Hind limb ischemia–reperfusion injury in diet-induced obese mice

Abstract

Background Obesity is a major risk factor for the development of diabetes. Limb ischemia reperfusion injury (IR) is a common clinical problem in diabetics who have compromised lower extremity perfusion. This study compared the histologic, metabolic and functional outcomes following hindlimb IR in diet-induced obese(DIO) and non-diabetic (ND) mice during the acute and the regenerative phases of IR. Methods DIO and ND mice were subjected to 1.5hrs unilateral hindlimb ischemia followed by 1 or 28 days IR. Muscle morphology, metabolic and genomic stress was evaluated at day 1 and 28 IR; Acute inflammation and thrombosis were only measured at day 1 IR. At day 28 IR, skeletal muscle contractility and maturation was also assessed. Results At day 1 IR, similar levels of acute muscle fiber necrosis were seen in both groups. DIO mice demonstrated substantially greater inflammatory, pro-thrombotic, and genomic stress responses which were also associated with a greater reduction in energy substrates and Akt phosphorylation. At 28 days, there was no difference in the peak forces generated in the hindlimbs for the two groups. DIO mice had reduced fatigue resistance compared to ND and larger areas of fat accumulation even though there was no significant difference in muscle fiber maturation. Conclusion DIO mice had an exacerbated acute response to IR with enhanced metabolic deficit, fat accumulation and defective functional recovery during the regenerative phase of IR. These changes in fatigue resistance reflect compromised functional recovery following IR injury and have relevance for the functional recovery of patients with metabolic syndrome and insulin resistance.

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

“…Age-matched male C57BL6 mice were acquired from Jackson Laboratory (Bar Harbor, ME) and were continuously fed with high fat diet consisted of 60% kcal derived from fat for 26 weeks to induce obesity, insulin resistance, and diabetes [21]. Male DIO mice were enrolled in this study since they are more susceptible to developing metabolic syndrome and diabetes compared to females [22–24].…”

“…Western blot analysis was performed as previously described [21]. Briefly, harvested hind limb muscle tissues were solubilized using lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Nonidet P-40, 5 mM Na 3 VO 4 , 20 mM NaF, 10 mM sodium pyrophosphate and a protease inhibitor cocktail.…”

Effect of limb demand ischemia on autophagy and morphology in mice

“…Age-matched C57BL6 male mice were acquired from Jackson Laboratory (Bar Harbor, ME) after being fed a diet consisting of 60% kcal from fat for 26 weeks to induce obesity, insulin resistance, and diabetes 16 . To induce hind limb ischemia, all mice were subjected to unilateral femoral artery ligation surgery (FAL) in one hind limb or sham surgery in the contralateral hind limb and then were allowed to recover for two weeks as previously described 17, 18 .…”

“…100µg soluble protein aliquots isolated from hind limb tissue using RIPA extraction buffer were subjected to Tris-HCl-SDS electrophoresis and electroblotting transfer followed by chemiluminescence detection as previously described 16 . To detect protein markers of autophagy, the membranes were incubated with an IgG against the microtubule-associated protein light chain-3B (LC3B, Cell Signaling, Danvers, MA), and rabbit monoclonal anti-Beclin-1 (Cell Signaling, Danvers, MA).…”

Cell Therapy and Critical Limb Ischemia: Evidence and Window of Opportunity in Obesity

“…Using hind limb ischemia model, Albadawi, et al [58] has shown that diet-induced obesity in mice demonstrated defective functional recovery during the regenerative phase of limb ischemia-reperfusion injury. This finding could be of strong relevance to perfusion of lower extremity in patients with metabolic syndrome and insulin resistance.…”