Ghrelin regulates adipose tissue metabolism: Role in hepatic steatosis

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Abstract

Fatty liver is the earliest and most common response of the liver to consumption of excessive alcohol. Steatosis can predispose the fatty liver to develop progressive liver damage. Chief among the many mechanisms involved in development of hepatic steatosis is dysregulation of insulin-mediated adipose tissue metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that ultimately results in hepatic steatosis. The adipose-liver axis is modulated by hormones, particularly insulin and adiponectin. In recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic β-cells. The consequent reduction in circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Because many tissues, including adipose tissue, express ghrelin receptor we hypothesized that ghrelin may directly affect energy metabolism in adipocytes. We have exciting new preliminary data which shows that treatment of premature 3T3-L1 adipocytes with ghrelin impairs adipocyte differentiation and inhibits lipid accumulation in the tissue designed to store energy in the form of fat. We further observed that ghrelin treatment of differentiated adipocytes significantly inhibited secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. These results were corroborated by our observations of a significant increase in serum adiponectin levels in ethanol-fed rats treated with a ghrelin receptor antagonist verses the un-treated ethanol-fed rats. Interestingly, in adipocytes, ghrelin also increases secretion of interleukin-6 (IL-6) and CCL2 (chemokine [C–C motif] ligand 2), cytokines which promote hepatic inflammation and progression of liver disease. To summarize, the alcohol-induced increase in serum ghrelin levels dysregulates adipose-liver interaction and promotes hepatic steatosis by increasing the free fatty acid released from adipose for hepatic uptake, and by altering adiponectin and cytokine secretion. Taken together, our data indicates that targeting the activity of ghrelin may be a powerful treatment strategy.

Introduction

Excessive alcohol use is a serious problem in US and worldwide. Among individuals with alcohol use disorder, 90% of the people develop hepatic steatosis [1,2] which is characterized by accumulation of lipids in the hepatocytes [3]. Although steatosis is often benign and reversible, it is widely believed to be the precursor to fibrosis, cirrhosis and cancer and therefore is a prime target for therapeutic intervention [4]. Central among the many mechanisms proposed to play a role in the development of alcoholic steatosis is dysregulation of adipose tissue lipid metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that contributes to the development of alcoholic steatosis [5,6].

Studies have demonstrated that the adipose-liver axis is modulated by hormones and cytokines, particularly adipokines [7,8]. The hormones, insulin and adiponectin play a major role in adipose-liver cross-talk [9,10]. Insulin profoundly affects both carbohydrate and lipid metabolism in both liver and adipose tissue. In the liver, insulin stimulates the export of fat as very-low density lipoproteins (VLDL) that are secreted into the circulation and taken up by various organs, including adipocytes to provide free fatty acids for their use [11,12]. Adipocytes use the free fatty acids to synthesize triglycerides (TG) which are stored in lipid droplets for energy needs. Insulin inhibits lipolysis in adipocytes thus allowing for increased lipid storage in these cells [13,14]. Adiponectin has both autocrine and paracrine functions. In adipocytes, adiponectin helps adipocytes expansion by improving insulin sensitivity for saving lipids in the form of lipid droplets to avoid lipotoxicity in peripheral organs [15,16]. In the liver, adiponectin reduces lipogenesis and promotes lipid oxidation [8,17,18].

In addition to peptide hormones, adipose-derived cytokines and chemokines play a major role in adipose-liver interplay, specifically progression of steatosis to advanced stage liver disease. Importantly, tumor-necrosis factor α (TNFα), interleukin-6 (IL-6) and a macrophage chemoattractant, CCL2 (chemokine [C–C motif] ligand 2), all play central roles in insulin sensitivity and lipid metabolism by paracrine and endocrine functions [[19], [20], [21], [22], [23]].

We and others have reported that alcohol administration decreases the circulating levels of both insulin [5,[24], [25], [26]] and adiponectin [[27], [28], [29], [30], [31], [32]] in humans and animal models while increasing circulating ghrelin levels [26,33,34]. Further, it has also been reported that alcohol administration increases TNF-α, IL-6 and CCL2 levels in adipose tissues [28,35].

In investigating further, we recently reported that the decrease in circulating insulin observed upon alcohol administration was due to increased serum ghrelin levels which impairs insulin secretion from pancreatic β-cells [25,26]. Adipose tissue expresses the ghrelin receptors, GHS-R1a [36,37] and previous studies have reported that ghrelin inhibits the differentiation of 3T3-L1 preadipocytes to mature adipocytes [38]. Based on these considerations, we hypothesized that the alcohol-induced ghrelin increase is responsible for the adipose lipid dysregulation observed during the development of alcoholic steatosis. Thus, the aim of this preliminary study was to investigate the direct role of ghrelin on adipocyte lipid breakdown and adipokine secretion which together can have a significant effect in the development of alcoholic fatty liver disease.

Section snippets

3T3-L1 preadipocyte cell Culture and differentiation

Mouse 3T3-L1 fibroblasts were purchased from ATCC. Cells were grown in DMEM media containing 10% FBS and 1% penicillin-streptomycin. At 70% confluence, cells were induced to differentiate by adding methylisobutylxanthine, dexamethasone and insulin to the cultured media as described previously [39]. After three days of treatment with differentiation media, cells were cultured in insulin supplemented media for 6 days. After that cells were maintained in FBS containing DMEM media until sufficient

Ethanol metabolizing enzyme expression

Differentiated 3T3-L1 adipocytes express both ADH (alcohol dehydrogenase) and CYP2E1 (cytochrome P450 2E1), the two major enzymes that metabolize ethanol (Fig. 1). The expression of both these enzymes was increased by ethanol exposure. These cells also express ghrelin receptor (GHS-R) (Fig. 1) as has been shown previously [43]. Collectively, these data indicate that 3T3-L1 adipocytes are an appropriate in vitro model to examine the effects of ghrelin and ethanol.

Ghrelin significantly inhibits adipose differentiation

Peroxisome

Discussion

Fatty liver, characterized by an accumulation of lipids in hepatocytes, is one of the earliest changes in the pathogenesis of both alcoholic fatty liver disease. Once the liver becomes steatotic, it is more prone to inflammatory mediators leading to progression to hepatitis, fibrosis and eventually cirrhosis and HCC [4]. It has been reported that adipose tissue plays a central role in development and progression of fatty liver disease. Impaired adipose metabolism, particularly lipid metabolism,

Grant

This research was supported by K01 AA024254 (KR) from the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health and the Merit Review grant BX004053 (KKK) from the U.S. Department of Veterans Affairs.

Author contribution

KR was responsible for the conception and design of the study, and analyzed the data; KR, JLK, KLK and LH performed the experiments; KR, CAC and KKK interpreted the results of experiments, analyzed the data, and co-wrote the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We thank Dr. Vasilis Vasiliou (Yale School of Public Health/Yale School of Medicine, New Haven, CT) for giving the opportunity to share our findings at the “Alcohol and Cancer 2019” meeting.

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