A controlled-release mitochondrial protonophore reverses hypertriglyceridemia, nonalcoholic steatohepatitis, and diabetes in lipodystrophic mice

Abstract

Lipodystrophy is a rare disorder characterized by complete or partial loss of adipose tissue. Patients with lipodystrophy exhibit hypertriglyceridemia, severe insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). Efforts to ameliorate NASH in lipodystrophies with pharmacologic agents have met with limited success. We examined whether a controlled-release mitochondrial protonophore (CRMP) that produces mild liver-targeted mitochondrial uncoupling could decrease hypertriglyceridemia and reverse NASH and diabetes in a mouse model (fatless AZIP/F-1 mice) of severe lipodystrophy and diabetes. After 4 wk of oral CRMP (2 mg/kg body weight per day) or vehicle treatment, mice underwent hyperinsulinemic-euglycemic clamps combined with radiolabeled glucose to assess liver and muscle insulin responsiveness and tissue lipid measurements. CRMP treatment reversed hypertriglyceridemia and insulin resistance in liver and skeletal muscle. Reversal of insulin resistance could be attributed to reductions in diacylglycerol content and reduced PKC-ε and PKC-θ activity in liver and muscle respectively. CRMP treatment also reversed NASH as reflected by reductions in plasma aspartate aminotransferase and alanine aminotransferase concentrations; hepatic steatosis; and hepatic expression of IL-1α, -β, -2, -4, -6, -10, -12, CD69, and caspase 3 and attenuated activation of the IRE-1α branch of the unfolded protein response. Taken together, these results provide proof of concept for the development of liver-targeted mitochondrial uncoupling agents as a potential novel therapy for lipodystrophy-associated hypertriglyceridemia, NASH and diabetes.-Abulizi, A., Perry, R. J., Camporez, J. P. G., Jurczak, M. J., Petersen, K. F., Aspichueta, P., Shulman, G. I. A controlled-release mitochondrial protonophore reverses hypertriglyceridemia, nonalcoholic steatohepatitis, and diabetes in lipodystrophic mice.

Keywords: NAFLD; NASH; insulin resistance; mitochondrial uncoupling; type 2 diabetes.

Figure 1.

Daily CRMP treatment did not alter whole-body energy balance in mice. A) Body weight. B) Respiratory exchange ratio (RER). C) Energy expenditure throughout the day. D) Food intake. Data are means ± sem (n = 6 per group). Statistical significance was determined by Student’s t test. NS, not significant.

Figure 2.

CRMP improved glucose tolerance and whole-body insulin sensitivity. A, B) Plasma glucose concentrations (A) and area under the curve (AUC) (B) during intraperitoneal glucose tolerance test. C, D) Plasma insulin concentrations (C) and AUC (D) during intraperitoneal glucose tolerance test. Mice were unfed for 6 h before the glucose tolerance test and euglycemic clamp study. Data are means ± sem (n = 7–8 per group). *P < 0.05, **P < 0.01 by 2-tailed, unpaired Student’s t test.

Figure 3.

CRMP improved whole-body insulin sensitivity. A, B) Glucose infusion rate (GIR) (A) and plasma glucose concentrations (B) during the hyperinsulinemic clamp. C) Glucose clearance. D) Insulin-stimulated glucose uptake in quadriceps. E) Hepatic glucose production. Food was withheld from mice for 6 h before the glucose tolerance test and hyperinsulinemic clamp study. Data are represented as means ± sem. Statistical significance determined by Student’s t test (n = 7–8 per group). EGP, endogenous glucose production.

Figure 4.

CRMP treatment reduced hepatic triglyceride and DAG content but did not alter hepatic ceramide content. A) Liver triglyceride (TAG) content and liver histology. Scale bars, 100 μm. B) Plasma TAG content. C, D) Liver cytosol (C) and membrane (D) DAG content. E) Liver ceramide content. F) PKC-ε translocation in liver. Data are represented as means ± sem (n = 8 per group). Statistical significance determined by Student’s t test.

Figure 5.

CRMP treatment reduced muscle triglyceride and DAG content but did not alter muscle ceramide content. A) Quadriceps TAG content. B, C) Quadriceps cytosol (B) and membrane (C) DAG content. D) Quadriceps ceramide content. E) PKC-θ translocation in muscle. Data are represented as means ± sem (n = 8 per group). Statistical significance determined by Student’s t test.

Figure 6.

CRMP treatment ameliorates liver inflammation and markers of apoptosis. A–C) Plasma aspartate aminotransferase (AST) (A), alanine aminotransferase (ALT) (B), and alkaline phosphatase (ALP) (C) concentrations. D–F) Hepatic protein expression of several proinflammatory and anti-inflammatory markers (D), CD69 (E), and caspase 3 (F). Data are means ± sem (n = 6–8 per group). Statistical significance was determined by Student’s t test. **P < 0.01.

Figure 7.

CRMP ameliorates activation of the IRE1-α branch of the unfolded protein response. A) Phosphorylation of IRE1-α. B) Phosphorylation of eIF2-α. C) GRP78 and GRP94 levels. Data are means ± sem (n = 6 per group). Statistical significance determined by Student’s t test. NS, not significant.