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

Highlights

• An enzyme-linked immunosorbent assay for human active glucagon-like peptide 1 (aGLP-1) was used to measure concentrations of this incretin hormone in equine plasma.

• Plasma aGLP-1 concentrations increased as expected during the oral sugar test and oral glucose tolerance test.

• No differences in aGLP-1 concentrations were detected when normal horses and those with equine metabolic syndrome (EMS) were compared.

• There was a trend toward higher percentage increases in aGLP-1 concentrations during the oral sugar test in horses with EMS, when compared with normal horses.

• Plasma aGLP-1 concentrations did not change significantly when horses with EMS were placed on a high-grain diet.

Abstract

Hyperinsulinemia is associated with laminitis in horses, and active glucagon-like peptide 1 (aGLP-1) stimulates insulin secretion. We hypothesized that plasma aGLP-1 concentrations measured during an oral sugar test (OST) would differ significantly between normal horses and those with equine metabolic syndrome (EMS) and that aGLP-1 concentrations would increase when EMS horses were placed on a high-grain diet. An enzyme-linked immunosorbent assay for aGLP-1 was validated with equine plasma and six horses with EMS, and 10 healthy Quarter horse crossbred mares were compared. Eleven months later, the same horses with EMS were placed on a high-grain diet for 8 weeks and aGLP-1 concentrations were measured during an oral glucose tolerance test at 0 and 8 weeks. Frequently sampled intravenous glucose tolerance tests were also performed. The assay 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 = .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 = .031) after 8 weeks when EMS horses were placed on a high-grain diet. Resting (fasted) insulin concentrations significantly increased (P = .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.

Introduction

Experimental evidence links hyperinsulinemia with laminitis in horses and ponies [1], [2], and high insulin concentrations 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 laminitis in equids [5]. Components of insulin dysregulation, including excessive insulin responses to oral sugars (postprandial hyperinsulinemia), fasting hyperinsulinemia, low C-peptide-to-insulin ratios, and tissue insulin resistance 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 statement, 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 secretion, 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 insulin 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 differential 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 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 pancreatic 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 hyperglycemia 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 antibody 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 inhibitor. 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 hormone 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 concentrations 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 concentrations would significantly increase as a result of obesity when EMS horses were placed on a high-grain diet to increase body fat mass.