Evaluation of fasting plasma insulin and proxy measurements to assess insulin sensitivity in horses.

BACKGROUND: Proxies are mathematical calculations based on fasting glucose and/or insulin concentrations developed to allow prediction of insulin sensitivity (IS) and ß-cell response. These proxies have not been evaluated in horses with insulin dysregulation. The first objective of this study was to evaluate how fasting insulin (FI) and proxies for IS (1/Insulin, reciprocal of the square root of insulin (RISQI) and the quantitative insulin sensitivity check index (QUICKI)) and ß-cell response (the modified insulin-to-glucose ratio (MIRG) and the homeostatic model assessment of ß-cell function (HOMA-ß)) were correlated to measures of IS (M index) using the euglycemic hyperinsulinemic clamp (EHC) in horses with insulin resistance (IR) and normal IS. A second objective was to evaluate the repeatability of FI and proxies in horses based on sampling on consecutive days. The last objective was to investigate the most appropriate cut-off value for the proxies and FI. RESULTS: Thirty-four horses were categorized as IR and 26 as IS based on the M index. The proxies and FI had coefficients of variation (CVs) ≤ 25.3 % and very good reliability (intraclass correlation coefficients ≥ 0.89). All proxies and FI were good predictors of the M index (r = 0.76-0.85; P < 0.001). The proxies for IS had a positive linear relationship with the M index whereas proxies for ß-cell response and FI had an inverse relationship with the M index. Cut-off values to distinguish horses with IR from horses with normal IS based on the M index were established for all proxies and FI using receiver operating characteristic curves, with sensitivity between 79 % and 91 % and specificity between 85 % and 96 %. The cut-off values to predict IR were < 0.32 (RISQI), < 0.33 (QUICKI) and > 9.5 µIU/mL for FI. CONCLUSIONS: All proxies and FI provided repeatable estimates of horses' IS. However, there is no advantage of using proxies instead of FI to estimate IR in the horse. Due to the heteroscedasticity of the data, proxies and FI in general are more suitable for epidemiological studies and larger clinical studies than as a diagnostic tool for measurement of IR in individual horses.

Acute phase response in lactating dairy cows during hyperinsulinemic hypoglycaemic and hyperinsulinemic euglycaemic clamps and after intramammary LPS challenge.

The link between energy availability, turnover of energy substrates and the onset of inflammation in dairy cows is complex and poorly investigated. To clarify this, plasma inflammatory variables were measured in mid-lactating dairy cows allocated to three groups: hyperinsulinemic hypoglycaemic clamp, induced by insulin infusion (HypoG, n = 5); hyperinsulinemic euglycaemic clamp, induced by insulin and glucose infusion (EuG; n = 6); control, receiving a saline solution infusion (NaCl; n = 6). At 48 h after the start of i.v. infusions, two udder quarters per cow were challenged with 200 µg of E. coli lipopolysaccharide (LPS). Individual blood samples were taken before clamps, before LPS challenge (i.e. 48 h after clamps) and 6.5 h after. At 48 h, positive acute phase proteins (posAPP) did not differ among groups, whereas albumin and cholesterol (index of lipoproteins), negative APP (negAPP), were lower (p < 0.05) in EuG compared to NaCl and HypoG. The concentration of IL-6 was greater in EuG (p < 0.05) but only vs. HypoG. At 6.5 h following LPS challenge, IL-6 increased in the NaCl and EuG clamps (p < 0.05), while TNF-α increased (p < 0.05) in the EuG only. Among the posAPP, haptoglobin markedly increased in EuG (p < 0.05), but not in NaCl (p = 0.76) and in HypoG; ceruloplasmin tended to decline during LPS challenge, the reduction was significant when all animals were considered (p < 0.05). Conversely, all the negAPP showed a marked reduction 6.5 h after LPS challenge in the three groups. In conclusion, EuG caused an inflammatory status after 48-h infusion (i.e. decrease of negAPP) and induced a quicker acute phase response (e.g. marked rise of TNF-α, IL-6) after the intramammary LPS challenge. These data suggest that the simultaneous high availability of glucose and insulin at the tissue-level makes dairy cows more susceptible to inflammatory events. In contrast, HypoG seems to attenuate the inflammatory response.

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS-induced mastitis in dairy cows.

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long-term manipulated glucose and insulin concentrations in combination with a LPS-induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT-qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha-lactalbumin, insulin-induced gene 1, κ-casein and acetyl-CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS-induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.

Hyperinsulinemic clamp modulates milk fat globule lipid composition in goats.

We determined the effect of insulin on milk fatty acid (FA) and lipid composition in goats. Four dairy goats, 150 d in milk, were subjected to hyperinsulinemic clamp (treatment) or saline (control) infusion for 4d in a crossover design study. Composition and concentration of plasma and milk FA, triglycerides, phospholipids, sphingolipids, and cholesterol were determined. Mammary gland biopsies were taken at the end of each experimental period and lipogenic gene expression was determined. Plasma insulin was elevated 3.5-fold, whereas plasma glucose remained constant during the treatment period. Feed intake decreased by 26% and fat yield decreased by 17% relative to controls. No change in nonesterified FA concentration was found between controls and treatment. Compared with controls, insulin decreased yield of long-chain saturated FA by 14%. Milk concentration of long-chain FA was reduced by 3%, whereas that of medium-chain FA increased by 5% during the treatment compared with controls. Hyperinsulinemic clamps increased the yields of milk phospholipids by 9% and cholesterol by 16%, whereas it only tended to decrease triglyceride yields (by 11%). Hyperinsulinemic treatment resulted in compositional changes in the milk fat globule membrane, as reflected by 15 and 9% decreases in phosphatidylethanolamine and phosphatidylcholine concentrations, respectively. Lipogenic gene expression of acyl coenzyme A carboxylase, stearoyl coenzyme A desaturase, and FA synthase did not change, whereas lipoprotein lipase gene expression tended toward an increase in the treatment period compared with controls. Hyperinsulinemic clamps reduce the availability of long-chain FA, which are considered to originate from the diet and adipose lipolysis for milk lipid synthesis by the mammary gland of goats. Under these conditions, long-chain FA might be preferentially channeled to phospholipid rather than triglyceride synthesis, hence increasing phospholipid yields. Mechanisms determining FA distribution among milk lipid components and the consequences of altered milk fat globule membrane lipid composition remained to be elucidated.

Pharmacodynamics of insulin detemir and insulin glargine assessed by an isoglycemic clamp method in healthy cats.

BACKGROUND: Insulin detemir and insulin glargine are synthetic long-acting insulin analogs. In people, insulin glargine is longer acting and has a relatively flat time-action profile, while insulin detemir has significantly less within-subject variability. Insulin detemir is also associated with less undesired weight gain and decreased frequency of hypoglycemic events. OBJECTIVES: To compare the pharmacodynamics of insulin detemir and insulin glargine in healthy cats. ANIMALS: Ten young, healthy, neutered, purpose-bred cats. METHODS: Randomized, cross-over design. Pharmacodynamics of insulin detemir and insulin glargine were determined by the isoglycemic clamp method after a 0.5 U/kg SC injection. RESULTS: The only significant difference in the pharmacodynamics of insulin detemir and insulin glargine was onset of action (1.8+/-0.8 and 1.3+/-0.5 hours for insulin detemir and insulin glargine, respectively, P=.03). End of action of insulin detemir was reached at 13.5+/-3.5 hours and for insulin glargine at 11.3+/-4.5 hours (P=.18). Time-to-peak action of insulin detemir was reached at 6.9+/-3.1 hours and for insulin glargine at 5.3+/-3.8 hours (P=.7). The time-action curves of both insulin analogs varied between relatively flat curves in some cats and peaked curves in others. CONCLUSION AND CLINICAL IMPORTANCE: Insulin detemir and insulin glargine have shorter durations of action than in people when assessed by the clamp method, but in some cats these insulin analogs could be useful as once-a-day drugs. Peak effects of both insulin analogs are pronounced in some cats.

Lamellar energy metabolism and perfusion in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Hyperinsulinaemia is associated with the development of endocrinopathic laminitis; however, the mechanisms remain unclear. OBJECTIVES: Evaluate the effects of hyperinsulinaemia on lamellar energy metabolism and perfusion during laminitis development. STUDY DESIGN: In vivo experiment. METHODS: Eight Standardbred horses were instrumented with a microdialysis probe in the lamellae of a forelimb. A 24 hours baseline period (BASELINE) was followed by 48 hours of a continuous euglycaemic hyperinsulinaemic clamp (EHC) from 24 to 72 hours (CLAMP). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate-to-pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Archived microdialysis samples from six identically instrumented Standardbred horses served as controls (CON). Variables were compared over time and between EHC and CON horses using a mixed-effects linear regression model. RESULTS: Glucose concentration decreased during the CLAMP period in CON and EHC horses (P < .001), but there was no difference between CON and EHC (P > .9). Lactate concentration increased during the CLAMP period in CON and EHC horses (P < .001), however, the rate of increase was significantly higher in EHC horses relative to CON (P = .014). There was a relative increase in pyruvate concentration in EHC horses compared with CON during the CLAMP period (P = .03). L:P increased significantly in CON horses during the CLAMP period (P < .001) but not in EHC (P = .1). Urea clearance did not change in CON (P = .9) or EHC (P = .05) during the CLAMP, but did increase in EHC relative to CON (P = .02). MAIN LIMITATIONS: The effects of microdialysis probe implantation on perfusion and metabolism remain unclear. The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: Laminitis developed without evidence of lamellar hypoperfusion or energy stress. Therapies to improve perfusion are unlikely to affect the initial development of endocrinopathic laminitis.

Repeatability of the hyperglycaemic clamp for assessment of ß-cell response and insulin sensitivity in horses.

BACKGROUND: The hyperglycaemic clamp has been used for measurement of insulin sensitivity (IS) but not for measurement of ß-cell response in the horse. However, the repeatability of this test has not been reported for horses. OBJECTIVES: To determine the repeatability and reliability of measures for IS and ß-cell response to glucose using the hyperglycaemic clamp in horses. STUDY DESIGN: Repeated measures, longitudinal study. METHODS: Six healthy Standardbred mares underwent a 120-min hyperglycaemic clamp on two occasions with a 10-day washout period. Indices of repeatability and reliability were calculated from measures of IS and ß-cell response to glucose derived from each hyperglycaemic clamp. RESULTS: Measures of ß-cell response to glucose, including area under the insulin response curve in the time interval 0-120 min and during steady state (60-120 min), as well as the mean insulin concentration during steady state had coefficient of variations (CV) of 9.5, 10.5 and 9.3 respectively and intraclass correlation coefficients (ICC) of 0.93, 0.93 and 0.95 respectively. The measure of glucose disposal (M) had lower CV (12.4) and ICC (0.71) compared to measure of IS (M/I-index; CV of 17.4 and ICC of 0.89). MAIN LIMITATIONS: The study was conducted in a small number of horses. CONCLUSIONS: The hyperglycaemic clamp is a diagnostic method for assessing ß-cell response to intravenous glucose with high repeatability and reliability. Insulin sensitivity can be determined with the same test but the repeatability for these measurements is lower compared to the measurements for ß-cell response.

Effect of digital hypothermia on lamellar inflammatory signaling in the euglycemic hyperinsulinemic clamp laminitis model.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear. HYPOTHESIS/OBJECTIVES: To determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis. ANIMALS: Eight Standardbred horses. METHODS: Prospective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL-11, cyclooxygenase 1 and 2, tumour necrosis factor-alpha [TNF-α], E-selectin, and intercellular adhesion molecule-1 [ICAM-1]) and immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1] and STAT3). RESULTS: Compared to AMB, lamellar messenger ribonucleic acid (mRNA) concentrations of CXCL6 (P =.02), CXCL8 (P = .008), IL-6 (P = .008), IL-1ß (P = .008), IL-11 (P = .008), and cyclooxygenase-2 (P = .008) were decreased in CDH. Cyclooxygenase-1 (P = .008) was increased in CDH, while CXCL1 (P = .15), MCP-1 (P = .05), MCP-2 (P = .46), TNF-α (P = .05), E-selectin (P = .15), and ICAM-1 (P = .15) mRNA were not significantly different. Compared to AMB, lamellar concentration of total STAT3 protein was decreased in CDH (P < .001), but there was no change in phosphorylated STAT3 (P-STAT3 [S727] P = .19; P-STAT3 [Y705] P = .05). There was no change in lamellar concentrations of total STAT1 (P = .75) or phosphorylated STAT1 (P-STAT1 [S727], P = .25; P-STAT1 [Y701], P = .64). CONCLUSIONS AND CLINICAL IMPORTANCE: These data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses.

Influence of litter size on insulin sensitivity in multiparous sows.

The objectives were to examine effects of litter size on insulin sensitivity in multiparous sows at the end of pregnancy. Twelve sows were allocated in two treatments after weaning: control (CTR) or ligature of the left oviduct (LIG). At 68 d of the subsequent pregnancy, catheters were implanted in a jugular vein, in a carotid artery, and in the main vein draining one uterine horn. A blood flow probe was fitted around the artery irrigating the same uterine horn. A meal test, a tolerance test, and an euglycemic hyperinsulinemic clamp test were performed at 108 ± 3 d of pregnancy. Serial blood samples were drawn simultaneously from the uterine vein and the carotid artery before and during the tests. The number of fetuses in the studied uterine horn was lower (3.7 vs. 8.0, P < 0.001), and piglets at birth were heavier (1.71 vs. 1.31 kg, P = 0.04) in the LIG sows than in the CTR sows. Treatment did not affect uterine blood flow (UBF), but UBF/fetus in the uterine horn was greater for the LIG treatment (0.67 vs. 0.34 L/min, P = 0.002). During meal test, glycemia, glucose uptake in the uterine horn and glucose uterine uptake/fetus were similar in both groups of sows, while insulin levels were higher in the LIG sows (P = 0.04). The decrease of NEFA concentrations was similar across treatments. Glucose half-life did not differ between treatments (13.4 min as a mean; P = 0.63) during tolerance test, but area under the insulin curve was greater in the LIG sows (P = 0.02). The glucose infusion rate during euglycemic hyperinsulinemic clamps was lower in the LIG sows than in CTR sows (6.1 ± 0.2 vs. 7.8 ± 0.1 mg glucose.kg-1 min-1; P = 0.01). The LIG sows are less sensitive to insulin than the CTR sows without adjustment of maternal glycemia and glucose tolerance. Insulin sensitivity adaptation to litter size in late pregnancy of sows would rather be connected to growth rate than to number of fetuses.

Comparison of pharmacodynamics and pharmacokinetics of insulin degludec and insulin glargine 300 U/mL in healthy cats.

Insulin glargine 300 U/mL (IGla-U300) and insulin degludec (IDeg) are synthetic insulin analogs designed as basal insulin formulations. In people, IGla-U300 is more predictable and longer acting compared with glargine 100 U/mL. The duration of action of IDeg in people is > 42 h, allowing flexibility in daily administration. We hypothesized that IDeg would have longer duration of action compared with IGla-U300 in healthy purpose-bred cats. Seven cats received 0.4 U/kg (subcutaneous) of IDeg and IGla-U300 on two different days, >1 wk apart. Exogenous insulin was measured and pharmacodynamic parameters were derived from glucose infusion rates during isoglycemic clamps and suppression of endogenous insulin. The Shapiro-Wilk test was used to assess normality, and normally distributed parameters were compared using paired t-tests. There was no difference between IDeg and IGla-U300 in onset, peak action, or total metabolic effect. On average, time to peak action (TPEAK) of IGla-U300 was 145 ± 114 min (95% confidence interval [CI] = 25-264) longer than TPEAK of IDeg (P = 0.03) and duration of action (TDUR) of IGla-U300 was 250 ± 173 min (95% CI = 68-432) longer than TDUR of IDeg (P = 0.02). The "flatness" of the time-action profile (as represented by the quotient of peak action/TDUR) was significantly greater for IGla-U300 compared with IDeg (P = 0.04). Overall, insulin concentration measurements concurred with findings from isoglycemic clamps. Based on these data, IDeg is not suitable for once-daily administration in cats. The efficacy of once-daily IGla-U300 in diabetic cats should be further investigated.

Association of sustained supraphysiologic hyperinsulinemia and inflammatory signaling within the digital lamellae in light-breed horses.

BACKGROUND: Hyperinsulinemia is associated with equine laminitis, and digital lamellar inflammation in equine metabolic syndrome-associated laminitis (EMSAL) is modest when compared with sepsis-associated laminitis. OBJECTIVES: To characterize digital lamellar inflammation in horses in a euglycemic-hyperinsulinemic clamp (EHC) model of laminitis. ANIMALS: Sixteen healthy adult Standardbred horses. METHODS: Prospective experimental study. Horses underwent EHC or saline infusion (CON) for 48 hours or until the onset of Obel grade 1 laminitis. Horses were euthanized, and digital lamellar tissue was collected and analyzed via polymerase chain reaction (pro-inflammatory cytokine and chemokine genes-CXCL1, CXCL6, CXCL8, IL-6, MCP-1, MCP-2, IL-1ß, IL11, cyclooxygenases 1 and 2, tumor necrosis factor alpha [TNF-α], E-selectin, and ICAM-1), immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1], STAT3, and p38MAPK), and immunohistochemistry (markers of leukocyte infiltration: CD163, MAC387). RESULTS: Lamellar mRNA concentrations of IL-1ß, IL-6, IL-11, COX-2, and E-selectin were increased; the concentration of COX-1 was decreased; and concentrations of CXCL1, CXCL6, MCP-1, MCP-2, IL-8, TNF-α and ICAM-1 were not significantly different in the EHC group compared to the CON group (P ≤ .003). Lamellar concentrations of phosphorylated STAT proteins (P-STAT1 [S727], P-STAT1 [Y701], P-STAT3 [S727], and P-STAT3 [Y705]) were increased in the EHC group compared to the CON group, with phosphorylated STAT3 localizing to nuclei of lamellar basal epithelial cells. There was no change in the lamellar concentration of P-p38 MAPK (T180/Y182), but the concentration of total p38 MAPK was decreased in the EHC samples. There was no evidence of notable lamellar leukocyte emigration. CONCLUSIONS AND CLINICAL IMPORTANCE: These results establish a role for lamellar inflammatory signaling under conditions associated with EMSAL.

Continuous digital hypothermia prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Continuous digital hypothermia can prevent the development and progression of laminitis associated with sepsis but its effects on laminitis due to hyperinsulinaemia are unknown. OBJECTIVES: To determine the effects of continuous digital hypothermia on laminitis development in the euglycaemic hyperinsulinaemic clamp model. STUDY DESIGN: Randomised, controlled (within subject), blinded, experiment. METHODS: Eight clinically normal Standardbred horses underwent laminitis induction using the euglycaemic hyperinsulinaemic clamp model (EHC). At initiation of the EHC, one forelimb was continuously cooled (ICE), with the other maintained at ambient temperature (AMB). Dorsal lamellar sections (proximal, middle, distal) were harvested 48 h after initiation of the EHC and were analysed using histological scoring (0-3) and histomorphometry. Cellular proliferation was quantified by counting epidermal cell nuclei staining positive with an immunohistochemical proliferation marker (TPX2). RESULTS: Severe elongation and disruption of SEL with dermo-epidermal separation (score of 3) was observed in all AMB feet at one or more section locations, but was not observed in any ICE sections. Overall 92% of the AMB sections received the most severe histological score (grade 3) and 8% were grade 2, whereas ICE sections were classified as either grade 1 (50%) or grade 2 (50%). Relative to AMB feet, ICE sections were 98% less likely to exhibit grades 2 or 3 (OR: 0.02, 95% CI 0.001, 0.365; P<0.01). Histomorphometry measurements of total and nonkeratinised primary epidermal lamellar length were significantly increased (P<0.01) in AMB limbs compared with ICE. TPX2 positive cell counts were significantly increased (P<0.01) in AMB limbs compared with ICE. MAIN LIMITATIONS: Continuous digital hypothermia was initiated before recognition of laminitis and therefore the clinical applicability requires further investigation. CONCLUSIONS: Continuous digital hypothermia reduced the severity of laminitis in the EHC model and prevented histological lesions compatible with lamellar structural failure.

Short communication: insulin alters hepatic progesterone catabolic enzymes cytochrome P450 2C and 3A in dairy cows.

High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500 mL of water (n = 10) or propylene glycol (a gluconeogenic substrate; n = 7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0 microg of insulin/kg of BW per h; n = 6 each) or remained as controls (saline infused; n = 7) for 96 h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.

Insulin sensitivity in Belgian horses with polysaccharide storage myopathy.

OBJECTIVE: To determine insulin sensitivity, proportions of muscle fiber types, and activities of glycogenolytic and glycolytic enzymes in Belgians with and without polysaccharide storage myopathy (PSSM). ANIMALS: 10 Quarter Horses (QHs) and 103 Belgians in which PSSM status had been determined. PROCEDURES: To determine insulin sensitivity, a hyperinsulinemic euglycemic clamp (HEC) technique was used in 5 Belgians with PSSM and 5 Belgians without PSSM. Insulin was infused i.v. at 3 mU/min/kg for 3 hours, and concentrations of blood glucose and plasma insulin were determined throughout. An i.v. infusion of glucose was administered to maintain blood glucose concentration at 100 mg/dL. Activities of glycogenolytic and glycolytic enzymes were assessed in snap-frozen biopsy specimens of gluteus medius muscle obtained from 4 Belgians with PSSM and 5 Belgians without PSSM. Percentages of type 1, 2a, and 2b muscle fibers were determined via evaluation of >or= 250 muscle fibers in biopsy specimens obtained from each Belgian used in the aforementioned studies and from 10 QHs (5 with PSSM and 5 without PSSM). RESULTS: Belgians with and without PSSM were not significantly different with respect to whole-body insulin sensitivity, muscle activities of glycogenolytic and glycolytic enzymes, or proportions of muscle fiber types. However, Belgians had an increased proportion of type 2a and decreased proportion of type 2b muscle fibers, compared with proportions in QHs, regardless of PSSM status. CONCLUSIONS AND CLINICAL RELEVANCE: PSSM in Belgians may be attributable to excessive glycogen synthesis rather than decreased glycogen utilization or enhanced glucose uptake into muscle cells.

Hypertriglyceridaemic insulin-resistant obese dog model: effects of high-fat diet depending on age.

In humans, obesity is closely associated with insulin resistance (IR) and dyslipidaemia. The purpose of this study was to explore the effect of age on metabolic disturbances related to obesity in dogs (n = 25). Three age-groups of dogs (puppies, young adults and mature adults) were overfed to induce obesity, and body composition, insulin sensitivity index (I(IS)) (euglycaemic-hyperinsulinaemic glucose clamp) and plasma lipids were measured. Fat mass was similar in the three obese groups (30 +/- 1% in puppies, 34 +/- 1% in young adults and 39 +/- 1% in mature adults). In mature adults, body weight (BW) increased (+45%, p < 0.001) and I(IS) decreased (-60%, p < 0.001) over 22 weeks. In young adults, BW gain was similar but slower (60 weeks) and I(IS) decreased to a lesser extent (-49%, p < 0.001). Overfed puppies weighed 30% more (p < 0.01) than normally-fed control puppies, but there was no change in I(IS). Unlike young and mature adults, obese puppies did not exhibit significant changes in triglycerides (TG) and free fatty acid concentrations. In conclusion, as in humans, obese dogs develop IR that is associated with high TG levels; however, younger animals may be better able to balance energy needs with energy consumption.

Pharmacodynamics and pharmacokinetics of insulin aspart assessed by use of the isoglycemic clamp method in healthy cats.

The objective of this study was to determine the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin aspart in healthy cats following intramuscular (IM) and subcutaneous (SC) injection. Eight healthy, purpose-bred cats were used in a randomized, crossover study design. Each cat had 2 isoglycemic clamps performed, one after receiving 0.25 IU/kg of insulin aspart by IM injection and one after receiving the same dose by SC injection. The two isoglycemic clamps were performed on different days, at least 48 h apart. The blood glucose, plasma endogenous insulin, and plasma insulin aspart concentrations were measured and the glucose infusion rate (GIR) was recorded during the clamp. The GIR over time was used to create a time-action curve for each clamp which was used to describe the PD of insulin aspart. Data that are normally distributed are reported as mean ± SD, while data that are not normally distributed are reported as median (25-75 percentile). When compared to the PD data that have been reported for regular insulin in healthy cats, insulin aspart had a more rapid onset (IM: 10 min [10-21.25 min], SC: 12.5 min [10-18.75 min]) and shorter duration of action (IM: 182.5 ± 34.33 min, SC: 159.38 ± 41.87 min). The onset of action (P = 0.795), time to peak action (P = 0.499), duration of action (P = 0.301), and total metabolic effect (P = 0.603) did not differ with route of administration; however, SC administration did result in a higher maximum plasma insulin aspart concentration (IM: 1,265.17 pmol/L [999.69-1,433.89 pmol/L], SC: 3,278.19 pmol/L [2,485.29-4,132.01 pmol/L], P = 0.000) and larger area under the insulin aspart vs time curve (IM: 82,662 ± 30,565 pmol/L, SC: 135,060 ± 39,026 pmol/L, P = 0.010). Insulin aspart has a rapid onset of action and short duration of effect in healthy cats when administered by IM and SC injection. Although it cannot be assumed that the PD and PK of insulin aspart will be the same in cats with diabetic ketoacidosis (DKA), our data support further investigation into the use of SC insulin aspart as an alternative to regular insulin for the treatment of DKA in cats.

Factors affecting clinical assessment of insulin sensitivity in horses.

Insulin resistance is thought to be involved in the pathogenesis of many equine conditions such as pars intermedia dysfunction, equine metabolic syndrome, diabetes mellitus, hyperlipaemia, laminitis, endotoxaemia and osteochondrosis dissecans (OCD); whereas polysaccharide storage myopathy in Quarter Horses and equine motor neuron disease (EMD) have been associated with increased insulin sensitivity. However, it is clear that there is not one ideal test, in terms of both practicality and accuracy, for evaluating insulin sensitivity in horses and improved diagnostic techniques are required. This review sets out the background to the subject and identifies current knowledge regarding the measurement of insulin sensitivity by tolerance testing and clamping techniques. Factors affecting insulin sensitivity, such as breed, pregnancy, lactation, obesity and nutritional factors are discussed. In addition, the relationship with training, nutritional supplementation and drug administration are considered.

Relationship Between ß-cell Response and Insulin Sensitivity in Horses based on the Oral Sugar Test and the Euglycemic Hyperinsulinemic Clamp.

BACKGROUND: A hyperbolic relationship between ß-cell response and insulin sensitivity (IS) has been described in several species including rodents, dogs, and humans. This relationship has not been elucidated in the horse. HYPOTHESIS/OBJECTIVES: To determine whether the hyperbolic relationship between ß-cell response and IS exists in horses by using indices of ß-cell response from the oral sugar test (OST) and IS measurements from the euglycemic hyperinsulinemic clamp (EHC). A second aim was to compare how well IS estimates from the OST and EHC correlate. ANIMALS: Forty-nine horses with different degrees of insulin regulation (normal-to-severe insulin dysregulation). METHODS: Cross-sectional study. Horses were examined with an OST and an EHC. RESULTS: Decreased IS was associated with increased ß-cell response in the horses. Nine of 12 comparisons between indices of ß-cell response and IS measures fulfilled the criteria for a hyperbolic relationship. Indices of IS calculated from the OST correlated highly with the insulin-dependent glucose disposal rate (M) and the insulin-dependent glucose disposal rate per unit of insulin (M/I) determined from the EHC (r = 0.81-0.87). CONCLUSIONS AND CLINICAL IMPORTANCE: A hyperbolic relationship between ß-cell response and IS exists in horses, which suggest that horses with insulin dysregulation respond not only with postprandial hyperinsulinemia but are also insulin resistant. The OST is primarily a test for ß-cell response rather than a test for IS, but calculated indices of IS from the OST may be useful to estimate IS in horses, especially when the horse is insulin resistant.

Estimating glucose requirements of an activated immune system in growing pigs.

Activated immune cells become obligate glucose utilizers, and a large i.v. lipopolysaccharide (LPS) dose causes insulin resistance and severe hypoglycemia. Therefore, study objectives were to quantify the amount of glucose needed to maintain euglycemia following an endotoxin challenge as a proxy of leukocyte glucose requirements. Fifteen fasted crossbred gilts (30.3 ± 1.7 kg) were bilaterally jugular catheterized and assigned 1 of 2 i.v. bolus treatments: control (CON; 10 mL sterile saline; = 7) or LPS challenge + euglycemic clamp (LPS-Eu; 055:B5; 5 µg/kg BW; 50% dextrose infusion to maintain euglycemia; = 8). Following administration, blood glucose was determined every 10 min and dextrose infusion rates were adjusted in LPS-Eu pigs to maintain euglycemia for 8 h. Pigs were fasted for 8 h prior to the bolus and remained fasted throughout the challenge. Rectal temperature was increased in LPS-Eu pigs relative to CON pigs (39.8 vs. 38.8°C; < 0.01). Relative to the baseline, CON pigs had 20% decreased blood glucose from 300 to 480 min postbolus ( = 0.01) whereas circulating glucose content in LPS-Eu pigs did not differ ( = 0.96) from prebolus levels. A total of 116 ± 8 g of infused glucose was required to maintain euglycemia in LPS-Eu pigs. Relative to CON pigs, overall plasma insulin, blood urea nitrogen, ß-hydroxybutrate, lactate, and LPS-binding protein were increased in LPS-Eu pigs (295, 108, 29, 133, and 13%, respectively; ≤ 0.04) whereas NEFA was decreased (66%; < 0.01). Neutrophils in LPS-Eu pigs were decreased 84% at 120 min postbolus and returned to CON levels by 480 min ( < 0.01). Overall, lymphocytes, monocytes, eosinophils, and basophils were decreased in LPS-Eu pigs relative to CON pigs (75, 87, 70, and 50%, respectively; ≤ 0.05). These alterations in metabolism and the large amount of glucose needed to maintain euglycemia indicate nutrient repartitioning away from growth toward the immune system. Glucose is an important fuel for the immune system, and data from this study established that the glucose requirements of an intensely and acutely activated immune system in growing pigs are approximately 1.1 g/kg BW/h.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves.

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.