1

This article is downloaded from

http://researchoutput.csu.edu.au

It is the paper published as: Author: K. Tinworth, R. Boston, P. Harris, M. Sillence, S. Raidal and G. Noble Title: The effect of oral metformin on insulin sensitivity in insulin-resistant ponies Journal: Veterinary Journal ISSN: 1090-0233 1532-2971 Year: 2012 Volume: 191 Issue: 1 Pages: 79-84 Abstract: Metformin may be an effective therapeutic option for insulin-resistant (I-R) horses/ponies because, in humans, it reportedly enhances insulin sensitivity (SI) of peripheral tissues without stimulating insulin secretion. To determine the effect of metformin on insulin and glucose dynamics in I-R ponies, six ponies were studied in a cross-over design by Minimal Model analysis of a frequently-sampled intravenous glucose tolerance test (FSIGT). Metformin was administered at 15 mg/kg bodyweight (BW), orally, twice-daily, for 21 days to the metformin-treated group. The control group received a placebo. A FSIGT was conducted before and after treatment. The Minimal Model of glucose and insulin dynamics rendered indices describing SI, glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and the disposition index (DI). The body condition score (BCS), BW and cresty neck score (CNS) were also assessed. There was no significant change in SI, Sg, AIRg, DI, BW, BCS or CNS in response to metformin, or over time in the control group. There were no measurable benefits of metformin on SI, consistent with recent work showing that the bioavailability of metformin in horses is poor, and chronic dosing may not achieve therapeutic blood concentrations. Alternatively, metformin may only be effective in obese ponies losing weight or with hyperglycaemia. Author Address: ktinworth@csu.edu.au drrayboston@yahoo.com pat.harris@effem.com martin.sillence@qut.edu.au sraidal@csu.edu.au gnoble@csu.edu.au CRO Number: 29685

2

Original article

The effect of oral metformin on insulin sensitivity in insulin-resistant ponies

Kellie D. Tinworth a, Ray C. Boston b, Patricia A. Harris c, Martin N. Sillence d,

Sharanne L. Raidal a, Glenys K. Noble a*

a School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW

2650, Australia

b Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett

Square, PA 19348

c Equine Studies Group, WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds,

Leicestershire, LE14 4RT, UK

d Faculty of Science and Technology, Queensland University of Technology, Brisbane 4001,

Australia

* Corresponding author: Tel.: +61 2 69 33 4242; fax: +61 2 69 33 2991.

E-mail address: gnoble@csu.edu.au (Glenys K. Noble)

3

Abstract

Metformin may be an effective therapeutic option for insulin-resistant (I-R)

horses/ponies because, in humans, it reportedly enhances insulin sensitivity (SI) of peripheral

tissues without stimulating insulin secretion. To determine the effect of metformin on insulin

and glucose dynamics in I-R ponies, six ponies were studied in a cross-over design by

Minimal Model analysis of a frequently-sampled intravenous glucose tolerance test (FSIGT).

Metformin was administered at 15 mg/kg BW, orally, twice-daily, for 21 days to the

metformin-treated group. The control group received a placebo. A FSIGT was conducted

before and after treatment. The Minimal Model of glucose and insulin dynamics rendered

indices describing SI, glucose effectiveness (Sg), acute insulin response to glucose (AIRg)

and the disposition index (DI). Bodyweight (BW), body condition score (BCS) and cresty

neck score (CNS) were also assessed. There was no significant change in SI, Sg, AIRg, DI,

BW, BCS or CNS in response to metformin, or over time in the control group. There were no

measurable benefits of metformin on SI, consistent with recent work showing bioavailability

of metformin in horses is poor, and chronic dosing may not achieve therapeutic blood

concentrations. Alternatively, metformin may only be effective in obese ponies losing weight

or with hyperglycaemia.

Keywords: Equine; Hyperinsulinaemia; Laminitis; Obesity; Pharmacology

4

Introduction

With an increasing appreciation of the pathophysiology of insulin resistance (IR) and

hyperinsulinaemia in horses/ponies (Hess et al., 2006; Asplin et al., 2007; Geor, 2008; de

Laat et al., 2010), the goal of owners and veterinarians is to prevent IR, or to treat it before

the consequences become manifest. While the correct management of energy intake and

exercise levels may be effective (Carter et al., 2010; Frank et al., 2010), there are

circumstances where pharmacological intervention may be warranted (Geor and Harris, 2009;

Frank et al., 2010).

With no licensed medications available for the treatment of IR in horses/ponies,

veterinarians increasingly prescribe the off-label use of medications used for IR in humans

(eg, metformin, levothyroxine sodium, glibenclamide) (Johnson et al., 2005; Frank et al.,

2008; Durham et al., 2009), therefore evaluation of efficacy of these drugs in horses/ponies is

necessary. The anti-hyperglycaemic drug metformin (dimethlybiguanide) is in widespread

human clinical use, where it enhances insulin sensitivity (SI) by increasing peripheral glucose

uptake. It also decreases blood glucose concentrations by inhibiting hepatic glucose

production and intestinal absorption of glucose (Saenz et al., 2005). Metformin promotes

weight loss and reduces lipid levels; adverse effects are rare (Salpeter et al., 2008).

Several reports describe the effects of metformin use in horses/ponies with mixed

results (Johnson et al., 2005; Vick et al., 2006; Durham et al., 2008; Durham et al., 2009;

Firshman et al., 2009; Hustace et al., 2009). A single dose of metformin (1.9 mg/kg

bodyweight (BW), orally) administered with an insulin secretagogue to a hyperglycaemic

horse reduced plasma glucose concentrations to values within the reference interval (Johnson

et al., 2005). Furthermore, metformin (2.8 mg/kg BW, orally, 12-hourly) administered to 14

5

obese mares for 30 days enhanced SI, measured with the hyperinsulinaemic-euglycaemic

clamp (HEC) (Vick et al., 2006). However, in the same study, metformin became ineffective

when given for longer or at an increased dose (Vick et al., 2006). In another study, at a dose

of 15 mg/kg BW orally, 12-hourly, metformin improved proxy measures of SI and beta-cell

function for up to 14 days in 18 insulin resistant (I-R) horses/ponies, compared with pre-

treatment values from the same animals (Durham et al., 2008). However, metformin given to

a hyperglycaemic mare at the same dose and frequency did not improve blood glucose or

serum insulin concentrations (Durham et al., 2009). In six non I-R horses, metformin therapy

(15 mg/kg BW, orally, 8-hourly, for 15 days) showed no effect on SI measured with the HEC

(Firshman et al., 2009). Pharmacokinetic studies of metformin in horses and I-R ponies show

low bioavailability of the drug in this species (Hustace et al., 2009), and that chronic dosing

may not achieve therapeutic blood concentrations (Tinworth et al., 2010).

The aim of this study was to determine the effect of oral metformin on insulin and

glucose dynamics in I-R ponies using Minimal Model analysis of a frequently-sampled

intravenous glucose tolerance test (FSIGT) conducted before and after 21 days of metformin

administration (15 mg/kg BW, orally, twice-daily), using six I-R ponies with no signs of

laminitis. The study was conducted as part of the aforementioned work that investigated the

pharmacokinetics of metformin (Tinworth et al., 2010).

We hypothesised that metformin treatment of IR in I-R ponies would enhance SI, that

glucose effectiveness (Sg) would remain unchanged, the acute insulin response to glucose

(AIRg) would be lowered, and that the disposition index (DI) would increase. We also

hypothesised that if SI was enhanced there would be a concurrent reduction in neck adiposity.

6

Materials and methods

Animals

Six female Welsh-cross and Shetland-cross ponies were used, with a (mean ± SD) age

of 12.00 ± 2.88 years, weighing 206 ± 53.5 kg. The ponies, purchased from local sales, were

not obese (BCS: 6 ± 0.89), but did display regional adiposity (Frank et al., 2010) and were

deemed to be I-R based on evaluation of neck crest adipose tissue (ie, cresty neck score

(CNS) ≥ 3) (Carter et al., 2009a) and results of a combined glucose-insulin test (CGIT)

(Frank et al., 2006). The ponies were otherwise healthy and not suffering from Pituitary Pars

Intermedia Dysfunction, based on results of a 19 h dexamethasone suppression test (Dybdal

et al., 1994), where testing was conducted during November (Southern Hemisphere late

Spring) (Donaldson et al., 2005). The study protocol was approved by the Charles Sturt

University Animal Care and Ethics Committee.

Study Design

The study was conducted as a 2 x 2 cross-over trial. The two treatments were control

(C) and metformin (M), and the six ponies were randomised to two treatment sequences MC

and CM, with three ponies in each group. The two treatment periods were separated by a 21-

day wash-out period with ponies kept in a communal paddock. Treatment periods continued

for 21 days with a FSIGT conducted on Day 0 and Day 22 on all ponies. At this time,

physical characteristics of BW, body condition score (BCS), CNS, neck circumference and

the height and thickness of the crest of the neck were also measured. Bodyweight was

measured using electronic scales; BCS was scored out of a total of 9 (Henneke et al., 1983).

The CNS was assigned according to Carter et al (2009a), with neck circumference, crest

height and thickness measured as described by Frank et al (2006). During treatment periods,

all ponies were stabled individually, with unrestricted access to water. Stables were cleaned

7

each morning while the ponies were allowed free exercise in a communal yard. Ponies were

fed maintenance rations of early-cut oaten hay at a rate of 1 to 2% of BW twice daily (NRC,

2007). The ponies also received 100 g rice bran pellets (Cool Conditioner, CopRice) twice

daily. Ponies were monitored for feeding behaviour at each meal, and received full health

checks daily. The C animals received exactly the same management as the M animals.

Metformin Administration

Metformin was given at 15 mg/kg BW, orally, twice-daily, with meals, at 8 am and 5

pm. For each pony, the dose of metformin was prepared by powdering the appropriate

number of metformin tablets (Metforbell 500 mg, CiplaGenpharm Australia, Pty Ltd), using a

mortar and pestle, and suspending the powder in 100 mL tap water. This solution was mixed

with 100 g rice bran pellets (Cool Conditioner, CopRice) for the ponies to eat. The entire

ration was readily consumed within 2 min.

FSIGT

The evening before the FSIGT, a 14G catheter was placed in the jugular vein of each

pony, after aseptic preparation of the site and the administration of local anaesthetic. On the

morning of the study, the ponies were fed oaten hay at a rate of 1% to 2% of BW. The FSIGT

was initiated at 9 am with a bolus of glucose (Dextrose solution 50%, Baxter Healthcare)

(300 mg/kg BW, IV) administered within 2 min. Twenty min later, an insulin bolus (Humulin

R, Eli Lilly and Co.) (20 mU/kg BW, IV) was administered within 30 s, as described by Yang

et al (1987). Basal blood samples (10 mL) were taken 60, 45, and 0 min before the glucose

dose. Blood samples were then drawn at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24,

25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 210, 240, 270, 300, 330, and 360

min after the glucose bolus. Blood samples were collected into lithium-heparin vacutainers

8

(Vacutainer, Becton-Dickinson), kept on ice and centrifuged at 4 °C (1,000 x g for 10 min)

within 20 min of collection.

Sample Analysis

Each plasma sample was divided into two aliquots and stored at -20 °C until assayed

for glucose and insulin. Plasma glucose concentrations were measured using an enzymatic

assay (Glucose Hexokinase, ThermoFisher Scientific Inc.) on a bench-top biochemistry

analyzer (Cobas Mira, Roche). Plasma insulin concentrations were determined using a

radioimmunoassay (RIA) (Coat-A-Count Insulin RIA, Siemens Medical Solutions

Diagnostics), validated for use in equines (Tinworth et al., 2009). Each sample was assayed

in duplicate, and intra-assay coefficients of variation < 5 % or < 10 % were required for

acceptance of glucose and insulin assay results, respectively.

Minimal Model Analysis

Glucose and insulin curves were interpreted according to the Minimal Model of

glucose and insulin dynamics (Bergman, 1989; Boston et al., 2003) using MINMOD

Millennium software (Boston et al., 2003). Application of this method in horses has been

described previously (Hoffman et al., 2003; Treiber et al., 2005a). All results are expressed as

mean ± SD.

Statistical Analyses

Values for SI, Sg, AIRg, DI and physical characteristics between C and M groups

were compared by calculating the difference in each variable between Day 22 and Day 0 for

each pony in each period, then conducting a Wilcoxon Matched Pairs Test comparing ponies

9

in the two treatment sequences. The null-hypothesis was rejected if P ≤ 0.05. Analyses were

carried out using GraphPad Prism version 5.00 for Windows (GraphPad Software).

Results

Pre-treatment physical characteristics of the six ponies are shown in Table 1. Pre-

treatment Minimal Model indices are shown in Table 2. No significant differences were

found.

Physical characteristics measured on Day 0 and Day 22, of the C group and the M

group at pre-treatment (Day 0) and post-treatment (Day 22) are shown in Figure 1. Results

for SI, Sg, AIRg and DI from Minimal Model analysis of the FSIGT of the C Group and the

M group at pre-treatment (Day 0) and post-treatment (Day 22) are shown in Figure 2.

Non-fasting basal insulin concentrations of the C Group were 8.67 ± 3.77 (mean ± SD)

on Day 0 and 7.80 ± 3.89 on Day 22, and of the M group were 9.43 ± 6.19 on Day 0 and 6.10

± 0.71 on Day 22. Data from the ponies were compared and no significant treatment effects

were observed.

Discussion

The results of this study were disappointing but not surprising, in light of

pharmacokinetic results obtained concurrently. The lack of apparent long-term efficacy of

metformin is attributable to its low bioavailability in the horse/pony. The bioavailability of

metformin in horses after oral administration was reportedly 3.9% to 7.1%, depending on

feeding status (Hustace et al., 2009). In contrast, bioavailability in humans is 40% to 60%

(Scheen, 1996). This bioavailability factor may be clinically relevant to metformin

10

concentrations at steady state (Css). From our study of the pharmacokinetics of metformin in

these I-R ponies, the Css of metformin administration (15 mg/kg BW orally, twice-daily) was

122 ng/mL (Tinworth et al., 2010), approximately 5 times lower than the Css of 500 to 1,000

ng/mL considered therapeutic in humans (Scheen, 1996). However, work by Stepensky et al

(1998) and Sambol et al (1996) in rodents and humans, have shown a lack of direct

correlation between the magnitude of glucose-lowering effect and blood metformin

concentrations, with a significant “first-pass” pharmacodynamic effect, in the liver and the

gastrointestinal wall, that contributes to the efficacy of metformin (Stepensky et al., 2002). It

is unknown if these pharmacodynamics are reflected in the horse/pony, but due to the

equivocal results obtained by other researchers using metformin to treat I-R horses/ponies,

the findings of the current study need to be presented.

The appeal of metformin as a therapeutic option for I-R horses/ponies is that it

reportedly enhances the SI of peripheral tissues (Salpeter et al., 2008), without stimulating

insulin secretion by pancreatic beta-cells (Klip and Leiter, 1990). In horses/ponies with a

history of endocrinopathic laminitis, such as the horses/ponies studied by Durham et al

(2008), their IR is characterised by basal hyperinsulinaemia (Treiber et al., 2005c; Walsh et

al., 2009). There seems to be another subgroup of horses/ponies, such as the ponies studied

here, with the pre-laminitic metabolic syndrome (Treiber et al., 2005c) where the animals

may not present with basal hyperinsulinaemia and do not show signs of laminitis, but are IR,

displaying hyperinsulinaemia in response to a glucose challenge. Prolonged

hyperinsulinaemia increases the risk for laminitis and other diseases (Hess et al., 2006;

Asplin et al., 2007; Geor, 2008; de Laat et al., 2010). Hence the aim to decrease insulin

secretion is paramount.

11

In humans, therapeutic concentrations of metformin decrease fasting plasma glucose

concentrations within 3 to 5 days of commencing therapy, stabilising within 1 to 2 weeks

(Sambol et al., 1996). Based on this, the 21 days allowed in the present study to assess the

efficacy of the drug should have been adequate.

Reports of metformin use in horses/ponies (Johnson et al., 2005; Vick et al., 2006;

Durham et al., 2008; Durham et al., 2009; Firshman et al., 2009; Hustace et al., 2009)

indicated some degree of effectiveness in treating IR and were valuable in suggesting an

optimal design for this study. Although the case study described by Johnson et al (2005)

showed metformin may be a therapeutic option for I-R horses/ponies, a randomised,

controlled study provides stronger clinical evidence. Our study furthered that of Firshman et

al (2009), by examining the response to metformin of ponies with IR. Studies by Durham et

al (2008; 2009) were important in assuring us that chronic administration of metformin to

horses/ponies was safe, using a dosing regimen comparable to that used in humans. Earlier

studies described were limited by the use of proxy measures of SI (Durham et al., 2008).

Proxy measures of SI, derived from basal insulin and glucose concentrations, have been

determined to improve the predictive ability of IR and the risk for subsequent disease

(Treiber et al., 2005b). Predicting IR by using these proxies in an inbred population, where a

major gene may be responsible for this tendency, gave a moderate predictive power of 78%.

The power of this method in the wider outbred population may be limited by greater

individual variability and may be inappropriate for assessing the general population of

horses/ponies. The use of basal blood samples to measure blood glucose concentrations

(Durham et al., 2009) in type 2 diabetes mellitus was appropriate to monitor hyperglycaemia

in the cases described. However, non-specific indicators of IR derived from basal blood

12

samples are limited in usefulness because they fail to provide values for SI, Sg or beta cell

function. Basal measurements of glucose and insulin are also subject to seasonal, diurnal,

stress, pain and feeding history influences. A dynamic challenge test, such as the HEC or the

FSIGT (Bailey et al., 2007), is a more discriminating and controlled method of determining

IR in horses/ponies (Frank et al., 2010). Currently, the gold-standard method of determining

IR, used in this study, is by Minimal Model analysis of the FSIGT (Monzillo and Hamdy,

2003). Minimal Model analysis has been used primarily to elucidate etiologies of diabetes in

humans and other species (Bergman, 1989), but has effectively estimated SI and Sg in horses

(Hoffman et al., 2003).

From the physiologic perspective, SI is the capacity of insulin to stimulate glucose

uptake into tissue (Bergman, 1989). We hypothesized that SI would be enhanced after 21

days of metformin administration. In humans, metformin enhances hepatic and muscle SI

(Saenz et al., 2005; Salpeter et al., 2008) through direct (Stith et al., 1996) and indirect effects

(Cusi et al., 1996). Our results failed to show a significant change in response to metformin in

this small population of ponies.

The fractional disposal rate of glucose (Sg) is the capacity of the cells to take up

glucose and suppress hepatic glucose production without insulin mediation (Bergman, 1989).

Metformin is reported to enhance SI of peripheral tissues (Saenz et al., 2005; Salpeter et al.,

2008), by pathways independent of insulin-mediated phosphoinositide-3 kinase and that may

involve 5'-AMP-activated kinase (Zhou et al., 2001), thus enhancing Sg. This action is

important in patients suffering from hyperglycaemia. In this study, as hypothesised, Sg

remained unchanged. It is likely hyperglycaemia is a pre-requisite to this pathway becoming

effective, and our ponies were not hyperglycaemic. Because Sg remained unaffected by

13

metformin treatment, and the ponies were normoglycaemic at baseline, it appears glucose

uptake into tissues via insulin-independent pathways is intact, and/or that elevated hepatic

gluconeogenesis is not a feature of the condition in these ponies. This is in contrast to the

horses studied by Durham et al (2009).

Responsiveness of beta-cells to the glucose load is described by the AIRg, the increase

in plasma insulin above basal concentration integrated from 0 to 10 min after the glucose

dose (Bergman, 1989). By enhancing SI (Saenz et al., 2005; Salpeter et al., 2008), metformin

was expected to decrease the insulin concentration required to metabolise the glucose load

and AIRg to be lowered. In this study, AIRg did not change significantly. Therefore, our

results suggest that insulin secretion was unaffected by metformin treatment and the control

management; corroborating our finding that SI was not significantly affected by the

metformin treatment.

The product of AIRg and SI determines the DI, the appropriateness of the beta-cell

response relative to the degree of IR in tissues (Bergman, 1989). In this study, DI did not

change. This suggests an adequate capacity of AIRg to compensate for limited SI. Our ponies

were able to compensate for their IR as they maintained euglycaemia, but secreted more

insulin than normal, to do so.

In I-R humans, metformin reduces BW (Salpeter et al., 2008). However, metformin

(2.8 mg/kg BW, orally, 12-hourly) in 14 obese mares for 30 days did not affect BW (Vick et

al., 2006). Similarly, in our study, the ponies’ BW was unchanged in response to metformin

administration.

14

Some adipocytes secrete endocrine factors that directly cause IR (Lyon et al., 2003).

These endocrinologically-active adipocytes are especially plentiful in visceral fat in humans.

Although there is a clear correlation between obesity and IR (Hoffman et al., 2003; Frank et

al., 2006; Vick et al., 2007), some I-R animals, such as our I-R ponies, are not obese on the

basis of BCS (ie, BCS < 7), but had enlarged fat deposits on the neck (a cresty neck) (Carter

et al., 2009b). Numerous endocrine signals produced by adipocytes in visceral fat are

collectively known as ‘adipokines’ (Dandona et al., 2004) and increased production of

adipokines by individuals with visceral fat is strongly associated with IR. Since it has been

speculated that visceral fat in humans may be equivalent to cresty necks in ponies (Carter et

al., 2009b), we expected any enhancement of SI may be accompanied by a reduction in neck

‘crestiness’. However, no significant change in apparent neck adiposity was evident.

Conclusions

In this study with non-obese, non-laminitic I-R ponies, our results do not support the

hypothesis that metformin is effective, but should be interpreted with caution due to the

limited sample size and large individual variation. However, in light of the pharmacokinetics

of metformin studied in these same I-R ponies (Tinworth et al., 2010), and the results

reported here, we speculate that metformin was not therapeutic, because the drug did not

circulate at concentrations required to enhance insulin-dependent glucose uptake; nor did it

affect insulin-independent glucose uptake pathways associated with hyperglycaemia.

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other

people or organizations that could inappropriately influence or bias the content of the paper.

15

Animal Care and Ethics statement

The protocol described was reviewed on Wednesday 5 March 2008 by the Charles

Sturt University Animal Care and Ethics Committee, and was approved with the approval

number 08/030. The ponies were owned by Charles Sturt University.

Acknowledgments

The authors would like to acknowledge WALTHAM Centre for Pet Nutrition,

Waltham-on-the-Wolds, U.K. and Rural Industries Research and Development Corporation,

Australia for their continuing support of Kellie Tinworth’s PhD candidature. We would also

like to express our gratitude to Naomie Tidd for her expert technical assistance and

intellectual support, and to the staff and students of the School of Animal and Veterinary

Sciences, Charles Sturt University, for their assistance with the pony studies, especially Dr

Chris Petzel for his clever advice on blood sampling.

References

Asplin, K.E., Sillence, M.N., Pollitt, C.C., McGowan, C.M., 2007. Induction of laminitis by

prolonged hyperinsulinaemia in clinically normal ponies. The Veterinary Journal 174,

530–535.

Bailey, S.R., Menzies-Gow, N.J., Harris, P.A., Habershon-Butcher, J.L., Crawford, C.,

Berhane, Y., Boston, R.C., Elliott, J., 2007. Effect of dietary fructans and dexamethasone

administration on the insulin response of ponies predisposed to laminitis. Journal of the

American Veterinary Medical Association 231, 1365-1373.

Bergman, R.N., 1989. Toward physiological understanding of glucose tolerance. Minimal-

model approach. Diabetes 38, 1512-1527.

16

Boston, R.C., Stefanovski, D., Moate, P.J., Sumner, A.E., Watanabe, R.M., Bergman, R.N.,

2003. MINMOD Millennium: a computer program to calculate glucose effectiveness and

insulin sensitivity from the frequently sampled intravenous glucose tolerance test.

Diabetes Technology and Therapeutics 5, 1003-1015.

Carter, R.A., Geor, R.J., Burton Staniar, W., Cubitt, T.A., Harris, P.A., 2009a. Apparent

adiposity assessed by standardised scoring systems and morphometric measurements in

horses and ponies. The Veterinary Journal 179, 204-210.

Carter, R.A., McCutcheon, L.J., Valle, E., Meilahn, E.N., Geor, R.J., 2010. Effects of

exercise training on adiposity, insulin sensitivity, and plasma hormone and lipid

concentrations in overweight or obese, insulin-resistant horses. American Journal of

Veterinary Research 71, 314-321.

Carter, R.A., Treiber, K.H., Geor, R.J., Douglass, L., Harris, P.A., 2009b. Prediction of

incipient pasture-associated laminitis from hyperinsulinaemia, hyperleptinaemia and

generalised and localised obesity in a cohort of ponies. Equine Veterinary Journal 41, 171-

178.

Cusi, K., Consoli, A., DeFronzo, R.A., 1996. Metabolic effects of metformin on glucose and

lactate metabolism in noninsulin-dependent diabetes mellitus. Journal of Clinical

Endocrinology and Metabolism 81, 4059-4067.

Dandona, P., Aljada, A., Bandyopadhyay, A., 2004. Inflammation: the link between insulin

resistance, obesity and diabetes. Trends in Immunology 25, 4-7.

de Laat, M., McGowan, C., Sillence, M., Pollitt, C., 2010. Equine laminitis: induced by 48 h

hyperinsulinaemia in Standardbred horses. Equine Veterinary Journal 42, 129-135.

Donaldson, M.T., McDonnell, S.M., Schanbacher, B.J., Lamb, S.V., McFarlane, D., Beech,

J., 2005. Variation in plasma adrenocorticotropic hormone concentration and

17

dexamethasone suppression test results with season, age, and sex in healthy ponies and

horses. Journal of Veterinary Internal Medicine 19, 217-222.

Durham, A.E., Hughes, K.J., Cottle, H.J., Rendle, D.I., Boston, R.C., 2009. Type 2 diabetes

mellitus with pancreatic B cell dysfunction in 3 horses confirmed with minimal model

analysis. Equine Veterniary Journal 41, 924-929.

Durham, A.E., Rendle, D.I., Newton, J.R., 2008. Use of metformin for the treatment of

insulin resistance in 18 horses and ponies. Equine Veterinary Journal 40, 1-8.

Dybdal, N.O., Hargreaves, K.M., Madigan, J.E., Gribble, D.H., Kennedy, P.C., Stabenfeldt,

G.H., 1994. Diagnostic testing for pituitary pars intermedia dysfunction in horses. Journal

of the American Veterinary Medical Association 204, 627-632.

Firshman, A., Hustace, J., Peterson, K., Mata, J., 2009. The effect of metformin on insulin

sensitivity in horses. In: Proceedings of American College of Veterinary Internal

Medicine, Montreal, Quebec, Canada.

Frank, N., Elliott, S.B., Boston, R.C., 2008. Effects of long-term oral administration of

levothyroxine sodium on glucose dynamics in healthy adult horses. American Journal of

Veterinary Research 69, 76-81.

Frank, N., Elliott, S.B., Brandt, L.E., Keisler, D.H., 2006. Physical characteristics, blood

hormone concentrations, and plasma lipid concentrations in obese horses with insulin

resistance. Journal of the American Veterinary Medical Association 228, 1383-1390.

Frank, N., Geor, R., Bailey, S., Durham, A., Johnson, P., 2010. Equine metabolic syndrome.

Journal of Veterinary Internal Medicine 24, 467-475.

Geor, R.J., 2008. Metabolic Predispositions to Laminitis in Horses and Ponies: Obesity,

Insulin Resistance and Metabolic Syndromes. Journal of Equine Veterinary Science 28,

753-759.

18

Geor, R.J., Harris, P., 2009. Dietary Management of Obesity and Insulin Resistance:

Countering Risk for Laminitis. Veterinary Clinics of North America: Equine Practice 25,

51-65.

Henneke, D.R., Potter, G.D., Kreider, J.L., Yeates, B.F., 1983. Relationship between

condition score, physical measurements and body fat percentage in mares. Equine

Veterinary Journal 15, 371-372.

Hess, T.M., Treiber, K.H., Kronfeld, D.S., Furr, M.O., 2006. Insulin resistance demonstrated

by a specific quantitative method in a hyperlipemic laminitic pony. Journal of Equine

Veterinary Science 26, 271-274.

Hoffman, R.M., Boston, R.C., Stefanovski, D., Kronfeld, D.S., Harris, P.A., 2003. Obesity

and diet affect glucose dynamics and insulin sensitivity in Thoroughbred geldings. Journal

of Animal Science 81, 2333-2342.

Hustace, J.L., Firshman, A.M., Mata, J.E., 2009. Pharmacokinetics and bioavailability of

metformin in horses. American Journal of Veterinary Research 70, 665-668.

Johnson, P.J., Scotty, N.C., Wiedmeyer, C., Messer, N.T., Kreeger, J.M., 2005. Diabetes

mellitus in a domesticated Spanish mustang. Journal of the American Veterinary Medical

Association 226, 584-588.

Klip, A., Leiter, L.A., 1990. Cellular mechanism of action of metformin. Diabetes Care 13,

696-704.

Lyon, C.J., Law, R.E., Hsueh, W.A., 2003. Minireview: adiposity, inflammation, and

atherogenesis. Endocrinology 144, 2195-2200.

Monzillo, L.U., Hamdy, O., 2003. Evaluation of insulin sensitivity in clinical practice and in

research settings. Nutrition Review 61, 397-413.

NRC, 2007. Nutrient Requirements of Horses, Vol. 6th rev. ed. National Academy Press,

Washington, D.C.

19

Saenz, A., Fernandez-Esteban, I., Mataix, A., Ausejo, M., Roque, M., Moher, D., 2005.

Metformin monotherapy for type 2 diabetes mellitus. Cochrane Database of Systematic

Reviews 3, CD002966.

Salpeter, S.R., Buckley, N.S., Kahn, J.A., Salpeter, E.E., 2008. Meta-analysis: Metformin

treatment in persons at risk for diabetes mellitus. The American Journal of Medicine 121,

149-157.

Sambol, N.C., Chiang, J., O'Conner, M., Liu, C.Y., Lin, E.T., Goodman, A.M., Benet, L.Z.,

Karam, J.H., 1996. Pharmacokinetics and pharmacodynamics of metformin in healthy

subjects and patients with noninsulin-dependent diabetes mellitus. Journal of Clinical

Pharmacology 36, 1012-1021.

Scheen, A.J., 1996. Clinical pharmacokinetics of metformin. Clinical Pharmacokinetics 30,

359-371.

Stepensky, D., Friedman, M., Raz, I., Hoffman, A., 2002. Pharmacokinetic-

pharmacodynamic analysis of the glucose-lowering effect of metformin in diabetic rats

reveals first-pass pharmacodynamic effect. Drug Metabolism and Disposition 30, 861-868.

Stepensky, D., Srour, W., Friedman, M., Raz, I., Hoffman, A., 1998. Dependency of

metformin hypoglycemic effect on mode of administration in diabetic rats: measurement

and kinetics of in vivo drug effects. In: Proceedings of 3rd International Symposium:

Advances in Simultaneous Pharmacokinetic/Pharmacodynamic Modeling, Amsterdam

Center for Drug Research, Leiden, The Netherlands.

Stith, B.J., Goalstone, M.L., Espinoza, R., Mossel, C., Roberts, D., Wiernsperger, N., 1996.

The antidiabetic drug metformin elevates receptor tyrosine kinase activity and inositol

1,4,5-trisphosphate mass in Xenopus oocytes. Endocrinology 137, 2990-2999.

20

Tinworth, K.D., Edwards, S., Harris, P.A., Sillence, M.N., Hackett, L.P., Noble, G.K., 2010.

Pharmacokinetics of metformin after enteral administration in insulin-resistant ponies.

American Journal of Veterinary Research 71, 1201-1206.

Tinworth, K.D., Wynn, P.C., Harris, P.A., Sillence, M.N., Noble, G.K., 2009. Optimising the

Siemens Coat-A-Count radioimmunoassay to measure insulin in equine plasma. Journal of

Equine Veterinary Science 29, 411-413.

Treiber, K.H., Boston, R.C., Kronfeld, D.S., Staniar, W.B., Harris, P.A., 2005a. Insulin

resistance and compensation in Thoroughbred weanlings adapted to high-glycemic meals.

Journal of Animal Science 83, 2357-2364.

Treiber, K.H., Kronfeld, D.S., Hess, T.M., Boston, R.C., Harris, P.A., 2005b. Use of proxies

and reference quintiles obtained from minimal model analysis for determination of insulin

sensitivity and pancreatic beta-cell responsiveness in horses. American Journal of

Veterinary Research 66, 2114-2121.

Treiber, K.H., Kronfeld, D.S., Hess, T.M., Byrd, B.M., Splan, R.K., 2005c. Pre-laminitic

metabolic syndrome in genetically predisposed ponies involves compensated insulin

resistance. Journal of Animal Physiology and Animal Nutrition 89, 430-431.

Vick, M.M., Adams, A.A., Murphy, B.A., Sessions, D.R., Horohov, D.W., Cook, R.F.,

Shelton, B.J., Fitzgerald, B.P., 2007. Relationships among inflammatory cytokines,

obesity, and insulin sensitivity in the horse. Journal of Animal Science 85, 1144-1155.

Vick, M.M., Sessions, D.R., Murphy, B.A., Kennedy, E.L., Reedy, S.E., Fitzgerald, B.P.,

2006. Obesity is associated with altered metabolic and reproductive activity in the mare:

effects of metformin on insulin sensitivity and reproductive cyclicity. Reproduction,

Fertility and Development 18, 609-617.

Walsh, D.M., McGowan, C.M., McGowan, T., Lamb, S.V., Schanbacher, B.J., Place, N.J.,

2009. Correlation of Plasma Insulin Concentration with Laminitis Score in a Field Study

21

of Equine Cushing's Disease and Equine Metabolic Syndrome. Journal of Equine

Veterinary Science 29, 87-94.

Yang, Y.J., Youn, J.H., Bergman, R.N., 1987. Modified protocols improve insulin sensitivity

estimation using the minimal model. American Journal of Physiology 253, E595-602.

Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., Wu, M., Ventre, J.,

Doebber, T., Fujii, N., Musi, N., Hirshman, M.F., Goodyear, L.J., Moller, D.E., 2001.

Role of AMP-activated protein kinase in mechanism of metformin action. Journal of

Clinical Investigation 108, 1167-1174.

22

Table 1. The baseline physical characteristics of the ponies at the frequently-sampled intravenous glucose tolerance test, before the treatment period. Each group comprised a sample size of six. Data are presented as mean ± SD.

Parameter (units) Metformin-Treated Ponies Control Ponies

Bodyweight

(kg) 209.50 ± 49.95 209.8 ± 54.05

Body Condition Score

(scale: 1 to 9) 6.00 ± 0.89 6.00 ± 0.89

Cresty Neck Score

(scale: 1 to 5) 3.17 ± 0.41 3.00 ± 0.00

Neck Circumference

(cm) 84.50 ± 6.03 85.67 ± 5.50

Crest Height

(cm) 10.53 ± 1.86 10.38 ± 1.29

Crest Thickness

(cm) 7.37 ± 1.31 7.73 ± 1.69

23

Table 2. The baseline values for insulin sensitivity (SI), glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and disposition index (DI) from Minimal Model analysis of the frequently-sampled intravenous glucose tolerance test of the ponies before treatment (Day 0). Each group comprised a sample size of six. Data are presented as mean ± SD.

Parameter (units) Metformin-Treated Ponies Control Ponies

SI ((mU/L)-1•min-1) 0.36 ± 0.31 0.32 ± 0.31

Sg (min–1•102) 0.98 ± 0.60 0.93 ± 0.58

AIRg (mU•L-1•min–1) 1079 ± 905.8 840.6 ± 557.2

DI 430.9 ± 406.9 355.3 ± 334.2

24

Figure legends

Figure 1: Physical characteristics of the six ponies, illustrating the change in these variables

over time, with or without metformin treatment. Day 0 () and Day 22 () illustrate the

change when no metformin treatment was applied. Day 0 () and Day 22 () illustrate the

change when the ponies also received metformin (15 mg/kg BW orally, twice-daily for 21

days). Horizontal bars represent the mean for each set of observations. There was no

significant effect of time or treatment on any of the variables measured. Significance was set

at P ≤ 0.05.

Figure 2: Insulin sensitivity (SI, A), glucose effectiveness (Sg, B), acute insulin response to

glucose (AIRg, C) and disposition index (DI, D) from Minimal Model analysis of the

frequently-sampled intravenous glucose tolerance test of the of the six ponies, illustrating the

change in these variables over time, with or without metformin treatment. Day 0 () and Day

22 () illustrate the change when no metformin treatment was applied. Day 0 () and Day

22 () illustrate the change when the ponies also received metformin (15 mg/kg BW orally,

twice-daily for 21 days). Horizontal bars represent the mean for each set of observations.

There was no significant effect of time or treatment on any of the variables measured.

Significance was set at P ≤ 0.05.

25

26