Limited Fatty Acid Oxidation (FAO) in CPT2 Knockout Myocytes Associates with Insulin Resistance and Cell Stress: possible role of acylcarnitine lipotoxicity

Abstract

Lipotoxicity (the accumulation of fat‐derived metabolites that activate cell stress pathways) has been implicated in muscle insulin resistance. We have proposed that lipotoxicity, and in particular accumulation of long‐chain acylcarnitines (LCACs), also contributes to the episodic myopathy and rhabdomyolysis that is evident in persons with inherited disorders of long‐chain FAO. Many studies in the arena of LCAC bioactivity have employed exogenous application of LCACs to cells; few studies have modified endogenously‐produced LCACs to determine myocyte outcomes. Thus, conclusions about the (patho)physiological relevance of LCAC‐associated lipotoxicity have been difficult to determine. Herein, we used CRISPR‐Cas9 technology to knock out carnitine palmitoyltransferase‐2 (CPT2) in murine C2C12 myotubes: as expected, this maneuver led to almost complete inhibition of FAO, plus significant cellular and media accumulation of palmitoylcarnitine in palmitate‐treated cells. In CPT2 KO myotubes, treatment with 500 μM palmitate led to ~2‐fold higher production of IL‐6 (a myocyte cell stress marker) and a ~1.5 to 2‐fold increased release of adenylate kinase (cell death and permeability marker) at 3 and 6 hr post‐treatment (when compared to palmitate‐treated wildtype control cells; n=4/treatment). Furthermore, CPT2 KO myotubes displayed higher sensitivity to palmitate‐induced insulin resistance: the % inhibition in terms of insulin‐induced p‐Akt abundance was 60%, 80%, and 95% in CPT2 KO cells (at 200, 500, and 750 μM palmitate, respectively), vs. 38%, 53%, and 67% in controls (p<0.01, n=8/treatment). The results indicate that limitation of long‐chain FAO by loss of CPT2 in muscle cells elicits cell stress and insulin resistance, consistent with a working model in which lipotoxicity and accumulation of LCAC trigger these outcomes. Further studies are needed to determine if endogenously‐produced LCACs contribute to the phenotypes observed in CPT2 KO myotubes, or if LCAC‐independent mechanisms are involved. It remains to be established whether these phenomena underlie the cardiac and skeletal muscle myopathies typical of inherited disorders of FAO, or contribute to the insulin resistance accompanying type 2 diabetes.Support or Funding InformationThis work was supported by USDA‐ARS Project 6026‐51000‐010‐05S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.