Acylated and desacyl ghrelin are associated with hepatic lipogenesis, β-oxidation and autophagy: role in NAFLD amelioration after sleeve gastrectomy in obese rats

Abstract

Bariatric surgery improves non-alcoholic fatty liver disease (NAFLD). Our aim was to investigate the potential role of ghrelin isoforms in the resolution of hepatic steatosis after sleeve gastrectomy, a restrictive bariatric surgery procedure, in diet-induced obese rats. Male Wistar rats (n = 161) were subjected to surgical (sham operation and sleeve gastrectomy) or dietary interventions [fed ad libitum a normal (ND) or a high-fat (HFD) diet or pair-fed]. Obese rats developed hepatosteatosis and showed decreased circulating desacyl ghrelin without changes in acylated ghrelin. Sleeve gastrectomy induced a dramatic decrease of desacyl ghrelin, but increased the acylated/desacyl ghrelin ratio. Moreover, sleeve gastrectomy reduced hepatic triglyceride content and lipogenic enzymes Mogat2 and Dgat1, increased mitochondrial DNA amount and induced AMPK-activated mitochondrial FFA β-oxidation and autophagy to a higher extent than caloric restriction. In primary rat hepatocytes, the incubation with both acylated and desacyl ghrelin (10, 100 and 1,000 pmol/L) significantly increased TG content, triggered AMPK-activated mitochondrial FFA β-oxidation and autophagy. Our data suggest that the decrease in the most abundant isoform, desacyl ghrelin, after sleeve gastrectomy contributes to the reduction of lipogenesis, whereas the increased relative acylated ghrelin levels activate factors involved in mitochondrial FFA β-oxidation and autophagy in obese rats, thereby ameliorating NAFLD.

Figure 1. Effect of sleeve gastrectomy on circulating ghrelin isoforms.

Bar graphs illustrate the impact of obesity and sleeve gastrectomy-induced weight loss on serum desacyl (A,B) and acylated (C,D) ghrelin levels as well as the acylated/desacyl ghrelin ratio (E,F). Differences were analyzed by Student’s t test or two-way ANOVA, where appropriate. *P < 0.05 vs lean control ND; ^bP < 0.05 effect of surgery.

Figure 2. Effect of acylated and desacyl ghrelin on the improvement of hepatic steatosis after sleeve gastrectomy.

Impact of obesity (A) and sleeve gastrectomy (C) on the mRNA expression levels of Pparg, Srebf1, Mogat2 and Dgat1 in liver samples of experimental animals. (B) Immunohistochemical detection of adipophilin in histological sections of rat liver (magnification 200x, scale bar = 100 μm). Effect of acylated (D,E) and desacyl (F,G) ghrelin on key lipogenic factors and intracellular triglycerides in rat hepatocyte cultures. The gene expression in lean rats, in the sham-operated groups fed a ND and in unstimulated hepatocytes was assumed to be 1. *P < 0.05; **P < 0.01 vs lean control ND or unstimulated hepatocytes; ^aP < 0.05 effect of diet; ^bP < 0.05 effect of surgery.

Figure 3. Impact of sleeve gastrectomy on hepatic mitochondrial biogenesis and FFA β-oxidation.

Bar graphs show the effect of obesity (A,B) and sleeve gastrectomy (D,E) on the hepatic gene expression of molecules involved in mitochondrial biogenesis (mtDNA content and Tfam) and FFA β-oxidation (Ppara and Cpt1a). Representative cropped blots and Western-blot analysis show the impact of obesity (C) and sleeve gastrectomy (F) on the phosphorylation/activation of AMPK in Thr172 and the phosphorylation/inactivation of ACC in Ser79 as well as the basal expression of AMPK, ACC, FAS and CPT1A enzymes in liver samples of the experimental groups. The gene and protein expression in lean or in the sham group fed a ND was assumed to be 1. *P < 0.05 vs lean control ND; ^aP < 0.05 effect of diet; ^bP < 0.05 effect of surgery.

Figure 4. Effect of acylated and desacyl ghrelin on mitochondrial biogenesis and FFA β-oxidation.

Bar graphs show the effect of acylated (A) and desacyl (D) ghrelin on the expression of molecules involved in mitochondrial biogenesis (Tfam) and FFA β-oxidation (Ppara and Cpt1a). Representative cropped blots and Western-blot analysis showing impact of acylated (B,C) and desacyl (E,F) ghrelin on P-AMPK/AMPK and P-ACC/ACC ratios as well as the basal expression of AMPK, ACC, FAS and CPT1A enzymes in primary cultures of rat hepatocytes. The gene and protein expression in unstimulated hepatocytes was assumed to be 1. *P < 0.05; **P < 0.01 vs unstimulated hepatocytes.

Figure 5. Impact of acylated and desacyl ghrelin on the activation of autophagy after sleeve gastrectomy.

Effect of obesity and sleeve gastrectomy on the expression of Atg5, Atg7 and Sqstm1 genes (A,D), autophagosome formation as evidenced by the LC3B-II/I ratio (B,E) as well as autophagy inhibition determined by p62 accumulation (C,F) in liver samples of the experimental groups. Representative cropped blots are shown at the top of the figures. Effect of acylated (G,H,I) and desacyl (J,K,L) ghrelin on key autophagy factors in rat hepatocyte cultures. The gene expression in lean rats, in the sham-operated groups fed a normal diet and in unstimulated hepatocytes was assumed to be 1. ^aP < 0.05 effect of diet; ^bP < 0.05 effect of surgery. *P < 0.05; **P < 0.01 vs unstimulated hepatocytes.