L. J. McCutcheon

Proinflammatory Cytokine and Chemokine Gene Expression Profiles in Subcutaneous and Visceral Adipose Tissue Depots of Insulin-Resistant and Insulin-Sensitive Light Breed Horses

BACKGROUND Insulin resistance has been associated with risk of laminitis in horses. Genes coding for proinflammatory cytokines and chemokines are expressed more in visceral adipose tissue than in subcutaneous adipose tissue of insulin-resistant (IR) humans and rodents. HYPOTHESIS/OBJECTIVES To investigate adipose depot-specific cytokine and chemokine gene expression in horses and its relationship to insulin sensitivity (SI). ANIMALS Eleven light breed mares. METHODS Animals were classified as IR (SI=0.58+/-0.31x10(-4) L/min/mU; n=5) or insulin sensitive (IS; SI=2.59+/-1.21x10(-4) L/min/mU; n=6) based on results of a frequently sampled intravenous glucose tolerance test. Omental, retroperitoneal, and mesocolonic fat was collected by ventral midline celiotomy; incisional nuchal ligament and tail head adipose tissue biopsy specimens were collected concurrently. The expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, plasminogen activator inhibitor-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) in each depot was measured by real-time quantitative polymerase chain reaction. Data were analyzed by 2-way analysis of variance for repeated measures (P<.05). RESULTS No differences in TNF-alpha, IL-1beta, IL-6, PAI-1, or MCP-1 mRNA concentrations were noted between IR and IS groups for each depot. Concentrations of mRNA coding for IL-1beta (P=.0005) and IL-6 (P=.004) were significantly higher in nuchal ligament adipose tissue than in other depots. CONCLUSIONS AND CLINICAL IMPORTANCE These data suggest that the nuchal ligament depot has unique biological behavior in the horse and is more likely to adopt an inflammatory phenotype than other depots examined. Visceral fat may not contribute to the pathogenesis of obesity-related disorders in the horse as in other species.

Rapid, simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin : use in various animals indicates functional role of myohemoproteins

A novel, simple, rapid, sensitive and reproducible microassay is described for determination of myoglobin and hemoglobin content of myocardial and skeletal muscle biopsy specimens from various mammals, birds and fish. As little as 50 mg of tissue is needed and myoglobin concentrations lower than 1 mg% can be detected. Myoglobin and hemoglobin are separated at alkaline pH by ammonium sulfate extraction followed by ultrafiltration. Heme content is determined by absorption of the Soret band when the hemoprotein extract is visibly colored or more sensitively by its peroxidase activity when the extract has low color. The heme reacts with tertiary-butyl hydroperoxide and orthotolidine to generate a blue color. Hemoglobin content is correlated with myoglobin content and is related to aerobic capacity and blood flow to the tissue. Myoglobin content varied over 5 orders of magnitude up to 7 per cent of the weight of tissue, whereas hemoglobin content varied over 2 orders of magnitude up to 6 per cent of tissue weight. Myoglobin content is increased in species with high basal metabolic rate, high physical activity, prolonged diving capacity, fatigue resistance, and red muscle, whereas it is decreased in white muscle, iron-deficient animals, animals with sedentary lifestyles, and in animals and tissues with small fiber diameters such as avian or fish hearts.

Respiratory and metabolic responses in the horse during moderate and heavy exercise

Thoroughbred horses were exercised to fatigue on a treadmill at 62% and 100% of their VO2max. Hypoxemia occurred at the onset of exercise under both exercise conditions. This hypoxemia persisted to fatigue during the heavy exercise but progressively diminished as the exercise continued and had disappeared by the end of exercise at the lighter load. As a result of the hypoxemia the oxygen content of arterial blood during exercise at VO2max was 17% below its carrying capacity. However, under both experimental conditions the CaO2 still exceeded that of rest owing to an elevation in hemoglobin concentration. The temperature of blood at the point of fatigue was similar, 41.0±0.2 ° C and 41.1±0.2 ° C, for exercise at 62% and 100% VO2max, respectively. Muscle samples collected at rest and at the termination of exercise did not demonstrate major differences between the exercise conditions except for a higher [lactate] and lower pH following the heavy exercise. From these results it can be suggested that the combined effects of an elevated body temperature, changes in muscle pH, and oxygen delivery may all be factors contributing to limit exercise capacity in the horse.

Myocardial myoglobin deficiency in various animal models of congestive heart failure.

Myoglobin is known to protect the mechanical function of the heart from hypoxia by acting as a sarcoplasmic oxygen reservoir and shuttle. We postulated a role for myoglobin in the pathogenesis of congestive heart failure. Several models of congestive heart failure were employed to test the hypothesis, including spontaneous inherited dilated cardiomyopathy in Doberman Pinschers, and heart failure produced by rapid ventricular pacing in dogs, volume overload in chickens and furazolidone toxicity in turkeys. Myocardial myoglobin was decreased by approximately 50% for all models (P less than 0.05). In Doberman Pinschers dogs which are predisposed to the development of dilated cardiomyopathy and have mild subclinical depression of cardiac performance, myocardial myoglobin (1.05 +/- 0.22 mg/g) is approximately 50% decreased compared to healthy mongrel dogs (2.15 +/- 0.52 mg/g), approximately twice as much as dobermans with heart failure (0.47 +/- 0.25 mg/g) but similar to the concentration found in dogs paced to heart failure (1.09 +/- 0.34 mg/g). Myocardium from poultry had remarkably decreased myoglobin compared to mammals (34 +/- 4 micrograms/g) with heart failure produced either by furazolidone or salt toxicity causing a further 50% reduction. In the canine models of heart failure, myocardial myoglobin concentration was demonstrated to be correlated with biochemical and physiological indicators of myocardial performance, namely, mitochondrial and sarcoplasmic reticular ATPase activities, and cardiac output, systemic vascular resistance, pulmonary capillary wedge pressure and mean arterial pressure, respectively. Our data implicates a role for myoglobin deficiency in the pathogenesis of congestive heart failure and in the predisposition of doberman pinschers to dilated cardiomyopathy.

Distribution of insulin receptor and insulin-like growth factor-1 receptor in the digital laminae of mixed-breed ponies: an immunohistochemical study.

REASONS FOR PERFORMING STUDY Hyperinsulinaemia has been implicated in the pathogenesis of laminitis; however, laminar cell types responding to insulin remain poorly characterised. OBJECTIVES To identify laminar cell types expressing insulin receptor (IRc) and/or insulin-like growth factor-1 receptor (IGF-1R); and to evaluate the effect of dietary nonstructural carbohydrate (NSC) on their expression. METHODS Mixed-breed ponies (n = 22) received a conditioning hay chop diet (NSC ∼6%); following acclimation, ponies were stratified into lean (n = 11, body condition score [BCS]≤4) or obese (n = 11, BCS ≥7) groups and each group further stratified to remain on the low NSC diet (n = 5 each for obese and lean) or receive a high NSC diet (total diet ∼42% NSC; n = 6 each for obese and lean) for 7 days. Laminar samples were collected at the end of the feeding protocol and stained immunohistochemically for IRc and IGF-1R. The number of IRc(+) cells was quantified; distribution of IGF-1R was qualitatively described. Laminar IRc content was assessed via immunoblotting. RESULTS The number of IRc(+) cells was greater in the laminae of high NSC ponies than low NSC ponies (P = 0.001); there was a positive correlation between the change in serum insulin concentration and number of IRc(+) cells (r(2) = 0.74; P<0.0001). No epithelial IRc(+) cells were observed; IRc(+) cells were absent from the deep dermis. Analysis of serial sections identified IRc(+) cells as endothelial cells. The distribution of IGF-1R was more extensive than that of IRc, with signal in vascular elements, epithelial cells and fibroblasts. CONCLUSIONS Increased dietary NSC results in increased laminar endothelial IRc expression. Laminar keratinocytes do not express IRc, suggesting that insulin signalling in laminar epithelial cells must be mediated through other receptors (such as IGF-1R). POTENTIAL RELEVANCE Manipulation of signalling downstream of IRc and IGF-1R may aid in treatment and prevention of laminitis associated with hyperinsulinaemia.

Effects of hyperinsulinemia on glucose and lipid transporter expression in insulin-sensitive horses

Plasma insulin concentrations are elevated (hyperinsulinemia) in horses with obesity-associated insulin resistance. In other species, insulin resistance is partly due to reduced levels of insulin receptor and the insulin-sensitive glucose transporter, and, in vitro, chronic hyperinsulinemic conditions reduce the expression of these proteins. Consumption of grain-based concentrate feeds results in postprandial hyperinsulinemia in horses, and adaptation to these diets is associated with insulin resistance. As such, it is possible that the repeated, chronic postprandial hyperinsulinemia associated with these diets could contribute to the development of insulin resistance. The purpose of the current study was to investigate the influence of a 6-h insulin infusion that increased plasma insulin concentrations to >1,000 mIU/L, on the expression of insulin receptor and glucose and lipid transporters in skeletal muscle and adipose tissue of lean, insulin-sensitive horses. Insulin infusion decreased transcript abundance of the glucose transporter 4 (P<0.05), glucose transporter 1 (GLUT1; P<0.09), and the insulin receptor (P<0.001) in adipose tissue, while increasing transcript abundance of GLUT1 (P<0.09) and decreasing protein abundance of the insulin receptor (P<0.09) in skeletal muscle. The acute, 6 hyperinsulinemic conditions achieved in this experiment resulted in alterations to mechanisms of glucose transport that could promote insulin resistance via reduced insulin-stimulated glucose disposal. Insulin infusion also reduced transcript abundance of the lipid transporters CD36 (P<0.001) and fatty acid transporter protein (FATP; P<0.05) in adipose tissue while increasing FATP (P<0.05) and lipoprotein lipase (P<0.01) in skeletal muscle. The reduction in adipose tissue lipid transporters could have been due to the decreased plasma lipid concentrations, whereas the increase in skeletal muscle may indicate that insulin stimulates lipid uptake into equine skeletal muscle. This report provides preliminary evidence that severe hyperinsulinemia alters glucose and lipid transporter expression that could promote an insulin-resistant state; these should be further investigated in horses consuming grain-based concentrates.

Effects of leucine or whey protein addition to an oral glucose solution on serum insulin, plasma glucose and plasma amino acid responses in horses at rest and following exercise

REASONS FOR PERFORMING STUDY Providing protein or amino acid mixtures in combination with glucose to post exercise in man has resulted in increases in the post feeding insulin response and in muscle glycogen and protein synthesis rates. However, whether protein and/or amino acids can modify the post exercise insulin responses in horses remains to be fully elucidated. OBJECTIVES To determine whether whey protein or leucine addition to a glucose solution affects the post gavage plasma insulin, glucose and amino acid responses in horses and whether these responses are different following a period of exercise vs. rest. METHODS Six mature, conditioned Thoroughbreds received a nasogastric gavage containing either 1 g/kg bwt glucose (G), G + 0.3 g/kg bwt whey protein (GW) or G + 0.3 g/kg bwt leucine (GL), following a period of either rest (R) or an exercise test on a high speed treadmill (EX). Each horse was studied under all 6 treatment conditions, separated by 10 day intervals. Blood samples were collected pre-exercise/rest, pregavage and at regular intervals up to 300 min post gavage. Plasma was analysed for glucose and amino acid concentrations and serum insulin concentrations were determined. RESULTS There was a significantly (P < 0.05) greater insulin response in GL-R and GL-EX when compared to the other treatments. When compared to rest, post exercise plasma glucose responses were lower in G and GW but unchanged following GL administration. Plasma alanine concentrations were elevated post exercise in all EX treatments. With the exception of markedly elevated plasma leucine concentrations after GL-R and GL-EX, the plasma concentrations of all indispensable amino acids decreased during the post gavage period. CONCLUSIONS Leucine but not whey protein augmented the serum insulin response to an oral glucose load. Leucine supplementation warrants further investigation as a means to increase the rate of post exercise muscle glycogen synthesis in horses.

De novo fatty acid synthesis and NADPH generation in equine adipose and liver tissue

The lipogenic capacities of equine liver and adipose tissue explants were investigated in vitro. Preference for glucose or acetate as the primary carbon source for de novo fatty acid synthesis was determined using (14)C labeled substrates. Additional aims included determining the relative contribution of NADPH generating pathways to reducing equivalent generation and comparing the lipogenic activity of two adipose depots, mesenteric and subcutaneous harvested from the crest region of the neck. Mesenteric adipose tissue had greater lipogenic activity than subcutaneous adipose tissue, and liver tissue showed minimal (14)C incorporation into fatty acids, indicating a low hepatic lipogenic capacity. Acetate was found to be the primary carbon source for fatty acid synthesis due to both the appearance of the (14)C label in the lipid fraction and the low activity of ATP-citrate lyase. Finally, the pentose phosphate and isocitrate dehydrogenase enzymes contributed to NADPH production in equine adipose tissue.

Effects of the insulin sensitizing drug, pioglitazone, and lipopolysaccharide administration on markers of systemic inflammation and clinical parameters in horses.

Equine metabolic syndrome (EMS) is a condition of obese horses characterized by insulin resistance, systemic inflammation, and an increased risk of laminitis. The pathogenesis of EMS is thought, in part, to be due to inflammatory proteins produced by adipose tissue. Reducing inflammation may decrease the incidence of laminitis in horses with EMS. Pioglitazone hydrochloride, a thiazolidinedione, has efficacy to reduce obesity associated inflammation in humans. Eight normal, adult, horses were administered 1mg/kg pioglitazone for 14 days, and eight horses served as controls. Physical examination and hematologic variables, transcript abundance of pro-inflammatory cytokines in skeletal muscle and adipose tissue, and circulating concentrations of the acute phase protein, serum amyloid A and pro-inflammatory cytokine, TNF-α were assessed prior to, and following, an LPS infusion (35 ng/kg). The objective was to determine if pre-treatment with pioglitazone would mitigate the development of inflammation and associated clinical markers of inflammation following LPS administration. Lipopolysaccharide administration induced systemic inflammation, as assessed by clinical and hematological aberrations, increased TNF-α, SAA and adipose tissue IL-6 mRNA abundance, however no mitigating effects of pioglitazone were detected. A longer treatment period or higher dose might be indicated for future experiments.

Effects of the insulin-sensitizing drug pioglitazone and lipopolysaccharide administration on insulin sensitivity in horses.

BACKGROUND Obesity and insulin resistance increase the risk of laminitis in horses. Pioglitazone (PG) is an insulin-sensitizing drug used in humans that is absorbed after oral administration to horses. HYPOTHESIS PG treatment will increase insulin sensitivity and transcript abundance of glucose and lipid transporters in adipose and skeletal muscle tissues. ANIMALS Sixteen lean, healthy horses. METHODS Eight horses were administered PG (1 mg/kg bodyweight PO) for 12 days before induction of insulin resistance through IV administration of lipopolysaccharide (LPS). Treated and untreated controls (CN; n = 8) were subjected to testing of peripheral insulin sensitivity and biopsies of both subcutaneous (nuchal ligament) adipose tissue and skeletal muscle before and after treatment, and 24 hours after LPS administration. RESULTS PG treatment did not improve basal insulin sensitivity (CNs: 1.4 ± 0.3, PG-treated: 1.9 ± 1.3; P > .4) or mitigate LPS-induced insulin resistance (CNs: 0.4 ± 0.3, PG-treated: 0.4 ± 0.3); however, transcript abundance of glucose and lipid transporters was altered in both skeletal muscle and subcutaneous adipose tissue. CONCLUSIONS AND CLINICAL IMPORTANCE Either a higher dose or longer treatment period might be required for physiological effects to be observed. PG is a novel therapeutic agent requiring further investigation in horses in order to determine treatment efficacy.