into the tail vein of pentobarbitol-anesthetized
rats and the blood samples were collected at 0, 1, 2, 5, 10, 30, and 60 minutes,
plasma was separated, and T G concentration was measured in each sample. The
different from W K Y rats (p<0.05) and + Significantly different from untreated SHR
(p<0.05).
92
Figure 9
93
Figure 10. Post heparin plasma lipase activity of 15-17 week old WKY and SHR rats. Heparin
(0.5U/g body weight) was injected into the tail vein of pentobarbitol-anesthetized rats
and the blood samples were collected at 0 and 20 minutes, plasma was separated, and
LPL activity was measured as a difference between total lipase and hepatic lipase
activities (See methods for details). The error bars represent standard error of means.
Figure 10
95
4.1.2. LPL regulation in fructose hypertensive rat hearts
Feeding fructose to Wistar rats, as a 10% solution in drinking water reduced food intake,
with a corresponding augmentation in fluid intake (Table 2). Weight gain over the acute and
chronic treatment periods was the same in both groups such that control and fructose-treated rats
had similar body weights at the time of death (Table 2). Whereas fructose feeding did not affect
plasma glucose concentrations (Table 2), plasma insulin (Figure 11A), and triglycerides (Figure
1 IB) were elevated in the acute and chronically treated rats. Blood pressure in fructose-treated
rats increased progressively, and was significantly different from the control group after 2 weeks
of treatment (Figure 11C). Prolonged fructose treatment for up to 6 weeks did not produce an
additional increase in blood pressure (Figure 11C). Moreover, withdrawal from fructose after a 6
week treatment period restored all of the above parameters to control values within 2 weeks
Retrograde perfusion of isolated whole hearts from control and fructose-treated rats with
heparin resulted in a rapid release of L P L into the coronary perfusate (Figure 12). At 2-weeks
after fructose treatment, there was no difference in heparin-releasable L P L activity between the
control and treated groups (Figure 12A). However, with prolongation of fructose treatment, a
significant reduction in heparin-induced release of LPL activity was observed (Figure 12B). This
to verify whether the decrease in functional L P L was due to a decreased synthesis at cardiac
myocytes. It appears neither myocyte surface L P L released by heparin over a 1-hour period, nor
intracellular L P L activity (measured in cell sonicate) was decreased in fructose treated rats
(Figure 13 A & B). This suggests that the decrease in functional L P L at coronary endothelium is
Fructose treatment induces hyperinsulinemia within 2 weeks in Wistar rats (Figure 11A).
Since in vitro insulin incubation was shown to release LPL from 3T3-L1 adipocytes (Chan et al
1988), we hypothesized that the decrease in endothelial L P L activity could be a result of greater
release of L P L by elevated insulin levels in these animals. However, our current results indicate
that retrograde perfusion of isolated whole hearts from control rats with insulin failed to release
L P L activity in control and chronic fructose-treated rats. Perfusion of isolated hearts from
control rats with nifedipine or CGS did not change the amount of heparin-releasable L P L activity
(Figure 15A). However, nifedipine administered for 10 min increased the basal, and heparin-
releasable L P L activity was observed in chronic fructose-treated rat hearts on prior perfusion of
hypertriglyceridemia. To test whether the reduction in L P L activity that we observed in the heart
is a generalized phenomena and a potential contributing factor towards the elevated plasma
triglycerides, pre- and post-heparin plasma L P L activity was determined from control and
chronic fructose-treated rats. Figure 16 shows that both basal and post-heparin plasma L P L
Acute Fructose Tx a
Chronic Fructose Tx a
The results are the mean ± S E M for the number of animals shown in parenthesis. Values are
those obtained before death. The acute treatment was for 2 weeks whereas the chronic treatment
was for a duration of 4-6 weeks.
99
Figure 11. Plasma insulin (A), triglyceride (B), and blood pressure (C) of acute (2 weeks) and
animals, datafromthese rats were pooled, and this group was hence defined as the
withdrawn) were subsequently maintained for an additional 2 weeks, after which they
hour fasted animals. Results are expressed as mean ± SEM. Probability values were
Figure 12. Heparin releasable LPL activity in perfused hearts from rats treated acutely (A) or
chronically (B) with fructose (Fructose Tx). Panel C represents animals that were
perfused with Joklik MEM as described in the methods. At the time indicated by the
arrow, heparin (5 U/ml) was added to the buffer, and LPL activity measured in the
coronary perfusate which was collected at the indicated times. Results are the mean
400 H
200 H
0
-2-0 0-2 2-4 4-6 6-10
a
u
400 H
200 H
0
-J
-2-0 0-2 2-4 4-6 6-10
PH
800 - i
C. Fructose-withdrawn Control (n=7)
Fructose (n=6)
600
400 H
200
0
-2-0 0-2 2-4 4-6 6-10
Time (min) Figure 12
103
Figure 13. A. Cell surface LPL activity on isolated cardiac myocytes from chronically fructose
treated (4-6 weeks) rats. To the cell suspension (0.4 x 10 cells), heparin (5U/ml)
6
was added to release surface bound LPL. 1.0 ml fractions were removedfromthe
addition of heparin. Medium was separated by centrifugation and the cell pellets
measured in cell sonicates of the cell pellets collected above at 0 and 10 minutes after
heparin incubation.
104
A . C e l l S u r f a c e LPL
Figure 13
105
Figure 14. Effect of increasing concentration of insulin (100, 200 and 500 ng/ml) and heparin (5
U/ml) perfusion on the release of LPL from isolated hearts of control Wistar rats.
Time (min)
Figure 14
107
Figure 15. In vitro effects of nifedipine and CGS-21680 on heparin releasable LPL activity in
removed and perfused with recirculating Joklik buffer containing nifedipine (100
nM) or CGS-21680 (10 pM) for 10 minutes. This was followed by perfusion with
9 0 0
1 A. Control Untreated (7)
+ Nifedipine (5)
+ CGS (5)
600
e 300 H
S
"o
s
e
08
0
u
- + Nifedipine (6)
+ CGS (4)
600 H
300
Figure 15
Time (min)
109
Figure 16. The effect of heparin (0.5 U/g) injection on the release of LPL in control and chronic
fructose treated rats. After collection of a blood sample for basal values, heparin was
injected into the tail vein and blood samples collected after 5 minutes. Blood was
centrifuged and plasma separated and used for the measurement of post heparin LPL.
Results are the mean ± SEM from the number of experiments indicated in
relative to control.
110
300
Control (n=4)
Fructose (n=5)
250 -A
200 H
150 H
20 -I
15
X
10
5 H
0
0 min 5 min
Figure 16
Ill
Induction of diabetes with 55 mg/kg STZ resulted in glycosuria (>4+). Body weight gain
over 2 weeks was reduced in D55 animals relative to controls, but there was no significant
difference in heart weight between control and diabetic rats (Table 3). The STZ injection caused
a reduction in plasma insulin levels that was accompanied by hyperglycemia. Both plasma T G
Retrograde perfusion of hearts from C O N and 2-week D55 rats with heparin resulted in
release of L P L activity into the coronary perfusate that was collected via the cannulated
pulmonary artery (Figure 17A). This heparin-induced L P L discharge was rapid, and peak
activity in both groups was observed within 1.5 min. On continuous perfusion of these hearts
with heparin, L P L activity returned to near basal levels. In D55 hearts, peak L P L activity in
coronary perfusate was 3-4 fold higher than C O N . To confirm that the elevated lipase activity
was specific to LPL, the assay was performed in the presence of 1M NaCl and in the absence of
apoCII, and under these conditions, peak activity from C O N and D55 was inhibited to 10-15% of
the total lipase activity measured above (CON = 253 [+LPL]; 29 [-LPL], D55 = 889 [+LPL]; 154
[-LPL] nmol/ml/h). The increase in L P L activity in the coronary perfusate of D55 rats was not
due to an increase in specific activity of the protein but was a consequence of a 4-fold increase in
L P L protein concentration as measured by ELISA (Table 4). Following heparin, the release of
L P L activity into the interstitial fluid was clearly different from that observed in coronary
1
112
perfusate (Figure 17B). Initially, the enzyme released from CON hearts was low, but gradually
increased over time. In D55 hearts, a peak release of enzyme was observed within 1-2 min
followed by gradual decline such that after 10 min, it was lower than CON.
Acute treatment of D55 rats with a rapid-acting insulin reduced hyperglycemia within 90
heparin-releasable LPL activity in the coronary perfusate was reduced, but remained higher than
control (Figure 17A). Although insulin had no effect on the prolonged release of LPL into the
interstitial fluid, the initial peak release observed in untreated diabetic rats was blunted by insulin
LPL regulation in the heart during diabetes was inconsistently reported in the literature.
The variability could be partly due to the experimental approach that did not distinguish various
LPL pools i.e., endothelial, interstitial, and myocyte-associated LPL pools. Therefore, we
determined total LPL activity in heart tissue homogenates. Indeed, we have shown that LPL
activity in whole heart homogenates was not different between CON and D55 groups (Figure 18)
observation that the augmented LPL in diabetic hearts was mainly localized at the endothelial
cells. Although the results are difficult to quantify, this technique provides information regarding
cellular localization of the LPL protein within the cardiac tissue. Whereas LPL
immunoreactivity was found throughout the control and diabetic myocardium (brown stain),
113
capillary blood vessels in the diabetic (Figure 19B) demonstrated a more intense LPL
immunoreactivity compared to control (Figure 19A). Staining was not seen when the primary
antibody was omitted or replaced by preirnmune chicken serum (data not shown).
Induction of diabetes with 100 mg/kg STZ produced the characteristic triphasic pattern of
changes in blood glucose and insulin in the 24-hour period immediately following injection
(Figure 20). An initial brief hyperglycemia was followed by a period of hypoglycemia that is
brought about by a release of insulin from damaged (3-cells. Blood glucose rose gradually with a
corresponding decline in plasma insulin, and hyperglycemia (>13 mmol/L) was usually attained
within 12-16 hours. Interestingly, even at this early stage of diabetes, peak (Figure 21) and total
(calculated as area under the curve over 10 min, Figure 21-inset) heparin-releasable LPL activity
in the coronary perfusate was increased when compared to control rats. Prolonging the
hyperglycemia for a further 3 or 6 hours (after glucose levels reached >20 mmol/L) maintained
Treatment of D100 rats for 1 week with a long-acting insulin resulted in an increase in
body weight and a normalization of fluid intake (Figure 22), plasma glucose and insulin (Figure
23) levels. Subsequent withdrawal of insulin produced an increase in plasma glucose from 24
hrs following the last injection. On reaching a glucose concentration of 13 mmol/L, diabetic
animals were kept for a further 6 (D100-6h) or 24 (D100-24h) hours before they were killed. At
termination, both the D100-6h ahd D100-24h groups were hyperglycemic and hypoinsulinemic
114
(Figure 23). As observed with the insulin depletion study, even a brief duration of 6 hours of
perfusate that remained elevated after 24 hours of hyperglycemia (Figure 24). Total LPL activity
was similarly high at these time points (Figure 24 inset). To examine whether the enhanced
coronary LPL activity in D100-24h rats was accompanied by a parallel increase in myocyte LPL,
isolated myocytes were incubated in the presence of heparin to measure both surface-bound and
secreted LPL. There was a significant reduction in heparin-releasable LPL from cardiac
myocytes (Figure 25) and a decrease in LPL activity in cell sonicates (Figure 25-inset) from
4.2.2.4. Fasting
Fasting is known to increase cardiac heparin releasable LPL activity without affecting
mRNA levels or protein synthesis. In the present study, overnight fasting for 16 hours reduced
plasma insulin to diabetic levels (CON 2.1±0.1, FAST 0.5±0.0 ng/ml), with no effect on plasma
glucose (CON 7.0±0.1, FAST 6.0±0.1 mmol/1). Fasting caused a two-fold increase in heparin-
releasable LPL activity at the coronary lumen (Figure 26A). However, unlike in acute diabetes,
overnight fasting had no effect on the release of enzyme into the interstitial fluid (Figure 26B).
115
Table 3. Characteristics of Diabetes (55 mg/kg STZ) at 2 Weeks after STZ Injection
Control STZ-Injected
Data are means 1 SEM for the 6-8 control and diabetic rats. Values are those obtained before
death. Plasma parameters were from fed rats. Blood was collected from the tail vein in
heparinized tubes that were centrifuged for the separation of plasma. Insulin was measured using
rat insulin standards. Probability values were calculated by a Student's t-test; * Significantly
coronary perfusate (A) and interstitial fluid (B) from C O N and D55 rat hearts. Hearts were
perfused (7-8 ml/min) with heparin (5 U/ml) in Krebs-Ringer Hepes buffer using a modified
Langendorff retrograde perfusion technique that separates the coronary perfusate from interstitial
fluid. The coronary perfusate samples (for 10 seconds) were collected at the indicated time
points. As interstitial fluid represents only 1-2% of the heart perfusate, samples were collected
over 60 seconds, at the indicated time points. Coronary and interstitial L P L activities were also
measured in insulin treated (90 minutes before killing) D55 rats. Results are the mean ± S E M for
6-8 rats in each group. Probability was calculated using multivariate A N O V A followed by
Newman-Keul's test, *Significantly different from control and insulin treated D55 rats, P<0.05.
117
A
800 -i
600
400
200
r n I i
0 2 8 10 12
B
A CON (n=8)
800 D55 (n=6)
O D55 +1 (n=6)
600
400 H
200 H
0 H
n i i 1 1 1
•1-0 1-2 3-4 5-6 7-8 9-10
Table 4. L P L Activity, Mass and Specific Activity in Coronary Perfusate From Hearts
(mU/ng)
Measurement of LPL catalytic activity (by in vitro hydrolysis of [ H]triolein) and mass
3
(by enzyme-linked immunosorbent assay) were done in pooled coronary perfusate samples (24
ml) collected between 1-3 min after heparin perfusion. For the ELISA, coronary perfusate
samples were lyophilized and resuspended in 0.2 ml H2O. 100 pl aliquots of the samples were
then diluted in PBS and added to the polystyrene microtiter plate wells coated with 100 pl of
anti-LPL antibody. 1 mU is defined as the amount of enzyme catalyzing the release of 1 nmol
oleate per min. Results are the mean ± SEM of 6-8 control and diabetic rats. Probability values
Figure 18. LPL activity in heart homogenates. Hearts from CON and D55 were frozen
immediately in liquid N2, after clearing the blood of the capillaries. Tissues were
homogenized in the presence of heparin and the clear supernatant was separated from
the tissue debris by centrifugation (see Methods section for details). Protein
activity was normalized for gram protein present in the samples and expressed as
Figure 18
121
following chronic diabetes induced with 55 mg/kg STZ. Cross sections of control
(A) and diabetic (B) ventricles were fixed and then immunolabeled using an antibody
specific for LPL. Low magnification image shows immunostaining over the entire
control and diabetic sections. However, in diabetic hearts, numerous capillaries were
heavily stained for LPL (arrow, B) in contrast to the observations in control heart
sections (A).
12
A. C O N T R O L
Figure
123
Figure 20. The chronological changes in plasma insulin (left panel) and glucose (right panel)
levels during a 24-hr period following an IV injection of 100 mg/kg STZ. The
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Figure 20
125
Figure 21. Peak heparin-releasable LPL activity in coronary perfusate at different time points
LPL activity in coronary perfusate from control and diabetic rats following the
hyperglycemia (13 mmol/L) within 12-16 hours. Some animals were killed at this
time (13mM+0h) or kept for a further three (13mM+3h) or six (13mM+6h) hours
before being used for the measurement of LPL activity. Hearts were perfused with
Krebs buffer containing heparin using the modified Langendorff procedure. The
inset shows the AUC of LPL released over 10 minute period. Results are the mean ±
SEM for 6-8 rats in each group. Probability values were calculated by ANOVA,
13 m M - O h 13 m M - 3 h 13 m M - 6 h
Figure 21
127
Figure 22. Effect of insulin treatment in STZ diabetic rats. Animals were made severely
diabetic with 100 mg/kg STZ (D100), and then treated subcutaneously with a long-
acting Ultralente insulin (D100+I) once daily. An insulin dose of-18-20 U/kg/day
was continued for 7 days. Body weight (A) and fluid intake (B) were measured
before injecting insulin. Results are the mean ± SEM for 6-8 rats in each group.
Figure 22
129
Figure 23. Plasma glucose (A) and insulin (B) levels of control (CON) and diabetic rats
animals had been well-controlled (D100+I), insulin treatment was stopped, and
plasma glucose monitored. Following the last insulin injection, plasma glucose
mmol/L was considered a hyperglycemic value, and when this level was reached,
diabetic animals were kept for a further 6 (D100-6h)or 24 (D100-24h) hours before
they were killed. Results are the mean ± S E M for 6-8 rats in each group. Probability
20 -,
4 -i
3
CO
C
1 H
o
CON D100+I D100-6h D100-24h
Duration of hyperglycemia
following withdrawal
Figure 23
131
Figure 24. Peak heparin-releasable LPL activity in coronary perfusate from control (CON) and
(100 mg/kg STZ) rats was measured after the animals had been well controlled with
hyperglycemia after insulin injection was stopped. Hearts were perfused with Krebs-
Results are the mean ± SEM for 6-8 rats in each group. Probability values were
900 -i
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i-
CON
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D100+I
D100
I 300
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83
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CON D100+I D100-6h D100-24h
Figure 24
133
Figure 25. LPL activity in cardiac myocytes from control (CON) and 24 hour hyperglycemic
rats following insulin withdrawal (D100-24h) are shown. Myocytes were prepared as
the time indicated by the arrow and the release of surface-bound LPL activity into the
medium determined at the indicated times of incubation. Results are the mean ±
SEM for 6-8 rats in each group. Probability values were calculated by multivariate
ANOVA. Inset shows intracellular LPL activity from CON and D100-24h groups.
control, PO.05.
134
Figure 25
135
Figure 26. Effect of fasting on heparin-releasable LPL activity in coronary perfusate (A) and
interstitial fluid (B). Control rats were fasted for 16-hrs (1800-1000) during which
food was withdrawn from the animals, but they had free access to water. Hearts were
perfusate from interstitial fluid. The coronary perfusate samples (over 10 seconds.)
were collected at the indicated time points. As interstitial fluid represents only 1-2%
of the heart perfusate, samples were collected over 60 seconds at the indicated time
points. Results are the mean ± SEM for 6-8 rats in each group. Significantly
+
Figure 26
137
4.2.3. H y d r o l y s i s o f V L D L i n the d i a b e t i c h e a r t
In fed control rats, 75% of total TG was present in TGRL (VLDL + CHYL), and the
remaining 25% was present in LDL and HDL fractions (Figure 27A,B&C). VLDL contributed
the least to the total cholesterol pool which was present predominantly in the HDL (61% of total
cholesterol) and LDL (30%) fractions. Diabetic rats had higher levels of TG and cholesterol
compared to control animals. When compared to CON, D55 showed higher concentrations of
TG as well as cholesterol in their TGRL fraction. In both LDL and HDL, there was no difference
in cholesterol and TG concentrations between CON and D55 groups indicating that the increase
in TG levels in D55 is mainly due to an increase in TGRL. However, fasting for 16 hours
Apolipoproteins in VLDL obtained from fed CON and D55 rats were analyzed by gel
electrophoresis, and a representative gel from a CON and a D55 rat is shown in Figure 33.
Smaller ApoB (>200 kDa), ApoE (~ 35 kDa), ApoA-I (-28 kDa) and ApoCs (-8-9 kDa) were
identified with the help of molecular weight standards (6.5 kDa to 220 kDa) that were
electrophoresed in parallel lanes 1, 5, and 6 (Figure 33). A sample comparison of CON and D55-
hypertriglyceridemia. To test whether the elevated plasma triglycerides in diabetic rats result
138
from a decrease in LPL activity, basal (before heparin injection) and post-heparin plasma was
obtained from CON and D55 rats at 2 weeks after diabetes induction. Neither basal nor post-
heparin plasma LPL activity was reduced relative to control at 2 weeks after diabetes induction
(Fig. 28).
20 mU of bLPL and the saturable substrate (VLDL-TG) concentration was 0.3 mM when
incubated for 30 minutes at 37°C (Figure 29A&B). Incubations for 2 hours indicate that VLDL-
TG hydrolysis by bLPL was not different between CON and D55. However, D55-TGRL
appeared to have a lower lipolysis towards the end of 2 hour incubation (data not shown). A
double reciprocal plot of 1/[V] versus 1/[S] is shown in Figure 30A. The intercept on the Y axis
gives V max whereas the X-axis intercept gives K values for this lipolytic reaction. The apparent
m
K values for CON and D55-VLDL were 0.15 mM, and 0.26 mM, respectively, whereas V
m max
values were 20.4 mU and 31.3 mU FFA released for CON and D55 VLDL, suggesting that
diabetic VLDL was hydrolyzed as effectively as VLDL obtained from control animals.
Similarly, K and V
m max for TGRL hydrolysis by bLPL, were the same using lipids from CON and
D55 rats (Figure 30B). As shown by Mamo et al (1992), in the present study, our preliminary
results indicate that VLDL obtainedfromCON and D55 rats may not be differentfromeach
other.
Given the possibility that the interaction between VLDL and rat LPL could be different
from that of bovine LPL, we purified LPL from CON and D55 post-heparin plasma. Figure 31 is
a representative elution profile of LPL and hepatic lipase (another major lipase released into
plasma in response to intravenous heparin injection) indicating that hepatic lipase elutes at
139
approximately 0.8 M NaCl while LPL elutes at 1.4 M NaCl. Fractions enriched with CON and
D55 LPL (>90%) were adjusted to have equivalent activities and subsequently used for in vitro
lipolysis of VLDL-TG obtained from CON and D55 rats. Our preliminary results indicate that
the lipolysis of diabetic VLDL-TG was not decreased (Figure 32), suggesting that at this duration
of diabetes, either LPL or VLDL characteristics with respect to their enzyme-substrate interaction
4.2.3.4. 3
H- VLDL clearance by isolated CON and D55 hearts
We next attempted to assess whether the D55 heart could clear [ H]VLDL faster than CON
3
hearts. Having established that control and diabetic VLDL were essentially similar, CON and
D55 rats were only perfused with control [ H]VLDL. Figure 34 compares the rate of [ H]VLDL-
3 3
TG clearance from re-circulating buffer containing [ H]VLDL-TG when perfused through CON
3
and D55 hearts by Langendorff method. Metabolism of [ H]VLDL by the isolated CON heart
3
was slow (net loss 17%; rate 0.50±0.17 nmol/min, n=4), but comparable to previously reported
values (O'Looney et al 1983). In contrast to CON rat hearts, the rate of disappearance of VLDL-
TG (over a perfusion period of 90 minutes) was more rapid from D55 rat hearts (net loss 44%;
rate 0.93±0.23 nmol/min, n=4). It has been suggested that LPL can detach from the endothelium
recirculating VLDL, enzyme activity was also determined in the buffer chamber after 30 minutes
of perfusion of unlabeled VLDL. Perfusion of control and diabetic hearts with unlabeled VLDL
did not result in detachment of LPL from its binding sites (n=l from CON and D55 group, data
not shown). The increased VLDL-TG hydrolysis was essentially abolished by prior perfusion of
the diabetic heart with heparin, implicating LPL in this process (Figure 35, inset). In all of the
140
above groups, heart rate remained unchanged (above 150 beats per minute) throughout the
perfusion period.
141
Table 5. Fed and fasted plasma parameters of control and diabetic rats
Fed Fasted
plasma free fatty acid. Results are average of 6 animals from each group. Probability values were
Figure 27. TG and cholesterol concentration in A. TGRL, B. LDL and C. HDL fractions
separated from the serum of fed control (CON) and diabetic (D55) rats. TG and
Results are an average of six animals from each group, error bars indicate SEM.
Figure 27
144
Figure 28. Post-heparin plasma LPL activity from CON and D55 rats. The LPL activity was
determined as a difference between total lipase (LPL + HL) activity and HL activity
Figure 28
146
Figure 29. In vitro lipolysis of (A) V L D L (0.3 m M TG) versus bovine L P L (increasing
concentrations of 10, 20, 50, or 100 mU) and (B) 20 m U of bovine L P L versus
Incubations were carried out for 30 minutes at 37°C and pH 8.2. (For details please
Figure 29
148
Figure 30. Double reciprocal plot of CON and D55 (A) VLDL-TG, (B) TGRL concentrations
(1/[S]) versus rate of lipolysis by LPL (1/V). 20 mU of LPL was exposed to various
concentrations of VLDL-TG (fasted serum) and TGRL (fed serum) obtained from
CON and D55 serum. Incubations were done at 37°C and at pH 8.2. Each data point
activity.
149
A. VLDL
B. TGRL
1/[S] ( m M )
1
Figure 30
150
Figure 31. A representative elution profile of L P L and H L from heparin sepharose affinity
columns. L P L elutes at higher salt concentrations (-1.4 M NaCl) and hepatic lipase
nmol/ml/min]
151
Figure 31
152
Figure 32. In vitro lipolysis of CON and D55 VLDL-TGs (0.3 mM) by rat LPL: (A) CON-LPL
and by (B) D55-LPL. FPLC fractions enriched with LPL (>90%) was adjusted to 20
earlier. Results are average of two separate experiments carried out in triplicate.
153
A. CONTROL LPL
80 -i
D55
60
CON
40 H
20 H
0
VLDL
PH
B. DIABETIC LPL
80 D55
60 H
CON
40 H
20 H
VLDL
Figure 32
154
C O N and D55-TGRL (obtained from fed rats). Lanes 1, 5, and 6 are molecular
weight standards. Lanes 2, 3, and 4 are D55-TGRL (triplicate) and lanes 7, 8, and 9
their protein content (3.2 mg/ml) and equal volumes were loaded into the wells.
Diabetic Control
1 2 3 4 5 6 7 8 9
Figure 33
156
hearts from C O N and D55 rats. Hearts were perfused with Krebs-Ringer-HEPES
mode for 90 minutes. Perfusate was collected at 0, 15, 30, 45, 60 and 90 minutes,
extracted for T G and the radioactivity was counted. Results are the average of 4
animals in each group. Error bars represent SEM. * Significantly different from
minutes (n=l).
Figure 34
158
5. D I S C U S S I O N
During hypertension, LPL activity is decreased in skeletal muscle and adipose tissue in
human patients (Pollare et al 1991, Marotta et al 1995) and Dahl salt-sensitive hypertensive rats
hypertensive SHR rat heart. To this end, we measured the rapid, heparin-releasable L P L fraction
in whole hearts obtained from W K Y and SHR rats. This rapidly releasable fraction is more
sensitive to altered physiological (e.g. feeding, fasting) and pathological (e.g. diabetes,
hyperthyroidism) states than total tissue activity. More importantly, the ability of the heart to
hydrolyze lipoprotein-TG is closely linked to changes in the rapidly releasable LPL, but not to
the remaining cellular fraction (Eckel 1989). Hence, any change in cardiac L P L activity during
hypertension may have significant implication in altering the supply of F F A to the heart. In this
report, we show for the first time that coronary heparin-releasable L P L activity is decreased in
15-16 week SHR rat hearts when compared to age-matched W K Y animals, and propose that this
Insulin has been cited as an important factor affecting L P L activity in vivo. For example,
in normal weight subjects, hyperinsulinemia combined with insulin resistance was associated
with a reduced L P L activity in skeletal muscle (Pollare et al 1991) and post-heparin plasma
(Knudsen et al 1995). In addition, Marotta et al. (Marotta et al 1995) recently observed a strong
correlation between serum insulin and post-heparin plasma L P L activity in mild hypertensive
patients. Since SHR rats have been characterized as hyperinsulinemic (Reaven and Chang 1991,
159
Verma et al 1994), we considered the possibility that the reduced heparin-releasable L P L activity
in SHR rat hearts was secondary to high insulin levels. However, we found that insulin levels in
the 5 hour fasted SHR rats were not significantly elevated when compared to W K Y rats of the
same age. Similarly, other studies have reported no evidence of hyperinsulinemia in SHR rats
contradictory findings in the literature with regard to the state of insulinemia in SHR animals
could be explained by differences such as the age group studied and procurement of animals
Christe and Rodgers 1995). Insulin was previously reported to directly release L P L from its
binding sites on 3T3-L1 adipocytes (Chan et al 1988), an effect suggested to involve the
membrane anchor. However, in this study, insulin did not displace endothelial-bound L P L into
the coronary perfusates. Similarly, LPL was not released by insulin from isolated control and
C from bovine aortic endothelial cells (Saxena et al 1991). Moreover, it was recently reported
Thus, it appears that neither a direct effect of insulin nor the presence of a hyperinsulinemic state
could account for the lowered heparin-releasable cardiac L P L activity in SHR rats.
activity may be due to decreased cellular L P L content or secretion. However, both cellular and
secreted L P L activities in this study do not indicate a diminished L P L synthesis in SHR cardiac
myocytes. A recent study observed that there was a duration dependent decrease in L P L m R N A
levels in SHR rat hearts (Masuzaki et al 1996). However, in this study, cardiac L P L activity and
160
protein mass were not measured. Since a dissociation between LPL-mRNA levels and enzyme
activity was observed previously (Bergo et al 1996a), it is not known whether the decrease in
LPL-mRNA level was actually accompanied by a decrease in LPL synthesis and activity.
Moreover, the above study was performed in whole heart homogenate that does not distinguish
the lower heparin-releasable LPL activity in SHR rat hearts could be related to the hypertensive
state per se. A positive correlation between plasma LPL activity and aortic flow velocity has
been reported in mild, uncomplicated hypertensive patients (Marotta et al 1995). Factors such as
poor peripheral blood flow, vascular hypertrophy and rarefaction of blood vessels have been
proposed to affect LPL action during hypertension by reducing vascular surface area for enzyme
binding sites and/or impeding the delivery of substrate (Lind and Lithell 1993). We examined
whether the increased perfusion of coronary vasculature could enhance the heparin-releasable
suggesting that flow through coronary blood vessels may regulate LPL activity. In support of
these findings, the reduced clearance of a circulating chylomicron-like emulsion in SHR rats was
improved with doxazosin (an a.\ blocker) (Mackintosh et al 1991), whereas a 4-fold increase in
post-heparin plasma LPL activity was observed in obese Zucker rats following treatment with the
SHR rats have previously been reported to be hypertriglyceridemic (Reaven and Chang
1991, Reaven and Ho 1991) a finding clearly opposite to that obtained in the present study. The
discrepancy in plasma TG between this and previous studies occurred despite identical
161
experimental parameters, i.e., the age of animals, and duration and time of fasting. Reduced
levels of serum T G and cholesterol in SHR rats relative to normotensive Wistar or W K Y rats
have similarly been reported (Iritani et al 1971, Tonooka et al 1985). The reasons for
hypotriglyceridemia in SHR rats are presently less clear. Plasma T G levels generally represent a
balance between production (from the liver and gut) and removal (lipolysis of TG-rich
coupled with a low P H P L A in older SHR rats. The decreased T G C R could be as a result of
vasoconstriction restricting L P L action in SHR rats (Ogawa et al 1991). Although, low TGSR
appeared to have greater influence on plasma T G levels in SHR rats, more studies need to be
Although plasma F F A levels are significantly higher in SHR rats, the approximate
contribution of different sources of F F A (from the circulation or intracellular sources) towards (3-
oxidation in the SHR rat heart is not known. We speculate that the reduced cardiac L P L action
coupled with low circulating T G levels, could significantly diminish F F A supply to the heart via
the exogenous pathway. In support of our hypothesis, it was demonstrated that the SHR heart
economy amidst adverse hemodynamic conditions (Christe and Rodgers 1994). Further studies
model like the fructose-hypertensive rat. As reported earlier, fructose feeding through drinking
aldehydes), activation of the renin-angiotensin system, etc., were shown to be some of the factors
responsible for elevated blood pressure in response to fructose feeding (Iyer et al 1996, Verma et
To our knowledge this is the first report which demonstrates that coronary functional LPL
Contrary to our chronic studies, short term fructose feeding for example, 4 days fructose
treatment did not affect LPL activityfromthe perfused heart or total cellular and surface-bound
LPL in isolated cardiac myocytes (Liu and Severson 1995). Similarly, acute fructose loading did
not alter the fasting induced increase in cardiac LPL activity (Pedersen and Schotz 1980).
Resultsfromthe present study confirm that in rats treated for 2 weeks with fructose, there was no
change in cardiac heparin-releasable LPL activity. Therefore, it appears that the decreased
cardiac heparin releasable LPL activity that was observed during chronicfructosefeeding in our
study may not be a direct effect of fructose but probably due to metabolic changes like
animals.
Since hyperinsulinemia combined with insulin resistance has been implicated in LPL
deficiency, we considered the possibility that the lower cardiac LPL infructose-fedrats was
secondary to hyperinsulinemia in these animals. However, our results indicate that 2 week
163
fructose treated rats do not demonstrate any change in LPL activity, despite being
hyperinsulinemia Further, similar to the SHR study, isolated whole heartsfromWistar rats did
not release LPL when perfused with insulin. Therefore, hyperinsulinemia may not have
treated rats paralleled the duration of the hypertensive state. This decrease in functional LPL
activity was not due to a decrease in LPL synthesis at the cardiac myocyte. Additionally, as
increase in blood pressure and decrease in cardiac LPL activity, an association between cardiac
LPL and hypertension can be suggested. Therefore, we performed in vitro treatment with
animals. Indeed, in vitro perfusion of fructose hypertensive rat hearts with nifedipine or CGS-
21680 increased heparin-releasable LPL activity. These results confirm our previous findings in
the SHR rat heart. Together, these results suggest that poor perfusion through coronary blood
hearts. Hence, it would appear that the hemodynamic state may be important in the regulation,
not only of the amount of LPL available for its physiologic function, but also for the delivery of
increased hepatic secretion of VLDL-TG (Herzberg and Rogerson 1988) or a decreased removal
result from structural changes in VLDL (Mamo et al 1991) or defective LPL activity. In a
164
previous study, post-heparin plasma lipolytic activity infructose-fedanimals was reported to be
similar to that of controls (Iwata et al 1990). However, Spirulina platensis (an agent which
significantly increases LPL activity in post heparin plasma) decreased fructose induced
fructose-fed animals. Our results clearly show thatfructosetreatment for 6 weeks reduced basal
and post heparin plasma lipolytic activity. Hence, impaired TG clearance after chronic fructose
treatment could be a combined effect of structural changes in VLDL together with altered post
heparin-releasable LPL activity in rats. Moreover, this effect is not due to a decreased synthesis
of the enzyme in the myocytes but probably due to the development of hypertension per se. The
impact of a reduced heparin-releasable LPL activity on FFA supply and utilization in the fructose
5.2. L P L d u r i n g diabetes
Recently, using the conventional Langendorff perfused heart, we determined that peak
heparin-releasable LPL activity was augmented in the diabetic rat (Rodrigues et al 1997). This
rapidly releasable LPL pool was believed to represent the endothelium-bound enzyme fraction.
However, heparin can diffuse through the arterial wall (Lovich and Edelman 1995). Thus, the
conventional heparin-perfused Langendorff heart may release LPL not only bound to the luminal
side of the endothelium but also the enzyme present within the endothelial cell, and at the
subendothelial, interstitial and myocyte cell surfaces. Indeed, using a modified Langendorff heart
165
perfusion that separates coronary from interstitial fluid, heparin was found to release L P L from
both compartments (Jansen et al 1980). In the present study, we used the modified Langendorff
heart to establish that in diabetes, the increase in LPL activity or protein originates mainly from
this finding and verified that despite widespread labeling of the enzyme within control and
blood vessels of the diabetic heart. In vivo administration of insulin to the diabetic rats 90
minutes before the experiment, reversed the coronary L P L activity to levels closer to that of
control. This short duration of insulin treatment however, did not increase the depressed
interstitial L P L but further obliterated the initial peak-release that was observed in untreated
diabetic rats. This reversal by insulin supports the fact that the altered L P L activity in the
diabetic heart is not due to a non-specific cytotoxic effect of STZ but due to the diabetic state per
se.
In the heart, capillary endothelial L P L is largely derived from cardiac myocytes that
we had hypothesized that the enhanced heparin-releasable L P L activity in diabetic rat hearts
could be due to an increased synthesis of the enzyme, myocyte L P L in these animals was found
of control hearts with heparin, there was a progressive increase in the discharge of L P L into the
interstitial fluid indicating that heparin could conceivably cross the capillary wall and release the
enzyme from the extracellular space and myocyte surface. Interestingly, in diabetic hearts, there
was a peak release of L P L into the interstitial fluid within 1 -2 min following heparin perfusion,
implying an accumulation of the enzyme at or near the endothelial cell, on the abluminal side.
Moreover, release of L P L into the interstitial fluid of the diabetic hearts was depressed following
166
prolonged heparin perfusion, an observation that is congruent with the previously reported
Insulin regulates LPL, but its effects vary depending on the tissue being investigated.
Thus, elevated levels of insulin in vivo (either postprandial or following an euglycemic clamp)
(Eckel 1987, Sadur and Eckel 1982) or in vitro (Ong et al 1988) increases L P L activity in adipose
tissue. In the heart, administration of insulin in vivo to control rats increases heparin-releasable
L P L activity in isolated cardiac myocytes within one hour. However, incubation of control
myocytes with insulin in vitro has no effect on L P L activity indicating that in the heart, additional
metabolic factors must be required for the regulation of L P L (Braun and Severson 1991 & 1992).
activity in cardiac cells. Indeed, in this study, a brief duration of hyperglycemia (24 hours)
reduced both heparin-releasable and intracellular LPL activity in cardiac myocytes. However, as
demonstrated previously in 2-week diabetic rats (Rodrigues et al 1997), even short-term diabetes
(within hours) increased capillary luminal L P L stores. These results indicate that in the heart, an
acute reduction in insulin has distinct and immediate regulatory effects on L P L at two levels: a
Hypoinsulinemia can also be induced by fasting which enhances cardiac and skeletal
muscle L P L activity but reduces L P L in adipose tissues (Rodrigues et al 1992, Semb and
posttranslational and did not involve altered mRNA levels, protein synthesis or specific activity
of the protein (Doolittle et al 1990). As previously reported using the modified Langendorff
reported that the primary effect of fasting on distribution of LPL occurred at the surface of
activity in the interstitial fluid of fasted rats remained unchanged and is consistent with the
finding that fasting does not alter LPL synthesis in the myocyte (Doolittle et al 1990). As the
degree of hypoinsulinemia was comparable between fasted and diabetic rats, it appears that
although hypoinsulinemia alone can enhance endothelial LPL activity, it may not entirely
influence the synthesis of LPL. Hence, other short-term diabetes-related factors may be
At present, the mechanism(s) by which insulin regulates LPL at the vascular endothelial
cell is not known. Endothelial LPL is regulated by detachmentfromits HSPG binding sites into
HSPGs associate with endothelial cells via their core proteins or a glycosylphosphatidylinositol
(GPI) linkage (Yanagishita and Hascall 1992) and cleavage of the GPI anchor by insulin-
sensitive phospholipases could release HSPGs, and hence LPL (Eckel et al 1978, Spooner et al
1979, Chan et al 1988). Provided this mechanism operates in vivo in the heart, hypoinsulinemia
would enable the enzyme to remain attached to its endothelial binding site. In perfused guinea
pig hearts, LPL moves from its site of synthesis in the parenchymal cells to the endothelial
surface within 30 min (Blanchette-Mackie et al 1989a, Liu and Olivecrona 1991). Thus, the
enhanced capillary LPL pool in diabetic rats could involve an accelerated translocation of LPL
from myocytes to the lumen (Rodrigues et al 1997, Stins et al 1992). It should be noted that the
reported that when the heparin-releasable LPL pool was allowed to recover for 1 hr after removal
168
of the enzyme, diabetic hearts continued to demonstrate a higher peak LPL activity after a second
heparin perfusion (Rodrigues et al 1997). Finally, the amount of luminal LPL can be regulated
by the endothelial cell. This process involves the internalization and recycling of LPL to the cell
surface, thereby allowing endothelial cells to maintain an additional pool of the enzyme at the
vascular endothelium (Saxena et al 1990). Alterations in pH can bring about dissociation of LPL
from its binding site, with less detachment of cell surface bound LPL at pH 5.5 compared with
pH 7.4 and 8.5 (Saxena et al 1990). Hence, the assumption is that inside the endothelial cell, an
acidic pH would permit LPL to remain bound to proteoglycans, and thus promote recycling of
(Feuvray and Lopaschuk 1997), it is possible that changes in pH in endothelial cells may
(due to a depleted post-heparin plasma lipolytic pool of LPL). However, TG secretion rates in
control and diabetic rats have been reported to be comparable (Chen et al 1979, Hirano et al
1991) and post heparin plasma lipolytic activity in this and other (Chen et al 1980) studies was
not significantly reduced in diabetic rats. The possibility remained that changes in structure of
lipoproteins could explain this hypertriglyceridemia. Several in vitro and in vivo studies in
clinical and experimental diabetes have shown that VLDL and chylomicrons undergo changes in
structure and composition such that they may become poorer substrates for LPL (Bar-On et al
1984, Hirano et al 1991, Levy et al 1985, Mamo et al 1992, O'Looney et al 1985). If VLDLs and
chylomicrons are altered during diabetes, another question of overriding importance is whether
169
or not the diabetic heart (with its augmented LPL activity) is in fact capable of metabolizing
these TG rich lipoproteins. In the present study, we were unable to detect any difference in the
between control and diabeic rats. Thus, VLDL prepared from diabetic rats appears to be a
normal substrate for LPL and impaired lipolysis of these particles may not be a causal factor
As the diabetic rats in the present study were hypertriglyceridemic in the fed but not in
the fasted state, it is likely that excessive accumulation of triglycerides occurs as a result of
lipoprotein. Mamo et al. (1992) have demonstrated that lipoproteins (plasma lipoprotein fraction
of density < 1.006 gm/ml that includes chylomicrons and VLDL) of diabetic origin are hydrolysed
by LPL at a significantly slower rate than lipoproteins from normal rats. However, as fasting for
16 hours prior to recovery of lipoproteins eliminated this difference in rates of lipolysis, the
authors concluded that during diabetes, the lipolytic defect might be specific for chylomicrons.
Interestingly, [ C]chylomicrons obtained from STZ diabetic rats when intravenously injected
14
into control animals disappeared from the circulation at a significantly slower rate than similarly
from diabetic animals in our study are able to clear chylomicrons efficiently remains to be
answered. However, an earlier study has reported that despite poor peripheral clearance of
chylomicrons, cardiac tissuefromdiabetic rats exhibit a 3-fold higher uptake of this lipoprotein
When radiolabeled VLDL-TG were perfused through the isolated heart and the decline in
triglyceride radioactivity monitored with time, diabetic hearts metabolized VLDL at a faster rate
than that observed in control rat hearts. This increased VLDL-TG clearancefromthe diabetic
170
heart is in agreement with the enhanced L P L activity and mass that was reported previously
removal of this lipoprotein by the diabetic heart (Wyne et al 1996). Moreover, the increased
clearance of V L D L from the diabetic heart was not a result of an increased lipolysis in the buffer
chamber due to detachment of LPL. We (Rodrigues et al 1992) and others (DeMan et al 1997)
have demonstrated that V L D L does not result in detachment of L P L from its HSPG complex.
In conclusion, our results demonstrate that during diabetes, rapid removal of V L D L from
the diabetic heart could be one mechanism for the provision of FFAs for energy production, to
compensate for the diminished contribution of glucose as an energy source. It should be noted
enzymes that catalyze the synthesis of T G promotes the accumulation of intracellular T G stores
during diabetes (Murthy et al 1983). Subsequent hydrolysis of this augmented T G store could
also lead to high tissue F F A levels (Kenno and Severson 1985). In view of these mechanisms
that enhance cardiac F F A levels, the relative significance of cardiac L P L activity in delivering
F F A to the diabetic heart is unknown. Interestingly, when the uptake pattern of albumin-bound
trophoblasts), the uptake of albumin-bound FFA after 24 hours was 4 to 6% of the initial amount
contained in the incubation medium. In contrast, when these cells were incubated with labeled
V L D L - T G , cellular TG-FFA content was many folds higher (Bonet et al 1992). Thus, at least in
some cell types, TG-rich lipoproteins may be a more efficient means to deliver F F A than are
albumin bound F F A and emphasizes the importance of L P L in the supply of F F A to the heart
(Bonet et al 1992, Levak-Frank et al 1995). A caveat is that this abnormally high capillary
171
endothelial L P L activity could provide excess F F A to the diabetic heart leading to a number of
metabolic, morphological and mechanical changes, and eventually to cardiac disease (Rodrigues
Hypertension and diabetes are two important risk factors of cardiovascular disease, a
leading cause of deaths in Western population. Cardiac dysfunction is one of the cardiovascular
diseases, which develops secondary to hypertension and diabetes. Although the underlying
mechanisms are not clear, it is proposed that early metabolic derangement could be causally
related to cardiac dysfunction. In this regard, we investigated LPL regulation, a key enzyme in
FFA supply to the heart, during hypertension and diabetes. Our results show that cardiac LPL
activity decreases at the capillaries (functional LPL pool) with the increase in blood pressure and
duration of hypertension in both SHR and fructose hypertensive rats. This decrease in functional
LPL activity was not due to a defective synthesis of the enzyme but probably due to poor
perfusion of coronary blood vessels of hypertensive hearts. Coronary vasodilators with divergent
mechanisms of action reversed the decreased LPL to normotensive levels by improving the
coronary perfusion. Therefore, it appears that poor capillary perfusion during hypertension per se
regulates cardiac LPL at the functional level. Based on the literature evidence that hypertensive
hearts utilize less FFA and more glucose for their energy transduction (Christe and Rodgers 1994
& 1995), we speculate that the decrease in LPL at the functional location could be linked to this
Capillary-bound and myocyte LPL are distinctly regulated during diabetes. Acute
hypoinsulinemia augments cardiac capillary LPL, presumably within or at the luminal and
abluminal surface of the endothelial cell (Figure 36). In contrast, the level of LPL in isolated
cardiac myocytes is low. So far, the mechanism for this selective augmentation of the enzyme at
the capillary is not known. We have identified that this enhanced recruitment of LPL is an
intrinsic ability of the diabetic heart. Various posttranslational mechanisms could play a role in
173
this regulatory process. Increased dimerization of LPL to the active form during its transit from
myocytes to capillary endothelium, a faster translocation of the enzyme from its site of synthesis,
and endothelial recycling, could be some of the mechanisms involved. Regulation at this
location is essential as it permits a rapid response to an acute demand for FFA in the absence of
glucose utilization. We have also demonstrated that the diabetic heart hydrolyzes significantly
higher amounts of VLDL-TG at a higher rate as compared to the non-diabetic heart. In fact, the
diabetic heart has elevated levels of FFAs and TGs. Hyperlipidemia combined with higher
functional LPL in the diabetic heart could possibly contribute to the accelerated supply of FFA to
the heart. Although various other sources like enhanced adipose tissue derived FFA, increased
intracellular TG synthesis and lipolysis of TG stores could contribute to these higher FFA levels
(Murthy et al 1983, Kenno and Severson 1985, Chattopadhyay et al 1990 ), a role of LPL in the
human LPL in skeletal and cardiac muscles in transgenic mice leads to elevated FFA uptake and
severe myopathy, characterized by muscle fiber degeneration, and extensive mitochondrial and
LPL in the diabetic heart could enhance FFA supply thereby playing a potential pathogenic role
in the development of diabetic cardiomyopathy. However, further studies are needed to examine
the fate of hydrolytic products of VLDL-TG when perfused through normal and diabetic hearts,
and the role of chylomicrons in normal and diabetic hearts. Also, future studies to prove that
increased LPL in the diabetic heart is causally related to the development of cardiomyopathy is of
great clinical significance. This awaits the discovery of a specific pharmacological agent that
rats have an augmented capillary LPL (Shepherd et al 1998). Thus the diabetes-induced
174
augmentation of capillary LPL counteracts the reduction in enzyme activity associated with
hypertension. We believe that these changes in cardiac LPL activity in the hypertensive-diabetic
heart, especially at the functional location, could play a key role in altering cardiac metabolism
Future studies would be pursued to investigate the contribution of TGRL derived FFA to
cardiac lipid metabolism in the diabetic heart in the absence or presence of NEFA. The question
of whether the FFAs are oxidized or re-esterified in the cardiac tissue subsequent to their release
Changes in insulin levels acutely regulate cardiac LPL during diabetes. The acute
changes in functional LPL appear to be an intrinsic ability of the heart to recruit more enzyme in
response to changes in insulin levels. Although our preliminary studies indicate there could be a
faster translocation of the enzyme from myocytes, more studies using S-methionine to measure
the rate of translocation of newly synthesized LPL will be carried out in the future. In addition,
the signaling events that bring about the faster translocation of LPL from its site of synthesis
(cardiac myocytes) to the functional site (coronary lumen) have yet to be uncovered (Figure 35).
Experiments are underway to determine whether a cAMP mediated mechanism could play a role
process, which involves PI3-kinase, tyrosine kinase, actin cytoskeleton rearrangement, and
SNARE-SNAP-NSF fusion processes. Whether this mechanism plays a role in LPL transport,
and if so, whether there is any augmentation of this process in the diabetic heart, has yet to be
discovered.
Another mechanism that could play a role in the acute regulation of coronary LPL is
endothelial recycling (Figure 35). Previous studies have shown that LPL attached to the HSPG
binding sites on the luminal side of the endothelial surface are constantly internalized and
177
recycled back. Therefore, it is possible that augmentation of this endothelial recycling could play
an additional role in the enhancement of capillary LPL of the diabetic heart. Endothelial
lipolytic products and internalizing proatherogenic lipoproteins like LDL in the vasculature
(Figure 35). Diabetes is associated with an increased incidence of atherosclerosis and coronary
heart disease. Since we observed an increased coronary LPL activity in the diabetic heart, it
would be of great clinical relevance to investigate whether this altered LPL activity could play a
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