c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway

Abstract

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.

Keywords: fatty acids; mitochondrial efficiency; nuclear factor E2-related factor-2.

Fig. 1.

CLA isomer differently affects coupling efficiency in the liver mitochondria of chow-fed rats. The liver mitochondrial respiration rates were evaluated in the presence of succinate (A) or palmitoyl-carnitine (B) as substrates. CS activity was measured in liver homogenate and mitochondrial fractions of differently treated rats; the mitochondrial protein mass was then calculated as the ratio between CS activity in the homogenate and the isolated mitochondria (C) and representative immunoblot of cytochrome C levels in liver mitochondria from differently treated rats (C, upper insert). The bands were quantified using densitometric analysis from triplicate experiments, and the values were expressed as average fold increase (± SD) as compared with control (CD). The effect of individual CLA isomers on mitochondria biogenesis or metabolism was evaluated by measuring the mRNA expression PGC-1α (D) or PGC-1β (E), respectively. Basal and palmitate-induced proton leaks were measured in the isolated hepatic mitochondria from C9 (green), C10 (blue), or CD animals (red) (F, G). The effects of CLA supplementation on the intracellular H₂O₂ yield (H) and basal aconitase/total aconitase ratio were reported (I). The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0.05).

Fig. 2.

9,11-CLA improves redox status and the Nrf2-mediated antioxidant and metabolic detoxifying defenses in the livers of CD-fed rats. Nrf2 activation in the livers of CD-fed rats with or without the CLA supplement was examined via measuring the mRNA expression of the phase 2 enzymes (GCL, GST, and NQO1) (A) and of genes involved in lipid metabolism (FGF21, PPARα, and PPARγ) (G–I). Representative immunoblot showing Nrf2 translocation in nuclear extracts (B) and GCL and NQO1 protein expression cytoplasmic extracts of the livers from differently treated rats (C) are shown. The bands were quantified by densitometric analysis and normalized against tubulin or β-actin. The values were expressed as average fold increase as compared with controls (CD). Liver GSH (D), GSSG content (E), and GSH/GSSG ratio (F) measured in differently treated rats. The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0.05).

Fig. 3.

CLA supplement improves liver accumulation and histopathological signs in HFD-fed rats. The total lipid (A), PC (B), and TBARS (C) contents in the livers of differently treated rats are shown. The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0.05). Representative photomicrographs of liver sections from CD, L, and L9 rats (D) stained with Sudan Black (upper panels) or HE (×400) (lower panels).

Fig. 4.

CLA isomers differently affect coupling efficiency in the liver mitochondria of L-fed rats. The liver mitochondrial respiration rates were evaluated in the presence of succinate (A) or palmitoyl-carnitine (B) as substrates. CS activity was measured in the liver homogenate and mitochondrial fractions of differently treated rats; the mitochondrial protein mass was then calculated as the ratio between CS activity in the homogenate and isolated mitochondria (C). The basal and palmitate-induced proton leak from L (black), L9 (green), L10 (blue), or CD animals (red) (D, E) were measured in isolated hepatic mitochondria. Representative immunoblot of cytochrome C and UCP2 levels in liver mitochondria (C and F, insert, respectively) from differently treated rats; the bands were quantified using densitometric analysis from triplicate experiments, and the values were expressed as average fold increase (± SD) as compared with CD (F). The CPT activity (G), intracellular H₂O₂ yield (H), and basal aconitase/total aconitase ratios were reported (I). The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0.05).

Fig. 5.

9,11-CLA improves the redox status and Nrf2-mediated antioxidant detoxifying defenses in the livers of L-fed rats. Nrf2 activation in the livers of CD- or L-fed rats supplemented or not with CLA supplement was examined by measuring the mRNA expression of downstream genes (GCL, GST, and NQO1) (A) and of genes involved in lipid metabolism (FGF21, PPARα, and PPARγ) (G–I). Representative immunoblot showing Nrf2 levels in nucleus (B) and the effects of the different treatments on enzyme GST and NQO1 activity are shown (C). GSH (D), GSSG content (E), and GSH/GSSG ratio (F) were measured in the livers of differently treated rats. The results are expressed as the means ± SD from triplicate analyses from n = 7 animals/group. Differing superscript letters indicate statistically significant differences (P < 0).