Salvatore P. Mangiafico

A PRIMARY DEFECT IN GLUCOSE PRODUCTION ALONE CANNOT INDUCE GLUCOSE INTOLERANCE WITHOUT DEFECTS IN INSULIN SECRETION

Increased glucose production is associated with fasting hyperglycaemia in type 2 diabetes but whether or not it causes glucose intolerance is unclear. This study sought to determine whether a primary defect in gluconeogenesis (GNG) resulting in elevated glucose production is sufficient to induce glucose intolerance in the absence of insulin resistance and impaired insulin secretion. Progression of glucose intolerance was assessed in phosphoenolpyruvate carboxykinase (PEPCK) transgenic rats, a genetic model with a primary increase in GNG. Young (4-5 weeks of age) and adult (12-14 weeks of age) PEPCK transgenic and Piebald Virol Glaxo (PVG/c) control rats were studied. GNG, insulin sensitivity, insulin secretion and glucose tolerance were assessed by intraperitoneal and intravascular substrate tolerance tests and hyperinsulinaemic/euglycaemic clamps. Despite elevated GNG and increased glucose appearance, PEPCK transgenic rats displayed normal glucose tolerance due to adequate glucose disposal and robust glucose-mediated insulin secretion. Glucose intolerance only became apparent in the PEPCK transgenic rats following the development of insulin resistance (both hepatic and peripheral) and defective glucose-mediated insulin secretion. Taken together, a single genetic defect in GNG leading to increased glucose production does not adversely affect glucose tolerance. Insulin resistance and impaired glucose-mediated insulin secretion are required to precipitate glucose intolerance in a setting of chronic glucose oversupply.

Impaired glucose metabolism and exercise capacity with muscle-specific glycogen synthase 1 (gys1) deletion in adult mice

Objective Muscle glucose storage and muscle glycogen synthase (gys1) defects have been associated with insulin resistance. As there are multiple mechanisms for insulin resistance, the specific role of glucose storage defects is not clear. The aim of this study was to examine the effects of muscle-specific gys1 deletion on glucose metabolism and exercise capacity. Methods Tamoxifen inducible and muscle specific gys-1 KO mice were generated using the Cre/loxP system. Mice were subjected to glucose tolerance tests, euglycemic/hyperinsulinemic clamps and exercise tests. Results gys1-KO mice showed ≥85% reduction in muscle gys1 mRNA and protein concentrations, 70% reduction in muscle glycogen levels, postprandial hyperglycaemia and hyperinsulinaemia and impaired glucose tolerance. Under insulin-stimulated conditions, gys1-KO mice displayed reduced glucose turnover and muscle glucose uptake, indicative of peripheral insulin resistance, as well as increased plasma and muscle lactate levels and reductions in muscle hexokinase II levels. gys1-KO mice also exhibited markedly reduced exercise and endurance capacity. Conclusions Thus, muscle-specific gys1 deletion in adult mice results in glucose intolerance due to insulin resistance and reduced muscle glucose uptake as well as impaired exercise and endurance capacity. In brief This study demonstrates why the body prioritises muscle glycogen storage over liver glycogen storage despite the critical role of the liver in supplying glucose to the brain in the fasting state and shows that glycogen deficiency results in impaired glucose metabolism and reduced exercise capacity.

Systemic VEGF-A Neutralization Ameliorates Diet-Induced Metabolic Dysfunction

The vascular endothelial growth factor (VEGF) family of cytokines are important regulators of angiogenesis that have emerged as important targets for the treatment of obesity. While serum VEGF levels rise during obesity, recent studies using genetic models provide conflicting evidence as to whether VEGF prevents or accelerates metabolic dysfunction during obesity. In the current study, we sought to identify the effects of VEGF-A neutralization on parameters of glucose metabolism and insulin action in a dietary mouse model of obesity. Within only 72 h of administration of the VEGF-A–neutralizing monoclonal antibody B.20-4.1, we observed almost complete reversal of high-fat diet–induced insulin resistance principally due to improved insulin sensitivity in the liver and in adipose tissue. These effects were independent of changes in whole-body adiposity or insulin signaling. These findings show an important and unexpected role for VEGF in liver insulin resistance, opening up a potentially novel therapeutic avenue for obesity-related metabolic disease.

Hepatocyte glutathione peroxidase-1 deficiency improves hepatic glucose metabolism and decreases steatohepatitis in mice

Aims/hypothesisIn obesity oxidative stress is thought to contribute to the development of insulin resistance, non-alcoholic fatty liver disease and the progression to non-alcoholic steatohepatitis. Our aim was to examine the precise contributions of hepatocyte-derived H2O2 to liver pathophysiology.MethodsGlutathione peroxidase (GPX) 1 is an antioxidant enzyme that is abundant in the liver and converts H2O2 to water. We generated Gpx1lox/lox mice to conditionally delete Gpx1 in hepatocytes (Alb-Cre;Gpx1lox/lox) and characterised mice fed chow, high-fat or choline-deficient amino-acid-defined (CDAA) diets.ResultsChow-fed Alb-Cre;Gpx1lox/lox mice did not exhibit any alterations in body composition or energy expenditure, but had improved insulin sensitivity and reduced fasting blood glucose. This was accompanied by decreased gluconeogenic and increased glycolytic gene expression as well as increased hepatic glycogen. Hepatic insulin receptor Y1163/Y1163 phosphorylation and Akt Ser-473 phosphorylation were increased in fasted chow-fed Alb-Cre;Gpx1lox/lox mice, associated with increased H2O2 production and insulin signalling in isolated hepatocytes. The enhanced insulin signalling was accompanied by the increased oxidation of hepatic protein tyrosine phosphatases previously implicated in the attenuation of insulin signalling. High-fat-fed Alb-Cre;Gpx1lox/lox mice did not exhibit alterations in weight gain or hepatosteatosis, but exhibited decreased hepatic inflammation, decreased gluconeogenic gene expression and increased insulin signalling in the liver. Alb-Cre;Gpx1lox/lox mice fed a CDAA diet that promotes non-alcoholic steatohepatitis exhibited decreased hepatic lymphocytic infiltrates, inflammation and liver fibrosis.Conclusions/interpretationIncreased hepatocyte-derived H2O2 enhances hepatic insulin signalling, improves glucose control and protects mice from the development of non-alcoholic steatohepatitis.

Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible “exonswitch” approach to knock in the constitutively active Pik3caH1047R mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3caH1047R mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild‐type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3caH1047R mutant mice died earlier (median survival 46.5 d post‐mutation induction) than wild‐type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.—Kinross, K. M., Montgomery, K. G., Mangiafico, S. P., Hare, L. M., Kleinschmidt, M., Bywater, M. J., Poulton, I. J., Vrahnas, C., Henneicke, H., Malaterre, J., Waring, P. M., Cullinane, C., Sims, N. A., McArthur, G. A., Andrikopoulos, S., Phillips, W. A. Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly. FASEB J. 29, 1426‐1434 (2015). www.fasebj.org

Metabolomic analysis of insulin resistance across different mouse strains and diets

Insulin resistance is a major risk factor for many diseases. However, its underlying mechanism remains unclear in part because it is triggered by a complex relationship between multiple factors, including genes and the environment. Here, we used metabolomics combined with computational methods to identify factors that classified insulin resistance across individual mice derived from three different mouse strains fed two different diets. Three inbred ILSXISS strains were fed high-fat or chow diets and subjected to metabolic phenotyping and metabolomics analysis of skeletal muscle. There was significant metabolic heterogeneity between strains, diets, and individual animals. Distinct metabolites were changed with insulin resistance, diet, and between strains. Computational analysis revealed 113 metabolites that were correlated with metabolic phenotypes. Using these 113 metabolites, combined with machine learning to segregate mice based on insulin sensitivity, we identified C22:1-CoA, C2-carnitine, and C16-ceramide as the best classifiers. Strikingly, when these three metabolites were combined into one signature, they classified mice based on insulin sensitivity more accurately than each metabolite on its own or other published metabolic signatures. Furthermore, C22:1-CoA was 2.3-fold higher in insulin-resistant mice and correlated significantly with insulin resistance. We have identified a metabolomic signature composed of three functionally unrelated metabolites that accurately predicts whole-body insulin sensitivity across three mouse strains. These data indicate the power of simultaneous analysis of individual, genetic, and environmental variance in mice for identifying novel factors that accurately predict metabolic phenotypes like whole-body insulin sensitivity.

Ferroportin expression in adipocytes does not contribute to iron homeostasis or metabolic responses to a high calorie diet

Background & Aims Iron has an increasingly recognized role in the regulation of adipose tissue function, including the expression of adipokines involved in the pathogenesis of nonalcoholic fatty liver disease. The cellular iron exporter, ferroportin, has been proposed as being a key determinant of adipocyte iron homeostasis. Methods We studied an adipocyte-specific ferroportin (Fpn1) knockout mouse model, using an Adipoq-Cre recombinase driven Fpn1 deletion and fed mice according to the fast food diet model of nonalcoholic steatohepatitis. Results We showed successful selective deletion of Fpn1 in adipocytes, but found that this did not lead to increased adipocyte iron stores as measured by atomic absorption spectroscopy or histologically quantified iron granules after staining with 3,3’-diaminobenzidine–enhanced Perls’ stain. Mice with adipocyte-specific Fpn1 deletion did not show dysregulation of adiponectin, leptin, resistin, or retinol-binding protein-4 expression. Similarly, adipocyte-specific Fpn1 deletion did not affect insulin sensitivity during hyperinsulinemic–euglycemic clamp studies or lead to histologic evidence of increased liver injury. We have shown, however, that the fast food diet model of nonalcoholic steatohepatitis generates an increase in adipose tissue macrophage infiltration with crown-like structures, as seen in human beings, further validating the utility of this model. Conclusions Ferroportin may not be a key determinant of adipocyte iron homeostasis in this knockout model. Further studies are needed to determine the mechanisms of iron metabolism in adipocytes and adipose tissue.

Dapagliflozin improves insulin resistance and glucose intolerance in a novel transgenic rat model of chronic glucose overproduction and glucose toxicity

To determine whether the excretion of glucose improves insulin resistance, impaired insulin secretion or both.

Erratum to: Hepatocyte glutathione peroxidase-1 deficiency improves hepatic glucose metabolism and decreases steatohepatitis in mice (Diabetologia, 10.1007/s00125-016-4084-3)

Unfortunately, ‘pyruvate dehydrogenase kinase 2’ was incorrectly written as ‘pyruvate kinase 2’ in the penultimate paragraph of this paper. The correct sentence reads: ‘We speculate that the oxidation of such PTPs would promote insulin signalling to regulate hepatic glucose metabolism, although we cannot exclude the contribution of other pathways given the growing number of metabolic proteins that can be oxidised, including pyruvate kinase M2 [49] and pyruvate dehydrogenase kinase 2 [50].’