Exploring the molecular mechanisms underlying the potentiation of exogenous growth hormone on alcohol-induced fatty liver diseases in mice

Abstract

Background: Growth hormone (GH) is an essential regulator of intrahepatic lipid metabolism by activating multiple complex hepatic signaling cascades. Here, we examined whether chronic exogenous GH administration (via gene therapy) could ameliorate liver steatosis in animal models of alcoholic fatty liver disease (AFLD) and explored the underlying molecular mechanisms.

Methods: Male C57BL/6J mice were fed either an alcohol or a control liquid diet with or without GH therapy for 6 weeks. Biochemical parameters, liver histology, oxidative stress markers, and serum high molecular weight (HMW) adiponectin were measured. Quantitative real-time PCR and western blotting were also conducted to determine the underlying molecular mechanism.

Results: Serum HMW adiponectin levels were significantly higher in the GH1-treated control group than in the control group (3.98 ± 0.71 μg/mL vs. 3.07 ± 0.55 μg/mL; P < 0.001). GH1 therapy reversed HMW adiponectin levels to the normal levels in the alcohol-fed group. Alcohol feeding significantly reduced hepatic adipoR2 mRNA expression compared with that in the control group (0.71 ± 0.17 vs. 1.03 ± 0.19; P < 0.001), which was reversed by GH therapy. GH1 therapy also significantly increased the relative mRNA (1.98 ± 0.15 vs. 0.98 ± 0.15) and protein levels of SIRT1 (2.18 ± 0.37 vs. 0.99 ± 0.17) in the alcohol-fed group compared with those in the control group (both, P < 0.001). The alcohol diet decreased the phosphorylated and total protein levels of hepatic AMP-activated kinase-α (AMPKα) (phosphorylated protein: 0.40 ± 0.14 vs. 1.00 ± 0.12; total protein: 0.32 ± 0.12 vs. 1.00 ± 0.14; both, P < 0.001) and peroxisome proliferator activated receptor-α (PPARα) (phosphorylated protein: 0.30 ± 0.09 vs. 1.00 ± 0.09; total protein: 0.27 ± 0.10 vs. 1.00 ± 0.13; both, P < 0.001), which were restored by GH1 therapy. GH therapy also decreased the severity of fatty liver in alcohol-fed mice.

Conclusions: GH therapy had positive effects on AFLD and may offer a promising approach to prevent or treat AFLD. These beneficial effects of GH on AFLD were achieved through the activation of the hepatic adiponectin-SIRT1-AMPK and PPARα-AMPK signaling systems.

Figure 1

Survival rates. The survival rate was 100% at baseline and decreased to 24.07 ± 3.21% in the alcohol-fed group and 66.96 ± 5.56% in the GH1-treated alcohol-fed group after 6 weeks of treatment. The survival rate was maintained at 100% in the other groups. n = 18 mice per group for the alcohol and GH1-treated alcohol groups; n = 6 mice per group for the other groups.

Figure 2

Food intake and body composition. (A) Daily food intake. (B) Body weight. (C) Hepatic index. (D) Visceral fat percentage. HI: hepatic index. VF%: visceral fat percentage. Error bars represent standard deviations. n = 6 mice per group. *P < 0.05 or **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 or ^###P < 0.001 vs. the alcohol group.

Figure 3

Liver histology. Accumulation of lipid droplets is evident in the liver of alcohol-fed mice, while relatively few lipid droplets were found in the hepatocytes of other groups (hematoxylin/eosin staining; original magnification, × 40). Serum ALT levels show similar trends to hepatic TG content in all groups. GH1 therapy reversed the alcohol-diet-induced increases in hepatic TG and serum ALT. ALT: alanine transaminase; TG: triglyceride. n = 6 mice per group. Means without a common letter differ at P < 0.05 vs. the control group.

Figure 4

GH1 therapy upregulated adiponectin and enhanced hepatic adipoR2 mRNA expression in alcohol-fed mice. (A) Serum HMW adiponectin concentrations. (B) Relative mRNA levels of adipoR1. (C) Relative mRNA levels of adipoR2. (D) Relative adipose tissue mRNA levels of adiponectin, TNFα, SIRT1 and FOXO1. AdipoR1: adiponectin receptor 1; AdipoR2: adiponectin receptor 2; FOXO1: forkhead transcription factor O 1; HMW adiponectin: high molecular weight adiponectin; SIRT1, sirtuin 1; TNFα, tumor necrosis factor-α. n = 6 mice per group. Means without a common letter differ at P < 0.05 vs. control group.

Figure 5

GH1 therapy stimulated hepatic AMPK activity in alcohol-fed mice. Western blotting of liver extracts was performed using anti-phosphorylated-AMP-activated protein kinase (AMPK)-α (anti-p-AMPKα), anti-AMPKα, anti-p- peroxisome proliferator activated receptor-α (PPARα), anti-PPAR-α, anti-phosphorylated acetyl CoA carboxylase (p-ACC) and anti-microsomal cytochrome P450, family 4, subfamily a, polypeptide 1 (Cyp4A1) antibodies. C: control group; GC: GH1-treated control group; A: alcohol group; GA: GH1-treated alcohol group; PI: pair-fed group I; PII: pair-fed group II. n = 6 mice per group. Means without a common letter differ at P < 0.05 vs. the control group.

Figure 6

GH administration upregulated hepatic SIRT1 expression in alcohol-fed mice. (A) Hepatic mRNA expression of SIRT1. (B) SIRT1 protein levels. (C) Relative PGC1α acetylation. Hepatic nuclear SIRT1 protein levels were determined using an anti-SIRT1 antibody. A nonspecific nuclear protein band was used to confirm equal loading and to normalize the data. PGC1α was immunoprecipitated from liver extracts and immunoblotted with either an anti-acetylated lysine (Ac-Lys) antibody to determine the extent of PGC1α acetylation or with an anti-PGC1α antibody to determine the total amount of PGC1α. C: control group; GC: GH1-treated control group; A: alcohol group; GA: GH1-treated alcohol group; PI: pair-fed group I; PII: pair-fed group II. AOX: acyl-CoA oxidase; CPT1a: carnitine palmitoyltransferase 1a; MCDA: medium chain acyl-Co-A dehydrogenase; PGC1α: PPARα coactivator; PPARγ: peroxisome proliferator activated receptor-γ; SIRT1: sirtuin 1. n = 6 mice per group. Means without a common letter differ at P < 0.05.

Figure 7

GH1 therapy suppressed SREBP-1c activity and reduced the mRNA levels of SREBP-1-regulated genes encoding lipogenic enzymes in the livers of alcohol-fed mice. (A) Nuclear SREBP-1c protein levels. (B) Relative mRNA levels of hepatic SREBP-regulated lipogenic enzymes. A nonspecific nuclear protein band in nuclear extracts was used to confirm equal loading and to normalize the data. C: control group; GC: GH1-treated control group; A: alcohol group; GA: GH1-treated alcohol group; PI: pair-fed group I; PII: pair-fed group II. ACCα: acetyl-CoA carboxylase-α; FAS: fatty acid synthase; GPAT1: glycerol-3-phosphate acyltransferase; ME: malic enzyme; nSREBP-1: nuclear sterol regulatory element binding protein 1; SCD1: stearoyl coenzyme A desaturase 1; SREBP-1, sterol regulatory element binding protein 1. n = 6 mice per group. Means without a common letter differ at P < 0.05.