Mitochondrial structural and functional dynamism in obesity-induced type 2 diabetes; Do exercise and weight loss regimens restore a healthy phenotype?

Abstract

Obesity is a common precursor to type 2 diabetes (T2DM); both reaching pandemic proportions and are associated with cardiovascular complications such as diabetic cardiomyopathy. Diabetic cardiomyopathy is linked with changes to mitochondrial function and dynamics; regulated by fission and fusion. However, the underlying mechanisms remain unknown. This project aimed to characterise cardiac function and investigate mitochondrial fission/fusion mechanisms in a diet-induced obesity (DIO) murine model. Here I employed eight-week-old C57BL/6J male mice, fed with either a 60% high-fat diet (HFD) or chow (10% fat) (n= 6-8) for 12 weeks to study mitochondrial function and dynamics remodelling that could contribute to the development of diabetic cardiomyopathy. A second trial group incorporated exercise (most common non-pharmacological management of obesity/T2DM) training (a daily swimming regimen) and/or diet exchange for chow after 12 weeks HFD feeding for 5 weeks to determine the reversible impact upon cardiac and mitochondrial function. Therefore, this study assessed the hypothesis that obesity-induced diabetes (DIO) alters mitochondrial dynamics in the heart, promoting changes to mitochondrial function and structure. HFD feeding led to weight gain, hyperglycaemia, hyperlipidaemia and insulin resistance in DIO mice, and echocardiography revealed signs of diastolic dysfunction. Histology staining showed evidence of apoptosis but no fibrosis or hypertrophy. Western blotting and RT-qPCR showed increased expression of the fission proteins Drp1 and Fis1 with a decreased in the phosphorylation of Drp1 at Ser637 suggest a shift towards fission. There was an upregulation of mitophagy proteins, PINK1 and Parkin. Employing serial block face scanning electron microscopy (SBF-SEM) I determined that there was a decrease in perinuclear mitochondria surface area and volume but with an increase in its surface area to volume ratio in HFD (DIO) compared to control. In addition, perinuclear mitochondria in control and HFD (DIO) were the smallest in size, and the largest surface area to volume ratio compared to subsarcolemmal and interfibrillar mitochondria. Diet exchange and exercise with diet exchange led to improve insulin sensitivity and lipid profile with restoration of normal fission-fusion axis, but the mice that underwent exercise only remained insulin resistant with alterations to the fission-fusion axis. I identified that HFD (DIO) feeding led to a decrease in sirtuin 5 (Sirt5) and so next investigated whether mechanistically there is a link to increased mitochondrial fission. siRNA-mediated Sirt5 knockdown in H9c2 cardiac myoblast cell line led to a similar alteration in mitochondrial dynamics as seen in HFD (DIO) model (increase in Drp1, decrease in P-Drp1(S637). I further demonstrated that there was no link between changes in mitochondrial dynamics in early-stage diabetic cardiomyopathy and ER stress, Prohibitins (PHB1 and PHB2) and insulin resistance. Finally, proteomic analysis comparing each permutation of exercise and diet showed perturbed inflammatory, cardiac contraction signalling and stress defence network in HFD (DIO) cardiac mitochondria with a shift in the major energy source from fatty acid oxidation to glycolysis. Moreover, PPARA was identified as one of the main inhibitory upstream regulators in HFD (DIO). Here I have identified changes in mitochondrial fission/fusion axis as a feature of diabetic cardiomyopathy and hence represent an early pathogenesis event, thus providing novel insights that can be incorporated into future therapeutic research. In addition, our studies on the reversible effect of exercise and/or diet highlight the role of healthy lifestyle in managing cardiac mitochondrial function. A putative mechanistic link between Sirt5 and mi