H. Albadawi1,3, R. Oklu2,3, J. D. Milner1, T. P. Uong1, H. Yoo1, M. T. Watkins1,3 1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA 2Massachusetts General Hospital,Department Of Imaging, Division Of Interventional Radiology,Boston, MA, USA 3Harvard School Of Medicine,Brookline, MA, USA
Introduction: Diabetes is a major risk factor for peripheral arterial disease, which frequently manifests as intermittent claudication due to lower limb demand ischemia (DI). In skeletal muscle, autophagy and mitochondrial biogenesis are known to mediate oxidative capacity and energy metabolism. In aging and diabetes, defective mitochondrial biogenesis, including mitochondrial fission/fusion events and autophagy, contributes to structural and metabolic derangements in muscle. This study evaluates the effect of exercise and DI on autophagy and mitochondrial biogenesis in a diabetic mouse model of diet induced obesity.
Methods: Two groups of C57BL6 male mice were fed a 60% high fat diet for 26 weeks to induce diabetes. Mice were subjected to unilateral femoral artery ligation (FAL, n=6) or sham surgery (n=6) and were allowed to recover for 14 days for stabilization. Subsequently, the FAL mice underwent 60 minutes of daily treadmill exercise (12m/min speed, 10° incline) for 4 weeks to induce DI in the ligated limb. The exercised contralateral (EX) limb acted as an internal control to assess the effect of exercise on muscle independent of DI. In contrast, sham (SH) mice remained sedentary. Hind limb perfusion was recorded at baseline before surgery and once a week thereafter. Muscle tissues from SH, DI, and EX hind limbs were analyzed by western blotting for markers of autophagy, LC3B type II/I ratio, and markers of mitochondrial biogenesis, which was quantified by the expression ratio of mitochondrial fusion (Opa-1) and fission (Drp-1) proteins. Specific band density ratios were calculated and expressed in arbitrary units (AU). Statistical analysis was performed using ANOVA with post hoc analysis.
Results: After 4 weeks of sedentary or exercise conditions, EX perfusion was significantly higher than in SH or DI (SH: 1249±100; EX: 2032± 307; DI: 998±207 Flux, p<0.01). There was significant enhancement in LC3BII/ I ratio in the EX muscle over SH (p<0.01), while DI exhibited an insignificant increase over SH (SH: 0.68±0.06; EX: 2±0.4; DI: 1.2±0.3 AU). In the DI muscle, we established a significant positive linear regression between limb perfusion and the LC3B II/I ratio (r2=0.81, p=0.014). In contrast, comparison of the Opa-1/Drp-1 ratio failed to yield significance between the three groups (SH: 1.1±0.3; EX: 2±0.7; DI: 1.8±0.5 AU, p=0.332).
Conclusion: Exercise upregulated autophagy in the tissue with normal limb perfusion, but it failed to enhance autophagy in the limb that experienced demand ischemia. Variable levels of autophagy were observed to positively correlate with limb perfusion in demand ischemia. Furthermore, exercise does not appear to significantly alter mitochondrial biogenesis in diabetic muscle regardless of the degree of perfusion. These results may have an implication for the evaluation of diabetic patients, who suffer from claudication.