Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice

Abstract

Hepatosteatosis, the ectopic accumulation of lipid in the liver, is one of the earliest clinical signs of alcoholic liver disease (ALD). Alcohol-dependent deregulation of liver ceramide levels as well as inhibition of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPAR-α) activity are thought to contribute to hepatosteatosis development. Adiponectin can regulate lipid handling in the liver and has been shown to reduce ceramide levels and activate AMPK and PPAR-α. However, the mechanisms by which adiponectin prevents alcoholic hepatosteatosis remain incompletely characterized. To address this question, we assessed ALD progression in wild-type (WT) and adiponectin knockout (KO) mice fed an ethanol-containing liquid diet or isocaloric control diet. Adiponectin KO mice relative to WT had increased alcohol-induced hepatosteatosis and hepatomegaly, similar modest increases in serum alanine aminotransferase, and reduced liver TNF. Restoring circulating adiponectin levels using recombinant adiponectin ameliorated alcohol-induced hepatosteatosis and hepatomegaly in adiponectin KO mice. Alcohol-fed WT and adiponectin KO animals had equivalent reductions in AMPK protein and PPAR-α DNA binding activity compared with control-fed animals. No difference in P-AMPK/AMPK ratio was detected, suggesting that alcohol-dependent deregulation of AMPK and PPAR-α in the absence of adiponectin are not primary causes of the observed increase in hepatosteatosis in these animals. By contrast, alcohol treatment increased liver ceramide levels in adiponectin KO but not WT mice. Importantly, pharmacological inhibition of de novo ceramide synthesis in adiponectin KO mice abrogated alcohol-mediated increases in liver ceramides, steatosis, and hepatomegaly. These data suggest that adiponectin reduces alcohol-induced steatosis and hepatomegaly through regulation of liver ceramides, but its absence does not exacerbate alcohol-induced liver damage.

Keywords: adiponectin; alcohol; ceramide; hepatosteatosis.

Fig. 1.

Liver and serum analysis after alcohol (Alc) feeding in wild-type (WT) and adiponectin (Adn) knockout (KO) mice. A: histological sections were scored for hepatosteatosis, and animals/score were plotted. B: quantitation of triglyceride (TG) from liver extracts. C: liver-to-body weight (B.W.) ratio. D: TNF-α levels were determined by ELISA using tissue lysates. E: mRNA extracted from livers of treated animals were assessed for IL-6, suppressor of cytokine signaling 3 (SOCS3), monocyte chemoattractant protein (MCP)-1 and TNF levels. F: alanine aminotransferase (ALT) measurements were determined in serum. Data presented as means ± SE. NS, not significant, *P < 0.05, ***P < 0.01.

Fig. 2.

Effect of recombinant adiponectin treatment in alcohol-fed adiponectin KO mice. A: daily serum samples from alcohol-fed adiponectin KO mice implanted with adiponectin-delivering Alzet pumps were measured for adiponectin by ELISA. #P < 0.05 compared with day 1. *P < 0.05 compared with day 3. B: serum high-molecular-weight (HMW) adiponectin assessed by ELISA at harvest. Sal, saline. C: representative liver histological sections from alcohol-fed adiponectin KO mice implanted with either saline- or adiponectin-delivering pumps stained with hematoxylin and eosin (top) or oil red O (bottom), scale bar = 100 μm. D: quantitation of triglyceride levels in liver extracts. E: liver-to-body weight ratio. F: mRNA extracted from livers of treated animals was analyzed for IL-6, SOCS3, MCP-1, and TNF levels by qRT-PCR. G: ALT activities in serum. Data presented as means ± SE. *P < 0.05, ***P < 0.01.

Fig. 3.

AMP-activated protein kinase (AMPK) phosphorylation, peroxisome proliferator-activated receptor α (PPAR-α) activation, and glucose tolerance test (GTT) after alcohol feeding. A: Western blot of representative samples probed with antibodies specific for AMPK-α subunit phosphorylated on Thr172 (P-AMPK), total AMPK-α protein (AMPK), and GAPDH. B: quantitation of AMPK protein levels from Western blots (n = 4/group). C: quantitation of P-AMPK relative to AMPK protein from Western blots in control and alcohol-fed animals. D: comparison of P-AMPK/AMPK ratio in chow-fed and liquid control diet-fed animals (n = 4/group). E: liver nuclear extracts assayed for PPAR-α-binding to PPAR DNA-binding element. F: GTT administered following alcohol feeding. AUC, calculated area under the curve. Data presented as mean ± SE. *P < 0.05, ***P < 0.01.

Fig. 4.

Effects of alcohol feeding and myriocin (Myr) treatment on alcohol-fed WT and adiponectin KO mice. A: liver lipid extracts were analyzed for ceramide levels by liquid chromatography/tandem mass spectrometry and normalized to protein content. B: serum adiponectin levels at harvest analyzed by ELISA. C: liver triglycerides at harvest. D: liver-to-body weight ratio at harvest. E: serum HMW adiponectin levels at harvest in WT animals analyzed by ELISA. F and G: quantitation of Western blots of liver lysates probed with specific antibodies for adiponectin receptor 1 (AdipoR1) and AdipoR2 (F) or acid sphingomyelinases (aSMase) (G) and normalized to GAPDH. H: mRNA extracted from livers of treated animals were assessed for sphingomyelinase (Smpd) isoforms (a), serine palmitate transferase (SPT) subunits (b), ceramide synthase (CerS) isoforms (c), and acid ceramidase (Asah) isoforms (d). Data presented as mean ± SE. (n ≥ 4) *P < 0.05, ***P < 0.01.