Mammary uptake of fatty acids supplied by intravenous triacylglycerol infusion to lactating dairy cows.

Supplementing dairy cows with n-3 fatty acid-rich feeds does not easily increase quantities in milk fat. Previous results demonstrated very long-chain n-3 fatty acids are primarily transported in the PL fraction of blood, making them largely unavailable to the mammary gland for enrichment of milk fat. Our objective was to compare mammary uptake of fatty acids of increasing chain length and unsaturation delivered intravenously as TAG emulsions. Late lactation dairy cows were assigned to a completely randomized block design. Treatments were intravenous TAG emulsions enriched with oleic acid (OLA), linoleic acid (LNA), alpha-linolenic acid (ALA), or docosahexaenoic acid (DHA) and were delivered continuously at 16 mL/h for 72 h. Each treatment supplied 30 g/day of the target fatty acid. Treatment did not affect feed intake, milk yield, or milk composition, but all treatments reduced intake and yield. The proportion of DHA increased in plasma FFA, TAG, and PL with infusion. Increases of n-3 fatty acids, ALA, EPA, and DHA, were evident in the plasma PL fraction, suggesting re-esterification in the liver. Transfer efficiencies were 37.8 ± 4.1, 27.6 ± 5.4, and 10.9 ± 4.1 %, and day 3 total milk fatty acyl yields were 37.0 ± 3.4, 10.8 ± 0.4, and 3.3 ± 0.3 g for LNA, ALA, and DHA. Variation in oleic acyl yield prevented calculation of OLA transfer efficiency. Mammary uptake of fatty acids was reduced with increased chain length and unsaturation. Both liver and mammary mechanisms may regulate transfer of long-chain polyunsaturates.

Adiposity, plasma insulin, leptin, lipids, and oxidative stress in mature light breed horses.

BACKGROUND: Increased blood insulin levels are associated with an increased risk of pasture-associated laminitis in equids. OBJECTIVE: To determine the relationship between plasma insulin, leptin, and lipid levels, and measures of oxidative stress with adiposity in mature light breed horses. ANIMALS: 300 randomly selected light breed horses, aged 4-20 years. METHODS: A random sample of horses (140 mares, 151 geldings, and 9 stallions) was drawn from the VMRCVM Equine Field Service practice client list. Evaluations occurred June 15 - August 15, 2006, with all sampling performed between 0600 and 1200 hours. Concentrate feed was withheld for at least 10 hours before sampling. Plasma was analyzed for insulin, glucose, leptin, triglycerides, nonesterified fatty acids, and measures of oxidative stress. Body condition score was determined as the average of 2 independent investigators. RESULTS: Overconditioned and obese horses had higher plasma insulin (P < .001) and leptin (P < .01) levels than optimally conditioned horses. Obese horses had higher triglyceride levels (P = .006) and lower red blood cell gluthathione peroxidase activities (P = .001) than optimally conditioned horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Maintaining horses at a BCS <7 might be important for decreasing the risk of pasture-associated laminitis.

2011 and 2012 Early Careers Achievement Awards: metabolic priorities during heat stress with an emphasis on skeletal muscle.

Environmental heat stress undermines efficient animal production resulting in a significant financial burden to agricultural producers. The reduction in performance during heat stress is traditionally thought to result from reduced nutrient intake. Recently, this notion has been challenged with observations indicating that heat-stressed animals may exploit novel homeorhetic strategies to direct metabolic and fuel selection priorities independent of nutrient intake or energy balance. Alterations in systemic physiology support a shift in metabolism, stemming from coordinated interactions at whole-body and tissue-specific levels. Such changes are characterized by increased basal and stimulated circulating insulin concentration in addition to the ostensible lack of basal adipose tissue lipid mobilization coupled with reduced adipocyte responsiveness to lipolytic stimuli. Hepatic and skeletal muscle cellular bioenergetics also exhibit clear differences in carbohydrate production and use, respectively, due to heat stress. The apparent dichotomy in intermediary metabolism between the 2 tissue types may stem from factors such as tricarboxylic acid cycle substrate flux and mitochondrial respiration. Thus, the heat stress response markedly alters postabsorptive carbohydrate, lipid, and protein metabolism through coordinated changes in fuel supply and use across tissues in a manner that is distinct from commonly recognizable changes that occur in animals on a reduced plane of nutrition. Perhaps most intriguing is that the coordinated systemic, cellular, and molecular changes appear conserved across physiological states and among different ruminant and monogastric species. Ultimately, these changes result in the reprioritization of skeletal muscle fuel selection during heat stress, which may be important for whole-body metabolism and overall physiological adaptation to hyperthermia.

An explant based-method for differentiating adipocytes from equine adipose tissue.

Culturing adipocytes enables fine control of experimental conditions and helps minimise animal use. This report describes an explant-based method for isolating stromal-vascular cells from equine adipose tissue that enables use of small amounts of tissue. Subcutaneous and mesenteric adipose tissues were harvested post mortem and stromal-vascular cells grown from explants, prior to testing the capacity of several differentiation media to induce lipid droplet formation and increase transcript abundance of adipocyte markers. Inclusion of rosiglitazone at 1 and 5 µmol/l concentrations, along with other media components, induced differentiation of cultured equine stromal-vascular cells derived from subcutaneous and mesenteric adipose tissues.

A 90-day adaptation to a high glycaemic diet alters postprandial lipid metabolism in non-obese horses without affecting peripheral insulin sensitivity.

High glycaemic feeds are associated with the development of insulin resistance in horses. However, studies that evaluated the effect of high glycaemic feeds used horses that either ranged in body condition from lean to obese or were fed to increase body condition over a period of months; thus, the ability of high glycaemic feeds to induce insulin resistance in lean horses has not been determined. This study evaluated the insulin sensitivity of 18 lean horses fed a 10% (LO; n = 6), 20% (MED; n = 6) or 60% (HI; n = 6) non-structural carbohydrate complementary feed for 90 days. Although both the MED and HI diets increased insulinaemic responses to concentrate feeding in relation to the LO diet (p > 0.05), neither induced insulin resistance, as assessed by glucose tolerance test, following the 90-day feeding trial. Interestingly, the post-feeding suppression of plasma non-esterified fatty acids was less pronounced in HI-fed horses (p = 0.054) on days 30 and 90 of the study, potentially indicating that insulin-induced suppression of adipose tissue lipolysis was reduced. As insulin-resistant animals often have elevated plasma lipid concentrations, it is possible that altered lipid metabolism is an early event in the development of insulin resistance. The effects of high glycaemic feeds that are fed for a longer duration of time, on glucose and lipid metabolism, should be investigated further.

Relationships between body condition score and plasma inflammatory cytokines, insulin, and lipids in a mixed population of light-breed horses.

BACKGROUND: Obesity and hyperinsulinemia increase the risk of laminitis in horses and ponies. In mares, obesity also has been associated with increased circulating concentrations of the proinflammatory cytokine, tumor necrosis factor (TNF)-α. The association of other proinflammatory cytokines with body condition score (BCS) and insulin requires further determination. HYPOTHESIS: Plasma concentrations of TNF, interleukin (IL)-1ß, IL-6, and serum amyloid A (SAA) will positively correlate with BCS or insulin or both in horses. Furthermore, inflammatory protein concentrations will correlate with age and variables associated with BCS, including plasma insulin, triglycerides, nonesterified fatty acids, and leptin concentrations. ANIMALS: One hundred and ten mixed light-breed horses, including mares, geldings, and stallions, aged 4-20 years. METHODS: Samples were selected from a larger population of plasma samples previously collected during June-July of 2006. Samples were analyzed for TNF, IL-1ß, IL-6, and SAA using commercially available ELISAs and simple correlations were used to determine relationships with BCS, insulin, age, and sex. RESULTS: Plasma TNF (P = .047) and IL-6 (P = .021) concentrations were higher in females than males, whereas IL-6 concentrations correlated (P = .001) with age. Plasma SAA concentrations correlated with both insulin (P < .001) and BCS (P = .007). CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides evidence for factors, including age and sex, that may be associated with plasma concentrations of inflammatory proteins. Concentrations of SAA correlated with BCS and insulin, independent of age or sex. Because BCS and insulin correlate with increased SAA, it is possible that SAA is a component of laminitis pathophysiology.

Regulation of lipid synthesis by liver X receptor α and sterol regulatory element-binding protein 1 in mammary epithelial cells.

The objectives of this experiment were to characterize the roles of the transcription factors liver X receptor α (LXRα) and sterol regulatory element-binding protein 1 (SREBP1) in the transcriptional regulation of lipid synthesis in a bovine mammary epithelial cell line. Whereas many lipid synthesis genes contain a response element in their promoters for SREBP1, a few also contain a response element for LXR, suggesting that both transcription factors could directly regulate transcription of these genes. However, the promoter of SREBP1 contains a response element for LXR, indicating the additional potential for indirect transcriptional regulation by LXR, through SREBP1, on lipogenic genes. To characterize these effects, small interfering RNA (siRNA) directed against LXRα and SREBP1 were used to knockdown gene expression, and then, in the presence of SREBP1 siRNA, T 4506585 (T09) was used to specifically activate LXRα. Reducing LXRα mRNA abundance in mammary alveolar T cells did not alter mRNA abundance of genes involved in de novo lipogenesis or the rate of de novo lipogenesis, suggesting that LXRα is not required for basal transcription of genes required for fatty acid synthesis. Knockdown of SREBP1 reduced the mRNA abundance of acetyl-coenzyme A (CoA) carboxylase, fatty acid synthase, diacylglycerol acyltransferase, and stearoyl-CoA desaturase-1, indicating that these genes are regulated in part by SREBP1. When SREBP1 was reduced, T09 increased the mRNA abundance of acetyl-CoA carboxylase, fatty acid synthase, and diacylglycerol acyltransferase, potentially indicating that these genes are directly regulated by LXR. The results of the present study provide insight into the transcriptional regulatory mechanisms involved in lipid synthesis by mammary epithelial cells, and suggest that several transcription factors may be required for full lipogenic activation.

Pharmacokinetics of pioglitazone after multiple oral dose administration in horses.

Pioglitazone is a thiazolidinedione class of antidiabetic agent with proven efficacy in increasing insulin sensitivity in humans with noninsulin-dependent diabetes mellitus, a syndrome of insulin resistance sharing similarities with equine metabolic syndrome. The purpose of this study was to determine the pharmacokinetics of pioglitazone in adult horses following multiple oral dose administration. Pioglitazone hydrochloride (1 mg/kg) was administered orally for 11 doses at 24-h intervals, and plasma samples were collected. Initially, a pilot study was performed using one horse; and thereafter the drug was administered to six horses. Samples were analyzed by liquid chromatography with tandem mass spectrometry, and pharmacokinetic parameters were calculated using noncompartmental modeling. The maximum plasma concentration was 509.1 ± 413.5 ng/mL achieved at 1.88 ± 1.39 h following oral administration of the first dose, and 448.1 ± 303.5 ng/mL achieved at 2.83 ± 1.81 h (mean ± SD) following the eleventh dose. Apparent elimination half-life was 9.94 ± 4.57 and 9.63 ± 5.33 h after the first and eleventh dose, respectively. This study showed that in healthy horses, pioglitazone administered at a daily oral dose of 1 mg/kg results in plasma concentrations and total drug exposure approximating, but slightly below, those considered therapeutic in humans.

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.

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 diet and weight gain on circulating tumour necrosis factor-α concentrations in Thoroughbred geldings.

Low-grade inflammation precedes the development of obesity-related metabolic disorders in humans, but whether the same is true in the horse is not known. The objective of this study was to examine the effects of weight gain and diet on the inflammatory state of horses as determined by serum concentrations of tumour necrosis factor-α (TNF), an inflammatory cytokine. Fifteen mature Thoroughbred geldings with an initial body weight (BW) of 519±12 kg and body condition score (BCS) of 4.3±0.1 were fed a diet of hay plus a concentrate that was either high in non-structural carbohydrates (NSC) (i.e. starch and sugar), similar to those commercially available (CON) or one that had the energy source replaced with fat and fibre (FAT) for 32 weeks. Weight gain was achieved by feeding an additional 20 Mcal/day in excess of digestible energy maintenance requirements and resulted in a final BW of 608±12 kg and BCS of 6.9±0.1. Horses were exercised twice daily at a walk during the weight gain period. Horses were assessed bi-weekly for BW and BCS. Serum TNF was analysed from blood samples collected at 4-week intervals. Although treatment groups began the study with similar mean serum TNF concentrations, 12 weeks of FAT feeding promoted a decrease in circulating TNF that was maintained throughout the study with the exception of weeks 20 and 32. For either diet, there were no linear correlations between serum TNF concentration and BCS when horses increased in BCS from four to seven. The higher level of TNF observed in horses fed the CON diet indicates an increase in some level of systemic inflammation that was independent of their weight gain from a moderately thin to fleshy condition. The influence of diet on serum TNF concentrations should be investigated in horses fed to maintain body condition.