Feeding dogs a high-fat diet induces metabolic changes similar to natural aging, including dyslipidemia, hyperinsulinemia, and peripheral insulin resistance.

OBJECTIVE: The goal of this study was to characterize changes induced by a high-fat diet in body composition, insulin levels and sensitivity, blood lipids, and other key biomarkers also associated with the metabolic dysfunction that occurs with natural aging. ANIMALS: 24 male Beagle dogs, 3 to 7 years of age, of mixed castration status. METHODS: Dogs were randomly assigned to continue twice daily feeding of the commercial adult maintenance diet (n = 12, including 2 intact) that they were previously fed or to a high-fat diet (12, including 2 intact) for 17 weeks between December 1, 2021, and April 28, 2022. Assessments included body composition (weight, body condition score, and adipose mass determined by deuterium enrichment), clinical chemistries, plasma fatty acid quantification, oral glucose tolerance test, and histology of subcutaneous and visceral adipose biopsy samples. RESULTS: The high-fat diet led to increased body weight, body condition score, fat mass and adipocyte size, hyperinsulinemia and peripheral insulin resistance, and elevations in serum lipids, including cholesterol, triglycerides, and several species of free fatty acids. Leptin levels increased in dogs fed a high-fat diet but not in control dogs. There were no significant changes in routine clinical chemistry values in either group. CLINICAL RELEVANCE: Feeding a high-fat diet for 17 weeks led to potentially deleterious changes in metabolism similar to those seen in natural aging in dogs, including hyperinsulinemia, insulin resistance, and dyslipidemia. A high-fat diet model may provide insights into the similar metabolic dysfunction that occurs during natural aging.

Effect of phenylbutazone on insulin secretion in horses with insulin dysregulation.

BACKGROUND: Phenylbutazone is often prescribed to manage pain caused by hyperinsulinemia-associated laminitis, but in diabetic people nonsteroidal anti-inflammatory drugs increase insulin secretion and pancreatic activity. HYPOTHESIS/OBJECTIVES: Investigate the effect of phenylbutazone administration on insulin secretion in horses. It was hypothesized that phenylbutazone will increase insulin secretion in horses with insulin dysregulation (ID). ANIMALS: Sixteen light breed horses, including 7 with ID. METHODS: Randomized cross-over study design. Horses underwent an oral glucose test (OGT) after 9 days of treatment with phenylbutazone (4.4 mg/kg IV q24h) or placebo (5 mL 0.9% saline). After a 10-day washout period, horses received the alternative treatment, and a second OGT was performed. Insulin and glucose responses were compared between groups (ID or controls) and treatments using paired t test and analyses of variance with P < .05 considered significant. RESULTS: In horses with ID, phenylbutazone treatment significantly decreased glucose concentration (P = .02), glucose area under the curve (2429 ± 501.5 vs 2847 ± 486.1 mmol/L × min, P = .02), insulin concentration (P = .03) and insulin area under the curve (17 710 ± 6676 vs 22 930 ± 8788 µIU/mL × min, P = .03) in response to an OGT. No significant effect was detected in control horses. CONCLUSION AND CLINICAL IMPORTANCE: Phenylbutazone administration in horses with ID decreases glucose and insulin concentrations in response to an OGT warranting further investigation of a therapeutic potential of phenylbutazone in the management of hyperinsulinemia-associated laminitis beyond analgesia.

Nesidioblastosis and Subsequent Diabetes Mellitus in a Dog with Hyperinsulinemic Hypoglycemia Treated with Partial Pancreatectomy.

A 6.5 yr old castrated male mixed-breed dog was presented for clinical signs associated with hypoglycemia. Hyperinsulinemic hypoglycemia was diagnosed as the cause of the persistent hypoglycemia. No obvious pancreatic mass was seen on abdominal computed tomography and exploratory laparotomy. A partial pancreatectomy was performed with the suspicion of an insulinoma-causing hyperinsulinemic hypoglycemia. Nesidioblastosis was diagnosed based clinical, biochemical, and histopathologic findings. There was beta cell hyperplasia and no evidence of neoplasia. The dog was euglycemic postoperatively after a partial pancreatectomy. Long-term follow-up after 2 yr revealed that the dog was diagnosed with diabetes mellitus.

Epidemiological investigation of insulin dysregulation in Shetland and Welsh ponies in Australia.

BACKGROUND: Insulin dysregulation (ID) is central to equine metabolic syndrome. There are limited epidemiological studies investigating dynamic testing of ID in ponies. OBJECTIVES: To evaluate prevalence and risk factors for ID through dynamic testing of hyperinsulinaemia (DHI) and insulin resistance (IR). STUDY DESIGN: Cross-sectional. METHODS: Sex, age, breed, height, cresty neck score (CNS), body condition score (BCS), laminitis, HMGA2:c.83G>A genotype and pituitary pars intermedia dysfunction (PPID) status were documented. Dynamic hyperinsulinaemia was diagnosed with an oral sugar test (OST) and IR with an insulin tolerance test (ITT). Owners completed surveys reporting activity, laminitis history and perception of body condition using a (1-9) visual analogue scale (VASo). Ordinal scores were converted to binary outcomes for CNS (≤2/5 or ≥3/5), BCS and VASo (≤6/9 or ≥7/9). Variables associated with insulin concentrations, glucose reduction after the ITT and laminitis were evaluated with mixed effects regression models accounting for random effects of farms. RESULTS: Among 167 ponies tested, median (range) age was 9 (4-21) years and BCS was 6 (4-8). Prevalence (95% confidence interval [CI]) of ID was 61 (53-68)%. Factors associated with insulin concentrations (estimate [95% CI]; µIU/mL) 60 min post-OST were: age (1.07 [1.02-1.11]), CNS (≥3/5, 1.52 [1.04-2.23]) and VASo (≥7/9, 1.75 [1.09-2.79]); and 90 min post-OST were: age (1.08 [1.03-1.12]), CNS (≥3/5, 1.80 [1.22-2.64]), VASo (≥7/9, 2.49 [1.52-4.08]) and sex (male, 0.64 [0.45-0.91]). Factors associated with glucose reduction after the ITT (estimate [95% CI]; %) were: age (-1.34 [-2.01 to -0.67]), sex (female, -6.21 [-11.68 to -0.74]) and VASo (≥7/9, -1.74 [-18.89 to -4.78]). Factors associated with laminitis (odds ratio [95% CI]) were DHI (4.60 [1.68-12.58]), IR (3.66 [1.26-10.61]) and PPID (11.75 [1.54-89.40]). MAIN LIMITATIONS: Single time-point sampling, laminitis definition and diet analysis. CONCLUSIONS: Ageing, being female and owner-perceived obesity were associated with ID.

The effect of pre-dosing with metformin on the insulin response to oral sugar in insulin-dysregulated horses.

BACKGROUND: A single dose of metformin administered 1 h prior to oral glucose challenge was previously shown to reduce insulinaemic responses in horses with experimentally-induced insulin dysregulation (ID). Targeted administration could be useful for controlling post-prandial hyperinsulinaemia in horses with naturally-occurring ID. OBJECTIVES: The objective was to compare the insulinaemic and glycaemic responses to oral sugar testing (OST) performed at different intervals after a single dose of metformin in horses with naturally-occurring ID. We hypothesised that pre-treatment with one dose of metformin would significantly decrease the insulinaemic response to OST. STUDY DESIGN: Randomised cross-over in vivo experiment. METHODS: Eight university-owned adult horses with naturally-occurring ID underwent OST 1, 2 and 6 h following a single oral dose of metformin (30 mg/kg) or 1 h after placebo (240 mL water) with a 7-day washout between treatments over a period of 3 weeks. Plasma insulin, C-peptide and glucose concentrations were measured at 0, 60 and 90 min after 0.45 mL/kg light corn syrup and the effect of treatment (and the interval since dosing) examined using a mixed effects linear regression model. RESULTS: Metformin treatment had no significant effect on plasma glucose, insulin or C-peptide concentrations at any time point compared with placebo (p > 0.05). For OST 1 h post metformin, median (IQR) plasma insulin was 91.3 (62.4-114.9) µIU/mL at 60 min versus 76.2 (59.1-134.5) for placebo (p = 0.8) and 62.7 (31.4-109.7) at 90 min versus 51.8 (29.2-126.3) for placebo (p = 0.9). MAIN LIMITATIONS: Small sample size may limit identification of more subtle decreases in insulin concentration with metformin pre-dosing. The results of this study are relevant only for one pre-treatment dose (30 mg/kg) which limits extrapolation to predictions about the effects of longer-term metformin administration on insulin and glucose dynamics in the horse. CONCLUSIONS AND CLINICAL IMPORTANCE: The results do not support the use of targeted metformin treatment to reduce post-prandial hyperinsulinaemia in horses with naturally-occurring ID.

Prolonged hyperinsulinemia increases the production of inflammatory cytokines in equine digital lamellae but not in striated muscle.

Hyperinsulinemia is the key feature of equine metabolic syndrome (EMS) which leads to debilitating sequelae. Hyperinsulinemia-associated laminitis (HAL) is one of the major sequelae of EMS, although the pathophysiological mechanisms are not well elucidated. Using an equine model, we hypothesized that expression of inflammatory markers would be increased in digital lamellae and striated muscle following prolonged hyperinsulinemia. Healthy Standardbred horses (5.4 ± 1.9 years) were alternately assigned to a prolonged euglycemic-hyperinsulinemic clamp (pEHC) or control group (n = 4 per group). Following a 48 h pEHC or a 48 h infusion of a balanced electrolyte solution (controls), biopsies were collected from digital lamellar tissue, skeletal muscle and cardiac muscle were obtained. All hyperinsulinemic horses developed laminitis regardless of previous health status at enrollment. Protein expression was quantified via Western blotting. A significant (P < 0.05) upregulation of the protein expression of heat shock protein 90 (HSP90), alpha 2 macroglobulin (A2M) and fibrinogen (α, ß isoforms), as well as inflammatory cytokines including interleukin-1ß were detected in digital lamellae following prolonged hyperinsulinemia. In contrast, protein expression of cytokines and acute phase proteins in heart and skeletal muscle was unchanged following hyperinsulinemia. Upregulation of inflammatory cytokines and acute phase proteins in digital lamellae during prolonged hyperinsulinemia may reveal potential biomarkers and novel therapeutic targets for equine endocrinopathic laminitis. Further, the lack of increase of inflammatory proteins and acute phase proteins in striated muscle following prolonged hyperinsulinemia may highlight potential anti-inflammatory and cardioprotective mechanisms in these insulin-sensitive tissues.

Obesity and obesity-associated metabolic disease conditions in Connemara ponies in Ireland.

BACKGROUND: Equine obesity combined with insulin dysregulation (ID) is a major risk factor associated with laminitis. Some pony breeds appear to be at increased risk. However, little is known regarding the prevalence of obesity or hyperinsulinaemia as evidence of ID in Irish ponies. OBJECTIVE: To investigate the prevalence of obesity and associated endocrine/metabolic disease conditions in Connemara ponies and to determine if hyperinsulinaemia in these ponies could be predicted by morphometric or metabolic markers. STUDY DESIGN: Cross-sectional study. METHODS: The study population included registered Connemara ponies recruited through public and veterinary social media posts. Ponies underwent a physical examination and information on their management and clinical history was obtained via owner questionnaire. The body condition score (BCS) was measured using the Henneke system; cresty neck score (CNS) and regionalised adiposity were also assessed. Hyperinsulinaemia was confirmed by measuring serum basal insulin concentration (BIC) or insulin concentration after an oral sugar test (OST). Blood glucose and triglyceride concentrations were measured. Characteristics of hyperinsulinaemic and insulin-sensitive ponies were compared by logistic regression. RESULTS: Two hundred ponies were included; 59 ponies (29.5%) had a BCS ≥7, 58 (29.0%) had a CNS ≥2.5 and 135 (67.5%) had regionalised adiposity; 137 (68.5%) ponies had at least one of these abnormalities. Owner-reported history or clinical evidence of chronic laminitis was found in 92 ponies (46.0%). Hyperinsulinaemia was confirmed in 32 ponies (16.0%), including 23 of 91 (25.3%) detected by OST and 9 of 109 (8.3%) by BIC. Hypertriglyceridaemia was observed in 12 of 198 ponies (6.1%) ponies and hyperglycaemia in 11 of 197 ponies (5.6%) ponies. The odds of hyperinsulinaemia increased by a factor of 6.53 (95% confidence interval: 2.95, 15.21) when BCS was ≥7. MAIN LIMITATIONS: The OST was not performed in all ponies. CONCLUSIONS: Increased adiposity, laminitis and metabolic derangements are prevalent in this native Irish pony breed.

Short-term induced hyperinsulinaemia and dexamethasone challenge do not affect circulating total adiponectin concentrations in insulin-sensitive ponies.

BACKGROUND: Hypoadiponectinaemia is a risk factor for endocrinopathic laminitis, but the directionality and nature of its association with insulin dysregulation is unclear. OBJECTIVES: To investigate the effects of short-term induced hyperinsulinaemia and dexamethasone challenge on circulating [total adiponectin] and whole blood expression of adiponectin (AdipoR1 and AdipoR2), insulin, and insulin-like growth factor 1 (IGF-1) receptors in insulin-sensitive ponies. STUDY DESIGN: In vivo experiment. METHODS: Six never-laminitic, insulin-sensitive, native-breed UK ponies first underwent a dexamethasone challenge (0.08 mg/kg i.v.) with blood samples collected every 15 min over 3 h. After a 14-day washout period, hyperinsulinaemia was induced for 9 h via a euglycaemic-hyperinsulinaemic clamp (EHC), with blood samples collected every 30 min. Serum [insulin], plasma [total adiponectin], and plasma [IGF-1] were measured using validated assays and receptor gene expression was assessed via quantitative polymerase chain reaction (qPCR). Finally, whole blood was incubated with 10-1000 ng/mL dexamethasone for 3 h at 37°C to investigate its direct effects on gene expression. RESULTS: There were no adverse effects observed during either protocol. Dexamethasone challenge did not alter circulating [insulin] or [total adiponectin] at any time-point, but significantly upregulated AdipoR1 and IGF-1R expression at 150 and 180 min. Ex vivo incubation of whole blood with dexamethasone did not alter expression of the genes examined. There was no change in [total adiponectin] or expression of the genes examined associated with EHC-induced hyperinsulinemia. MAIN LIMITATIONS: This was a small sample size that included only native-breed ponies; total adiponectin was measured rather than high-molecular-weight adiponectin. CONCLUSIONS: Short-term induced hyperinsulinaemia and dexamethasone challenge did not affect circulating [total adiponectin] in insulin-sensitive ponies. However, dexamethasone administration was associated with upregulation of two receptors linked to adiponectin signalling, suggesting that a physiological response occurred possibly to counteract dexamethasone-associated changes in tissue insulin sensitivity.

A glucagon-like peptide-1 receptor antagonist reduces the insulin response to a glycemic meal in ponies.

High plasma concentrations of insulin can cause acute laminitis. Ponies and horses with insulin dysregulation (ID) exhibit marked hyperinsulinemia in response to dietary hydrolyzable carbohydrates. Glucagon-like peptide-1 (GLP-1), an incretin hormone released from the gastrointestinal tract, enhances insulin release, and is increased postprandially in ponies with ID. The aim of this study was to determine whether blocking the GLP-1 receptor reduces the insulin response to a high glycemic meal. Five adult ponies were adapted to a cereal meal and then given two feed challenges 24 h apart of a meal containing 3 g/kg BW micronized maize. Using a randomized cross-over design all ponies received both treatments, where one of the feeds was preceded by the IV administration of a GLP-1 receptor blocking peptide, Exendin-3 (9-39) amide (80 µg/kg), and the other feed by a sham treatment of peptide diluent only. Blood samples were taken before feeding and peptide administration, and then at 30-min intervals via a jugular catheter for 6 h for the measurement of insulin, glucose, and active GLP-1. The peptide and meal challenge caused no adverse effects, and the change in plasma glucose in response to the meal was not affected (P = 0.36) by treatment: peak concentration 9.24 ±â€…1.22 and 9.14 ±â€…1.08 mmol/L without and with the antagonist, respectively. Similarly, there was no effect (P = 0.35) on plasma active GLP-1 concentrations: peak concentration 14.3 ±â€…1.36 pM and 13.7 ±â€…1.97 pM without and with the antagonist, respectively. However, the antagonist caused a significant decrease in the area under the curve for insulin (P = 0.04), and weak evidence (P = 0.06) of a reduction in peak insulin concentration (456 ±â€…147 µIU/mL and 370 ±â€…146 µIU/mL without and with the antagonist, respectively). The lower overall insulin response to the maize meal after treatment with the antagonist demonstrates that blocking the GLP-1 receptor partially reduced insulin production in response to a high starch, high glycemic index, diet. Using a different methodological approach to published studies, this study also confirmed that GLP-1 does contribute to the excessive insulin production in ponies with ID.

Horses and ponies are prone to suffer from laminitis if they produce too much insulin after eating a high-sugar/starch meal. Laminitis associated with high insulin is very painful and can result in the affected animals having to be put down. The reason why some ponies over-produce insulin is not known. However, we do know that small molecules produced in the upper intestine contribute to the problem. In this study we blocked the action of these molecules, to see if we could reduce the insulin released after a meal that was high in soluble carbohydrate (starch and sugar) content, in ponies. Using a specially designed drug, we were able to reduce insulin responses to the meal by over 20%. None of the ponies had any clinical problems in this study. This study helped us to explain why some animals produce excessive insulin; this compound may even have potential as a future therapy. However, whilst a promising finding, this effect was not as strong as it needs to be to help prevent laminitis in all animals. The next step is to test the drug at different doses, and under varying conditions, to see whether we can improve its performance.

Short-term effects of canagliflozin on glucose and insulin responses in insulin dysregulated horses: A randomized, placebo-controlled, double-blind, study.

BACKGROUND: Decreasing hyperinsulinemia is crucial in preventing laminitis in insulin dysregulated (ID) horses. Complementary pharmacological treatments that efficiently decrease postprandial hyperinsulinemia in ID horses are needed. OBJECTIVES: Compare short-term effects of canagliflozin vs placebo on glucose and insulin responses to an oral sugar test (OST) as well as the effects on body weight and triglyceride concentrations in horses with ID. ANIMALS: Sixteen privately-owned ID horses. METHODS: A single-center, randomized, double-blind, placebo-controlled, parallel design study. The horses were randomized (ratio 1:1) to either once daily PO treatment with 0.6 mg/kg canagliflozin or placebo. The study consisted of an initial 3-day period for obtaining baseline data, a 3-week double-blind treatment period at home, and a 3-day follow-up period similar to the initial baseline period but with continued double-blind treatment. Horses were subjected to an 8-sample OST in the morning of the third day on both visits. RESULTS: Maximal geometric least square (LS) mean insulin concentration (95% confidence interval [CI]) during the OST decreased after 3 weeks of canagliflozin treatment compared with placebo (83.2; 55.4-125.0 vs 215.2; 143.2-323.2 µIU/mL). The geometric LS mean insulin response (insulin AUC0-180 ) for canagliflozin-treated horses was >66% lower compared with placebo. Least square mean body weight decreased by 11.1 (4-18.1) kg and LS mean triglyceride concentrations increased by 0.99 (0.47-1.5) mmol/L with canagliflozin treatment. CONCLUSIONS AND CLINICAL IMPORTANCE: Canagliflozin is a promising drug for treatment of ID horses that requires future studies.

Hypertriglyceridemia in equines with refractory hyperinsulinemia treated with SGLT2 inhibitors.

Background: Sodium-Glucose CoTransporter-2 (SGLT2) inhibitors, the -flozin group of drugs, which block glucose reuptake in the renal proximal tubule, are being increasingly used off-label to treat horses with refractory hyperinsulinemia. After 2 years of use by animals in our group, a horse on canagliflozin was incidentally noted to be hyperlipemic. Case Description: We have been following a cohort of equines (n = 20) treated with SGLT2 inhibitors due to refractory hyperinsulinemia. The animals are owned by members of the Equine Cushing's and Insulin Resistance Group and treated by their attending veterinarians. The index case was a 23 years old gelding with a 2 years history of recurring laminitis that began canagliflozin therapy to control hyperinsulinemia which was no longer responsive to metformin. Between 6 and 10 weeks post start of therapy, significant weight loss was noticed. Two days later he was hospitalized with colic symptoms and hyperlipemia but was bright, alert, and eating well throughout. Canagliflozin was discontinued and triglycerides returned to normal reference values within 10 days. A subsequent study of 19 other horses on SGLT2 inhibitors revealed varying degrees of hypertriglyceridemia, all asymptomatic. Conclusion: While this class of drugs holds great promise for cases of refractory hyperinsulinemia and laminitis that do not respond to diet or metformin therapy, hypertriglyceridemia is a potential side effect. In our experience, animals remained asymptomatic and eating well. Further study of hypertriglyceridemia in horses on SGLT2 inhibitors and the possible mitigating effect of diet is indicated. To our knowledge, this is the first report of hypertriglyceridemia with canagliflozin treatment in equines.

Equine metabolic syndrome: Role of the enteroinsular axis in the insulin response to oral carbohydrate.

Equine insulin dysregulation (ID) comprises amplified insulin responses to oral carbohydrates or insulin resistance, or both, which leads to sustained or periodic hyperinsulinaemia. Hyperinsulinaemia is important in horses because of its clear association with laminitis risk, and the gravity of this common sequela justifies the need for a better understanding of insulin and glucose homoeostasis in this species. Post-prandial hyperinsulinaemia is the more commonly identified component of ID and is diagnosed using tests that include an assessment of the gastrointestinal tract (GIT). There are several factors present in the GIT that either directly, or indirectly, enhance insulin secretion from the endocrine pancreas, and these factors are collectively referred to as the enteroinsular axis (EIA). A role for key components of the EIA, such as the incretin peptides glucagon-like peptide-1 and 2, in the pathophysiology of ID has been investigated in horses. By comparison, the function (and even existence) of many EIA peptides of potential importance, such as glicentin and oxyntomodulin, remains unexplored. The incretins that have been examined all increase insulin responses to oral carbohydrate through one or more mechanisms. This review presents what is known about the EIA in horses, and discusses how it might contribute to ID, then compares this to current understanding derived from the extensive studies undertaken in other species. Future directions for research are discussed and knowledge gaps that should be prioritised are suggested.

Identifying possible thresholds for nonstructural carbohydrates in the insulin dysregulated horse.

BACKGROUND: Identifying intake levels of nonstructural carbohydrates (NSCs) that limit the postprandial insulinaemic response in the insulin dysregulated (ID) horse may help reduce hyperinsulinaemia-associated laminitis (HAL) risk. OBJECTIVE: To determine if ID horses have thresholds for pure sources of starch and sugar, above which there is an augmented insulin response. STUDY DESIGN: Randomised crossover experiment. METHODS: Fourteen adult horses (6 ID and 8 noninsulin dysregulated, NID; matched for bodyweight) were randomly fed eight dietary treatments. Dietary treatments were formulated using a base of low-nonstructural carbohydrate pellet (LNSC; 0.04 g of water-soluble carbohydrates (WSCs)/kg bwt and 0.01 g of starch/kg bwt), to which pure sugar (dextrose) or starch (50:50 mix of waxy-maize and oat starch powder) sources were titrated to create diets with increasing amounts of either WSC (0.06-0.17 g WSC/kg bwt), or starch (0.03-0.1 g starch/kg bwt). Horses were fed each dietary treatment at a rate of 1 g/kg bwt once over 12 weeks. Serial blood samples were collected pre- and up to 240 min postprandially. Insulin was determined via RIA and diet analytes were determined via wet chemistry. Statistical analysis was performed with a mixed effect model. Positive incremental area under the curve for insulin (IAUCi) was calculated for all horses and dietary treatments. RESULTS: There was no significant effect of diet in NID horses but diets with NSC >0.1 g/kg bwt produced an augmented response in ID horses compared with the LNSC (p < 0.05). ID horses IAUCi were also significantly different to all NID IAUCi for diets with NSC >0.1 g/kg (p < 0.04). Apparent thresholds for sugar and starch addition varied. CONCLUSIONS: Based on this study, using supplemental pure starch and sugar sources, ID horses seem to have an apparent threshold for NSC of around 0.1 g/kg bwt/meal, above which significantly increased insulin responses are seen compared with NID horses.

Effect of intravenous lipid infusion on biomarkers of insulin resistance and immune functions of dry and nonpregnant dairy cows.

During the transition period, dairy cows often experience negative energy balance, which can induce metabolic and immunological disturbances. Previous work has shown that there is a relationship between the dysfunction of immune cells and the increase in blood nonesterified fatty acid (NEFA) concentration. Nevertheless, it is difficult to determine the exact effect of NEFA on the immune system, as other metabolic and hormonal perturbations occur simultaneously during the transition period. In the present study, we have determined the effect of NEFA on immune functions using an experimental model designed to assess the effects independently of energy balance, as well as hormonal and metabolic changes due to parturition. Six dry and nonpregnant cows were infused with either sterile water (control treatment) or a lipid emulsion (Intralipid 20%, Frenesius Kabi, lipid treatment) at a rate of 1 mL/kg per hour for 6 h according to a crossover design. Blood concentrations of NEFA, ß-hydroxybutyrate (BHB), and glucose were measured every hour throughout the infusion period, and 1 and 18 h after the end of infusion. Proliferation and interferon-γ secretion of lymphocytes, phagocytosis, and oxidative burst of neutrophils and blood insulin concentration were evaluated before, during, and at the end of the infusion. For NEFA, BHB, and glucose, treatment × time interactions were present. When compared with the control condition, NEFA and BHB levels were greater in the plasma of cows infused with lipids from 1 h after the start of infusion until 1 h after the end of infusion. Glucose level also increased in response to lipid infusion from 2 h of infusion until 1 h after the end of treatment. For sterile water and lipid infusions, respectively, maximal concentrations were 0.06 ± 0.10 mM and 1.39 ± 0.10 mM for NEFA, 0.70 ± 0.05 mM and 1.06 ± 0.05 mM for BHB, and 4.56 ± 0.27 mM and 6.90 ± 0.27 mM for glucose. For all blood metabolites, there were no differences between treatments 18 h postinfusion. Lipid infusion significantly increased blood insulin concentration at 3 and 6 h of infusion. However, it returned to its basal concentration 18 h after the end of the infusion. Lymphoproliferation declined as early as 3 h after the start of the lipid infusion. At 3 and 6 h of infusion, lipid treatment significantly reduced INF-γ concentration in the culture cell supernatant. The lipid infusion did not affect neutrophil phagocytosis. Nevertheless, the efficacy of the response was affected by a reduction of neutrophils' oxidative burst. These results confirm that NEFA inhibits immune functions independently of energy balance and other changes that occur during the transition period. They also indicate that high blood lipid concentration causes insulin resistance.

Comparison of production-related responses to hyperinsulinemia and hypoglycemia induced by clamp procedures or heat stress of lactating dairy cattle.

Hyperinsulinemia concurrent with hypoglycemia is one of a myriad of physiological changes typically experienced by lactating dairy cows exposed to heat stress, the consequences of which are not yet well defined or understood. Therefore, the objective of this experiment was to separate the production-related effects of hyperinsulinemia with hypoglycemia from those of a hyperthermic environment. Multiparous lactating Holstein cows (n = 23; 58 ± 4 d in milk, 3.1 ± 0.3 lactations) were housed in temperature-controlled rooms and all were subjected to 4 experimental periods as follows: (1) thermoneutral (TN; temperature-humidity index of 65.1 ± 0.2; d 1-5), (2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; insulin infused at 0.3 µg/kg of BW per h, glucose infused to maintain 90 ± 10% of baseline blood glucose for 96 h; d 6-10), (3) heat stress (HS; temperature-humidity index of 72.5 ± 0.2; d 16-20), and (4) HS + euglycemic clamp (EC; glucose infused to reach 100 ± 10% of TN baseline blood glucose for 96 h; d 21-25). Cows were fed and milked twice daily. Feed refusals were collected once daily for calculation of daily dry matter intake, and milk samples were collected at the beginning and end of each period for component analyses. Circulating insulin concentrations were measured in daily blood samples, whereas glucose concentrations were measured more frequently and variably in association with clamp procedures. Rectal temperatures and respiration rates were greater during HS than TN, as expected, and states of hyperinsulinemia and hypoglycemia were successfully induced by the HHC and high ambient temperatures (HS and EC). Feed intake differed based upon thermal environment as it was similar during TN and HHC periods, and declined for HS and EC. Milk production was not entirely reflective of feed intake as it was greatest during TN, intermediate during HHC, and lowest during HS and EC. All milk components differed with the experimental period, primarily in response to the thermal environment. Interestingly, TN baseline glucose concentrations were highly correlated with the change in glucose from TN to HS, and were related to glycemic status during HS. Furthermore, although few in number, those cows that failed to become hypoglycemic during HS tended to have a greater reduction in milk yield. The work presented here addresses a critical knowledge gap by broadening our understanding of the physiological response to heat stress and the related changes in glycemic state. This broadened understanding is fundamental for the development of novel, innovative management strategies as the dairy industry is compelled to become increasingly efficient in spite of global warming.

Use of the SGLT2 inhibitor canagliflozin for control of refractory equine hyperinsulinemia and laminitis.

Background: Hyperinsulinemia associated with pituitary pars intermedia dysfunction (PPID) and/or equine metabolic syndrome is well documented to put horses at high risk of laminitis. While dietary control of simple sugars and starch is the most effective therapy to control hyperinsulinemia, some horses fail to respond. Case Descriptions: Ten horses with hyperinsulinemia refractory to diet control, metformin, levothyroxine, and pergolide (if diagnosed with PPID) were treated with sodium-glucose cotransporter-2 inhibitor canagliflozin (Invokana®). Nine horses were hyperglycemic (>5.5 mmol/l) or had a history of hyperglycemia. Before instituting therapy, renal function was assessed by determining serum creatinine and blood urea nitrogen concentrations. Canagliflozin was administered orally once a day, with food. Dipstick urinalysis was performed every 2 weeks to confirm glucosuria and screen for proteinuria. Owners were also instructed regarding clinical signs consistent with urinary tract infection. All horses responded with a substantial decrease in serum insulin concentrations to normal or near normal values. Laminitis pain resolved in all cases, with regression of fat deposits. Owner satisfaction with outcomes was 100%. Conclusion: Once daily administration of the SGLT2 inhibitor canagliflozin corrected hyperglycemia, reduced insulin to normal or near normal levels, and was 100% effective in reversing or reducing abnormal fat pads and eliminating laminitis pain in horses with refractory hyperinsulinemia and laminitis. The core aspects of therapy-diet control, exercise when possible, and adequate treatment of PPID-must also be maintained if using canagliflozin. Canagliflozin should be reserved for refractory cases. Further controlled trials to investigate canagliflozin pharmacokinetics, pharmacodynamics, efficacy, and safety are needed.

Demographic, morphologic, hormonal and metabolic factors associated with the rate of improvement from equine hyperinsulinaemia-associated laminitis.

BACKGROUND: Although several studies have investigated factors associated with the onset and occurrence of hyperinsulinaemia-associated laminitis (HAL), few have examined the factors associated with the rate of improvement during recovery from an acute bout of the disease. This observational study sought to discover if a range of demographic, morphologic, hormonal and metabolic variables are associated with the improvement rate from HAL in 37 naturally-occurring cases identified by 16 clinics across Germany. Each case was evaluated for laminitis severity on the day of inclusion in the trial (d 0), then after 4, 9, 14, 25 and 42 d. The horses were managed according to best clinical practice including restricting exercise and prescribing a diet of hay-only, for a minimum of 9 d. Blood samples were also collected during each evaluation, except on d 9, and analysed for glucose, insulin, ACTH and leptin. RESULTS: Based on individual clinical laminitis scores plotted against time, most horses improved markedly within 2 weeks, with a 'fast group' (n = 27) having a median (interquartile range) score on a 12-point scale of 0 (0-2) by d 14. However, there was a clear disparity within the total cohort, as ~ 1 in 4 horses demonstrated much slower improvement, with a median score of 5 (4-7) by d 14, or a marked relapse thereafter ('slow group', n = 10). Horses in the slow improvement group were younger (12.5 (8.8-16.3) vs 17 (14-24) yr; P = 0.008), but were not more likely to be heavier, male, very fat, to have presented with a previous history of laminitis or elevated ACTH concentrations, or to be receiving pergolide treatment. Of the hormonal and metabolic parameters measured, glucose and insulin concentrations were within the normal range following transition to the hay-only diet, but were higher in the group that failed to improve quickly, with a small but significant difference being evident on d 4, 14 and 25 for glucose (11 to 16%; P < 0.05), and a larger difference for insulin on d 14 and 25 (51 to 55%; P < 0.05). There was no difference between the groups in ACTH or leptin concentrations throughout the study. The main limitations of this study were the small number of slow-improvement horses and an inability to control or measure certain variables, such as feed quality. CONCLUSIONS: Young age and a modest increase in blood glucose and insulin concentrations are associated with delayed laminitis improvement.

Postprandial insulin responses to various feedstuffs differ in insulin dysregulated horses compared with non-insulin dysregulated controls.

BACKGROUND: Controlling postprandial hyperinsulinaemia is important in insulin dysregulated (ID) horses to reduce the risk of laminitis. OBJECTIVES: To evaluate postprandial insulin responses of ID versus non-insulin dysregulated (NID) horses to feedstuffs varying in nonstructural carbohydrate (NSC) and crude protein (CP). STUDY DESIGN: Randomised crossover. METHODS: Eighteen adult mixed-breed horses (13.3 ± 2.2 years; 621 ± 78.8 kg) were individually fed [~1 g/kg body weight (BW)] specific feedstuffs within two crossover studies. Eight ID and eight NID were used in Study A, and 11 ID and 5 NID in Study B. In Study A, all horses were randomly fed once: cracked corn (CC: ~74% NSC & ~9% CP), ration balancer with low protein (RB-LP: ~15% NSC & ~17% CP), ration balancer with high protein (RB-HP: ~14% NSC and ~37% CP) and 50:50 mixture of RB-LP:RB-HP (MIX-P). In Study B, horses were randomly fed once: CC, RB-HP, steam-flaked corn (SF: ~73% NSC & ~10% CP), oat groats (OG: ~64% NSC & ~14% CP) and a low NSC pellet (L-NSC: ~6% NSC & ~12% CP). Blood was collected for insulin determination [radioimmunoassay (RIA)] before and 30, 60, 75, 90, 105, 120, 150, 180, 210 and 240-minute post-feeding in Study A and at 60-minute in Study B. Data were analysed via analysis of variance (ANOVA) for repeated measures after any required transformations. RESULTS: ID horses had significantly greater insulin responses (AUCi) than NID for all diets in both studies (P < .001; ID 22 362 ± 10 298 µIU/mL/min & NID 6145 ± 1922 µIU/mL/min). No effect of diet on AUCi for NID (P = .2), but in ID, the CC (32 000 ± 13 960 µIU/mL/min) AUCi was higher than RB-LP (P = .01; 18 977 ± 6731 µIU/mL/min). ID insulin (T60) was lower for the L-NSC (57.8 ± 18.5 µIU/mL) versus all other diets (P < .02; 160.1 ± 91.5 µIU/mL). MAIN LIMITATIONS: Small numbers of horses; no ponies. CONCLUSIONS: NSC appears to be the main driver of the postprandial insulin response. ID horses respond disproportionately to feeding even small amounts of low/moderate NSC feedstuffs. Data on possible dietary thresholds for postprandial insulin responses cannot be extrapolated from NID horses.

Continuous digital hypothermia reduces expression of keratin 17 and 1L-17A inflammatory pathway mediators in equine laminitis induced by hyperinsulinemia.

The euglycemic hyperinsulinemic clamp model (EHC) of equine endocrinopathic laminitis induces rapid loss of lamellar tissue integrity, disrupts keratinocyte functions, and induces inflammation similar to natural disease. Continuous digital hypothermia (CDH) blocks tissue damage in this experimental model, allowing identification of specific genes or molecular pathways contributing to disease initiation or early progression. Archived lamellar tissues (8 horses, 48 h EHC treatment, including CDH-treated front limbs) were used to measure relative expression levels of genes encoding keratin 17 (KRT17), a stress-induced intermediate filament protein, and genes upregulated downstream of keratin 17 and/or interleukin 17A (IL-17A), as mediators of inflammation. Compared to front or hind limbs at ambient temperature, CDH resulted in significantly lower expression of KRT17, CCL2, CxCL8, PTGS2 (encoding COX2), IL6, TNFα, S100A8 and MMP1. By immunofluorescence, COX2 was robustly expressed in lamellar keratinocytes from ambient limbs, but not in CDH-treated limbs. Genes not significantly reduced by CDH were IL17A, DEFB4B, S100A9 and MMP9. Overall, 8 of 12 genes were expressed at lower levels in the CDH-treated limb. These 8 genes are expressed by wounded or stress-activated keratinocytes in human disease or mouse models, highlighting the role of keratinocytes in equine laminitis.