Adiponectin is a 30-kDa adipocyte-specific protein consisting of four distinct domains: a signal peptide at the N terminus, followed by a short variable region, a collagenous domain, and a C-terminal globular domain. Adiponectin is the most abundant gene product in adipose tissue and circulating adiponectin concentrations account for ∼0.01% of total plasma protein. Full-length adiponectin can undergo proteolytic processing to liberate a fragment containing the C-terminal globular adiponectin domain (gAd). Adiponectin circulates in the serum as three major oligomeric forms: a low molecular weight (LMW) trimer, a medium molecular weight (MMW) hexamer, and a high molecular weight (HMW) multimer.
Rodents and humans display sexual dimorphism in circulating levels of adiponectin and adiponectin isoforms, with females having higher levels of circulating adiponectin and HMW forms. Sex hormones such as estrogen and testosterone may regulate the production, release, and oligomerization of adiponectin. For recent reviews on adiponectin, (see refs.1–4.
Role of Adiponectin in Alcoholic Fatty Liver
Liver is one of the main target organs for adiponectin. Hypoadiponectinemia is associated with the development of various types of liver injury including steatosis, inflammation, and fibrosis in rodents and humans (reviewed in refs.4, 5). Alcoholic fatty liver disease (AFLD) is characterized by excessive accumulation of fat in the liver in response to chronic alcohol consumption. Considerable evidence has shown that adiponectin plays a vital role in alcoholic fatty liver in several animal models. Chronic ingestion of an ethanol-containing, high-fat diet by mice or rats significantly decreases circulating adiponectin concentrations, which correlates closely with the accumulation of hepatic lipid (6–10). Delivery of full-length recombinant adiponectin into ethanol-fed animals alleviates liver steatosis (6).
Several lines of investigation have provided evidence that dietary modulation of adiponectin prevents alcoholic fatty liver in animals. For example, it is well known that diets high in either saturated fatty acids or medium chain triglycerides prevent the development of AFLD. Feeding mice with a high polyunsaturated fat and ethanol-containing diet resulted in decreased circulating adiponectin and a substantial increase in hepatic lipid accumulation (7). Conversely, mice fed with a high-saturated fat and ethanol-containing diet showed elevated circulating adiponectin and blockage of lipid accumulation in the liver, suggesting that elevated adiponectin levels mediate protective effects of saturated fat against alcoholic fatty liver (7).
Abnormal homocysteine/methionine metabolism in liver as well as in adipose tissue plays an integral role in the pathogenesis of alcoholic liver steatosis (10, 11). Chronic ethanol feeding inhibits methionine synthase, which disturbs the synthesis of methionine, causes hyperhomocysteinemia, and reduces the synthesis of S-adenosylmethionine (SAM) in rodents and humans. Feeding Yucatan micropigs with an ethanol-containing folate-deficient diet caused a significant reduction in circulating adiponectin concentrations and led to development of liver steatosis (11). Supplementation of ethanol-containing diets with SAM normalized circulating adiponectin levels and reduced liver triglycerides in these animals (11). Consistent with these findings, betaine supplementation to ethanol-fed mice increased circulating adiponectin levels and attenuated ethanol-induced liver steatosis (10).
Resveratrol (trans-3,5,4′-trihydroxystilbene) is a dietary polyphenol found in a wide variety of plant species including grapes, berries, and peanuts (reviewed in ref.12). We recently found that resveratrol treatment blocked the inhibitory effects of chronic ethanol feeding on adiponectin production and prevented the accumulation of hepatic lipid in mice (J. M. Ajmo and M. You, unpublished observations). The mechanisms of induction of adiponectin by SAM, betaine, and resveratrol will be further discussed in subsequent sections.
Two major adiponectin receptors (AdipoRs), designated adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2), have been identified. AdipoR1 and AdipoR2 are structurally related integral plasma membrane proteins with seven membrane-spanning domains. AdipoR1 possesses high affinity for the globular form of adiponectin and low affinity for full-length adiponectin, whereas AdipoR2 exhibits intermediate binding affinity for both the globular and the full-length adiponectin. AdipoR1 is ubiquitously expressed, most abundantly in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. T-cadherin has been identified as a third AdipoR and is selective for the HMW and MMW forms of adiponectin (13). However, the role of T-cadherin in adiponectin signaling is not clear. Factors such as insulin, nuclear receptors, growth hormone, dietary fatty acids, and fasting/feeding states regulate AdipoRs' expression in rodents. For recent reviews on AdipoRs, (see refs. (1–4).
Ethanol Regulation of Adiponectin Receptors
Regulation of hepatic AdipoRs by ethanol is not well understood. AdipoR1 mRNA expression was found to be diminished in livers of ethanol-fed Yucatan micropigs, and this was associated with attenuation of adiponectin signaling and liver steatosis (11). In the same micropigs, the levels of AdipoR1 were restored to control levels by SAM supplementation of the ethanol diet, and liver steatosis was alleviated. In a separate study, we found that hepatic AdipoR2, but not AdipoR1, was downregulated by chronic ethanol feeding in mice (14).
HEPATIC ADIPONECTIN RECEPTORS-MEDIATED SIGNALING
Circulating adiponectin interacts with AdipoRs in the liver and thereby exerts effects on lipid metabolism through a number of signaling molecules including AMP-activated kinase (AMPK), PPAR-gamma coactivator alpha (PGC-1α), peroxisome proliferator-activated receptor alpha (PPARα), and sterol regulatory element-binding protein 1 (SREBP-1) (15–17). Adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif 1 (APPL1) is a newly identified AdipoR interacting protein (18). Interaction of APPL1 with AdipoRs could provide a vital molecular link between adiponectin binding and subsequent signaling events in liver.
Adiponectin and AMPK
AMPK is a heterotrimeric protein kinase, which is known to act as a major regulator of lipid metabolism through direct phosphorylation of its substrates and indirect control over gene transcription. Subject to activation by elevated intracellular AMP/ATP ratio, AMPK activity is also dramatically stimulated by phosphorylation of threonine 172 on the alpha subunit by upstream AMPK kinases including tumor suppressor LKB-1 and calmodulin-dependent protein kinase kinase. Activation of the AMPK signaling pathway constitutes the main mechanism of action of adiponectin in the regulation of lipid metabolism in liver.
Adiponectin stimulates hepatic AMPK which in turn phosphorylates acetyl-CoA carboxylase (ACC) on Ser-79 and attenuates ACC activity. Inhibition of ACC directly reduces lipid synthesis and indirectly enhances fatty acid oxidation by blocking the production of malonyl-CoA, an allosteric inhibitor of carnitine palmitoyltransferase I (CPT I). CPT I mediates fatty acid transport into the mitochondria and is the rate limiting enzyme in fatty acid oxidation. Thus, adiponectin-mediated AMPK activation favors lipid catabolism and opposes lipid deposition in liver. For recent reviews on AMPK and adiponectin, (see refs. (1–4, 19, 20).
Effects of Ethanol on AMPK
Dysregulation of hepatic AMPK signaling in response to chronic ethanol exposure represents a crucial mechanism for development of alcoholic fatty liver in animals. Chronic ethanol exposure inhibited AMPK activity in cultured rat hepatocytes (21). Hepatocytes isolated from rats fed with ethanol confirmed impaired AMPK activity (22). In livers of ethanol-fed animals such as mice, rats, or micropigs, AMPK activity was significantly decreased (10, 11, 21, 23, 24). The ethanol-mediated inhibition of AMPK was associated with enhanced ACC activity, increased malonyl-CoA concentrations, reduced activity of CPTI, and development of liver steatosis in these animals. Treatment with AICAR, a known activator of AMPK, prevented alcohol-induced fatty liver in rats (22). However, although much of the data in the literature suggests that ethanol inhibits AMPK activity, there are some reports where such inhibitory effects are not observed (25, 26). These studies suggest that the effects of ethanol on AMPK may be quite variable, and perhaps might depend on the animal model of ethanol exposure utilized.
Effects of Adiponectin on Ethanol-Mediated AMPK Inhibition
Adiponectin relieves ethanol-mediated inhibition of AMPK in cultured hepatic cells and in animal liver. In cultured rat hepatocytes, the inhibition of AMPKα phosphorylation by ethanol was reversed by the presence of full-length recombinant adiponectin (7). Consistent with in vitro findings, replenishment of adiponectin to ethanol-fed mice relieved the ethanol-mediated impairment of AMPK signaling components although AMPK activity was not measured in this study (6). In ethanol-fed animals, the increase in circulating adiponectin caused by a diet high in saturated fat, SAM, or betaine restored hepatic AMPK activity (7, 10, 11).
Adiponectin and SREBP-1
SREBPs are transcription factors that regulate cholesterol and lipid synthesis. SREBPs are synthesized as inactive precursors (∼125 kDa) embedded in the endoplasmic reticulum (ER). Processing of SREBP protein by proteolytic cleavage generates an active, mature form of SREBP (∼68 kDa) which is then transported to the nucleus. The nuclear SREBP initiates transcription by binding to sterol response elements (SRE) in promoters of its target genes. There are three major SREBP isoforms, SREBP-1a, SREBP-1c, and SREBP-2. In animal liver, the SREBP-1c transcript predominates and mainly regulates lipid synthesis (reviewed in ref.20). Activation of AMPK by adiponectin in the liver leads to decreased mRNA and protein expression of SREBP-1c (15). Reduction of SREBP-1c activity by adiponectin via AMPK activation results in downregulation of SREBP-1 regulated genes encoding lipid synthesizing enzymes in liver, thereby contributing to an adiponectin-mediated reduction in hepatic lipid synthesis (15–18).
Effects of Ethanol on SREBP-1
In several cultured hepatic cell lines (H4IIEC3, McA RH7777 or Hep G2 cells), ethanol or acetaldehyde, a major metabolite of ethanol, caused an increase in the amount of nuclear SREBP-1 (27, 28). Likewise, chronically feeding ethanol to animals such as mice, rats, or micropigs resulted in a significant increase in the level of nuclear SREBP-1c (11, 26–29). The increased SREBP-1c activity was associated with triglyceride accumulation in the liver and increased mRNA levels of genes encoding lipogenic enzymes including fatty acid synthase, stearoyl-coenzyme A desaturase 1, mitochondrial glycerol-3-phosphate acyltransferase, ATP citrate lyase, and ACC. Ethanol feeding to SREBP-1c knockout mice largely prevented development of liver steatosis, confirming the critical role of SREBP-1c in the development of alcoholic liver steatosis (26).
The molecular mechanism by which ethanol activates SREBP-1 remains unclear. A known activator of AMPK, AICAR, has been shown to abolish the ability of ethanol to stimulate SREBP-1 activity, suggesting that ethanol-induced SREBP-1 activity may partially occur through inhibiting AMPK activity (21, 23). Interestingly, metformin, which is recognized as an activator of AMPK, alleviated alcoholic liver steatosis in animals via downregulation of plasminogen activator inhibitor-1, a mechanism independent of AMPK activation (25). Although the basis for this apparently paradoxical action is unknown, taken together, these studies suggest that targeting various pathways might be beneficial in designing treatments for AFLD.
SIRT1 is a NAD+-dependent class III protein deacetylase. SIRT1 regulates gene expression by deacetylation of modified lysine residues on several crucial hepatic transcriptional regulators (reviewed in ref.30). In both cultured rat hepatoma H4IIEC3 cells and in mice, ethanol exposure inhibited SIRT1 activity leading to SREBP-1c acetylation and activation (31–34). The effect of ethanol on SREBP-1 was abolished by expression of wild-type SIRT1 or treatment with resveratrol, a known potent SIRT1 agonist, suggesting that the effect of ethanol on SREBP-1 is partially mediated through SIRT1 inhibition (31). Intriguingly, growing evidence suggests an association between SIRT1 and AMPK signaling (35–37). Thus, impairment of such a SIRT1-AMPK axis by chronic ethanol exposure could be responsible for the increase in SREBP-1 activity found to occur in the livers of several ethanol-fed animal models.
Effects of Adiponectin on Ethanol-Mediated SREBP-1 Activation
To date, there is no direct evidence that adiponectin can abolish ethanol-mediated SREBP-1 activation. However, as discussed earlier, adiponectin-mediated activation of AMPK signaling would lead to the suppression of SREBP-1c activity in ethanol-fed animals. Indeed, normalization of adiponectin levels by supplementing ethanol diets with SAM or with highly saturated fat inhibited hepatic SREBP-1 signaling, as determined by reduced expression of mRNAs and proteins of SREBP-1 regulated lipogenic enzymes, as well as reduced liver triglycerides in ethanol-fed animals (7, 11).
It is important to note that treatment of primary human myotubes with globular adiponectin increases SIRT1 protein expression levels (38). Recently, we have observed that treatment of rat Kupffer cells (resident hepatic macrophages) with globular adiponectin increased SIRT1 protein levels (39). These studies suggest that adiponectin may suppress SREBP-1 activity in ethanol-fed animals partially through upregulating hepatic SIRT1.
Adiponectin and PGC-1α/PPARα
PPARα is a transcription factor that mainly controls transcription of a panel of genes encoding fatty acid oxidation enzymes (reviewed in ref.40). Adiponectin stimulates PPARα activity in hepatocytes and in animal liver, thus inducing a battery of hepatic enzymes which are involved in fatty acid oxidation, thereby leading to increased oxidation of fatty acids (15–18). It is not entirely clear how adiponectin regulates hepatic PPARα function. PPARγ coactivator-1α (PGC-1α) may provide a critical signaling link between adiponectin and PPARα. In cultured rat hepatocytes, adiponectin stimulated activities of both PGC-1α and PPARα (7). More importantly, the activation of the PPARα-responsive promoter activity by adiponectin required coexpression of PGC-1α, suggesting that adiponectin-induced PPARα activity is mediated through upregulation of PGC-1α (7).
Effects of Ethanol on PGC-1α/PPARα Function
Ethanol inhibited the transcriptional activity of PGC-1α/PPARα in rat H4IIEC3 hepatoma cells or rat hepatocytes (41). Chronic ethanol feeding of mice or rats led to decreased PGC-1α/PPARα expression and reduced mRNAs of a panel of PGC-1α/PPARα regulated genes encoding fatty acid oxidation enzymes (42–44). In PPARα null mice, ethanol-induced liver injuries including steatosis were more extensive than in wild-type mice (43). Treatment of ethanol-fed animals with known PPARα activators (WY14 643, or clofibrate) ameliorated ethanol-induced fatty liver through increasing PGC-1α/PPARα activity and upregulating expression of many PGC-1α/PPAR-α regulated enzymes including long-chain acyl-CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, acyl-CoA oxidase (AOX), and very-long-chain acyl-CoA synthetase (42, 44).
Effects of Adiponectin on Ethanol-Induced PGC-1α/PPARα Impairment
Adiponectin relieves ethanol-inhibition on PGC-1α/PPARα signaling. Treatment with adiponectin (1 μg/ml) partially restored the ethanol-inhibited PPARα activity in rat hepatoma cells (7). Ethanol-fed mice receiving diets high in saturated fat experienced elevated plasma adiponectin levels; greater mRNA and protein expression of PGC-1α; and increased mRNA levels of hepatic AOX, a target of both PGC-1α and PPARα (7).
REGULATION OF ADIPONECTIN BY ETHANOL
Although it is well known that adipose tissue is the ultimate source of circulating adiponectin, the factors that influence plasma adiponectin levels are still being investigated. While cytokines, such as IL-6 and TNFα, have been shown to have inhibitory effects on adiponectin levels, assorted nutritional factors (including dietary fatty acids) regulate adiponectin levels in a myriad of ways. Additionally, various nuclear transcription factors including peroxisome proliferator-activated receptor γ (PPARγ), SREBP-1c, forkhead transcription factor O1 (FOXO1), and SIRT1 are apparently involved in transcriptional regulation of adiponectin. Truly, the regulation of adiponectin's gene expression, secretion into and clearance from the circulation is highly complex. For recent reviews on regulation of adiponectin, (see refs.1–4, 45, 46.
An example of the intricate nature of these control mechanisms is the seemingly self-contradictory role that SIRT1 plays. Notably, in one study, it was shown that activation of SIRT1 in differentiated 3T3-L1 adipocytes lead to a FOXO1-dependent increase in adiponectin transcription (47). Meanwhile, in another study, SIRT1, through its inhibition of PPARγ, was shown to downregulate circulating adiponectin levels by suppressing the secretion of HMW adiponectin from 3T3-L1 adipocytes (48). Thus, although the precise mechanisms remain unclear, these studies suggest that up or downregulation of adiponectin by SIRT1 could be the result of distinct signaling pathways. Molecular dissection of such pathways will presumably elucidate SIRT1's specific involvement more fully.
Effects of Ethanol on Adiponectin Expression and Secretion
Several studies investigating the effects of chronic ethanol exposure on adiponectin production have provided insights into the changes in adiponectin expression and secretion in adipocytes following chronic ethanol exposure. In differentiated 3T3-L1 adipocytes, coincubation of ethanol with unsaturated fatty acids (either oleic or linoleic acid) decreased adiponectin secretion, suggesting that ethanol may interact with the specific fatty acids in the cultured adipocytes media to inhibit adiponectin expression (7). Chronic ethanol feeding to mice, rats, and micropigs significantly decreased adiponectin mRNA levels in adipose tissues (6–11). Accordingly, protein levels of adiponectin in both adipose tissue and plasma were also decreased in those animals. Moreover, in chronically ethanol-fed rats, decreased adiponectin secretion from subcutaneous adipocytes was associated with a disruption in the intracellular trafficking of adiponectin (9). All of these studies suggest that chronic alcohol feeding has inhibitory effects on circulating adiponectin levels in animals.
Role of TNFα in Ethanol Regulation of Adiponectin
Adiponectin and tumor necrosis factor α (TNFα) suppress each others' gene expression and production. Circulating adiponectin levels have been found to be inversely related to serum TNFα concentrations in several ethanol-fed animal models (6–8, 11). Therefore, ethanol's depression of adiponectin might be the consequence of elevated TNFα. However, it remains unclear whether increased TNFα resulting from ethanol feeding causes adiponectin reduction or whether suppression of adiponectin production by ethanol leads to TNFα induction.
Role of Homocysteine in Inhibition of Adiponectin Production by Ethanol
As discussed earlier, chronic ethanol exposure induces hyperhomocysteine levels in alcoholics and in ethanol-fed rodents, due to abnormal hepatic or adipose methionine metabolism. Homocysteine has recently been suggested as a regulator of adiponectin levels (10). In cultured primary adipocytes, exogenous homocysteine treatment resulted in decreased mRNA expression and protein levels of adiponectin. In ethanol-fed mice, an elevated homocysteine level in adipose tissue correlates closely with decreased adiponectin production from adipose tissue. Both betaine and SAM can lower homocysteine levels in ethanol-fed animals by correcting abnormal methionine-homocysteine metabolism. In ethanol-fed animals, betaine or SAM supplementation lowered the homocysteine levels, blocked ethanol-induced reduction of circulating adiponectin levels, and prevented the development of liver injuries including steatosis (10, 11). Taken together, these studies suggest that ethanol-induced hyperhomocysteinemia contributes to decreased adiponectin production and subsequent increased lipid accumulation in liver.
Role of PPARγ in Ethanol Regulation of Adiponectin
Several studies have indicated that activation of the transcription factor PPARγ increases adiponectin expression and production in adipocytes. The thiazolidinedione (TZD) class of insulin-sensitizing drugs (e.g., rosiglitazone and pioglitazone) are known potent ligands for PPARγ. The TZD-mediated increase in levels of gene expression, total protein, and HMW isoform of adiponectin have been attributed to their ability to stimulate PPARγ activity in adipocytes (reviewed in refs.45, 46).
Several lines of investigation have demonstrated that pioglitazone prevents alcohol-induced liver injury in rodents, implying that upregulation of adiponectin by pioglitazone via activation of PPARγ may contribute to its hepatic protective effects (49–51). Although chronic ethanol feeding did not alter adipose mRNA expression of PPARγ in animals (11), PPARγ ligand binding function could be suppressed by ethanol. Indeed, ethanol exposure significantly reduced both basal and clofibrate-stimulated PPARγ-dependent transcriptional activities in rat hepatic cells (41). Therefore, ethanol's inhibitory effects on adiponectin may be mediated through a direct downregulation of PPARγ activity in adipocytes.
Involvement of SIRT1 in Ethanol Regulation of Adiponectin
We recently found that resveratrol (a potent agonist of SIRT1) treatment of ethanol-fed mice markedly increased serum adiponectin concentrations with prevention of hepatic fat accumulation (J. M. Ajmo and M. You, unpublished observations). Moreover, this upregulation of adiponectin was associated with increased mRNA levels of SIRT1 and FOXO1 in adipose tissues, suggesting that an ethanol-dependant decrease in the expression of SIRT1 and/or FOXO1 in adipose tissues may contribute to the diminished adiponectin levels associated with AFLD.
On the other hand, SIRT1 has been shown to downregulate adiponectin through its inhibition of PPARγ (48, 52). Accordingly, ethanol-mediated inhibition of SIRT1 should result in increased PPARγ activity and adiponectin levels. However, as chronic ethanol treatment is known to suppress plasma adiponectin levels, this is clearly not the case. Thus, it seems unlikely that ethanol's inhibition of adiponectin involves the SIRT1-PPARγ axis.
Adiponectin, an adipose-derived hormone, exerts its effects through interactions with AdipoRs and plays a vital role in lipid homeostasis in liver. Hypoadiponectinemia and altered hepatic AdipoR expression are associated with the development of alcoholic fatty liver in several animal models. Adiponectin exerts a protective effect against alcoholic liver steatosis by coordinating multiple signaling pathways mediated by SIRT1, AMPK, PGC-1α, PPARα, and SREBP-1 leading to diminished lipogenesis, increased fat oxidation and prevention of hepatic lipid accumulation. The exact mechanism by which ethanol downregulates adiponectin remains to be determined. Available evidence suggests that chronic alcohol feeding has inhibitory effects on both gene and protein expression and secretion of adiponectin by adipose tissue. Moreover, ethanol-mediated hyperhomocysteinemia, or alteration of adipose signaling molecules such as SIRT1 and/or FOXO1 may contribute to reduced adiponectin levels associated with chronic ethanol exposure. The current understanding of adiponectin and its receptors in relationship to alcoholic fatty liver is summarized in Fig. 1.
In addition to playing a crucial role in the regulation of hepatic lipid metabolism, adiponectin also has potent antiinflammatory activity. Adiponectin treatment reduces the levels of both TNFα and reactive oxygen species (ROS) in Kupffer cells of animals chronically fed ethanol (reviewed in refs.53, 54). Signaling of TNFα and ROS has a significant impact on the activities of hepatic transcriptional regulators involved in the development of AFLD including SIRT1, AMPK, and SREBP-1 (55–57). It will be of great interest to determine whether the protective effect of adiponectin against alcoholic liver steatosis is also mediated through its anti-inflammatory effect.
Ultimately, more research on the relationship between ethanol and adiponectin is needed to clarify the role of adiponectin in AFLD. A multifaceted approach is suggested here. First, it will be important to determine the physiological significance of individual adiponectin oligomeric forms and how chronic ethanol exposure affects their expression. Second, investigators should study how ethanol interacts with various dietary factors to cause changes in the expression of adiponectin and its receptors. Third, it would be beneficial to determine whether the well-known, gender-related differences in susceptibility to AFLD are the result of ethanol's effects on adiponectin, its isoforms, and receptors. Fourth, in order to make our knowledge of AFLD more relevant, studies with human subjects must be undertaken.
Finally, given the beneficial effects of adiponectin on AFLD, the development of pharmacological and/or nutritional strategies for increasing adiponectin signaling is desirable. Specifically, upregulation of adiponectin, either by treatment with PPARγ agonists or by dietary supplementation with resveratrol, select saturated fatty acids, medium chain triglycerides, SAM or betaine may provide novel and effective remedies for AFLD.
This work was conducted with support from grants AA015951 and AA013623 (to M. You). We are indebted to the outstanding technical and intellectual contributions of Xiaomei Liang, Zheng Shen, Brandi Pennock, and Drew Rideout.