Effect of limb demand ischemia on autophagy and morphology in mice

Abstract

Background Obesity is a major risk factor for diabetes and peripheral arterial disease (PAD), which frequently leads to lower limb demand ischemia. Skeletal muscle autophagy and mitochondrial biogenesis are important processes for proper oxidative capacity and energy metabolism which are compromised in diabetes. This study compares autophagy, mitochondrial biogenesis, energy metabolism, and morphology in the hind limbs of obese diabetic mice subjected to demand or sedentary ischemia. Materials and Methods Unilateral hind limb demand ischemia was created in a group of diet induced obese mice following femoral artery ligation (FAL) and 4 weeks of daily exercise. A parallel group of mice underwent FAL but remained sedentary for 4 weeks. Hind limbs muscles were analyzed for markers of autophagy, mitochondrial biogenesis, ATP, and muscle tissue morphology. Results At the end of the 4-week exercise period, demand ischemia increased the autophagy mediator Beclin-1, but it did not alter the autophagy indicator LC3B-II/I ratio, or markers of mitochondrial biogenesis, Opa-1/Drp-1. In contrast, exercise significantly increased the level of mitochondrial protein-SDHA and reduced adipocyte accumulation and the percentage of centrally nucleated myofibers in the demand ischemia limb. In addition, demand ischemia resulted in decreased uncoupling protein-3 levels without altering muscle ATP or pS473-Akt levels. Conclusions Limb demand ischemia markedly decreased adipocyte accumulation and enhanced muscle regeneration in obese mice but it did not appear to enhance autophagy, mitochondrial biogenesis, energy metabolism, or insulin sensitivity. Future studies aimed at evaluating novel therapies that enhance autophagy and mitochondrial biogenesis in diabetes with PAD are warranted.

Revascularization and muscle adaptation to limb demand ischemia in diet-induced obese mice

“…This is consistent with the prior work from our laboratory showing that significant adipocyte accumulation in sedentary ischemia that was reduced by exercise [10]. UCP-1 is expressed predominantly by brown adipocytes and has been described to regulate thermogenesis.…”

“…Adversely, demand ischemia reduced myofiber CSA (Figure 2) despite the enhanced proangiogenic response indicating substantial myofiber atrophy. However, the enhanced capillary density in our study was not expected because laser Doppler perfusion imaging in prior work from our laboratory showed that limb perfusion in demand ischemia remained diminished after one month of exercise and was not different from sedentary ischemia [10]. One possible explanation for this apparent disparity may be that the hind limb muscle which had chronic ischemia after femoral artery ligation had a distal compensatory angiogenic response in the ischemia tissue without a global increase in limb perfusion that can be detected with laser Doppler imaging.…”

“…Male DIO mice were enrolled in this study since they are more susceptible to developing metabolic syndrome and diabetes compared to females [22–24]. To induce hind limb ischemia, all mice were subjected to unilateral femoral artery ligation surgery (FAL) and were then allowed to recover for two weeks as previously described [10]. Subsequently, mice were divided into two groups: an exercise induced demand ischemia group or a sedentary ischemia group.…”

“…To simulate PAD in human, our laboratory investigated the effect chronic hind limb ischemia using a murine model of diet-induced obesity (DIO) under sedentary or exercise induced demand ischemia conditions [10]. Using this model, we demonstrated that chronic sedentary ischemia causes diminished energy metabolism associated with increased adipocyte infiltration and increase expression of uncoupling protein-3 (UCP-3) protein in the hind limb muscles indicating mitochondrial uncoupling and defective energy metabolism [10].…”

“…Using this model, we demonstrated that chronic sedentary ischemia causes diminished energy metabolism associated with increased adipocyte infiltration and increase expression of uncoupling protein-3 (UCP-3) protein in the hind limb muscles indicating mitochondrial uncoupling and defective energy metabolism [10]. Subsequent analysis of a parallel group of mice which were subjected to exercise induced limb demand ischemia showed reduced adipocyte infiltration and decreased levels of mitochondrial uncoupling protein, UCP-3, without an improvement in metabolic substrate levels indicating persistent metabolic deficit.…”