Journal of Equine Veterinary Science 40 (2016) 16–25

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Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Original Research

Comparison of Plasma Active Glucagon-Like Peptide

1 Concentrations in Normal Horses and Those With
Equine Metabolic Syndrome and in Horses Placed on
a High-Grain Diet
Kelly A. Chameroy a,1, Nicholas Frank b, *, Sarah B. Elliott a, Raymond C. Boston c
a
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN
b
Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA
c
Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA

a r t i c l e i n f o a b s t r a c t

Article history: Hyperinsulinemia is associated with laminitis in horses, and active glucagon-like peptide 1
Received 13 October 2015 (aGLP-1) stimulates insulin secretion. We hypothesized that plasma aGLP-1 concentrations
Received in revised form 20 January 2016 measured during an oral sugar test (OST) would differ significantly between normal horses
Accepted 20 January 2016
and those with equine metabolic syndrome (EMS) and that aGLP-1 concentrations would
Available online 1 February 2016
increase when EMS horses were placed on a high-grain diet. An enzyme-linked immu-
nosorbent assay for aGLP-1 was validated with equine plasma and six horses with EMS,
Keywords:
and 10 healthy Quarter horse crossbred mares were compared. Eleven months later, the
Incretin hormone
Glucose same horses with EMS were placed on a high-grain diet for 8 weeks and aGLP-1 con-
Insulin centrations were measured during an oral glucose tolerance test at 0 and 8 weeks.
Hyperinsulinemia Frequently sampled intravenous glucose tolerance tests were also performed. The assay
Laminitis was validated with equine plasma and concentrations increased during the OST. Area
under the aGLP-1 curve did not differ between normal and EMS horses. There was a weak
trend (P ¼ .097) toward a higher maximum percentage increase in aGLP-1 concentrations
during the OST in EMS horses compared with normal horses. Body weight increased (P ¼
.031) after 8 weeks when EMS horses were placed on a high-grain diet. Resting (fasted)
insulin concentrations significantly increased (P ¼ .012), but plasma aGLP-1 concentrations
did not change significantly. Our hypotheses were not supported because plasma aGLP-1
concentrations did not differ significantly between normal horses and those with EMS
and did not increase when EMS horses were placed on a high-grain diet for 8 weeks.
However, a trend toward higher percentage increases in aGLP-1 concentrations during the
OST was detected in EMS horses, compared with normal horses, and this warrants further
investigation.
Ó 2016 Elsevier Inc. All rights reserved.

Presented in part at the 2010 American College of Veterinary Internal
Medicine Forum, Anaheim, California, and published in the proceedings
of that meeting.
* Corresponding author at: Dr. Frank, Department of Clinical Sciences,
Cummings School of Veterinary Medicine at Tufts University, North
Grafton, MA 01536.
E-mail address: nicholas.frank@tufts.edu (N. Frank).
1
Dr. Chameroy’s present address is 4921 Fleetwood Drive, Knoxville,
TN 37921.
1. Introduction
Experimental evidence links hyperinsulinemia with
laminitis in horses and ponies [1,2], and high insulin con-
centrations predict the development of pasture-associated
laminitis in ponies [3,4]. Altered insulin metabolism, which
is referred to as insulin dysregulation, is a key component
of the equine metabolic syndrome (EMS), a group of
endocrine and metabolic conditions associated with

0737-0806/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jevs.2016.01.009
 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
laminitis in equids [5]. Components of insulin dysregula-
tion, including excessive insulin responses to oral sugars
(postprandial hyperinsulinemia), fasting hyperinsulinemia,
low C-peptide-to-insulin ratios, and tissue insulin resis-
tance have all been detected in horses and ponies at risk for
laminitis [6]. When EMS was defined in the 2010 American
College of Veterinary Internal Medicine consensus state-
ment, emphasis was placed on measuring fasting insulin
concentrations to detect insulin dysregulation because this
is common method of assessing insulin status in humans.
Accordingly, fasting hyperinsulinemia was recommended
for the diagnosis of EMS and defined by a fasting insulin
concentration >20 mU/L in serum or plasma [5]. However,
fed insulin concentrations may be more relevant to the
development of laminitis when horses are grazing on
pasture and feeding on a near continual basis. Sugars and
amino acids contained within grass stimulate insulin
secretion and subsequent hyperinsulinemia by stimulating
the release of incretin hormones from the intestine [7].
Postprandial hyperinsulinemia therefore requires further
study in horses, and the role of the incretin hormones,
glucagon-like peptide 1 (GLP-1), and glucose-dependent
insulinotropic polypeptide (GIP) warrants investigation.
Direct measurement of insulin sensitivity is required for
studies of insulin dysregulation in horses, and both the
euglycemic-hyperinsulinemic clamp procedure and insulin-
modified frequently sampled intravenous tolerance test
(FSIGTT) with minimal model analysis have been used in
horses [8]. Both tests provide values for tissue insulin
sensitivity, in the form of the metabolized glucose per unit of
serum insulin ratio (M/I ratio) for the clamp procedure and
sensitivity to insulin (SI) for the FSIGTT. An additional benefit
of the FSGITT is the assessment of pancreatic insulin secre-
tion, which is represented by the acute insulin response to
glucose (AIRg) value in the minimal model [9]. In the same
model, the capacity of glucose to mediate its own disposal is
referred to as glucose effectiveness (Sg), and the ability of
islet cells to secrete insulin normalized to the degree of in-
sulin resistance is represented by the disposition index (DI),
which is calculated by multiplying AIRg and SI values. The
insulin-modified FSIGTT with minimal model analysis was
selected here so that pancreatic insulin secretion could be
assessed in addition to insulin sensitivity.
Glucagon-like peptide 1 is a 30 amino acid hormone
secreted from L-cells of the small intestine that stimulates
insulin secretion and inhibits glucagon release in response
to feeding [10]. This hormone is formed through differen-
tial posttranslational processing of proglucagon and is
referred to as GLP-1 7-37 or active GLP-1 (aGLP-1) in
humans. Rodents and pigs also secrete an amidated form of
the hormone called GLP-1 7-36 amide [10]. Both active
forms of GLP-1 are rapidly degraded by the enzyme
dipeptidyl peptidase 4 (DPP4), which cleaves two amino
acids from the amino terminus, resulting in the formation
of GLP-1 (9-37) or GLP-1 9-36 amide (also referred to as
GLP-1m), respectively. These metabolites were previously
considered to be inactive, but now appear capable of
suppressing hepatic glucose production and exerting
insulin-like actions [11]. Hormone degradation begins in
the intestine, so less than 25% of newly secreted GLP-1
enters the liver and only 10%–15% reaches the systemic
17
circulation in humans [10]. The circulating half-life of GLP-1
in plasma is only 2 minutes in humans [12].
Glucagon-like peptide 1 and GIP play important roles in
limiting postprandial hyperglycemia by inducing insulin
secretion as glucose enters the small intestine [13]. Both
GLP-1 and GIP also exert insulinotropic effects on pancre-
atic beta cells that vary with the amount and availability of
glucose and other carbohydrates [14]. Effects of incretin
hormones on insulin secretion can be demonstrated by
administering glucose PO and IV at the same dosage and
measuring insulin or C-peptide concentrations [10,15]. This
“incretin effect” was demonstrated in horses and ponies by
Duhlmeier et al [16] with plasma GIP concentrations
increasing during the oral glucose tolerance test (OGTT).
Glucagon-like peptide 1 also limits postprandial hypergly-
cemia by inhibiting glucagon secretion, slowing gastric
emptying and intestinal motility, and inducing satiety [10].
Assays are available to measure aGLP-1 and total GLP-1
in human plasma. Total GLP-1 assays measure all forms of
the hormone, including the metabolites GLP-1 9-36 amide
in humans and GLP-1 (9-37) in rodents and pigs. An anti-
body directed against the C-terminus of the molecule is
used for total GLP-1 assays, whereas the amino terminus is
targeted for the aGLP-1 assay. Active GLP-1 concentrations
are measured in plasma samples containing a DPP4 inhib-
itor. Measurements of aGLP-1 in equine plasma were
recently described by de Laat et al [17] in ponies subjected
to PO and IV glucose challenges, but aGLP-1 concentrations
have not been reported for horses.
This study was undertaken to validate a commercial
aGLP-1 assay for use with plasma from horses and to
determine whether concentrations of this incretin hor-
mone increase during an oral sugar test (OST) in horses. We
hypothesized that aGLP-1 concentrations would increase in
response to PO administered glucose and other sugars, as
they do in humans [18] and that an incretin effect could be
demonstrated. The specific hypothesis that aGLP-1 con-
centrations measured during the OST would be higher in
EMS horses, compared with normal horses was tested. It
was further hypothesized that insulin and aGLP-1 concen-
trations would significantly increase as a result of obesity
when EMS horses were placed on a high-grain diet to in-
crease body fat mass.
2. Materials and Methods
2.1. Assay Validation
Blood samples collected during the OST from one horse
with EMS and three healthy adult mares were used for the
aGLP-1 validation procedure. Plasma samples were thawed
on ice and assay components prepared according to kit
(Glucagon-like peptide-1 (Active) ELISA EGLP-35K, Millipore
Corp, Billerica, MA) directions. All samples were analyzed in
duplicate. The standard curve for the assay consisted of the
following concentrations: blank (assay buffer alone), 2, 5, 10,
20, 50, and 100 pmol/L, plus quality controls for low (5.1–
10.5 pmol/L) and high (27.8–57.7 pmol/L) aGLP-1 concen-
trations. Dilution and linearity were assessed by diluting
(1:5, 2:5, 3:5, and 4:5 ratios) pooled plasma from samples
collected at 30, 45, and 60 minutes during the OST
18 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
performed in the obese hyperinsulinemic horse. This pooled
plasma was diluted with assay buffer or plasma collected
from the same horse before sugar administration. Dilutions
were performed to determine the lowest limit of detection.
Spike and recovery were performed using plasma from one
fasted healthy horse and the high aGLP-1 standard solution
(100 pmol/L) from the kit. Intraassay variability was calcu-
lated from quadruplicate measures of aGLP-1 in three sam-
ples collected at three time points (0, 15, and 30 minutes)
during the OST in three healthy mares. Interassay variability
was calculated from values obtained for the two quality
controls across three different plates.
2.2. Animals
Six horses (three mares and three geldings) with EMS
were acquired through donation. Equine metabolic syn-
drome was defined by increased adiposity, hyper-
insulinemia, and laminitis [5]. Hyperinsulinemia was
defined by a resting (fasted) insulin concentration >20 mU/
L or peak insulin concentration >60 mU/L during the OST
[19]. Horses ranged in age from 8 to 22 years (median,
16 years) and Arabian (1), Tennessee Walking Horse (1),
Paso Fino (2), Morgan (1), and Azteca (1) breeds were
represented. At the time of the first experiment, all horses
were obese, as defined by a body condition score (BCS) 7/
9 according to the one to nine scoring system reported by
Henneke et al [20]. Horses ranged in body weight from 429
to 559 kg (mean, 475  59 kg), and BCSs ranged from 7/9 to
9/9. Ten Quarter horse crossbred mares aged 7 to 15 years
(median, 8 years) were also evaluated. Body weight ranged
from 445 to 507 kg (median, 469  25 kg), and BCSs ranged
from 4/9 to 6/9 for this group. The 10 mares in this group
had fasted insulin concentrations <20 mU/L and OST in-
sulin concentrations <60 mU/L and are referred to as
normal. All horses were owned by the institution. Horses
with EMS were housed in individual stalls (2.75 m  3.5 m)
with attached outdoor drylots (3.7 m  10 m) for the first
experiment, and normal mares were housed together on a
pasture with run-in sheds at the same facility.
Eleven months later, EMS horses (n ¼ 6) were placed on a
high-grain diet for 8 weeks. Body condition scores ranged
from 4/9 to 8.5/9 at the beginning of this experiment; two
horses were obese, and four horses were not obese. Body
weight ranged from 381 to 533 kg (median, 423 kg) at week
0. Horses were housed in individual grass paddocks
(approximately 15 m  55 m) with run-in sheds for this
experiment and had been transferred there 2 months earlier.
2.3. Experimental Design
Plasma aGLP-1 concentrations were measured in equine
plasma and compared between nonobese normal mares
and horses with EMS. The first experiment was performed
in July and the second experiment was performed
11 months later, and this separation was a result of summer
research project scheduling. Horses in the EMS group were
managed to decrease body fat mass and lower insulin
concentrations during the 11-month period between ex-
periments. In the second experiment, the horses in the EMS
group (n ¼ 6) were placed on a high-grain diet for 8 weeks
to induce weight gain. Glucose and insulin dynamics were
assessed at 0 and 8 weeks by performing the FSIGTT and
OGTT procedures. Tests were performed on consecutive
days with the FSIGTT performed on the first day and the
OGTT performed the following day. The OGTT was per-
formed instead of the OST to allow for comparison of
glucose and insulin concentrations when dextrose was
administered IV and PO. Active GLP-1 concentrations were
measured in plasma collected during the OGTT. Horses
were transported to the teaching hospital in pairs 2 days
beforehand in order for them to acclimate to their sur-
roundings. Morphometric measurements were also
collected at 0 and 8 weeks. Studies were approved by the
University of Tennessee Institutional Animal Care and Use
Committee.
2.4. High-Grain Diet
Horses consumed grass while housed in paddocks and
also received additional feed to provide twice the digestible
energy requirement for maintenance for each horse. Forty
percent of digestible energy was provided as Timothy/Or-
chard grass hay, containing 1,100 kcal/lb on a dry matter
basis and 60% from a commercial sweet feed that provided
1,330 kcal/lb on a dry matter basis. Hay contained 13.7%
water-soluble carbohydrate, 0.9% starch, and 10.8%
ethanol-soluble carbohydrate on a dry matter basis and
sweet feed provided 8.8% water-soluble carbohydrate,
17.4% starch, and 6.2% ethanol-soluble carbohydrate on a
dry matter basis, as determined by a commercial feed
testing laboratory (Equi-Analytical, Inc, Ithaca, NY). Horses
were transitioned to the diet over a span of 1 week by
incrementally feeding 0.25  target amount twice daily on
day 1, 0.5  target amount on days 2, 3, and 4, 0.75  target
amount on days 5, 6, and 7, and the full amount by day 8.
Water was available ad libitum via automatic waterer, and
grain and hay were provided between 07:00 and 08:00 and
again between 15:00 and 15:30.
2.5. Morphometric Measurements
Morphometric measurements included neck circum-
ference, body weight, BCS, girth circumference, and rump
fat thickness. Neck circumference was measured perpen-
dicular to the crest of the neck at 0.25, 0.50 (midneck), and
0.75 of the distance from the withers to the poll with a
measuring tape as previously described [21]. Mean neck
circumference values were calculated. Rump fat thickness
was measured by placing the ultrasound probe 5 cm lateral
to the dorsal midline at the center of the pelvic bone, as
previously described by Carter et al [22].
2.6. Oral Sugar Test
A 14-gauge polypropylene catheter was placed in the
jugular vein the night before testing. All horses were
remained in stalls overnight and received a single flake of
hay at 20:00, with no additional feed provided until after
testing was completed the next day. Patency of the catheter
was maintained overnight by injecting 100 units heparin
into a heparin lock infusion cap. Baseline blood samples
 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
were collected at 08:00, and then, corn syrup (Karo Light
Corn Syrup, ACH Food Companies, Inc, Memphis, TN) was
administered via dose syringe at a dosage of 0.15 mL/kg bwt
to provide 150 mg total digestible sugar per kg bwt, as
previously described [19]. The manufacturer of this product
will not provide compositional analysis results to the
public. Blood samples were then collected from the cath-
eter at 15, 30, 45, 60, 75, 90, 105, and 120 minutes relative to
sugar administration. At each time point, 3 mL of blood was
withdrawn from the infusion line and discarded. Blood was
collected into tubes containing ethylenediaminetetraacetic
acid (EDTA), and then, 5 mL heparinized saline was infused.
2.7. Frequently Sampled Intravenous Glucose Tolerance Test
Fourteen-gauge polypropylene catheters were inserted
into jugular veins the night before testing using aseptic
technique. Patency of IV catheters was maintained by
infusion of 5 mL saline solution containing heparin (4 U/
mL) into the catheter every 6 hours. Horses were fasted
overnight and throughout the test. Water was available at
all times. A 50% (wt/vol) dextrose solution (Dextrose 50%
Injection, Abbott Laboratories, North Chicago, IL) was
infused IV at a concentration of 150 mg/kg bwt, followed by
20 mL heparinized saline. Blood samples were collected
from the other jugular catheter 15 minutes before dextrose
infusion (15 minutes) and at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15,
16, 19, 22, 23, 24, 25, 27, 30, 35, 45, 50, 60, 75, 90, 105, 120,
150, and 180 minutes relative to dextrose infusion. The
dextrose infusion catheter was removed after completion
of the FSIGTT, whereas the other catheter was maintained
until the next day when the OGTT was performed.
2.8. Minimal Model Analysis
Minimal model parameters for SI, Sg, and AIRg were
calculated as previously described with computer software
(MinMod Millennium, version 6.10, Raymond Boston, Uni-
versity of Pennsylvania, Kennet Square, PA) (Stata 9.2, Stata
Corp, College Station, TX) [9]. Disposition index was
calculated by multiplying AIRg and SI values.
2.9. Oral Glucose Tolerance Test
Dextrose powder (Sigma-Aldrich Co, St. Louis, MO) was
weighed to provide 150 mg/kg bwt dextrose. Powder was
mixed with water and gently heated to bring dextrose into
a solution that was administered to horses via dose syringe.
Blood samples were collected 15 minutes before dextrose
administration (15 minutes) and at 15, 30, 45, 60, 75, 90,
105, 120, 135, 150, 165, and 180 minutes relative to PO
dextrose administration.
2.10. Sample Processing
Blood samples for glucose and insulin measurements
were collected into tubes containing a glycolytic inhibitor
(potassium oxalate/sodium fluoride) and tubes without
anticoagulant, respectively. A sample processing protocol
developed by the Cherrington Laboratory (Vanderbilt Uni-
versity, Nashville, TN) for measuring aGLP-1 concentrations
19
in dogs [23] was used. Blood was collected into prechilled
EDTA-treated tubes and centrifuged immediately (3,500g
for 3 minutes) after collection. After centrifugation, 1 mL of
plasma was added to 10 mL DPP4 (DPP 4 Inhibitor, Millipore
Corp, Billerica, MA) in 1.5-mL microcentrifuge tubes that
were kept on ice. Samples were inverted to mix thoroughly,
and samples were then flash frozen on dry ice and stored at
20 C until further analysis.
2.11. Assays
Plasma glucose concentrations were measured by use of
a colorimetric assay (Glucose, Roche Diagnostic Systems
Inc, Somerville, NJ) on an automated discrete analyzer
(Cobas Mira, Roche Diagnostic Systems Inc, Somerville, NJ).
Serum insulin concentrations were determined by use of a
radioimmunoassay (Coat-A-Count insulin radioimmuno-
assay, Siemens, Los Angeles, CA) previously validated for
use in horses [24]. Plasma non-esterified fatty acids (NEFA)
concentrations were measured using an enzymatic colori-
metric test kit (NEFA C; Wako Chemicals USA, Richmond,
VA) using acyl CoA synthetase, acyl CoA oxidase, and
ascorbate oxidase reactions that has previously been vali-
dated in our laboratory for use with equine plasma. Each
sample was assayed in duplicate, and intraassay co-
efficients of variation (CV) <5% or <10% were required for
acceptance of glucose and insulin or NEFA assay results,
respectively. Active GLP-1 was measured by use of an
enzyme-linked immunosorbent assay (ELISA) as described
previously. Each sample was assayed in duplicate, and CV%
of <10% was required for acceptance of aGLP-1 results.
2.12. Statistical Analysis
Peak aGLP-1 concentration during the OST or OGTT was
determined, and maximum percent change was calculated.
Area under the curve values for glucose (AUCg), insulin
(AUCi), and aGLP-1 (AUCglp) concentrations were calculated
using the trapezoidal method and computer software
(GraphPad Prism 5.03, GraphPad Software, Inc, La Jolla,
CA.). These data did not fit a normal distribution, and
nonparametric Mann–Whitney tests were performed to
compare groups using the same software. Median (range)
values are reported. Oral sugar test aGLP-1 data were nor-
mally distributed, and repeated measures analysis of vari-
ance (ANOVA) analysis was performed, with the main
effects of group and time examined. In the second study, 0-
and 8-week results were compared using the Wilcoxon
matched-pairs signed rank test. Spearman correlation co-
efficients were also calculated. Significance was defined as
a value P < .05.
3. Results
3.1. Assay Validation
A quadratic curve with r2 ¼ 0.9997 was obtained with
standards provided in the kit (range 2–100 pmol/L). Dilu-
tion and linearity testing with pooled OST plasma and assay
buffer resulted in a linear plot of GLP-1 concentrations with
an observed slope of 5.0 (r2 ¼ 0.99) compared with an
20 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
expected slope of 4.9. When baseline (time ¼ 0) plasma was
used as a diluent, aGLP-1 concentrations were linear with
an observed slope of 3.8 (r2 ¼ 0.99), compared with an
expected slope of 3.6. Mean  standard deviation (SD)
observed-to-expected ratio for dilution and linearity was
99.2  4.5% for the assay buffer and 99.9  4.3% for baseline
plasma. The lowest limit of detection stated by the manu-
facturer (2.0 pmol/L) was applied for the assay.
Spike and recovery produced an observed slope of 85.1
(r2 ¼ 0.98) compared with the expected value of 97.6 (r2 ¼
0.99). Mean observed-to-expected ratio for spike and re-
covery was 97.6  10.5%. Mean  SD intraassay CV was 9.5
 5.1%, and interassay CV for the two quality controls was
2.3% and 2.6%, respectively.
3.2. Comparison of Normal and EMS Horses
Median (range) fasting serum insulin concentrations
were 3.0 mU/L (1.7–10.4 mU/L) for normal mares and 25.5
mU/L (9.4–111.0 mU/L) for EMS, and there was a significant
difference between groups (P ¼ .010; Fig. 1). Peak OST in-
sulin values of 24.5 mU/L (5.8–48.3 mU/L) and 240.1 mU/L
(104.7–269.6 mU/L) were detected for normal and EMS
groups, respectively, and groups differed significantly (P <
.001), as would be expected because horses were assigned
to the EMS group on the basis of a resting (fasted) insulin
concentration >20 mU/L or peak insulin concentration >60
mU/L during the OST. Peak insulin correlations were posi-
tively correlated with baseline insulin concentrations (rs ¼
0.92; P < .001).
Resting (fasted) aGLP-1 concentrations ranged from
below the limit of detection to 6.5 pmol/L (median, 3.3
pmol/L) at time ¼ 0 in normal Quarter horse crossbred
mares and from 2.2 to 6.3 pmol/L (median, 3.4 pmol/L) in
EMS horses (Fig. 2), with no difference detected between
groups (P ¼ .744). Active GLP-1 concentrations increased
over time during the OST, and peak values ranged from
3.3 to 12.7 pmol/L (median, 4.7 pmol/L) in normal mares
and from 3.8 to 18.3 pmol/L in EMS horses (median, 9.2
pmol/L), with wide individual horse variability observed
Fig. 1. Median (range) plasma insulin concentrations in normal Quarter
horse crossbred mares (n ¼ 10; circles) and horses with equine metabolic
syndrome (EMS; n ¼ 6; squares) during an oral sugar test. Baseline (P ¼ .010)
and peak (P < .001) insulin concentrations and area under the insulin curve
values (P < .001) differed between groups.
Fig. 2. Median (range) active glucagon-like peptide 1 (aGLP-1) concentra-
tions in 10 normal Quarter horse crossbred mares (circles; dashed line) and
six horses with equine metabolic syndrome (EMS; squares; solid line) during
an oral sugar test. Area under the aGLP-1 curve values did not differ between
groups (P ¼ .744).
and no difference in peak values detected between
groups (P ¼ .147; Fig. 3). A significant effect of time (P <
.001), but not group (P ¼ .389) or group  time (P ¼ .715),
was detected by repeated measures ANOVA for OST aGLP-
1 concentrations. Median (range) maximum percentage
increase in aGLP-1 concentrations from baseline was 68%
(15% to 193%) in normal mares and 148% (36%–286%) in
EMS horses, and there was a trend (P ¼ .097) toward
higher values in EMS horses (Fig. 4). A trend was also
detected for the correlation between percentage increase
in aGLP-1 concentration and peak insulin concentration
(rs ¼ 0.44; P ¼ .091). Area under the curve values for
plasma aGLP-1 concentrations did not differ (P ¼ .254)
between groups. Active GLP-1 concentrations were below
the lowest limit of detection (2.0 pmol/L) at time ¼ 0 for
one horse and at 0 and 15 minutes for a second horse,
and both horses were in the normal group. All other
aGLP-1 concentrations measured in this study were
greater than or equal to 2.0 pmol/L.
Fig. 3. Scatterplot showing peak aGLP-1 values for normal Quarter horse
crossbred mares (n ¼ 10; circles) and horses with equine metabolic syn-
drome (n ¼ 6; squares) during an oral sugar test. Horizontal line represents
median value. aGLP-1, active glucagon-like peptide 1; EMS, equine metabolic
syndrome.
 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
Fig. 4. Scatterplot showing percentage increase in aGLP-1 values for normal
Quarter horse crossbred mares (n ¼ 10; circles) and horses with equine
metabolic syndrome (n ¼ 6; squares) during an oral sugar test. Horizontal
line represents median value. aGLP-1, active glucagon-like peptide 1; EMS,
equine metabolic syndrome.
3.3. High-Grain Diet
When EMS horses were placed on a high-grain diet for
8 weeks, mean body weight significantly increased (P ¼
.031), with individual horse percentage weight gain over
8 weeks ranging from 2% to 10% (Table 1). Body condition
score, mean neck circumference, neck-to-height ratio,
girth, and rump fat thickness also increased significantly
over 8 weeks. One horse was removed from the study after
4 weeks because clinical signs consistent with laminitis
were observed and testing was performed at this time for
this horse. Mild bilateral forelimb lameness consistent with
laminitis was detected in two other horses at 8 weeks.
Affected horses were treated with phenylbutazone and
lameness resolved over time. Resting (fasted) glucose
concentrations did not change significantly over 8 weeks (P
¼ .101), but resting (fasted) insulin concentrations
increased (P ¼ .012; Table 2). Plasma NEFA concentrations
did not differ significantly over time.
Glucose and insulin concentrations increased in
response to dextrose administration during the OGTT, but
AUCg and AUCi did not differ significantly between 0 and
8 weeks (Figs. 5 and 6). Plasma aGLP-1 concentrations
increased during the OGTT (Fig. 7) and peaked at 45 mi-
nutes, but AUCglp did not change significantly between
0 and 8 weeks (P ¼ .438). Glucose and insulin concentra-
tions increased in response to IV administered dextrose in
Table 1
Median (range) physical measurement values at 0 and 8 weeks for six horses with equine metabolic syndrome that were placed on a high-grain diet.a
Variable Period (wk)
0 8
Body weight (kg) 423 (381–533)
BCS (1–9) 5.75 (4.0–8.50)
Mean neck circumference (cm) 82.0 (89.0–98.0)
Neck-to-height (ratio) 0.60 (0.56–0.66)
Girth circumference (cm) 180.5 (171.0–187.0)
Rump fat thickness (cm) 1.78 (1.11–3.09)
Abbreviation: BCS, body condition score.
P < .05.
a
Measurements performed at 4 weeks in one horse.
21
the FSIGTT (Figs. 8 and 9) with AUCg decreasing over
8 weeks (P ¼ .031). There was a trend toward higher AUCi
values (P ¼ .063) at week 8, compared with week 0. Median
AUCi values for OGTT curves at 0 and 8 weeks were 6,934
and 10,952 mU/L$min, respectively, whereas FSIGTT AUCi
values were 9,072 and 16,702 mU/L$min, respectively.
Minimal model analysis revealed a trend toward higher
AIRg values at week 8 (P ¼ .063), but SI, Sg, and DI values
did not differ significantly between 0 and 8 weeks. Insulin
sensitivity values ranged from 0.04 to 1.10 
104 L$min1$mU1 at the beginning of the study, and
horses that developed clinical signs consistent with lami-
nitis had initial SI values of 0.12 (laminitis at 4 weeks), 0.04,
and 0.68  104 L$min1$mU1.
4. Discussion
A commercial ELISA for measuring aGLP-1 was validated
for use with plasma from horses and concentrations
increased during the OST and OGTT. Active GLP-1 concen-
trations did not differ significantly between healthy horses
and those with EMS in this study when area under the
curve values were compared, and our hypothesis was not
supported. However, there was a weak trend toward higher
percent increases in aGLP-1 concentrations during the OST
in EMS horses compared with normal horses. A trend was
also detected in the correlation between maximum percent
increase in aGLP-1 concentration and peak insulin con-
centration. Marked individual horse variability was
observed, and additional studies must now be performed
with larger populations of horses, subdivided by age, sex,
and breed. When EMS horses were placed on a high-grain
diet for 8 weeks, fasting insulin concentrations increased,
FSIGTT area under the glucose curve decreased, and there
was a trend toward higher FSIGTT area under the insulin
curve values. Three horses developed forelimb lameness
consistent with laminitis. Plasma aGLP-1 concentrations
did not change in EMS horses that were placed on a high-
grain diet for 8 weeks.
We report measurements of aGLP-1 concentrations in
plasma from horses using a commercially available ELISA
for measuring human aGLP-1. One limitation of this study is
that equine-specific GLP-1 was not available for the spike
and recovery component of the validation, so aGLP-1 con-
centrations are human equivalents of immunoreactive
aGLP-1. It is also assumed that the antibody used in the
Treatment
P
461 (397–550) .031
8.0 (7.0–9.0) .036
89.0 (96.5–103.0) .031
0.64 (0.61–0.70) .034
184.5 (174.0–194.0) .035
2.59 (1.34–3.88) .031
22 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25

Table 2
Median (range) blood values at 0 and 8 weeks for six horses with equine metabolic syndrome that were placed on a high-grain diet.a

Variable Period (wk) P

0 8
Resting glucose (mg/dL) 85.7 (75.4–114.5) 80.7 (69.8–93.9) .110
OGTT AUCg ([mg/dL] min)  103 18.4 (15.3–19.5) 16.2 (14.4–18.3) .438
FSIGTT AUCg ([mg/dL] min)  103 22.0 (19.9–26.2) 18.7 (16.9–20.6) .031
Resting insulin (mU/L) 8.5 (3.2–18.1) 14.9 (4.8–52.1) .012
OGTT AUCi ([mU/L] min)  103 6.9 (1.7–13.0) 10.9 (4.2–15.4) .313
FSIGTT AUCi ([mU/L] min)  103 9.1 (4.5–12.3) 16.7 (9.3–23.7) .063
Baseline aGLP-1 concentration (pmol/L) 5.1 (4.0–9.8) 4.9 (3.6–9.3) .094
OGTT AUCglp ([pmol/L] min) 692 (566–1,636) 610 (347–1,483) .438
SI (L$min1$mU1)  104 0.62 (0.04–1.1) 0.42 (0.25–6.5) .688
Sg (min1)  102 0.015 (0.001–0.020) 0.011 (0.007–0.019) .688
AIRg ([mU/L]$min) 630.3 (503.0–943.1) 1,121 (575.7–1,525) .063
DI  102 373.6 (26.0–653.9) 548.2 (179.7–5,203) .219

Abbreviations: aGLP-1, active glucagon-like peptide 1; AIRg, acute insulin response to glucose; AUCg, area under the glucose curve; AUCglp, area under the
aGLP-1 curve; AUCi, area under the insulin curve; DI, disposition index; FSIGTT, frequently sampled intravenous tolerance test; OGTT, oral glucose tolerance
test; Sg, glucose effectiveness; SI, sensitivity to insulin index.
a
Measurements performed at 4 weeks in one horse.

assay bound to GLP-1 and did not cross-react with other
proteins in equine plasma. The increase in aGLP-1 con-
centrations detected after oral administration of corn syrup
or dextrose strongly suggests that the assay was detecting
GLP-1 because incretin hormones are expected to increase
after feeding. It must be determined whether aGLP-1 exists
as GLP-1 7-37 in horses as it does in humans or as the
amidated form, GLP-1 7-36 amide found in rodents and
pigs [10]. Other forms of GLP-1 were not measured in this
study, so future studies should include measurement of
total GLP-1 concentrations.
Horses were allocated to the EMS group on the basis
of resting (fasted) insulin concentrations and OST results.
A 60 mU/L cutoff value is commonly used when the OST
is performed as a diagnostic test, so all horses included
were considered positive [19]. Horses in the EMS group
were obese at the time of the first experiment, were
exhibiting regional adiposity, and had histories of lami-
nitis before donation. Direct measurements of insulin
sensitivity were obtained in the second experiment, and SI
values <1.0  104 L$min1$mU1 were detected in five of
six horses. Insulin resistance has not been defined by a
specific cutoff value in horses, but Treiber et al [25] re-
ported a range of 0.14 to 0.78  104 L$min1$mU1 for
the lowest quintile of SI values for 46 horses (10 mature
Thoroughbred geldings, 12 pregnant Thoroughbred
mares, 12 Arabian geldings, and 12 Thoroughbred wean-
lings). Tissue insulin resistance would be expected to in-
crease the magnitude of the insulin curve during the OST
and OGTT as insulin clearance decreases in liver, skeletal
muscle, and adipose tissues. Plasma C-peptide concen-
trations should be measured in the future to assess the
relative effects of increased insulin secretion and
decreased insulin clearance on OST and OGTT results.
Lower C-peptide-to-insulin ratios have been detected in
insulin-resistant horses [26], indicating that both reduced
hepatic insulin clearance and increased insulin secretion
contribute to hyperinsulinemia.
Plasma aGLP-1 concentrations did not differ signifi-
cantly between groups in this study, but marked individual
horse variability was observed and this affected group
comparisons. Potential factors affecting individual horse

Fig. 5. Median (range) plasma glucose concentrations during oral glucose
tolerance tests performed at 0 (circles; dashed line) and 8 (squares; solid
line) weeks in six horses with equine metabolic syndrome that were placed
on a high-grain diet. One horse was tested at 4 weeks before being removed
from the study.
Fig. 6. Median (range) serum insulin concentrations during oral glucose
tolerance tests performed at 0 (circles; dashed line) and 8 (squares; solid
line) weeks in six horses with equine metabolic syndrome that were placed
on a high-grain diet. One horse was tested at 4 weeks before being removed
from the study.
 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
Fig. 7. Median (range) plasma aGLP-1 concentrations during oral glucose
tolerance tests performed at 0 (circles; dashed line) and 8 (squares; solid
line) weeks in six horses with equine metabolic syndrome that were placed
on a high-grain diet. One horse was tested at 4 weeks before being removed
from the study. GLP-1, glucagon-like peptide 1.
incretin hormone responses include genetic variability,
breed, adiposity, age, and diet. Genetic differences in the
GLP-1 receptor have been detected in humans and explain
individual responses to GLP-1 agonists [27]. Differences in
microbial flora within the stomach of individual horses
might also have altered the relative abundance of different
sugars arriving at L-cells within the small intestine and
affected glucose bioavailability. Individual variability in
fasting total GLP-1 concentrations has been observed in
dogs [28], and further studies are required to determine
whether differences in aGLP-1 concentrations among in-
dividual horses result from variation in glucose absorption
into L-cells, GLP-1 secretion, or DPP4-mediated activation
of GLP-1.
Two trends were observed with respect to percent in-
crease in aGLP-1 concentrations during the OST and should
be revaluated in larger populations of horses. Results sug-
gest that EMS horses have higher percentage increases in
aGLP-1 concentrations during the OST than normal horses,
and horses with higher peak insulin concentrations during
Fig. 8. Median (range) plasma glucose concentrations during frequently
sampled intravenous glucose tolerance tests performed at 0 (open circles;
dashed line) and 8 (squares; solid line) weeks in six horses with equine
metabolic syndrome that were placed on a high-grain diet. One horse was
tested at 4 weeks before being removed from the study.
23
Fig. 9. Median (range) serum insulin concentrations during frequently
sampled intravenous glucose tolerance tests performed at 0 (open circles;
dashed line) and 8 (squares; solid line) weeks in six horses with equine
metabolic syndrome that were placed on a high-grain diet. One horse was
tested at 4 weeks before being removed from the study.
the OST have higher percentage increases in aGLP-1 con-
centrations. These findings are supported by results of a
recent study of ponies in which plasma aGLP-1 concen-
trations measured during an oral glucose test were signif-
icantly higher in hyperinsulinemic ponies, when compared
with normoinsulinemic ponies [17]. However, P values
were close to .10 in the study reported here, and larger
populations must be studied in the future to determine
whether these preliminary findings have biological signif-
icance in horses.
According to compositional analysis results provided by
the National Data System for Research, corn syrup of the
type administered to horses in this study contains glucose,
maltose, and starch in approximately 19%, 14%, and 67%
proportions [29]. Effects of specific sugars on aGLP-1 con-
centrations have not been determined in horses, and this is
relevant because studies performed in rats and dogs have
shown that aGLP-1 concentrations vary according to food
type. Massimino et al [30] determined that chronic inges-
tion of high-fermentable dietary fiber in dogs significantly
increased GLP-1 concentrations, compared with feeding
low-fermentable fiber. However, the opposite results were
reported when dogs were fed high- or low-fermentable
fiber diets, possibly as a result of lower voluntary feed
intake in the high-fermentable fiber group [31]. A decrease
in GLP-1 concentrations was also detected when rats were
fed a high-fat diet compared with a basic chow diet [32].
Oral glucose tolerance tests were performed using dextrose
powder in the second phase of the study so that only one
type of sugar was administered. Glucose solution was
successfully prepared and administered via dose syringe in
this study, but corn syrup is easier to administer and is
therefore recommended for future studies.
Higher fasting plasma GLP-1 concentrations have been
associated with obesity and increased rates of fat oxidation
in humans [33]. Horses in the EMS group were obese for the
first experiment, but lost body fat mass in the 11-month
period that followed as a result of intensive management.
Obesity affects total GLP-1 concentrations in cats, with
lower AUCglp values detected in obese cats when the OGTT
is performed [34]. Nonalcoholic fatty liver disease is also
24 K.A. Chameroy et al. / Journal of Equine Veterinary Science 40 (2016) 16–25
associated with obesity in humans, and this condition
lowers aGLP-1 concentrations by increasing the activity of
DPP4, the enzyme responsible for rapid degradation of
aGLP-1 in circulation [35]. Our research group has detected
mild hepatic lipidosis in obese horses on postmortem ex-
amination, so fatty infiltration of the liver may contribute to
individual horse variability in aGLP-1 concentrations.
Future studies should include measurements of DPP4 ac-
tivity and total GLP-1 concentrations and correlations
among these measures and the degree of hepatocyte lipid
infiltration or liver enzyme activities.
Horses with EMS gained body fat mass when placed on
a high-grain diet for 8 weeks that was providing twice the
digestible energy requirement for maintenance. This was
the approach used by Carter et al [22] to induce weight gain
in Arabian geldings, and body mass increased by 20% over a
period of 16 weeks. An association exists between fat
accumulation in the neck (cresty neck) and insulin sensi-
tivity in horses [21], and mean neck circumference-to-
height ratio is used as a measure of neck adiposity [36]. A
neck-to-height ratio of 0.63  0.1 or cresty neck score of
3/5 increases the risk of pasture-associated laminitis [36].
Ratios >0.63 were detected after weight gain in this study,
and three horses exhibited bilateral forelimb lameness
consistent with laminitis. These clinical signs of laminitis
abated once grain was removed from the diet, and medical
treatment was provided. No additional diagnostic tests
were performed to confirm that laminitis had developed,
so this was a presumptive diagnosis based on the onset of
bilateral forelimb lameness.
Resting (fasted) insulin concentrations increased over
8 weeks when EMS horses were placed on a high-grain
diet, with a trend toward higher AUCi values during the
FSIGTT. Feeding EMS horses a high-grain diet therefore
exacerbated hyperinsulinemia, and this finding supports
current recommendations to limit sugar intake in affected
horses. Our study provides evidence that plasma aGLP-1
concentrations increase in response to orally adminis-
tered dextrose or corn syrup in horses, which supports a
role for aGLP-1 in postprandial hyperinsulinemia. However,
an incretin effect was not detected in this study because
AUCi values were higher for the FSIGTT than the OGTT. This
finding may be explained by differences in time points
between the tests, the limited duration (180 minutes) of
the OGTT used here, or slower absorption of glucose from
the intestinal tract, compared with IV administration of
dextrose as a bolus. de Laat et al [17] also failed to
demonstrate an incretin effect in ponies, and results of both
studies suggest that equids differ from humans in this re-
gard [18]. Evolutionary adaptation to forage diets and en-
ergy production through hindgut fermentation may
account for this difference between species.
Plasma aGLP-1 concentrations did not change signifi-
cantly when six EMS horses were placed on a high-grain
diet for 8 weeks, and our hypothesis was not supported.
Effects of obesity on GLP-1 concentrations in humans have
differed among studies. In one study, obese male subjects
had lower AUCglp values than lean subjects after a 2.5 MJ
test meal, and values increased after weight loss [37]. In
contrast, GLP-1 did not differ between obese and lean
young men receiving an intraduodenal infusion of glucose
and fat for 120 minutes (2.86 kcal/min) on 2 separate days
[38]. Reductions in GLP-1 secretion are attributed to
diminished L-cell responsiveness as free fatty acid con-
centrations rise with obesity [39], but this relationship is
not straightforward because free fatty acid concentrations
are not correlated with GLP-1 secretion in humans with
diabetes mellitus [40]. One limitation of the study reported
here is that effects of feeding grain and increasing body fat
mass on aGLP-1 concentrations could not be separated. A
high-grain diet might alter incretin hormone concentra-
tions during the OGTT, independent of obesity as more
sugars, proteins, and fats enter the small intestine. Dogs fed
a high-fat diet for 12 weeks had higher resting and post-
prandial aGLP-1 concentrations than those fed a normal
diet for 3 weeks [41]. A second limitation of the study was
the absence of normal horses in the second experiment.
Glucose, insulin, and aGLP-1 responses to a high-grain diet
and obesity could not be compared between EMS and
normal horses, so it remains possible that normal horses
respond differently to the same intervention.
5. Conclusions
A commercially available ELISA for aGLP-1 was used
with plasma from horses and measured concentrations
increased during the OST and OGTT, as one would expect
for an incretin hormone. Plasma aGLP-1 concentrations did
not differ significantly between normal horses and those
with EMS and did not increase after EMS horses were
placed on a high-grain diet for 8 weeks. Wide individual
variability was detected, so larger populations of horses
must be studied to more fully investigate the role of
incretin hormones in postprandial hyperinsulinemia in
horses with EMS. Differences in genetics, age, or previous
diet may contribute to this variability. A trend toward
higher percentage increases in aGLP-1 concentrations
during the OST was detected in EMS horses, compared with
normal horses, and this warrants further investigation.
Acknowledgments
Funding was provided by the University of Tennessee
College of Veterinary Medicine Center of Excellence. The
authors declare no conflicts of interest.
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