Glucodynamics of long-acting basal insulin peglispro compared with insulin glargine at steady state in patients with type 1 diabetes: substudy of a randomized crossover trial.

AIMS: To compare, in an open-label, randomized, crossover phase II substudy, the glucodynamics of insulin glargine and those of basal insulin peglispro (BIL) in patients with type 1 diabetes. METHODS: Patients (n = 23) underwent 24-h euglycaemic clamps after 8 weeks of treatment with glargine or with BIL. Clinically-titrated basal insulin doses (BIL group 16-64 U; glargine group 19-60 U) were administered on the morning of the clamp. RESULTS: At baseline, the patients' mean ± standard deviation (s.d.) body mass index was 26.78 ± 4.20 kg/m2 and glycated haemoglobin was 7.69 ± 0.99%. The mean ± s.d. endpoint dose for the BIL group was 0.42 ± 0.13 U/kg and for the glargine group was 0.42 ± 0.10. The daily mean ± s.d. blood glucose concentration was 7.7 ± 1.2 in the BIL group and 7.9 ± 1.2 mmol/l in the glargine group (p = 0.641). The mean ± s.d. total and nocturnal hypoglycaemia rates/30 days were 2.7 ± 2.3 and 0.5 ± 0.8, respectively, for the BIL group, and 3.0 ± 2.4 and 0.7 ± 1.1, respectively, for the glargine group (p = 0.112 and 0.428). The mean glucose infusion rate (GIR) normalized to insulin unit was lower for BIL than for glargine. One patient in the glargine group and eight patients in the BIL group had minimal (<0.8 g/kg) GIRs over 24 h. The mean ± s.d. total glucose infused over 24 h (GTOT(0-24) ) was 1.22 ± 0.82 g/kg in the BIL group and 1.90 ± 1.01 g/kg in the glargine group (p = 0.002). The mean ± s.d. total glucose infused during hours 0-6 (GTOT(0-6) ) was 0.21 ± 0.22 in the BIL group and 0.41 ± 0.22 g/kg in the glargine group (p < 0.001), while the mean total glucose infused during hours 18-24 (GTOT(18-24) ) in the BIL group was 0.28 ± 0.18 g/kg and in the glargine group was 0.35 ± 0.23 g/kg (p = 0.198). The peak-to-trough ratio was 1.41 for BIL versus 2.22 for glargine. CONCLUSIONS: BIL has a flatter profile than glargine, with potentially more stable metabolic control. The lower GTOT(0-24) observed in the BIL group is consistent with BIL's reduced peripheral action.

Counter-regulatory hormone responses to hypoglycaemia in people with type 1 diabetes after 4 weeks of treatment with liraglutide adjunct to insulin: a randomized, placebo-controlled, double-blind, crossover trial.

AIMS: To investigate the effect of glucagon-like peptide 1 receptor agonist liraglutide on the counter-regulatory hormone response to hypoglycaemia in type 1 diabetes. METHODS: We conducted a randomized, double-blind, placebo-controlled, single-centre trial, in which a total of 45 adults with type 1 diabetes [mean ± standard deviation age 34.5 ± 11.2 years, BMI 23.9 ± 2.4 kg/m(2) , glycated haemoglobin (HbA1c) 7.6 ± 0.8%, diabetes duration 16.6 ± 9.4 years] underwent a hypoglycaemic clamp after 4 weeks' crossover treatment with once-daily liraglutide/placebo added to insulin in one of three liraglutide dose groups: 0.6 mg (n = 15); 1.2 mg (n = 14); and 1.8 mg (n = 16). The main outcome measure was glucagon concentration at nadir plasma glucose (2.5 mmol/l). Clinical outcomes were also evaluated. Five participants were withdrawn from the trial; three because of adverse events. All participants were included in the analysis. RESULTS: Glucagon concentration at nadir plasma glucose was modest, trending towards lower concentrations at increasing liraglutide dose versus placebo: 34.7 versus 38.1 pg/ml, p = 0.555 (0.6 mg); 28.8 versus 37.2 pg/ml, p = 0.126 (1.2 mg); and 28.4 versus 37.5 pg/ml, p = 0.092 (1.8 mg). There was no difference, however, between liraglutide and placebo in incremental change in glucagon during hypoglycaemia. Other counter-regulatory hormone levels increased during hypoglycaemia with no systematic differences between groups. Glucose infusion rates were significantly lower with liraglutide versus placebo during the clamp. After 4 weeks' treatment, HbA1c remained unchanged in the liraglutide and placebo groups. Greater reductions in insulin dose and body weight were seen with liraglutide versus placebo. CONCLUSIONS: Liraglutide did not compromise hypoglycaemic responses in type 1 diabetes after 4 weeks' treatment.

Association between specific adipose tissue CD4+ T-cell populations and insulin resistance in obese individuals.

BACKGROUND & AIMS: An increased number of macrophages in adipose tissue is associated with insulin resistance and metabolic dysfunction in obese people. However, little is known about other immune cells in adipose tissue from obese people, and whether they contribute to insulin resistance. We investigated the characteristics of T cells in adipose tissue from metabolically abnormal insulin-resistant obese (MAO) subjects, metabolically normal insulin-sensitive obese (MNO) subjects, and lean subjects. Insulin sensitivity was determined by using the hyperinsulinemic euglycemic clamp procedure. METHODS: We assessed plasma cytokine concentrations and subcutaneous adipose tissue CD4(+) T-cell populations in 9 lean, 12 MNO, and 13 MAO subjects. Skeletal muscle and liver samples were collected from 19 additional obese patients undergoing bariatric surgery to determine the presence of selected cytokine receptors. RESULTS: Adipose tissue from MAO subjects had 3- to 10-fold increases in numbers of CD4(+) T cells that produce interleukin (IL)-22 and IL-17 (a T-helper [Th] 17 and Th22 phenotype) compared with MNO and lean subjects. MAO subjects also had increased plasma concentrations of IL-22 and IL-6. Receptors for IL-17 and IL-22 were expressed in human liver and skeletal muscle samples. IL-17 and IL-22 inhibited uptake of glucose in skeletal muscle isolated from rats and reduced insulin sensitivity in cultured human hepatocytes. CONCLUSIONS: Adipose tissue from MAO individuals contains increased numbers of Th17 and Th22 cells, which produce cytokines that cause metabolic dysfunction in liver and muscle in vitro. Additional studies are needed to determine whether these alterations in adipose tissue T cells contribute to the pathogenesis of insulin resistance in obese people.

Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Obeticholic acid (OCA; INT-747, 6α-ethyl-chenodeoxycholic acid) is a semisynthetic derivative of the primary human bile acid chenodeoxycholic acid, the natural agonist of the farnesoid X receptor, which is a nuclear hormone receptor that regulates glucose and lipid metabolism. In animal models, OCA decreases insulin resistance and hepatic steatosis. METHODS: We performed a double-blind, placebo-controlled, proof-of-concept study to evaluate the effects of OCA on insulin sensitivity in patients with nonalcoholic fatty liver disease and type 2 diabetes mellitus. Patients were randomly assigned to groups given placebo (n = 23), 25 mg OCA (n = 20), or 50 mg OCA (n = 21) once daily for 6 weeks. A 2-stage hyperinsulinemic-euglycemic insulin clamp was used to measure insulin sensitivity before and after the 6-week treatment period. We also measured levels of liver enzymes, lipid analytes, fibroblast growth factor 19, 7α-hydroxy-4-cholesten-3-one (a BA precursor), endogenous bile acids, and markers of liver fibrosis. RESULTS: When patients were given a low-dose insulin infusion, insulin sensitivity increased by 28.0% from baseline in the group treated with 25 mg OCA (P = .019) and 20.1% from baseline in the group treated with 50 mg OCA (P = .060). Insulin sensitivity increased by 24.5% (P = .011) in combined OCA groups, whereas it decreased by 5.5% in the placebo group. A similar pattern was observed in patients given a high-dose insulin infusion. The OCA groups had significant reductions in levels of γ-glutamyltransferase and alanine aminotransferase and dose-related weight loss. They also had increased serum levels of low-density lipoprotein cholesterol and fibroblast growth factor 19, associated with decreased levels of 7α-hydroxy-4-cholesten-3-one and endogenous bile acids, indicating activation of farnesoid X receptor. Markers of liver fibrosis decreased significantly in the group treated with 25 mg OCA. Adverse experiences were similar among groups. CONCLUSIONS: In this phase 2 trial, administration of 25 or 50 mg OCA for 6 weeks was well tolerated, increased insulin sensitivity, and reduced markers of liver inflammation and fibrosis in patients with type 2 diabetes mellitus and nonalcoholic fatty liver disease. Longer and larger studies are warranted. ClinicalTrials.gov, Number: NCT00501592.

Risk factors for postoperative hyperglycemia in neonates.

OBJECTIVE: Postoperative hyperglycemia has been shown to be associated with higher morbidity and mortality in pediatric patients. Data on risk factors for neonatal patients is limited. The objective of this study was to identify pre- and intraoperative risk factors associated with postoperative glucose in neonates. METHODS: We conducted a retrospective cohort study of neonates after surgical procedures between January and December 2016 in a quaternary neonatal intensive care unit. The primary outcome was hyperglycemia defined as serum glucose ≥8.3 mmol/L during the first 4 hours postoperatively. Secondary outcomes included death and length of stay. We assessed the association of risk factors with the postoperative glucose. RESULTS: In total, 206 surgical procedures (171 patients) were evaluated, among which 178 had serum glucose values during the first 4 hours postoperatively available. The incidence of hyperglycemia was 54% (n = 96). The median (IQR) glucose during the first 4 hours in NICU was 8.4 (6.52-10.65) mmol/L. Risk factors for postoperative hyperglycemia were intraoperative glucose infusion rate (GIR) and gestational age. There was a non-linear relationship between gestational age and postoperative hyperglycemia. Mortality occurred in 6 (7%) in the no-hyperglycemia group and 3 (3%) in the hyperglycemia group (p = 0.31). CONCLUSIONS: Among the risk factors, intraoperative GIR was identified as a modifiable factor that can reduce postoperative hyperglycemia. A non-linear relationship of gestational age with postoperative glucose provides new insights that may help advance our understanding of the complex mechanisms of glucose homeostasis in neonates.

Hypoglycemia attenuates acute amylin-induced reduction of food intake in male rats.

The ability of amylin to inhibit gastric emptying and glucagon secretion in rats is reduced under hypoglycemic conditions. These effects are considered part of a fail-safe mechanism that prevents amylin from further decreasing nutrient supply when blood glucose levels are low. Because these actions and amylin-induced satiation are mediated by the area postrema (AP), it is plausible that these phenomena are based on the co-sensitivity of AP neurons to amylin and glucose. Using hyperinsulinemic glucose clamps in unrestrained and freely-feeding rats, we investigated whether amylin's ability to inhibit food intake is also reduced by hypoglycemia (HYPO). Following an 18 h fast, rats were infused with insulin and glucose for 45 min to clamp blood glucose at baseline levels (between 90 and 100 mg/dL). HYPO (approximately 55 mg/dL) was induced between 45 and 60 min and then maintained for the remainder of the clamp. Rats were injected with amylin (20 µg/kg) or saline and offered normal chow at 85 min. Food intake was measured at 30 and 60 min after amylin. Control hyperinsulinemic/euglycemic (EU) rats were maintained at approximately 150 mg/dL (which is a physiological periprandial glucose level) before and after amylin injection. Terminal experiments tested the effect of amylin to induce the phosphorylation of ERK, a marker of amylin action in the AP, in EU and HYPO conditions. Amylin significantly reduced 30- and 60-min food intake in EU rats, but the effect at 60-min was attenuated in HYPO rats. Interestingly, glucose infusion rate had to be dramatically reduced at meal onset in saline-treated, but not in amylin-treated, EU or HYPO rats; this suggests that meal-related glucose appearance in the blood was inhibited by amylin under both EU and HYPO. Finally, amylin induced a similar pERK response in the AP in EU and HYPO rats. We conclude that amylin's action to decrease eating is blunted in hypoglycemia, and this effect seems to be downstream from amylin-induced pERK in AP neurons. These data allow us to extend the idea of a hypoglycemic brake on amylin's actions to its food intake-reducing effect, but also demonstrate that amylin can buffer meal-induced glucose appearance at EU and HYPO levels.

The glucose infusion rate of parenteral nutrition in the first week of life in preterm infants: an observational study.

BACKGROUND: Most preterm infants require a continuous glucose infusion in the early postnatal period due to the interruption of the transplacental glucose supply after birth to promote better neurodevelopmental outcomes. AIMS: To investigate the glucose infusion rate (GIR) on parenteral nutrition (PN) in the first week of life administered in preterm infants and its effect on neonatal morbidity and mortality. METHODS: This study included 97 infants aged < 37 gestational weeks and weighed < 2500 g at birth. Infants recruited in this study were classified into 3 groups based on the GIR usage in parenteral nutrition as follows: GIR usage of 5- < 7 g/kg/day (Group I), GIR usage of 7-13 g/kg/day (Group II), and GIR usage of > 13-15 g/kg/day (Group III). Univariate and multivariate logistic regression analyzes were carried out to investigate whether the GIR usage in the three groups was associated with selected neonatal morbidities and mortality. Neonatal morbidities analyzed included respiratory distress syndrome (RDS), necrotizing enterocolitis, sepsis, retinopathy of prematurity, pulmonary hypertension, hypoglycemia, and hyperglycemia. RESULT: Of 97 preterm infants included, 51.5% infants had a gestational age of 34- < 37 weeks, and 54.6% infants had a birth weight of 1500- < 2500 g. The multivariate logistic regression analysis showed that the GIR usage of 5- < 7 g/kg/day was an independent variable that significantly increased the risk of hypoglycemia (Adjusted Odds Ratio [AOR] = 4.000, 95% Confidence Interval [CI] = 1.384-11.565, P = 0.010) and reduced the risk of sepsis (AOR = 0.096, 95% CI = 0.012-0.757, P = 0.026). The GIR usage in all three groups did not increase the risk of mortality. For neonatal morbidity analyzed in this study, RDS (AOR = 5.404, 95%CI = 1.421-20.548, P = 0.013) was an independent risk factor of mortality. CONCLUSION: The GIR usage of < 7 g/kg/day in PN in the first week of life administered to preterm infants was an independent variable in increasing hypoglycemia, but in contrast, reducing the risk of sepsis.

Time-action profiles of insulin degludec in healthy dogs and its effects on glycemic control in diabetic dogs.

Insulin degludec (IDeg) is a new insulin formulation that facilitates long-term control of glucose level in humans. In this study, we investigated the effects of IDeg on glycemic control in dogs. Its time-action profiles were monitored in healthy dogs using an artificial pancreas apparatus under euglycemic conditions. At 9.0-13.5 hr post-IDeg injection, an indistinct peak of glucose level was detected. Moreover, the action of IDeg was persistent for >20 hr. Both IDeg and neutral protamine Hagedorn insulin (NPH) lowered blood glucose concentrations in diabetic dogs, but IDeg caused postprandial hyperglycemia and a somewhat lower preprandial glucose level than that caused by NPH. IDeg might be ineffective in concurrently preventing postprandial hyperglycemia and preprandial hypoglycemia in a single-agent administration.

Correlation of maternal A1c with glucose infusion rate requirements in the newborn.

OBJECTIVE: Fetal hyperinsulinemia and neonatal hyperglycemia are complications of poor maternal glycemic control and may result in increased glucose infusion rate (GIR) requirements in infants of diabetic mothers (IDMs). The objectives of this study were to correlate maternal A1c levels with GIR requirements in IDMs, establish an A1c threshold predictive for GIR requirements, and identify associations between A1c levels and complications in IDMs. STUDY DESIGN: A retrospective review of paired maternal A1c values and GIR requirements of IDMs were compared via logistic regression analysis. A likelihood ratio was calculated to correlate A1c levels with GIR requirements, and identify a maternal A1c threshold. RESULTS: Increasing A1c values were significantly correlated with GIR≥5 mg/kg/min (OR, 1.37; 95% CI 1.04-1.79, p = 0.021). Macrosomia was the most frequent complication (OR, 1.31; 95% CI 1.04-1.67, p = 0.022) and A1c > 6.8% was predictive for increased GIR requirements. CONCLUSION: Increased A1c values were significantly associated with GIR requirements≥5 mg/kg/min. Increased maternal A1c is significantly associated with complications in newborns, specifically macrosomia. A maternal A1c of 6.8% was identified as a threshold predictive of increased GIR requirements.

Hypoglycemia: When to Treat?

Hypoglycemia is the most common metabolic disorder encountered in neonates. The definition of hypoglycemia as well as its clinical significance and management remain controversial. Most cases of neonatal hypoglycemia are transient, respond readily to treatment, and are associated with an excellent prognosis. Persistent hypoglycemia is more likely to be associated with abnormal endocrine conditions, such as hyperinsulinemia, as well as possible neurologic sequelae. Manifestations of hypoglycemia include seizures which can result in noteworthy neuromorbidity in the long haul. Thus, hypoglycemia constitutes a neonatal emergency which requires earnest analytic assessment and prompt treatment. In this review, we have tried to cover the pathophysiology, the screening protocol for high-risk babies, management, long-term neurologic sequelae associated with neonatal hypoglycemia, with evidence-based answers wherever possible, and our own practices.

Repetitive hyperbaric oxygen treatment increases insulin sensitivity in diabetes patients with acute intracerebral hemorrhage.

AIM: The role of hyperbaric oxygen therapy (HBOT) in the treatment of acute ischemic stroke is controversial. This study aims to investigate whether the peripheral insulin sensitivity of type 2 diabetes patients suffering from intracerebral hemorrhage can be increased after HBOT. METHODS: Fifty-two type 2 diabetes participants were recruited after being diagnosed with intracerebral hemorrhage in our hospital. Insulin sensitivity was measured by the glucose infusion rate during a hyperinsulinemic euglycemic clamp (80 mU m-2 min-1) at baseline and 10 and 30 days after HBOT sessions. Serum insulin, fasting glucose, and hemoglobin A1C were measured in fasting serum at baseline and after HBOT sessions. In addition, early (∼10 days after onset) and late (1 month after onset) outcomes (National Institutes of Health Stroke Scale, NIHSS scores) and efficacy (changes of NIHSS scores) of HBOT were evaluated. RESULTS: In response to HBOT, the glucose infusion rate was increased by 37.8%±5.76% at 1 month after onset compared with baseline. Reduced serum insulin, fasting glucose, and hemoglobin A1C were observed after HBOT. Both early and late outcomes of the HBOT group were improved compared with baseline (P<0.001). In the control group, there was significant difference only in the late outcome (P<0.05). In the assessment of efficacy, there were statistically significant differences between the groups when comparing changes in NIHSS scores at 10 days and 1 month after onset (P<0.05). CONCLUSION: Peripheral insulin sensitivity was increased following HBOT in type 2 diabetes patients with intracerebral hemorrhage. The HBOT used in this study may be effective for diabetes patients with acute stroke and is a safe and harmless adjunctive treatment.

Hypoglycemia During the Temporary Interruption of Parenteral Nutrition Infusion in Pediatric Hematopoietic Stem Cell Transplantation.

BACKGROUND: Parenteral nutrition (PN) is required with pediatric procedures such as hematopoietic stem cell transplantation (HSCT). However, risks associated with temporary PN infusion interruption remain unclear. MATERIALS AND METHODS: We retrospectively analyzed in 22 children undergoing HSCT receiving PN with the same daily routine: temporary PN infusion interruption before breakfast for administering a saline-diluted acyclovir drip. After correcting patients' glucose levels, we examined minimum blood glucose levels between preparative regimen initiation and post-HSCT day 30. Patients were divided into 2 groups according to a minimum glucose cutoff of 60 mg/dL. Patient background characteristics and hypoglycemia risk factors were compared between both groups. RESULTS: The hypoglycemia group had a significantly lower body surface area, higher glucose infusion rate (GIR), lower cholinesterase levels, and higher zinc levels at the onset of the minimum blood glucose level ( P < .05). Multivariate analyses revealed an association only between higher GIR (≥5 mg/kg/min) and hypoglycemia during the temporary PN infusion interruption. A time course analysis of blood glucose and immunoreactive insulin (IRI) levels in 1 patient revealed a combined high-caloric and saline flush before acyclovir initiation, causing temporary increased IRI, as the etiology for hypoglycemia. CONCLUSIONS: Particular attention and several precautions are required to prevent complications associated with temporary PN infusion interruption in children with higher GIR.

Inhibition of citrate cotransporter Slc13a5/mINDY by RNAi improves hepatic insulin sensitivity and prevents diet-induced non-alcoholic fatty liver disease in mice.

OBJECTIVE: Non-alcoholic fatty liver disease is a world-wide health concern and risk factor for cardio-metabolic diseases. Citrate uptake modifies intracellular hepatic energy metabolism and is controlled by the conserved sodium-dicarboxylate cotransporter solute carrier family 13 member 5 (SLC13A5, mammalian homolog of INDY: mINDY). In Drosophila melanogaster and Caenorhabditis elegans INDY reduction decreased whole-body lipid accumulation. Genetic deletion of Slc13a5 in mice protected from diet-induced adiposity and insulin resistance. We hypothesized that inducible hepatic mINDY inhibition should prevent the development of fatty liver and hepatic insulin resistance. METHODS: Adult C57BL/6J mice were fed a Western diet (60% kcal from fat, 21% kcal from carbohydrate) ad libitum. Knockdown of mINDY was induced by weekly injection of a chemically modified, liver-selective siRNA for 8 weeks. Mice were metabolically characterized and the effect of mINDY suppression on glucose tolerance as well as insulin sensitivity was assessed with an ipGTT and a hyperinsulinemic-euglycemic clamp. Hepatic lipid accumulation was determined by biochemical measurements and histochemistry. RESULTS: Within the 8 week intervention, hepatic mINDY expression was suppressed by a liver-selective siRNA by over 60%. mINDY knockdown improved hepatic insulin sensitivity (i.e. insulin-induced suppression of endogenous glucose production) of C57BL/6J mice in the hyperinsulinemic-euglycemic clamp. Moreover, the siRNA-mediated mINDY inhibition prevented neutral lipid storage and triglyceride accumulation in the liver, while we found no effect on body weight. CONCLUSIONS: We show that inducible mINDY inhibition improved hepatic insulin sensitivity and prevented diet-induced non-alcoholic fatty liver disease in adult C57BL6/J mice. These effects did not depend on changes of body weight or body composition.

Skeletal muscle salt inducible kinase 1 promotes insulin resistance in obesity.

OBJECTIVE: Insulin resistance causes type 2 diabetes mellitus and hyperglycemia due to excessive hepatic glucose production and inadequate peripheral glucose uptake. Our objectives were to test the hypothesis that the proposed CREB/CRTC2 inhibitor salt inducible kinase 1 (SIK1) contributes to whole body glucose homeostasis in vivo by regulating hepatic transcription of gluconeogenic genes and also to identify novel SIK1 actions on glucose metabolism. METHODS: We created conditional (floxed) SIK1-knockout mice and studied glucose metabolism in animals with global, liver, adipose or skeletal muscle Sik1 deletion. We examined cAMP-dependent regulation of SIK1 and the consequences of SIK1 depletion on primary mouse hepatocytes. We probed metabolic phenotypes in tissue-specific SIK1 knockout mice fed high fat diet through hyperinsulinemic-euglycemic clamps and biochemical analysis of insulin signaling. RESULTS: SIK1 knockout mice are viable and largely normoglycemic on chow diet. On high fat diet, global SIK1 knockout animals are strikingly protected from glucose intolerance, with both increased plasma insulin and enhanced peripheral insulin sensitivity. Surprisingly, liver SIK1 is not required for regulation of CRTC2 and gluconeogenesis, despite contributions of SIK1 to hepatocyte CRTC2 and gluconeogenesis regulation ex vivo. Sik1 mRNA accumulates in skeletal muscle of obese high fat diet-fed mice, and knockout of SIK1 in skeletal muscle, but not liver or adipose tissue, improves insulin sensitivity and muscle glucose uptake on high fat diet. CONCLUSIONS: SIK1 is dispensable for glycemic control on chow diet. SIK1 promotes insulin resistance on high fat diet by a cell-autonomous mechanism in skeletal muscle. Our study establishes SIK1 as a promising therapeutic target to improve skeletal muscle insulin sensitivity in obese individuals without deleterious effects on hepatic glucose production.

Enhanced insulin sensitivity in skeletal muscle and liver by physiological overexpression of SIRT6.

OBJECTIVE: Available treatment for obesity and type 2 diabetes mellitus (T2DM) is suboptimal. Thus, identifying novel molecular target(s) exerting protective effects against these metabolic imbalances is of enormous medical significance. Sirt6 loss- and gain-of-function studies have generated confounding data regarding the role of this sirtuin on energy and glucose homeostasis, leaving unclear whether activation or inhibition of SIRT6 may be beneficial for the treatment of obesity and/or T2DM. METHODS: To address these issues, we developed and studied a novel mouse model designed to produce eutopic and physiological overexpression of SIRT6 (Sirt6BAC mice). These mutants and their controls underwent several metabolic analyses. These include whole-blood reverse phase high-performance liquid chromatography assay, glucose and pyruvate tolerance tests, hyperinsulinemic-euglycemic clamp assays, and assessment of basal and insulin-induced level of phosphorylated AKT (p-AKT)/AKT in gastrocnemius muscle. RESULTS: Sirt6BAC mice physiologically overexpress functionally competent SIRT6 protein. While Sirt6BAC mice have normal body weight and adiposity, they are protected from developing high-caloric-diet (HCD)-induced hyperglycemia and glucose intolerance. Also, Sirt6BAC mice display increased circulating level of the polyamine spermidine. The ability of insulin to suppress endogenous glucose production was significantly enhanced in Sirt6BAC mice compared to wild-type controls. Insulin-stimulated glucose uptake was increased in Sirt6BAC mice in both gastrocnemius and soleus muscle, but not in brain, interscapular brown adipose, or epididymal adipose tissue. Insulin-induced p-AKT/AKT ratio was increased in gastrocnemius muscle of Sirt6BAC mice compared to wild-type controls. CONCLUSIONS: Our data indicate that moderate, physiological overexpression of SIRT6 enhances insulin sensitivity in skeletal muscle and liver, engendering protective actions against diet-induced T2DM. Hence, the present study provides support for the anti-T2DM effect of SIRT6 and suggests SIRT6 as a putative molecular target for anti-T2DM treatment.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

Knockout of STAT3 in skeletal muscle does not prevent high-fat diet-induced insulin resistance.

OBJECTIVE: Increased signal transducer and activator of transcription 3 (STAT3) signaling has been implicated in the development of skeletal muscle insulin resistance, though its contribution, in vivo, remains to be fully defined. Therefore, the aim of this study was to determine whether knockout of skeletal muscle STAT3 would prevent high-fat diet (HFD)-induced insulin resistance. METHODS: We used Cre-LoxP methodology to generate mice with muscle-specific knockout (KO) of STAT3 (mKO). Beginning at 10 weeks of age, mKO mice and their wildtype/floxed (WT) littermates either continued consuming a low fat, control diet (CON; 10% of calories from fat) or were switched to a HFD (60% of calories from fat) for 20 days. We measured body composition, energy expenditure, oral glucose tolerance and in vivo insulin action using hyperinsulinemic-euglycemic clamps. We also measured insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake technique. RESULTS: STAT3 protein expression was reduced ∼75-100% in muscle from mKO vs. WT mice. Fat mass and body fat percentage did not differ between WT and mKO mice on CON and were increased equally by HFD. There were also no genotype differences in energy expenditure or whole-body fat oxidation. As determined, in vivo (hyperinsulinemic-euglycemic clamps) and ex vivo (2DOG uptake), skeletal muscle insulin sensitivity did not differ between CON-fed mice, and was impaired similarly by HFD. CONCLUSIONS: These results demonstrate that STAT3 activation does not underlie the development of HFD-induced skeletal muscle insulin resistance.

The AMPK activator R419 improves exercise capacity and skeletal muscle insulin sensitivity in obese mice.

OBJECTIVE: Skeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects. METHODS: Glucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK ß1ß2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation. RESULTS: There was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice. CONCLUSIONS: Treatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity.

Defective insulin secretory response to intravenous glucose in C57Bl/6J compared to C57Bl/6N mice.

OBJECTIVE: The C57Bl/6J (Bl/6J) mouse is the most widely used strain in metabolic research. This strain carries a mutation in nicotinamide nucleotide transhydrogenase (Nnt), a mitochondrial enzyme involved in NADPH production, which has been suggested to lead to glucose intolerance and beta-cell dysfunction. However, recent reports comparing Bl/6J to Bl/6N (carrying the wild-type Nnt allele) under normal diet have led to conflicting results using glucose tolerance tests. Thus, we assessed glucose-stimulated insulin secretion (GSIS), insulin sensitivity, clearance and central glucose-induced insulin secretion in Bl/6J and N mice using gold-standard methodologies. METHODS: GSIS was measured using complementary tests (oral and intravenous glucose tolerance tests) and hyperglycemic clamps. Whole-body insulin sensitivity was assessed using euglycemic-hyperinsulinemic clamps. Neurally-mediated insulin secretion was measured during central hyperglycemia. RESULTS: Bl/6J mice have impaired GSIS compared to Bl/6N when glucose is administered intravenously during both a tolerance test and hyperglycemic clamp, but not in response to oral glucose. First and second phases of GSIS are altered without changes in whole body insulin sensitivity, insulin clearance, beta-cell mass or central response to glucose, thereby demonstrating defective beta-cell function in Bl/6J mice. CONCLUSIONS: The Bl/6J mouse strain displays impaired insulin secretion. These results have important implications for choosing the appropriate test to assess beta-cell function and background strain in genetically modified mouse models.