Free Radical Biology & Medicine, Vol. 5, pp. 113-124, 1988 0891-5849/88 $3.00 + .

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Printed in the USA. All rights reserved. © 1988 Pergamon Press plc

Review Article

FREE RADICALS AND DIABETES

L A R R Y W . OBERLEY
Radiation Research Laboratory, Department of Radiology, 14 Medical Laboratories, The University of Iowa,
Iowa City, IA 52242

Abstract--The role of active oxygen species in diabetes is discussed in this review. Type I diabetes is caused by
destruction of the pancreatic beta cells responsible for producing insulin. In humans, the diabetogenic process
appears to be caused by immune destruction of the beta cells; part of this process is apparently mediated by white
cell production of active oxygen species. Diabetes can be produced in animals by the drugs alloxan and strepto-
zotocin; the mechanism of action of these two drugs is different, but both result in the production of active oxygen
species. Scavengers of oxygen radicals are effective in preventing diabetes in these animal models. Not only are
oxygen radicals involved in the cause of diabetes, they also appear to play a role in some of the complications
seen in long-term treatment of diabetes. Changes in antioxidants in the diabetic state and their consequences are
discussed.
Keywords--Diabetes, Alloxan, Streptozotocin, Superoxide dismutase, Catalase, Glutathione

I. I N T R O D U C T I O N ("adult onset"). The prevalence in the U.S. of type I

insulin-dependent diabetes mellitus (IDDM) is 1.3 per
Diabetes is a general term referring to disorders char-
thousand, and 35 per thousand for type II, non-insulin
acterized by excessive urine excretion. When used by
dependent diabetes mellitus (NIDDM).I
itself, this term refers to diabetes mellitus. Diabetes
While insulin and other medical treatments can con-
mellitus is a metabolic disorder characterized by high
trol many aspects of diabetes, numerous complications
blood glucose. The major symptoms are thirst, hunger,
are common. Peripheral vascular disease is a major
emaciation, and weakness, eventually resulting in coma.
factor in diabetic complications. Other health problems
These symptoms can be treated with insulin in certain
seen in diabetics are renal failure, coronary heart dis-
types of diabetes. Two general types of diabetes mel-
ease, blindness, infections, and atherosclerosis. Be-
litus are recognized: type I and type II. Type I patients
cause diabetics often live a long time after onset of
are totally dependent on exogenous insulin, while type
disease, the cost of care for these patients is enormous.
II patients can be treated with dietary changes, exercise
Clearly, much research is needed to prevent diabetes
and oral medication. There is not a clear separation
and to treat the ill effects of the diabetic state.
between type I and type II diabetes. In general, type
Recently, oxygen free radicals have been implicated
I diabetics tend to be young and lean ("juvenile-on-
set"), while type II diabetics tend to be older and obese in this disease. It is the purpose of this paper to review
the role of active oxygen species in diabetes.

Larry W. Oberley received his undergraduate education in physics

at Northwestern University. He received the M.S. and Ph. S. degrees
in physics from the University of Iowa. His Ph.D. dissertation was
on the Mossbauer effect of biological compounds. This research
sparked his interest in biology and biochemistry which has been his
focus since. In 1975, Dr. Oberley joined the Radiation Research
Laboratory at the University of Iowa where he has remained. In
1985, he was appointed Professor of Radiology. Dr. Oberley's main
research interest is the role of oxygen free radicals and antioxidant
enzymes in cancer; in recent years, this research has gravitated to-
ward the molecular biology of the antioxidant enzymes in tumor
cells. Dr. Oberley has edited three volumes entitled Superoxide Dis-
mutase for CRC Press.
II. R O L E O F F R E E R A D I C A L S IN T H E C A U S E
OF DIABETES
A. Immune response, virus, or environmental agent?
The cause of diabetes is still unknown. With type I
diabetes, it has been demonstrated that destruction of
the pancreatic beta cells (which are found in the islets
of Langerhans) causes the disease by destroying the
cells that produce insulin. Type I diabetes has been

113
114 L.W. OBERLEY
mainly associated with some type of destructive im-
mune response: a large fraction of type I diabetics have
antibodies against their own beta cells. Several other
factors also implicate autoimmune disease in the de-
velopment of type I diabetes, including lymphocyte
infiltration of islets in patients with recent disease on-
set, correlation of certain human leukocyte antigens
and incidence of diabetes, increased K-cell rosette for-
mation and antibody- or complement-mediated cyto-
toxicity, and increased numbers of activated T lym-
phocytes. 2 In one study, cell-mediated cytotoxicity to
islet cells was identified in 11 of 14 patients with type
I diabetes, while antibody-mediated cytotoxicity was
seen in half the patients. 2'~
Autoimmune disease has also been implicated in
diabetogenesis in non-obese diabetic (NOD) mice, 4
db/db mice, 5 and BB-Wistar rats.6 Subdiabetogenic doses
of streptozotocin (STZ) also gave rise to diabetes via
a T-cell-mediated immune response, v'8 Moreover, NOD
mice destroyed transplants of treated allogeneic islet
tissue by a recurrence of the immune disease process
that destroyed the original islet tissue. This was pre-
vented by treatment of the animals with combined des-
ferrioxamine and nicotinamide (a DNA repair inhibi-
tor). 9 Transplanted animals became normoglycemic and
remained so for the duration of the treatment.
There are indications for other factors besides the
immune response in the cause of type I diabetes. Viral
infection and environmental agents have also been im-
plicated. 2 One possibility to tie the three agents to-
gether is that injury due to viruses or toxins kill some
islet cells, resulting in the expression of antigens that
cause an immune response to the remaining islet cells,
The cause of type II diabetes is even more of a
mystery. Type II diabetes is associated with obesity,
is seen in older patients, and appears to be more of a
heterogenous disease than type I diabetes. Insulin is
usually produced by type II diabetics, but their cells
may not respond to the hormone.
1. Free radical attack on beta cells. Type I diabetes
can be produced in animals by the action of chemicals
or by an immune attack on the pancreatic beta cells.
In both instances, oxygen free radicals are produced
and appear to play a role in the beta cell killing. The
pancreatic beta cell appears to be susceptible for a
number of reasons, as will be discussed later. A major
finding is that the pancreatic beta cell appears to be
low in protective substances. However, all of the major
antioxidant enzymes are present in pancreatic beta cells
in reasonable amounts. Thus, in animals copper and
zinc containing superoxide dismutase (Cu-ZnSOD), ~°-13
manganese containing superoxide dismutase (MnSOD),~3
catalase (CAT), ~2 glutathione peroxidase (GPX), 13~4
and glutathione reductase (GRy 5 have been reported
to be present in reasonable amounts in islets of beta
cells. However, as will be discussed later, the total
antioxidant enzyme activity of the beta cell may be
low. Moreover, the antioxidant enzyme status of the
islet cells may be more susceptible to certain forms of
stress. Asayama et al.~6 have investigated the effect of
vitamin E deficiency, selenium deficiency, and com-
bined deficiency on rat islet function and free radical
scavenging systems. SOD levels in liver, heart, and
skeletal muscles were within 20% of control levels in
all the deficient groups. However, the MnSOD levels
in islets were significantly lower in the vitamin E,
selenium, and combined deficiency animals than in the
controls. Combined deficiency appeared to have an
additive effect. In contrast, Cu-ZnSOD levels were
higher in the deficient groups than the controls. GPX
activity was markedly decreased in selenium-deficient
animals in all tissues studied. Catalase activity did not
change significantly with deficiency. Islets had the lowest
GPX and CuZnSOD levels among tissues studied. These
changes in antioxidant enzyme status were reflected in
decreased islet cell function. Insulin secretory reserve
was decreased in each of the three deficient groups.
The decrease was reflected as glucose intolerance only
in the group with combined deficiency.
B. Alloxan
Alloxan (2,4,5,6-tetraoxohexahydropyrimidine) is
an unstable and reactive chemical substance that exists
in several tautomeric forms. Alloxan is readily reduced
to dialuric acid, which is the toxic form of the com-
pound. 17The drug is now widely used for the induction
of type I diabetes in a variety of animals. ~v,18It should
be emphasized at this point that there is no evidence
linking this drug to diabetes in humans.
1. Free radical production. The alloxan-dialuric acid
couple caused oxygen consumption and produced hy-
drogen peroxide when a reducing agent was present. ~9
The production of H202 was attributed to the quinone
structure of alloxan. ~8 The autooxidation of dialuric
acid yielded H202, O2, O27 , and "OH. 2° Hydroxyl rad-
ical production detected by ethylene formation was
inhibited by SOD, CAT, and the hydroxyl radical scav-
engers benzoate and ethanol, suggesting the Haber-
Weiss reaction as the source of hydroxyl radicals. 18
Alloxan-mediated production of free radicals has
been investigated by measuring luminol lumines-
cence. 2~ Reduced glutathione (GSH) and iron sulfate
were needed for maximum luminescence. The metal
chelators diethylenetriamine-pentaacetic acid (DETA-
PAC), ethylenediaminetetraacetic acid (EDTA), and
 Free radicals and diabetes
desferrioxamine, the sugars 3-0-methyl-D-glucose, L-
and D-glucose, D-fructose, and D-mannose, and NAD,
NADH, NADP, and NADPH all prevented lumines-
cence. 2~ From these results, it was suggested that al-
loxan acts via a thiol group (due to the need for GSH),
that hydroxyl radical was needed for the production of
luminescence (since metal chelators blocked the ef-
fects), that NAD(P) and NAD(P)H can scavenge 02 ~,
H202, and .OH, and hexoses can scavenge free radicals. 21
2. Free radical production in in vitro cells. Many stud-
ies support a role for oxygen free radicals as mediators
in the toxic effect of alloxan to cells in vitro. DETA-
PAC protected isolated mouse islet cells from alloxan-
induced inhibition of rubidium accumulation. 22 Cop-
per(II), a known catalyst of the Haber-Weiss reaction,
caused a similar inhibition of rubidium accumulation
as alloxan, and was additive to the effect of alloxan,
suggesting a similar mechanism for each compound.
Isolated rat pancreatic islets were protected against the
cytotoxic effects of alloxan and dihydroxyfumarate by
the hydroxyl radical scavenger dimethylurea.Z3 Isolated
mouse islet cells were protected against the toxic ef-
fects of alloxan on rubidium uptake and trypan blue
exclusion by the hydroxyl radical scavengers mannitol,
dimethylsulfoxide, and butanol, while the singlet ox-
ygen scavenger histidine had no effect. 24 Addition of
CAT, SOD, or DETAPAC protected rat islet cells in
vitro from the toxic actions of alloxan, as measured
by glucose-induced insulin release, 2~ while SOD and
CAT protected mouse islet cells in vitro from the toxic
effect of alloxan using rubidium uptake and trypan blue
exclusion as indicators of cytotoxicity. 24 These data
were interpreted to suggest that islet cells are vulner-
able to the effect of alloxan due to a metabolic spe-
cialization that facilitates reduction of the drug. 2~
Chemiluminescence in the presence of luminol and
alloxan has also been measured in isolated pancreatic
islet cells. 26 Alloxan-induced chemiluminescence ap-
peared very rapidly and lasted more than five minutes.
On the other hand, streptozotocin failed to produce
chemiluminescence. SOD and/or CAT markedly sup-
pressed alloxan-induced chemiluminescence. Islets
produced substantially more chemiluminescence than
either red cells or hepatocytes, 27 suggesting that islet
cells produced more free radicals in the presence of
alloxan.
The site in the cell where alloxan produces free
radicals is still unknown. Many have suggested it is
the cell membrane. For instance, Grankvist and
Marklund zs have shown that damage caused by xan-
thine oxidase-hypoxanthine to isolated pancreatic beta
cells is similar to that caused by alloxan. Thus, xan-
thine oxidase/hypoxanthine caused increased trypan
115
blue uptake by cells. SOD or CAT, but not hydroxyl
radical scavengers, protected against the increase in
cell injury. Moreover, both L- and D-glucose pro-
tected. The authors hypothesized that extracellular free
radicals generated by alloxan or xanthine oxidase caused
toxicity by damaging the cell membrane. This hypoth-
esis of the cell membrane as the site of alloxan action
is hard to reconcile with the fact that alloxan causes
DNA strand breaks. 29,3°Moreover, SOD and CAT which
are thought not to enter cells, protected isolated rat
islet cells from the DNA strand breaks caused by al-
loxan. 29,3° Further work is needed to elucidate how
these DNA strand breaks are produced.
Sugars have also been shown to protect against al-
loxan-induced cytotoxicity to in vitro cells. Isolated
rat hepatocytes were sensitive to alloxan as measured
by GSH depletion and potassium, lactate dehydrogen-
ase, or glutamate-pyruvate transaminase leakage. 3~ The
sugars D-glucose (but not L-glucose), D-galactose, D-
fructose, 2-deoxyglucose, and D-mannitol protected
these hepatocytes from damage caused by alloxan, 3~
Mannoheptulose also protected, although it failed to
protect isolated islet cells, and blocked the protective
effect of glucose on isolated islet cells. 32
Three theories have been proposed to explain the
protective action of sugars against alloxan toxicity.
One theory is that sugars might act as free radical
scavengers, as both D-mannito124 and D-glucose 33have
been shown to scavenge free radicals. Two other the-
ories have been proposed to explain the protective ac-
tion of sugars against alloxan beta toxicity. 34 One hy-
pothesis is that alloxan interacts with the membrane
glucose receptor site or the site of glucose-stimulated
insulin release. Sugars would block because they would
prevent alloxan binding. A third hypothesis is that sug-
ars augment the generation rate of reducing equivalents
(especially NADPH) and thus allows the recycling of
GSH via GR. This latter hypothesis has been cham-
pioned forcefully by Malaisse. 34 Malaisse observed that
the protective action of D-glucose and 2-ketoisoca-
proate against the alloxan-induced inhibition of glu-
cose-stimulated insulin release was impaired when the
exposure to alloxan was performed in a medium con-
taining NH4C1 or menadione. The effects of the latter
two agents were attributed mainly to a lowering of islet
content of NADH or NADPH. Malaisse argued that it
is unlikely that the protective effect of glucose was due
to a decreased uptake of alloxan, since D-glucose has
been reported to have no effect on [2-t4C] alloxan up-
take. 34 Rather, the protective action of the sugars de-
pends on their capacity to be metabolized via glycol-
ysis. Different hexoses show different capacities to
protect and this capacity seems to correlate well with
the capacity of the compounds to undergo glycolysis
and hexose oxidation, thus forming NADH and NADPH.
116 L.W. OBERLEY
3. Free radical production in in vivo cells. Numerous
studies in vivo implicate oxygen-derived free radicals
in the diabetogenic action of alloxan. Heikkila et al.
found that the hydroxyl radical scavengers methanol,
ethanol, n-propanol, n-butanol, and thiourea protected
against alloxan-induced diabetes in mice. 35 The pro-
tective action of these agents correlated with their hy-
droxyl radical scavenging ability. The diabetogenic ac-
tion of alloxan in mice was also blocked by pretreatment
with the hydroxyl radical scavengers monomethylurea,
monoethylurea, and diethylurea, 36 dimethylsulfox-
ide, 37 dimethylurea, 38 and amygdalin, 39'4° as well as the
free radical scavenger butylated hydroxyanisole. 4~ Vi-
tamin E supplementation also protected rats from the
diabetogenic action of alloxan. 42
Injection of polyethylene glycol-SOD into mice 12
hours before alloxan blocked the diabetogenic action
of the drug. 43 Moreover, exogenous Cu-ZnSOD pro-
tected rat pancreatic beta cells against morphological
damage by alloxan as determined by ultrastructural
examination and immunostaining for insulin. 44 In this
study, alpha and delta cells appeared unaffected by
administration of alloxan and Cu-ZnSOD or either
agent alone. Autoradiography after injection of
~25I-CuZnSOD into normal rats showed no evidence
that the enzyme enters viable islet cells, suggesting
an extracellular site of protection against alloxan.
Certain metal chelators have also been shown to
block alloxan-induced diabetes. DETAPAC adminis-
tered prior to alloxan blocked the formation of diabetes
in mice, apparently by removing metals necessary to
catalyze the Haber-Weiss reaction. 45 The metal che-
lator EDTA, which does not prevent chelated metal
from producing hydroxyl radicals, afforded no protec-
tion. 45 In a similar study, it was found that DETAPAC
inhibited while desferrioxamine stimulated the hyper-
glycemic response of mice to alloxan. 46 The anticancer
agent ICRF-187 has also been shown to protect against
alloxan induced diabetes in mice .47.48This drug is struc-
turally similar to EDTA and is thought to bind metals,
but has no free radical scavenging ability itself. 48
Thus, O2 ~, H202, and .OH are all produced in the
presence of metals. This data taken together suggests
that alloxan causes diabetes in vivo by producing an
iron-catalyzed Haber-Weiss reaction in the pancreatic
13 cells.
4. Basis of selected toxicity of alloxan. Why is alloxan
selectively toxic to the pancreatic beta cells compared
to other cells in the body? A number of hypotheses
have been presented. One possibility is selective up-
take of alloxan by the beta cells. Another is an un-
usually high efficiency for reducing alloxan to dialuric
acid. A third possibility is a high production of active
oxygen species or low protection against those oxygen
species produced. The second and third hypothesis are
obviously related since the production of dialuric acid
is asociated with active oxygen species production;
however, the two hypotheses should be considered sep-
arate since there may not be a constant stoichiometry
between the two processes.
The first possibility seems unlikely since in vivo
liver took up alloxan as rapidly and extensively as islet
cells, yet did not suffer alloxan-induced toxicity. 22 There
is at present no good evidence for the second possi-
b i l i t y - t h a t islet cells produce more dialuric acid from
alloxan. There is strong evidence for the third possi-
b i l i t y - - a high level of active oxygen species in the
beta cells after alloxan exposure. Thus, upon compar-
ing five different tissues (islet, muscle, exocrine pan-
creas, kidney, and liver), Malaisse et al. 14 observed a
tight correlation between the activity of GPX and the
minimal concentration of tert-butyl hydroperoxide re-
quired to alter glucose oxidation, the lowest values for
both variables being found in the pancreatic islets and
the highest values in liver. A comparable, although not
identical, hierarchy in the activity of GPX in various
tissues was reported by Grankvist et al. ~3These authors
also measured Cu-ZnSOD, MnSOD, and CAT activity
in these tissues. When the tissues were ranked from
highest to lowest with regard to the activity of the
enzymes, the average rank values, which were used as
crude indices of the enzymatic protection against active
oxygen species, followed an order close to that of Ma-
laisse et al., t4 namely, 1.5 for liver, 2.3 for kidney,
4.0 for exocrine pancreas, 5.5 for islets, and 8.0 for
muscle.
Malaisse 34 has concluded that both the capacity to
take up alloxan and the sensitivity to active oxygen
species must be taken into account when examining
susceptibility to alloxan. Malaisse feels that: (a) muscle
could be protected against alloxan, despite high sen-
sitivity to peroxides, because of slow or poor penetra-
tion into muscle cells; (b) liver could be resistant to
alloxan, despite rapid accumulation, because of its re-
sistance to peroxide; and (c) islet beta cells could be
highly vulnerable to alloxan because of its rapid uptake
and its sensitivity to active oxygen species.
In conclusion, the reason why the beta cells of
the pancreas are most vulnerable remains unknown.
Uptake, metabolism, and antioxidant status may all be
involved; the role of active oxygen species appears to
have the most conclusive evidence in its favor.
C. Streptozotocin
Streptozotocin (STZ), the common name for 2-deoxy-
2-(3-methyl-3-nitrosoureido)-D-glucopyranose, is an
 Free radicals and diabetes
antibiotic isolated from Streptomyces achromogenes.
This agent has antibacterial and antitumor properties,
as well as being carcinogenic and diabetogenic. Strep-
tozotocin is structurally closely related to 1,3-bis(2-
chloroethyl)-l-nitrosourea (BCNU), a nitrogen mus-
tard derivative commonly used in cancer therapy. It
has been assumed in the past that STZ is cytotoxic
because it acts as an alkylating agent. 49'5°'5~ However,
there is growing evidence for involvement of active
oxygen species in the diabetogenic action of STZ.
1. Metabolism and specificity. The glucose moiety of
STZ confers specificity for beta cell killing. Without
the glucose moiety, little beta cell killing occurs. For
instance, there was a four fold greater uptake of STZ
into is lets when compared to methylnitrosourea ;52nearly
3.5-fold more methylnitrosourea than STZ was needed
to produce diabetes.52 In vivo uptake studies of labeled
STZ showed that it accumulated in the pancreas. 53'54
When STZ was labeled at the 1'- or 2'-position, 70
and 80%, respectively, of the labe| appeared in the
urine after 6 hours, while only 40% of the label at the
3' methyl position appeared in the urine after 6 hours.
STZ was rapidly cleared from the blood, mostly by the
kidney, with some hepatic uptake. Further studies in-
dicated that the only labeled compound found in the
urine after 3'-methyl labeled STZ was administered
was the unmetabolized STZ. 54 It was proposed that
STZ is metabolized by scission between the 2'-carbon
and the methyl nitrogen, with the N-nitrosoureido moiety
being retained in the cell and causing damage. 54'55
2. Toxic-action of streptozotocin--nonfree radical
mechanisms. It has been proposed that STZ is toxic to
cells because it causes depression of NAD and NADP
levels in cells. Islet NAD levels were depressed to 12%
of normal by STZ administration.52.56 The cause of this
depletion may be from several origins. The N-nitro-
soureido moiety of STZ is believed to break down into
the methyl carbonium ion 49 or methyl radical, 3° which
can alkylate and cross-link DNA and inhibit incorpo-
ration of precursors into D N A . 49'5° The methyl car-
bonium ion has been found to methylate nicotinamide
moieties and caused the appearance of methylated de-
rivatives in the urine. ~7'58 Nicotinamide is a precursor
for both NAD and NADP in cells and thus this may
be one mechanism for their depletion. Administration
of nicotinamide was shown to prevent the diabetogenic
action of alloxan, possibly by elevating the levels of
NAD precursors in the c e l l ; 59'6° nicotinamide did not
effect the anti-leukemic affect of STZ. 6° Studies of
STZ-induced NAD depletion using 5-methylnicotin-
amide, a nicotinamide analogue that inhibits NAD deg-
radation, but is not a precursor for NAD synthesis,
ll7
demonstrated that synthesis of NAD was not impaired,
but that degradation of NAD was increased after STZ
exposure .61
A second mechanism that could cause NAD deple-
tion involves DNA damage. The alkylating action of
STZ can cause DNA cross linkage and strand scis-
sion, 29'30'62which was not blocked by SOD or CAT. In
contrast, alloxan induced DNA strand breakage was
blocked by these enzymes. 3° Regardless of cause, this
DNA damage activates the enzyme poly(ADP-ribose)
synthetase, which is involved in DNA repair and re-
quires NAD. 29 Activation of this enzyme may be re-
sponsible for depletion of NAD in STZ-treated islets.29
Picolinamide and nicotinamide, two inhibitors of this
nuclear enzyme, blocked the depression of NAD levels
in STZ-treated tissues. 62 The depression of cytosolic
NAD has been shown to lead to an inhibition of proin-
sulin synthesis, but allowed for repair of DNA damage,
while inhibition of poly(ADP-ribose) synthetase inhib-
ited DNA repair, but allowed proinsulin synthesis to
continue. 62
These studies led Uchigata et al. 3° to propose a
mechanism of inhibition of proinsulin synthesis by al-
loxan and streptozotocin. Both agents were hypothe-
sized to cause DNA strand breaks, alloxan by active
oxygen species (since breakage is inhibited by SOD
and CAT), and STZ by an alkylating species. The DNA
breaks were then proposed to cause an increase in
poly(ADP-ribose) synthetase, an enzyme requiring
NAD. NAD will then be depleted and since this co-
factor is needed for proinsulin synthesis, an inhibition
will occur. It was hypothesized that poly(ADP-ribose)
synthetase inhibitors prevented the inhibition of proin-
sulin synthesis by preventing NAD depletion. Later
work by Okamoto and Yamamoto has shown that
poly(ADP-ribose) synthetase inhibitors did not keep
DNA strand breaks from occurring, but these inhibitors
prevented diabetes .63 Therefore, beta-cells may survive
with the residual DNA damage within their genome.
About one year after the combined administration to
rats of alloxan or STZ with poly(ADP-ribose) synthe-
tase inhibitors, diabetes still had not developed but islet
beta-cell tumors were frequently f o u n d . 63 This suggests
that insulin-dependent diabetes and beta cell tumors
are closely related; the tumor cell arises when the DNA
cannot be repaired, whereas diabetes arises when the
DNA can be repaired.
3. Active oxygen species and streptozotocin action.
There is evidence that active oxygen species are in-
volved in the diabetogenic action of STZ. There was
a 21% decrease in GSH in human erythrocytes exposed
to STZ in vitro and a 74% increase in oxidized glu-
tathione (GSSG) levels.64 The GSH levels of rat islets
118 L.W. OBERLEY
incubated with STZ in vitro also decreased 33% and
GSSG levels increased 95%. 64 Nicotinamide admin-
istration prevented the decrease in GSH levels in hu-
man erythrocytes exposed to S T Z . 65 This suggested
that nicotinamide is used to synthesize NADPH, which
is then used to maintain GSH levels probably via the
GR recycling system.
A variety of free radical scavengers protected ani-
mals from the diabetogenic effects of STZ, including
S O D , 66'67 SOD linked to polyethylene glycol, 68 the SOD-
mimetic compound copper(II)(3,5-diisopropylsalicy-
late), 69 the hydroxyl radical scavenger dimethyl-
u r e a , 7°.7j and vitamin E. 42 Vitamin E or selenium-de-
ficient rats were sensitive to doses of STZ that were
normally subdiabetogenic. 42 Moreover, administration
of acetyl-homocysteine-thiolactone caused a 200% in-
crease in the Cu-ZnSOD activity of rat pancreatic islets
and protected against STZ-induced beta cell damage, v2
All of the results are not consistent, however. Ex-
ogenous SOD did not protect against STZ-induced DNA
strand breaks or inhibition of proinsulin synthesis in
isolated rat cells, which led to the proposal that the
diabetogenic action of STZ was mediated through methyl
radicals, not active oxygen species. 3° Another study
reported a single injection of Cu-ZnSOD failed to pro-
tect from the diabetogenic action of multiple injections
of STZ; 73'74 however, this group subsequently dem-
onstrated protection with CuZnSOD using a modified
procedure. 74 It has also been shown that 1,10-phen-
anthroline, a metal chelator that inhibits hydroxyl rad-
ical formation, reduced the diabetogenic effects of al-
loxan in rats but not S T Z . 75
Thus, while evidence demonstrating a role for active
oxygen species in STZ-induced diabetes is present, it
is difficult to find a unified picture. The best guess at
present is that STZ interferes with GSH metabolism,
leading to an increase in H202 and its reaction products.
lIl. EFFECTS OF DIABETES ON ACTIVE OXYGEN
SPECIES METEABOLISM
Even though diabetes can be treated and thus life
preserved in the short run, many diabetics suffer severe
complications during long term therapy. The reason
for these complications are still unknown. One possible
hypothesis is that the diabetic state leads to increases
in active oxygen species production. Indeed, changes
in SOD and CAT activity, GSH metabolism, vitamin
E levels and increases in lipid peroxides have been
observed. This data is summarized below.
A. Superoxide dismutase
SOD activity has been measured in tissues of ani-
mals with chemically-induced diabetes. Matkovics et
al. found that rats with alloxan or STZ-induced dia-
betes of two months duration had decreased total SOD
activity in liver, kidney, spleen, heart, testis, pancreas,
skeletal muscle and erythrocytes. 76'77Total SOD activ-
ity was not changed in brain or lung. 77 Crouch et al.
found that rats with STZ-induced diabetes of 5 days
duration had decreased Cu-ZnSOD activity in eryth-
rocytes and retina. 78 This study found no change in
SOD activity in lung, liver, brain, aorta, kidney, whole
eye, and lens of rats with STZ-induced diabetes, and
that insulin treatment for 3 days failed to restore SOD
activity in retina or erythrocytes. 78
A third group of investigators found Cu-ZnSOD ac-
tivity was depressed in renal cortex and large and small
bowel mucosa of rats with STZ-induced diabetes of 9
or 10 days duration. 79 Loven et al. found that MnSOD
activity was unchanged in intestinal mucosa, but was
increased in the renal cortex of diabetic animals. 79 These
investigators found that treatment of diabetic animals
for 5 or 6 days with insulin restored intestine and renal
cortex Cu-ZnSOD activity and renal cortex MnSOD
activity to the levels found in normal tissues. 79 These
effects of diabetes and of insulin treatment were found
both in diabetic animals fed ad libitum and in pair-fed
animals. 79 The amount of food ingested in these ex-
periments is a worry because diabetic animals eat less
and because starvation has been shown to affect SOD,
CAT, and GPX in tissues of rats. s° Cu-ZnSOD activity
was found to be decreased in renal cortex and jejunum
of STZ-induced diabetic rats fed ad libitum. Sl Both oral
glutathione and intramuscular insulin elevated the Cu-
ZnSOD activity, st Renal cortex exhibited elevated
MnSOD activity, which was normalized by insulin or
glutathione. Cu-ZnSOD activity was also decreased in
kidney, erythrocytes, and liver of pair-fed rats with
STZ-induced diabetes of 9 to 10 days duration. ~2 In-
sulin treatment restored activity in liver, but not eryth-
rocytes, s2 Oral glutathione treatment restored the SOD
activity of renal cortex and liver, but not erythrocytes
of rats with STZ-induced diabetes but did not lower
the blood glucose levels. 82 The reason why oral GSH
has this effect is still unknown. It is not clear whether
oral GSH can be absorbed from the stomach and enter
cells intact. One possibility is that GSH is broken down
into its constituents, which can then enter the cell and
serve as building blocks for further GSH synthesis.
More studies are needed to elucidate the mechanism
of action of oral glutathione.
Two other studies have examined SOD activity in
STZ-induced diabetic rats. Nishida et al. s3 found that
SOD activity was decreased in retinas of these rats;
however, no changes were found in the cornea, lens,
blood, liver, or kidney. Electrophoretic studies showed
no qualitative alteration of the Cu-ZnSOD protein in
the diabetic rat retina. Lammi-Keefe et al. studied SOD
 Free radicals and diabetes
activity of various tissues of rats 80 days after STZ
injection.84 SOD activity was not altered in the skeletal
muscle or erythrocytes of diabetic rats but was some-
what decreased in the heart.
The basis for the various discrepancies of these re-
ports is not clear. The loss of SOD activity may be a
function of the duration of diabetes; these studies range
in time from 5 days to nearly 3 months after onset of
diabetes. The severity of diabetes also varies in these
studies and may well affect the SOD activities.
A number of studies have also been done on the
levels of SOD in human diabetics. There was no change
in erythrocyte Cu-ZnSOD activity, or lymphocyte Cu-
Zn or MnSOD activity in patients with type I diabetes
maintained on insulin for at least two years. 85 In con-
trast, subjects with type II diabetes maintained on oral
hypoglycemic agents had a 97% decrease in erythro-
cyte Cu-ZnSOD activity. 76 It has recently been found
that human erythrocytes contain glucosylated and non-
glucosylated Cu-ZnSODs.86 The percentage of the glu-
cosylated form, which had a lower enzymatic activity,
was significantly increased in the erythrocytes of pa-
tients with diabetes as compared to normal erythro-
cytes. 86 Incubation of Cu-ZnSOD with D-glucose in
vitro led to glucosylation, suggesting this was what
occurred in vivo. Thus, a mechanism for the inacti-
vation of Cu-ZnSOD seen in vivo during diabetes was
suggested. Other studies observed that erythrocyte Cu-
ZnSOD was diminished in diabetic children when com-
pared to controls. 87 Kaji et al. observed no changes in
erythrocyte SOD activity in 60 women with type II
diabetes when compared to 71 healthy control women.88
Marklund has measured the newly discovered S O D - -
extracellular SOD or E C - S O D - - i n plasma from chil-
den with type I diabetes. 89The specimens were obtained
from 8 patients with disease of recent onset and from
15 patients with disease of longer duration. There was
no significant difference in EC-SOD between the pa-
tients and controls.
The substrate for SOD, the superoxide radical
(O2~), has been measured in the white cells of diabetics
in a number of studies. Nath et al. found 02: was
significantly elevated in polymorphonuclear leuko-
cytes (PMNs) from diabetic patients as compared to
normal subjects. 9° This elevation was attributed to the
reduction in the activities of the cytoplasmic and mi-
tochondrial superoxide dismutases, the effect being more
pronounced in the cytoplasmic fraction. The content
of copper decreased considerably in the diabetic PMN,
the decrease in the zinc content was less significant
and the manganese levels were unchanged. PMNs ob-
tained from insulin treated diabetic patients showed
considerable elevation of SOD levels. Wiernusz et al.
have measured PMN function of patients with diabe-
tes. 91 Phagocytosis, bactericidal capacity and O2 ~ pro-
119
duction were determined in 30 patients with well-con-
trolled insulin-dependent diabetes (type I) and in 50
patients with non-insulin-dependent diabetes (type II).
Data was also obtained on 20 elderly patients without
glucose intolerance. The phagocytosis of viable staph-
ylococci was the same in all groups. However, the
bactericidal capacity in all diabetic patients was sig-
nificantly reduced. The 02 ~ production in patients with
type I diabetes was similar to control values, whereas
0 2 ; production in patients with type II diabetes as well
as in elderly patients was significantly reduced and
correlated with bactericidal capacity impairment. A last
study used chemiluminesce and 02 ~ production in PMNs
as a measure of oxidative metabolism. 92 Using phorbol
myristate acetate or oponsonized zymosan as a stim-
ulus, leukocytes from diabetic patients had a markedly
depressed chemiluminesce and 02 ~ response com-
pared to controls. Insulin or glucose had no significant
effect on control cell response. It has been demon-
strated that glucose itself has no effect on PMN func-
tion. 93 Thus, the elevation of glucose seen in diabetes
cannot be responsible for the impaired white cell func-
tion.
In any case, it is tempting to hypothesize that the
impaired respiratory burst seen in PMNs from diabetic
patients is at least partly to blame for the increased
morbidity and mortality due to infection seen in dia-
betic patients. This subject matter deserves much fu-
ture study.
B. Catalase and peroxidase
CAT activity of rats with alloxan- or STZ-induced
diabetes was increased in liver, kidney, testis, and
erythrocyte hemolysate, but decreased in spleen. 76 He-
patic CAT activity of female rats with STZ-induced
diabetes was not significantly depressed at 30 or 60
days after induction of diabetes, but was decreased at
90 days. 94 CAT activity was decreased in all muscles
examined in diabetic rats 80 days after STZ-adminis-
tration.84 The authors suggested the response was due
to the rats eating less, as starvation caused similar
changes in CAT activity. 8°
In humans, there was no change in the CAT activity
of erythrocytes of subjects with type I diabetes, 76,88 or
in erythrocytes of subjects with type II diabetes on
insulin maintenance. 85 However, Wataa et al. have re-
ported an elevation in CAT activity in erythrocytes of
diabetic children. 87
The guaiacol peroxidase activity of several organs
of animals with alloxan- or STZ-induced diabetes has
also been measured. 76 Peroxidase activity was in-
creased in liver, spleen, pancreas and skeletal muscle,
decreased in erythrocyte, hemolysate, heart muscle,
and testis and unchanged in kidney, lung, and brain.
120 L.W. OBERLEY
C. Glutathione levels
There was a transient decrease in serum glutathione
levels in rats after administration of alloxan, but serum
glutathione levels were normal after the diabetic state
had been induced. 95 Hepatic glutathione levels de-
creased but small bowel jejunal mucosal glutathione
content did not change in male rats with STZ-induced
diabetes of 10 days duration. 82 This effect was not due
to starvation as the animals were pair-fed. Starvation
does reduce GSH in liver. 8° Insulin, but not oral glu-
tathione, restored hepatic glutathione concentrations to
normal levels in STZ-induced diabetic rats. 82 Female
rats with STZ-induced diabetes had a 27% decrease in
both hepatic GSH and GSSG levels. 96 Depletion of
glutathione by administration of acetaminophen caused
a more rapid drop and subsequent restoration of glu-
tathione in female rats with STZ-induced diabetes than
in controls, suggesting the glutathione pool is turned
over more rapidly in diabetic animals. 96 In contrast to
these findings, a 20% increase in hepatic glutathione
content of male rats with STZ-induced diabetes, and
an increased resistance to acetaminophen-induced
damage in diabetic animals was reported. 97 The dis-
crepancies in these studies may be related to sex or
strain differences.
Changes in glutathione have been noted in humans
also. Total glutathione content in normal human eryth-
rocytes decreased with the age of the cells and a similar
decrease occurred with cells from type II diabetics,
whether untreated or treated with oral hypoglycemic
agents. 98 However, when cells of each age group were
compared, erythrocytes from nontreated diabetic pa-
tients had 30% less glutathione than did normal sub-
jects. 98 Erythrocytes from the treated patients had 20%
less gluathione when compared to normal controls. 98
Others have also noted reduced glutathione levels in
erythrocytes of patients with type II diabetes.99 Serum
glutathione levels in subjects with type II diabetes were
also lowered.'°° Moreover, reduced glutathione levels
in PMNs of diabetic patients were significantly de-
creased. 101
D. Glutathione peroxidase and reductase
In one study, GPX activity was increased in eryth-
rocytes from type II diabetics, 76 but decreased in an-
other study, 99 and showed no changes in another. 88
GPX activity in subjects with type I diabetes main-
tained on insulin was not significantly changed. 85 GPX
but not GR was found to be reduced in human PMNs,. 101
Plasma GPX levels were elevated in type II diabetes. 88
Thus, there appears to be no consistent changes in
GPX.
Likewise, there appear to be no consistent changes
in GR activity. GR levels were reported to be increased
20% in erythrocytes from patients with either form of
diabetes treated with insulin or tolbutamide. 502Another
group reported a decreased GR activity in type I dia-
betics, which could be restored by adding riboflavin
to the diet.l°3 Children with type I diabetes do have a
greater incidence of riboflavin deficiency which could
lead to GR reduction as this cofactor is needed for GR
activity.J°3 There was no significant difference in the
activity of erythrocyte or hepatic GR activity in ge-
netically diabetic KK mice when compared to Swiss
albino mice on riboflavin-supplemented diets. ~04When
riboflavin deficiency was induced, similar decreases
in GR activity occurred in both tissues of normal and
diabetic animals. 104 This suggests that normal and di-
abetic mice are equally sensitive to the effects of ri-
boflavin deficiency on GR activity. It thus appears that
GR activity is decreased in diabetes only in riboflavin
deficiency, a condition which might be more prevalent
in diabetes.
E. Vitamin E
Platelet vitamin E levels were depressed 66% in rats
with STZ-induced diabetes, an effect which was re-
versed by dietary supplentation with vitamin E. 105 Other
studies found no change in serum vitamin E in rats
with STZ-induced diabetes of 1 or 2 weeks.duration,
but a 26% decrease in rats with STZ-induced diabetes
of 20 weeks duration.l°6 Hepatic vitamin E was also
decreased 92% in rats with STZ-induced diabetes of
20 weeks duration.l°6
Plasma from patients with type II diabetes had a
25% increase in total tocopherol, 31% increase in a -
tocopherol, and no change in ~/-tocopherol.l°7 Platelet
total tocopherol was increased 28%, and ot-tocopherol
36%, while ~/-tocopherol was unchanged.l°7 A second
report found a 10% increase in a-tocopherol in type lI
diabetes.~°6 These reports suggested that the demand
for the antioxidant vitamin E is increased in diabetes,
and that dietary vitamin E can be sufficient to meet
this demand.
F. Lipid peroxides
Patients with type II diabetes and angiopathy had
9 1% more thiobarbituric (TBA) reactive material than
controls, while patients without angiopathy had no in-
crease.lOS This observation led to the suggestion that
the increased TBA-reactive material originated from
the intima of the blood vessel and might be related to
the development of atherosclerosis.108
Serum TBA-reactive material was increased 61% in
adult subjects with poorly controlled type I or II dia-
 Free radicals and diabetes
betes, but u n c h a n g e d in patients with well-controlled
diabetes. ~09The serum T B A reactive material was con-
centrated in the low density lipoprotein serum fraction
in both normal and diabetic patients, but the increase
in T B A reactive material occurred in the high density
lipoprotein fraction, which showed a 57% increase in
TBA reactive substance in diabetes.~l° Triglyceride levels
were increased 83% in the high density lipoprotein
serum fraction of subjects with both forms of diabe-
tes. H0 The source of these lipid peroxides was proposed
to be the end product of lipoprotein metabolism from
m e m b r a n e s of peripheral tissues, suggesting a mem-
brane damage in diabetes. H0 Other reports found a 61%
increase in TBA-reactive material in subjects with
diabetes of unstated origin, 1°6 and a 153% increase in
the blood of subjects with type II diabetes. 88'99 Others
have found an increase in plasma and erythrocyte lipid
peroxides 99 in type II diabetics.
Plasma TBA-reactive material was increased in rats
with S T Z - i n d u c e d diabetes.~°5 Rats with S T Z - i n d u c e d
diabetes of 1 week duration had a 31% increase in
serum T B A reactive material, but no significant change
in diabetes of 20 weeks duration. ~°6 Hepatic TBA-re-
active material was increased 24% 1 week after in-
duction of diabetes, and 21% 20 weeks after induction.
Rats with untreated STZ- or alloxan-induced diabetes
of unspecified duration have 333% more malondialde-
hyde (MDA) in liver and 98% more M D A in brain,
but 73% less in kidney and 57% less in spleen. 76
IV. CONCLUSIONS
The role of active oxygen species in the develop-
ment of diabetes seems secure. More work needs to be
done to correlate what happens in the h u m a n disease
compared to the animal models. In particular, how
closely does alloxan- or S T Z - i n d u c e d diabetes corre-
spond to the i m m u n e condition seen in h u m a n s ?
The role of active oxygen species in the diabetic
state is more unclear. In the future, the effect of an-
tioxidants on the complications of diabetes need to be
e x a m i n e d in greater detail. Only in this way will we
be able to definitively answer whether any of these
complications are due to active oxygen species.
Acknowledgements--This work was supported by NIH grant R01
CA41267. I thank Ms. Susan Barnett and Ms. Linda Davies for their
help in manuscript preparation. I would also like to acknowledge
the earlier work done by Dr. Dean Loven in a review on this subject.
This review formed the basis for the present work.
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