Controlled-release mitochondrial protonophore reverses diabetes and steatohepatitis in rats

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a major factor in the pathogenesis of type 2 diabetes (T2D) and nonalcoholic steatohepatitis (NASH). The mitochondrial protonophore 2,4 dinitrophenol (DNP) has beneficial effects on NAFLD, insulin resistance, and obesity in preclinical models but is too toxic for clinical use. We developed a controlled-release oral formulation of DNP, called CRMP (controlled-release mitochondrial protonophore), that produces mild hepatic mitochondrial uncoupling. In rat models, CRMP reduced hypertriglyceridemia, insulin resistance, hepatic steatosis, and diabetes. It also normalized plasma transaminase concentrations, ameliorated liver fibrosis, and improved hepatic protein synthetic function in a methionine/choline-deficient rat model of NASH. Chronic treatment with CRMP was not associated with any systemic toxicity. These data offer proof of concept that mild hepatic mitochondrial uncoupling may be a safe and effective therapy for the related epidemics of metabolic syndrome, T2D, and NASH.

Fig. 1. CRMP improves insulin sensitivity in high-fat–fed rats

(A) Hepatic V_TCA from fat oxidation (solid bars) and glucose oxidation (striped bars) in chow fed rats. (B to D) Fasting plasma glucose, NEFA, and insulin concentrations. (E and F) Plasma glucose and insulin concentrations during an intraperitoneal glucose tolerance test. *P < 0.05, **P < 0.01, ***P < 0.001 by means of Student's t test. (G) Glucose infusion rate during a hyperinsulinemiceuglycemic clamp. (H) Basal (solid bars) and clamped (striped bars) rates of hepatic glucose production. (I to K) Liver, quadriceps, and plasma triglyceride concentrations. (L) Liver VLDL export. In (A) to (L), data are mean ± SEM of n = 6 to 8 rats per group.

Fig. 2. CRMP improves glucose tolerance in diabetic rats

(A) Random plasma glucose concentrations in vehicle-treated (black circles) and CRMP-treated rats (red squares). (B to D) Fasting plasma glucose, triglyceride, and insulin concentrations. (E and F) Glucose and insulin concentrations during an intraperitoneal glucose tolerance test. (G and H) Plasma ALT and AST concentrations. (I) Liver histology (hematoxylin and eosin stain). Scale bars, 100 μm. In (A) to (I), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by means of Student's t test. Data are mean ± SEM of n = 6 to 7 rats per group.

Fig. 3. CRMP ameliorates liver disease in a rat model of NASH

(A) Liver triglyceride content. (B and C) Plasma ALTand AST. (D) Liver inflammatory cytokine concentrations, normalized to total protein. n = 4 rats per group. (E) Liver histology. Scale bars, 100 μm. (F) Fibrosis score. (G) Liver collagen mRNA. (H) Liver smooth muscle actin protein. (I) Hepatic hydroxyproline content. (J and K) Liver caspase 3 and caspase 9 protein. (L) Plasma albumin. Unless otherwise specified, n = 6 to 8 rats per group. Data are mean ± SEM, with comparisons by means of analysis of variance.