PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity

Abstract

Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.

Figure 1

PRAS40 prevents HFD-induced cardiac dysfunction. A  Echocardiographic assessment of Chow-fed control or HFD fed mice for fractional shortening (FS), ejection fraction (EF) and end-diastolic dimension (LVID) and left anterior wall dimension (LVAW). **p < 0.03 versuscontrol HFD, ***p < 0.008 versus control HFD. One-way anova n = 4 for Chow, n = 10 for HFD group. Values are mean ± SEM. B  Heart weight to tibia length ratio (HW/TL) in the indicated groups. (***p < 0.01 vs. control chow; ^###p < 0.01 vs. control HFD). CSA in control and PRAS40 mice. ***p < 0.01 versus control chow; ^###p < 0.05 versus control HFD. n = 4 for Chow, n = 10 for HFD group. One-way anova. Values are mean ± SEM. C  Nppa, Nppb. Col1 and SERCA2a gene expression. *p < 0.01 versus control chow; ^#p < 0.05 versus control HFD. n = 4 for Chow, n = 10 for HFD group 1-way anova. Values are mean ± SEM. D  Masson-trichrome staining. HFD fed mice show distinct perivascular fibrosis. Bar = 150 μm. E  Representative confocal scans for the Flag-tag and actinin (green and blue, top panel) and pRibS6 and actinin (blue and red, bottom panel) in the indicated groups Bar = 30 μm. F  Body weight and white adipose tissue (WAT) weight. n = 4 for Chow, n = 10 for HFD group ***p < 0.01 versus control chow. One-way anova. Values are mean ± SEM. G  Glucose tolerance test and Area under the curve (A.U.C.) in Chow-fed control mice, and AAV-control or AAV-PRAS40 mice fed HFD for 25 weeks. n = 4 for Chow, n = 5 for HFD group ***p < 0.01 versus control chow. One-way anova. Values are mean ± SEM.

Figure 2

Metabolic remodelling induced by HFD is prevented by mTORC1 inhibition. A  Lower glycolytic (hexokinase) activity but increased oxidative (citrate synthase and β-hydroxy-acyl-CoA dehydrogenase) in HFD-fed AAV controls compared to AAV-PRAS40 mouse hearts. ***p < 0.01 versuscontrol chow; ^###p < 0.01 versus control HFD. n = 4 for Chow, n = 5 for HFD group. One-way anova. Values are mean ± SEM. B  TG content in the heart *p < 0.01 versus control chow; ^#p < 0.05 versuscontrol HFD. n = 3 for Chow, n = 3 for HFD group. One-way anova. Values are mean ± SEM. Lipid accumulation evidenced by Oil Red O staining. Bar = 100 μm. C  The expression levels of genes from glucose metabolism and oxidatative phosphorylation were measured by qRT-PCR. *p < 0.05 versus control chow; ^#p < 0.05 versus control HFD. n = 4 for Chow, n = 5 for HFD group. One-way anova. Values are mean ± SEM. D  The expression levels of genes from FA metabolism and lipid and sterol biosynthesis were measured by qRT-PCR. Expression levels of HIF1a and SREBP *p < 0.05 versus control chow; ^#p < 0.05 versus control HFD. n = 4 for Chow, n = 5 for HFD group. One-way anova. Values are mean ± SEM. E  Glucose uptake, visualized by fluorescence imaging of Xenolight RediJect 2-DG 750 in cardiac and skeletal muscle. ***p < 0.01 versus control chow; ^###p < 0.01 versus control HFD. n = 4 for Chow, n = 4 for HFD group. One-way anova. Values are mean ± SEM. F  Immunoblots of proteins involved in insulin signalling. G  IRS-1 gene expression levels in the indicated groups.

Figure 3

Hepatic insulin sensitivity is improved by PRAS40. A  Body weight, white adipose tissue (WAT) weight and baseline glucose levels in Chow-fed control mice, and Ad-control or Ad-PRAS40 mice fed HFD for 10 weeks. ***p < 0.01 versus control chow. n = 4 for Chow, n = 6 for HFD group. One-way anova. Values are mean ± SEM. B  H&E staining of WAT bar = 50 μm. C  Glucose tolerance test and area under the curve (AUC) in Chow-fed control mice, and Ad-control or Ad-PRAS40 mice fed HFD for 10 weeks. *p < 0.01versus control chow; ^#p < 0.01 versus control HFD. n = 4 for Chow, n = 6 for HFD group. One-way anova. Values are mean ± SEM. D  Paraffin-embedded sections from control livers and PRAS40 treated liver stained for FLAG-tag (green) and nuclei (blue). Bar = 10 μm. E  Immunoblots of proteins involved in insulin and mTORC1 signalling. F  Paraffin-embedded sections from control livers and PRAS40 treated liver stained for pRibS6 (red), FLAG (green) and albumin (blue). Bar = 50 μm. G  Serum cholesterol and triglycerides levels in the indicated groups *p < 0.01versus control. n = 4 for Chow, n = 4 for HFD group. One-way anova. Values are mean ± SEM. H  mTORC1 signalling attenuates AKT activation through feedback mechanisms. PRAS40 blocks mTORC1 activation, improves insulin sensitivity, resulting in decreased growth and improved metabolic function.