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Published in final edited form as: Chem Biol Interact. 2020 Mar 11;322:109059. doi: 10.1016/j.cbi.2020.109059

Ghrelin regulates adipose tissue metabolism: Role in hepatic steatosis

Karuna Rasineni a,b,*, Jacy L Kubik a,b, Kurt L Knight a,b, Lukas Hall c, Carol A Casey a,b,d,1, Kusum K Kharbanda a,b,d,1
PMCID: PMC7716754  NIHMSID: NIHMS1645292  PMID: 32171850

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.

Keywords: Alcoholic fatty liver, Ghrelin, Adipose tissue

1. 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 [19–23].

We and others have reported that alcohol administration decreases the circulating levels of both insulin [5,24–26] and adiponectin [27–32] 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.

2. Methods

2.1. 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-strep-tomycin. 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 lipid droplet formation. We had two different treatment modalities; ghrelin or ethanol treatments were either included during differentiation, or these treatments were done after differentiation. For measuring a ghrelin or ethanol effect on the differentiation of preadipocytes, we added 5–10 nm ghrelin or 50–100 mM ethanol at the same time in the incubation medium with methylisobutylxanthine, dexamethasone and insulin. For determining the effect after differentiation, ghrelin or ethanol was added only after 90% of the cells maintained in FBS containing medium exhibited lipid droplet formation. Post-treatment, the spent media was collected for measuring adiponectin, free glycerol and cytokines levels. The cells were washed with PBS and lysed for the following determinations:

2.2. Western Blot analysis

Adipocytes lysed in ice-cold RIPA buffer (250μl/well in 6-well plates) were subjected to 12% SDS-PAGE, transferred to nitrocellulose and proteins were detected by incubation with primary antibodies and their respective HRP-conjugated secondary antibodies.

2.3. Cellular triglycerides

Lipids were extracted from adipocyte lysate by the Folch method [40]. Triglycerides (TG) in the lipid extracts were measured using the diagnostic kit # TR22421 from Thermo Fisher Scientific (Middletown, VA). An aliquot of the RIPA lysate was also used to determine total DNA [41].

2.4. Gene expression analysis

RNA was isolated from cell pellets using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA) as described previously. One μg of RNA was used to make cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems). Quantitative PCR (qPCR) was performed using mouse-specific primers for PPARγ (Sense 5′-ACCACTCGCATTCCTTT GAC-3’; Antisense 5′-TCAGCGGGAAGGACTTTATG-3′) and 36B4 (Sense 5′-GAGGAATCAGATG AGGATATGG GA-3’; Antisense 5′-AAGCAGGCT GACTTGGTTGC-3′) from Integrated DNA Technologies with iTaq Universal SYBR Green Supermix (Biorad). The ΔΔCt method was used to determine the fold change in adipocyte using ribosomal phosphoprotein (36B4) for normalization.

2.5. Free glycerol, adiponectin and cytokine measurement in the spent media

Free glycerol levels were determined in the media using a free glycerol kit (Sigma, St. Louis, MO; Cat #F6428). Specific ELISA kits were used for measuring adiponectin (Cat# DY1119; R&D Systems, Inc., Minneapolis, MN) and cytokines (BD Biosciences, San Jose, CA; IL-6 Cat# 555240; CCL2 Cat# 555260) following the manufacturer’s instructions.

2.6. In vivo study with ghrelin antagonist

To examine whether the inhibition of ghrelin activity can improve alcohol-induced impairment of adipose and hepatic metabolism, we administered a ghrelin receptor antagonist to alcohol-fed rats. For this, male Wistar rats were weight matched and pair-fed with Lieber-DeCarli control and ethanol diet for 6 weeks as previously described [42]. The total calories consisted of 11% carbohydrate, 18% protein, 35% fat and 36% ethanol. In the control diet, the ethanol was replaced isocalorically with maltodextrose. After 6 weeks feeding, intraperitoneal injections of ghrelin receptor antagonist, [D-Lys-3] GHRP-6 (DLys; Tocris Bio-Techne brand, Minneapolis, MN), were administered to half of the rats in the control and ethanol groups at a dose of 9 mg/kg BW for 5 days. The other half of the rats received saline injections. During the 5 days of injections, all the rats were pair-fed to the ethanol rats injected with the antagonist. Rats were sacrificed after treatment. The rats were given fresh control or ethanol diet 2 h prior to sacrifice to maintain the “fed” condition. At the time of sacrifice by anesthesia with isoflurane, blood and tissues were collected for analysis. All animals received humane care as directed by the American Association for the Accreditation of Laboratory Animal Care guidelines. All protocols were approved by the Institutional Animal Care and Use Committee at the NWIHCS VA Research Service.

2.7. Statistical analysis

The results were presented as mean ± SEM. Data were analyzed by one-way ANOVA, followed by Student’s Newman-Keuls post-hoc test. p-Values of < 0.05 were considered significant.

3. Results

3.1. 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.

Fig. 1. Alcohol metabolizing enzymes and ghrelin receptor (GHS-R) in differentiated 3T3-L1cells.

Fig. 1.

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After three days of differentiation, cells were treated with growth media supplemented with insulin along with ethanol (50 mM). After 6 days of treatment, the cell lysates were prepared and subjected to Western Blot analysis using appropriate antibodies.

3.2. Ghrelin significantly inhibits adipose differentiation

Peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor highly expressed in adipose tissue, is a key regulator for adipocyte differentiation and lipid storage [44–46]. Since PPARγ is commonly used as an adipocyte differentiation marker, we measured its expression in 3T3-L1 cells exposed to ghrelin and ethanol. In these preliminary experiments, we found a significant decrease in PPARγ expression in ghrelin and ethanol-treated 3T3-L1 adipocytes (Fig. 2), indicating that both, ghrelin and ethanol, inhibit adipose differentiation to mature adipocytes, which have the capacity to store the lipids.

Fig. 2. Ghrelin decreases the PPARγ expression in adipocytes.

Fig. 2.

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3T3-L1 pre-adipocytes were exposed to differentiation media containing ghrelin (5 nm) or ethanol (50 mM) as described in the method section. After three days of differentiation, cells were treated with growth media supplemented with insulin along with ghrelin (5 nm) or ethanol (50 mM). After 6 days of treatment, RNA was isolated and PPARγ gene expression determined. The ΔΔCt method was used to determine the fold change in adipocyte using ribosomal phosphoprotein (36B4) for normalization. Results presented are from 3 independent experiments with 2 samples/condition and expressed as mean ± SEM. Values not sharing a common letter are statistically different, p < 0.05.

3.3. Ghrelin significantly inhibits adipocyte lipid accumulation and promotes lipolysis

Since chronic alcohol consumption is associated with increased adipose tissue lipolysis, we investigated whether ghrelin has any direct effect on fat accumulation and breakdown. For this we measured intracellular TG in the cell lysates and free glycerol, the product of TG breakdown, in the spent media to estimate lipolysis. Both these determinations were normalized to total cellular DNA. As shown in Fig. 3A, ghrelin as well as ethanol treatment to adipocytes significantly reduced intracellular triglyceride accumulation compared with non-treated cells. Concomitantly, both ghrelin and ethanol treatments enhanced lipolysis as indicted by increased free glycerol in the media (Fig. 3B). While ghrelin’s effect on glycerol secretion was similar to ethanol treatment, total cellular triglycerides were statistically much less than ethanol treatment alone. These data suggest that ghrelin could be affecting the lipogenesis or fatty acid oxidation accounting for the much more reduced TG accumulation compared with ethanol alone treatment.

Fig. 3. Effect of ghrelin on adipocyte lipid accumulation and lipolysis.

Fig. 3.

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After 3 days of differentiation of the 3T3-L1 pre-adipocytes, the cells were exposed to insulin-containing growth media supplemented with ghrelin (10 nM) or ethanol (50 mM) for 6 days. After that the cells were maintained in FBS containing DMEM media along with ghrelin or ethanol until sufficient lipid droplet formation (which takes 4–6 days). At termination, A) cellular TG and B) free glycerol levels in the spent media was determined and normalized to cellular DNA. The results presented are from 3 independent experiments with 4 samples/condition and expressed as mean ± SEM. Values not sharing a common letter are statistically different, p < 0.05.

3.4. Ghrelin significantly inhibits adiponectin secretion

Several studies have reported decreased serum adiponectin levels with chronic alcohol consumption [5,27,29,30]. To examine the direct effect of ghrelin on adiponectin secretion, we analyzed the adipocyte spent media. As shown in Fig. 4A, ghrelin treatment of differentiated adipocytes significantly (~75%) reduced adiponectin secretion compared to non-treated adipocytes. This decrease by ghrelin was more profound than the ~50% reduction in adiponectin secretion by ethanol treatment alone. The data showing the direct role of ghrelin in inhibiting adiponectin secretion from adipose tissue was corroborated by our in vivo studies showing reduction in serum adiponectin levels by ethanol administration and increased serum adiponectin levels to control levels in Dlys-treated ethanol diet-fed rats) (Fig. 4B). Interestingly, significantly higher adiponectin levels were seen in the DLys treated control diet-fed rats compared with the controls.

Fig. 4. Effect of ghrelin on adiponectin secretion in vitro and in vivo.

Fig. 4.

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A) After differentiation and insulin treatments of the 3T3-L1 pre-adipocytes, cells were treated with ghrelin (5 nM) or ethanol (100 mM) in FBS contained DMEM for 48 h. After treatment, the adiponectin level in media was measured and normalized to cellular DNA. Results are from 3 independent experiments with 4 samples/condition and expressed as mean ± SEM. B) Serum adiponectin levels expressed as mean ± SEM in ghrelin antagonist, [D-Lys-3] GHRP-6 (DLys) treated control and ethanol-fed rats. Values not sharing a common letter are statistically different, p < 0.05.

3.5. Ghrelin role on adipokine secretion

It has been reported that increased cytokine levels play a key role in inflammation and progression of both alcoholic and non-alcoholic liver diseases [9,35]. Thus, we examined whether ghrelin could directly affect CCL2 and IL-6 secretion by adipocytes. We observed that compared to un-treated controls, ghrelin significantly increased secretion of CCL2 and IL-6 up to 11.2- and 1.5-fold, respectively (Fig. 5). These increases by ghrelin were much more profound than the increases seen in CCL2 and IL-6 secretion by ethanol exposure (Fig. 5).

Fig. 5. Effect of ghrelin on cytokine secretion from adipocytes.

Fig. 5.

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After 3 days of differentiation, the 3T3-L1 pre-adipocytes were exposed to ghrelin (5 nM) or ethanol (50 mM) in the growth media supplemented with insulin for 6 days. After that, the cells were maintained in FBS containing DMEM media along with ghrelin or ethanol until sufficient lipid droplet formation (4–6 days). At termination, A) CCL2 and B) IL-6 levels were measured in the spent media and normalized to cellular DNA. Results are from 3 independent experiments with 2 samples/condition and expressed as mean ± SEM. Values not sharing a common letter are statistically different, p < 0.05.

4. 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, lead to increased free fatty acid mobilization from adipose tissue to liver, thereby contributing to hepatic steatosis [5,6,47]. In addition, it is also reported that chronic alcohol consumption is associated with altered serum levels of adipokines that play a central role in energy metabolism and inflammation by autocrine and paracrine effects [35,48]. Importantly, adiponectin helps the adipocyte expansion to store excess lipids in the form of lipid droplets to avoid lipotoxicity in peripheral organs [15,16]. Further, adiponectin has a paracrine effect on the liver decreasing lipogenesis and increasing beta oxidation [8,17,18]. Cytokines, particularly TNF-α and IL-6, play a detrimental role on adipocyte insulin signaling by decreasing tyrosine phosphor-ylation of insulin receptor substrates leading to impaired insulin-mediated lipolysis and increased free fatty acid release from the adipose tissue [9,22,49]. At the same time, the adipose-derived TNF-α and IL-6 contribute to increased hepatic inflammation and development of fibrosis [35,48].

In our recent studies, we reported that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic β-cells. The consequent reduction in the circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. In addition to inhibiting insulin secretion, ghrelin directly promoted fat accumulation in hepatocytes [26]. Based on the direct and indirect effects of ghrelin in promoting alcoholic steatosis through the pancreas-adipose-liver axis, this study was conducted to examine whether ghrelin has a direct effect on the adipose tissue especially since ghrelin receptor is present on the adipocytes. In this preliminary study, we observed that ghrelin decreases expression of PPARγ, a key regulatory factor for adipocyte proliferation and maturation [44,45] indicating that ghrelin can directly influence adipose metabolism. Many studies reported that PPARγ ligands the thiazolidinediones (TZD), can ameliorate hepatic steatosis by improving adipose tissue insulin sensitivity, triglyceride storage and free fatty acid flux. It is also reported that thiazolidinediones suppress hepatic inflammation by inhibiting secretion of cytokines such as TNF-α and IL-6 [50,51].

Importantly, in this study, along with decreased PPARγ, we also observed decreased fat accumulation and increased free glycerol levels in the media of ghrelin treated mature adipocytes indicating an enhancement of adipose lipolysis by ghrelin. However, we do not know whether decreased lipid storage and increased lipolysis by ghrelin is mediated via decreased PPARγ, expression, or if it directly promotes lipolysis. Ethanol treatment also decreased both PPARγ and increased adipose lipolysis, indicating, as has been reported before, that ethanol has a direct effect on adipose tissue lipid metabolism which may be non-ghrelin mediated. Alternatively, ethanol may be inducing ghrelin secretion from adipocytes, which can now in an autocrine manner affect adipose homeostasis. Indeed, ghrelin has been reported to be present in 3T3-L1 adipocytes [38] and adipose tissue [52], in addition to the stomach, the traditionally known major site for ghrelin synthesis [53]. In our recently published study, we reported decreased expression of adipose PPARγ in ethanol-treated rats which was increased in the ghrelin receptor antagonist, Dlys, treated ethanol rats. Further, improved PPARγ levels correlated with normalized serum non-esterified free fatty acid levels in ghrelin receptor antagonist treated alcohol-fed rats compared to increased levels seen in the alcohol-fed rats [25]. Whether the increased PPARγ expression is due to normalized insulin levels seen in the ghrelin receptor antagonist treated alcohol-fed rats [25] or due to a direct effect of alleviation of ghrelin inhibition is being currently investigated.

Our studies also revealed that both ghrelin and ethanol significantly decreased adiponectin secretion and increased CCL2 and IL-6 secretion from mature adipocytes. Many studies have reported that chronic alcohol consumption decreases adiponectin and increases inflammatory markers IL-6 and CCL2 in adipose tissue [54,55]. This altered adipokine secretion is due to the combination of a ghrelin and/or ethanol effect. However, when we treated the ethanol-fed rats with ghrelin antagonist, we observed that adiponectin levels were normalized to control levels. However, the control-diet fed rats treated with the ghrelin antagonist showed a significantly increased serum adiponectin above the control levels (Fig. 4B). These data collectively indicate that ghrelin can directly affect adiponectin synthesis and/or secretion and that the ethanol-induced decrease in circulating adiponectin is possibly mediated via ghrelin, since similar effects on decreased adiponectin synthesis [28] and secretion [27] after chronic ethanol consumption have been reported.

In conclusion, alcohol-induced increases in serum ghrelin levels impairs insulin secretion from pancreatic β-cells. The consequent reduction in the circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Our preliminary data shows that in addition to its indirect activity via decreased pancreatic insulin, ghrelin can directly increase adipocyte lipolysis and concomitantly inhibit secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. Further, ghrelin directly increases IL6 and CCl2 secretion from adipose tissue, leading to impaired insulin mediated lipid metabolism and further contributes to the progression of fatty liver disease. Currently, we are investigating the mechanistic pathways by which ghrelin directly on alcohol-induced adipose tissue dysfunction and the development of alcoholic steatosis.

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.

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.

Footnotes

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.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cbi.2020.109059.

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