Publications by authors named "Salvatore P Mangiafico"

10 Publications

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E2f8 and Dlg2 genes have independent effects on impaired insulin secretion associated with hyperglycaemia.

Diabetologia 2020 07 30;63(7):1333-1348. Epub 2020 Apr 30.

Department of Medicine (Austin Health), Austin Hospital, University of Melbourne, Level 7, Lance Townsend Building, Studley Road, Heidelberg, VIC, 3084, Australia.

Aims/hypothesis: Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia.

Methods: Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference.

Results: Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10) and Dlg2 loci (p = 3.83 × 10) on chromosome 7 to be significantly associated with hyperglycaemia susceptibility. Fine mapping the implicated regions using congenic mice demonstrated that these two loci have independent effects on insulin secretion in vivo. Significantly, our results revealed that increased E2F8 and DLG2 gene expression are correlated with enhanced insulin secretory function in human islets. Furthermore, loss-of-function studies in MIN6 cells demonstrated that E2f8 is involved in insulin secretion through an ATP-sensitive K channel-dependent pathway, which leads to a 30% reduction in Abcc8 expression. Similarly, knockdown of Dlg2 gene expression resulted in impaired insulin secretion in response to glucose and non-glucose stimuli.

Conclusions/interpretation: Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases.
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http://dx.doi.org/10.1007/s00125-020-05137-0DOI Listing
July 2020

Insulin regulates POMC neuronal plasticity to control glucose metabolism.

Elife 2018 09 19;7. Epub 2018 Sep 19.

Metabolism, Diabetes and Obesity Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia.

Hypothalamic neurons respond to nutritional cues by altering gene expression and neuronal excitability. The mechanisms that control such adaptive processes remain unclear. Here we define populations of POMC neurons in mice that are activated or inhibited by insulin and thereby repress or inhibit hepatic glucose production (HGP). The proportion of POMC neurons activated by insulin was dependent on the regulation of insulin receptor signaling by the phosphatase TCPTP, which is increased by fasting, degraded after feeding and elevated in diet-induced obesity. TCPTP-deficiency enhanced insulin signaling and the proportion of POMC neurons activated by insulin to repress HGP. Elevated TCPTP in POMC neurons in obesity and/or after fasting repressed insulin signaling, the activation of POMC neurons by insulin and the insulin-induced and POMC-mediated repression of HGP. Our findings define a molecular mechanism for integrating POMC neural responses with feeding to control glucose metabolism.
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http://dx.doi.org/10.7554/eLife.38704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170188PMC
September 2018

Metabolomic analysis of insulin resistance across different mouse strains and diets.

J Biol Chem 2017 11 5;292(47):19135-19145. Epub 2017 Oct 5.

From the Charles Perkins Centre, School of Life and Environmental Sciences, the University of Sydney, Sydney NSW 2006, Australia,

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.
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http://dx.doi.org/10.1074/jbc.M117.818351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702658PMC
November 2017

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

Diabetes Obes Metab 2017 08 6;19(8):1135-1146. Epub 2017 Apr 6.

Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Victoria, Australia.

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

Materials And Methods: Appropriate methods were used to assess insulin sensitivity (euglycaemic-hyperinsulinaemic clamp) and insulin secretion (hyperglycaemic clamp) in insulin-resistant and hyperglycaemic phosphoenolpyruvate carboxykinase (PEPCK) transgenic rats after treatment with the sodium-glucose co-transporter-2 (SGLT2) inhibitor dapagliflozin.

Results: In 14-week-old rats with hyperglycaemia, insulin resistance and glucose intolerance, 6 weeks of dapagliflozin treatment resulted in lower weight gain, plasma glucose and insulin levels, and improved glucose tolerance, associated with enhanced insulin sensitivity (rate of glucose disappearance: 51.6 ± 2.3 vs 110.6 ± 3.9 µmol/min/kg; P < .005) and glucose uptake in muscle (0.9 ± 0.1 vs 1.7 ± 0.3 µmol/min/100 g; P < .05) and fat (0.23 ± 0.04 vs 0.55 ± 0.10 µmol/min/100 g, P < .05). Additionally, adipose tissue GLUT4 protein levels were increased (0.78 ± 0.05 vs 1.20 ± 0.09 arbitrary units; P < .05), adipocyte count was higher (221.4 ± 17.7 vs 302.3 ± 21.7 per mm fat area; P < .05) and adipocyte size was reduced (4631.8 ± 351.5 vs 3397.6 ± 229.4 µm , P < .05). There was no improvement, however, in insulin secretion. To determine whether earlier intervention is necessary, 5-week-old PEPCK transgenic rats were treated with dapagliflozin for 9 weeks and insulin secretion assessed. Dapagliflozin resulted in improved plasma glucose and insulin levels, and lower weight gain but, again, insulin secretion was not improved.

Conclusions: In this transgenic model of low-grade chronic hyperglycaemia, SGLT2 inhibitor treatment resulted in reduced blood glucose and insulin levels and enhanced glucose tolerance, associated with improved muscle and fat insulin resistance but not improved insulin secretory function.
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http://dx.doi.org/10.1111/dom.12923DOI Listing
August 2017

Erratum to: Hepatocyte glutathione peroxidase-1 deficiency improves hepatic glucose metabolism and decreases steatohepatitis in mice.

Diabetologia 2016 12;59(12):2729

Metabolic Disease and Obesity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, 3800, Australia.

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http://dx.doi.org/10.1007/s00125-016-4124-zDOI Listing
December 2016

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

Diabetologia 2016 12 15;59(12):2632-2644. Epub 2016 Sep 15.

Metabolic Disease and Obesity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, 3800, Australia.

Aims/hypothesis: In 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 HO to liver pathophysiology.

Methods: Glutathione peroxidase (GPX) 1 is an antioxidant enzyme that is abundant in the liver and converts HO to water. We generated Gpx1 mice to conditionally delete Gpx1 in hepatocytes (Alb-Cre;Gpx1 ) and characterised mice fed chow, high-fat or choline-deficient amino-acid-defined (CDAA) diets.

Results: Chow-fed Alb-Cre;Gpx1 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;Gpx1 mice, associated with increased HO 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;Gpx1 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;Gpx1 mice fed a CDAA diet that promotes non-alcoholic steatohepatitis exhibited decreased hepatic lymphocytic infiltrates, inflammation and liver fibrosis.

Conclusions/interpretation: Increased hepatocyte-derived HO enhances hepatic insulin signalling, improves glucose control and protects mice from the development of non-alcoholic steatohepatitis.
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http://dx.doi.org/10.1007/s00125-016-4084-3DOI Listing
December 2016

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

Mol Metab 2016 Mar 21;5(3):221-232. Epub 2016 Jan 21.

University of Melbourne, Department of Medicine (Austin Health), Heidelberg, Victoria, 3084, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molmet.2016.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770268PMC
March 2016

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

FASEB J 2015 Apr 30;29(4):1426-34. Epub 2014 Dec 30.

*Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia; University of Melbourne Department of Medicine, Austin Health, Heidelberg, Victoria, Australia; Translational Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; St. Vincent's Institute of Medical Research and University of Melbourne Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria, Australia; Bone Research Program, ANZAC Research Institute, The University of Sydney, Sydney, New South Wales, Australia; Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; **Department of Pathology, University of Melbourne, Parkville, Victoria, Australia; Molecular Oncology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; and University of Melbourne Department of Surgery, St. Vincent's Hospital, Fitzroy, Victoria, Australia

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 "exon-switch" approach to knock in the constitutively active Pik3ca(H1047R) mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3ca(H1047R) 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. Pik3ca(H1047R) 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.
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http://dx.doi.org/10.1096/fj.14-262782DOI Listing
April 2015

Systemic VEGF-A neutralization ameliorates diet-induced metabolic dysfunction.

Diabetes 2014 Aug 2;63(8):2656-67. Epub 2014 Apr 2.

Diabetes and Obesity Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, AustraliaCharles Perkins Centre, School of Molecular Bioscience, The University of Sydney, Sydney, Australia

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.
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http://dx.doi.org/10.2337/db13-1665DOI Listing
August 2014

A primary defect in glucose production alone cannot induce glucose intolerance without defects in insulin secretion.

J Endocrinol 2011 Sep 23;210(3):335-47. Epub 2011 Jun 23.

Department of Medicine, Heidelberg Repatriation Hospital, University of Melbourne, 300 Waterdale Road, Heidelberg Heights, Melbourne, Victoria 3081, Australia.

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.
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http://dx.doi.org/10.1530/JOE-11-0126DOI Listing
September 2011