Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model

Abstract

Background & aims: In this study, we sought to determine the temporal relationship between hepatic mitochondrial dysfunction, hepatic steatosis and insulin resistance, and to examine their potential role in the natural progression of non-alcoholic fatty liver disease (NAFLD) utilising a sedentary, hyperphagic, obese, Otsuka Long-Evans Tokushima Fatty (OLETF) rat model.

Methods: OLETF rats and their non-hyperphagic control Long-Evans Tokushima Otsuka (LETO) rats were sacrificed at 5, 8, 13, 20, and 40 weeks of age (n=6-8 per group).

Results: At 5 weeks of age, serum insulin and glucose and hepatic triglyceride (TG) concentrations did not differ between animal groups; however, OLETF animals displayed significant (p<0.01) hepatic mitochondrial dysfunction as measured by reduced hepatic carnitine palmitoyl-CoA transferase-1 activity, fatty acid oxidation, and cytochrome c protein content compared with LETO rats. Hepatic TG levels were significantly elevated by 8 weeks of age, and insulin resistance developed by 13 weeks in the OLETF rats. NAFLD progressively worsened to include hepatocyte ballooning, perivenular fibrosis, 2.5-fold increase in serum ALT, hepatic mitochondrial ultrastructural abnormalities, and increased hepatic oxidative stress in the OLETF animals at later ages. Measures of hepatic mitochondrial content and function including beta-hydroxyacyl-CoA dehydrogenase activity, citrate synthase activity, and immunofluorescence staining for mitochondrial carbamoyl phosphate synthetase-1, progressively worsened and were significantly reduced at 40 weeks in OLETF rats compared to LETO animals.

Conclusions: Our study documents that hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance in the OLETF rats. This evidence suggests that progressive mitochondrial dysfunction contributes to the natural history of obesity-associated NAFLD.

Fig. 1. Serum glucose (A), serum insulin (B), and HbA1c (C) levels at 5, 8, 13, 20, and 40 weeks of age

Values (means ± SE, n = 6–8) with different letter superscripts within each animal group are significantly different (p <0.01). *Significantly different from LETO at respective ages (p <0.01).

Fig. 2. Representative images of H&E (A), Oil-Red O (B), and picrosirius red staining, TGF-β staining, and inflammatory cell infiltration (C)

Note the large lipid vacuoles (A) and macro- and micro-vesicular steatosis (B) and the progression from 8 to 40 weeks in the liver of the OLETF rats. Staining for fibrosis, TGF-β, and inflammatory cell infiltration did not differ between OLETF and LETO animals at 5, 8, 13, or 20 weeks of age (data not shown); however by 40 weeks, there was significantly greater staining for picrosirius red and TGF-β (brown staining) and inflammatory cells (black arrows) in the OLETF animals compared with LETO controls (C). Quantification of hepatic TG, serum ALT, and hepatic picrosirius red staining (40 weeks only) are shown in (D). Values (means ± SE, n = 5–8) with different letter superscripts within each animal group are significantly different (p <0.01). No significant differences existed within the LETO groups for any measured parameter (p >0.05). *Significantly different from LETO at respective ages (p <0.01).

Fig. 3. Hepatic mitochondrial CPT-1 activity (A), β-HAD activity (B), and citrate synthase activity (C)

Values (means ± SE, n = 5–8) with different letter superscripts within each animal group are significantly different (p <0.05). *Significantly different from LETO at respective ages (p <0.05).

Fig. 4. Complete hepatic palmitate oxidation (CO₂ production; A), total hepatic palmitate oxidation (CO₂ + ASMs; B), and total cytochrome c protein content (C)

Values are means ± SE, n = 6–8. *Significantly different from LETO at respective ages (p <0.01). ^†Significantly reduced in the 13, 20, and 40 week old compared with 5 and 8 week old animals (p <0.05).

Fig. 5. Representative immunofluorescent photomicrographs from liver for mitochondrial marker CPS-1 (red) shows granular staining patterns in liver sections (A)

While there were no differences between animal groups at 5, 8, 13, and 20 weeks of age (not shown), the OLETF livers (left) had visibly decreased CPS-1 staining compared to the LETO animals (right) at 40 weeks of age. Also shown are representative electron micrographs (EM) from liver of OLETF (left) and LETO (right) rats at low (B) and high (C) magnifications. While no apparent differences were observed at 5, 8, or 13 weeks of age (data not shown), the mitochondria in the OLETF animals at 20 and 40 weeks show signs of rounding (B) and disruptions in cristae and outer and inner mitochondrial membranes (C). LD = lipid droplet.

Fig. 6. Hepatic UCP-2 protein content in LETO and OLETF rats

Values are means ± SE, n = 6–8. ^†Significantly greater in 8 week old compared with 5 week old animals (p < 0.05). ^‡Significantly greater in the 13, 20, and 40 week old compared with 5 and 8 week old animals (p <0.001). *Significantly greater in OLETF compared with LETO animals (p <0.05).