The results reported here demonstrate what we believe to be a novel biochemical and pharmacological mechanism for alcohol-induced fatty liver, a common medical problem. Our results demonstrate that, consistent with results of prior in vitro and in vivo studies in animals and humans, ethanol promotes hepatic adenine nucleotide release (1
), which is subsequently dephosphorylated extracellularly to adenosine by the action of CD73 (5
). As previously reported, adenosine levels are further increased as a result of diminished adenosine uptake in the liver (32
). Chronic alcohol-stimulated adenosine release stimulates adenosine A1
receptors, which promote the development of fatty liver, since blockade or deletion of these receptors in vivo and blockade of these receptors in vitro diminishes hepatic triglyceride accumulation and development of fatty liver.
Adenosine and its receptors regulate a variety of hepatic and hepatocellular functions, including glucose release (34
), protein synthesis (36
), glutathione synthesis (37
), hepatic regulation of renal Na+
and water excretion, and portal blood flow (18
). Moreover, ethanol- and acetate-induced adenosine release mediates many of these effects. Thus, although the effects of adenosine (whether exogenous or released in response to either ethanol or acetate) and its receptors on hepatic triglyceride metabolism have not previously been explored, the demonstration that adenosine and its receptors mediate ethanol-induced changes in hepatic function is not without precedent.
Previous studies provide indirect support for a role for adenosine and its receptors in the pathogenesis of fatty liver. Muroyama et al. (41
) found that ingestion of a mixture of caffeine, arginine, thiamine, and citric acid reduced body fat, triceps skinfold thickness, and serum triglyceride levels in healthy human subjects with a high percentage of body fat and was effective in reducing visceral fat, including liver fat, in obese subjects. Caffeine, a nonselective adenosine receptor antagonist, in combination with vitamins and arginine, significantly suppressed an increase in hepatic lipid content in fasted and refed diabetic KK mice (42
). Thus, adenosine receptors may also play a role in the pathogenesis of nonalcoholic fatty liver as well.
Recently, Osei-Hyiaman et al. (44
) found that endocannabinoid activation of hepatic cannabinoid receptors (CB1
receptors) is associated with development of diet-induced hepatic steatosis in mice, and others have reported that ethanol ingestion leads to stellate cell production of endocannabinoids, which activate hepatic CB1
receptors on hepatocytes, leading to hepatic steatosis (45
). Treatment of rats with cannabinoid receptor antagonists prevents the development of fatty liver in animal models as well (46
). In the CNS, adenosine A1
receptors are tightly linked to CB1
receptors and these receptors cross-activate and cross-desensitize each other (48
), although their interaction in the liver has not been explored. The results reported here are consistent with and expand upon the known link between cannabinoid receptors and adenosine receptors and further suggest that adenosine receptors play a role in nonalcoholic hepatic steatosis.
Prior studies, as well as the results reported here, clearly demonstrate that adenosine A1
, and A3
receptors are expressed on hepatocytes (11
) and chronic ethanol ingestion increases A1
, and A2B
receptor expression in the liver. Adenosine receptors are expressed ubiquitously and other cell types in the liver express adenosine receptors as well: stellate cells express adenosine A1
, and A2B
); Kupffer cells and sinusoidal endothelial cells express adenosine A2A
). Since ethanol has a variety of CNS effects, many of which are due to increased CNS adenosine levels with resulting A2A
receptor activation (56
), it is also possible that extrahepatic effects of adenosine may lead to hepatic steatosis by, for example, production of neuroendocrine mediators or increased food intake. However, the demonstration that adenosine A1
receptors directly stimulate steatosis in AML-12 hepatocytic cells is more consistent with the hypothesis that adenosine receptors directly regulate hepatocyte metabolism.
Alterations in 3 major regulatory pathways in the hepatocyte contribute to the development of fatty liver: stimulation of SREBP1 activation, inhibition of AMPK, and diminished PPARα activation/increased PPARγ activation. SREBP1 is a member of the basic helix-loop-helix leucine zipper (bHLH-ZIP) family of transcription factors that is synthesized as a 125-kDa precursor attached to the nuclear envelope and endoplasmic reticulum (58
). In sterol-depleted cells, the membrane-bound precursor is cleaved to generate a soluble NH2-terminal fragment that translocates to the nucleus (60
). SREBP1 plays an active role in regulating the transcription of genes involved in hepatic triglyceride synthesis (including ACC
, stearoyl-CoA desaturase-1, ACL
, and l
-α-glycerophosphate acyltransferase; refs. 61
). Ethanol and its metabolites activate SREBP1 (63
), and alcohol-induced fatty liver correlates with activation and induction of SREBP1 (30
). In our studies ethanol and adenosine A1
receptor occupancy significantly increased nuclear SREBP1 and downstream expression of ACL
mRNA expression, both deletion and antagonism of adenosine A1
receptors significantly diminished their expression in vivo and in vitro (Figures and ). These results are consistent with the hypothesis that adenosine A1
receptors regulate SREBP1 expression and activation to increase expression of fatty acid synthetic enzymes (Figure ).
Ethanol-induced adenosine release provokes fatty change via adenosine A1 and A2B receptor–mediated stimulation of increased fatty acid synthesis and diminished fatty acid utilization, respectively.
PPARα, -β/δ, and -γ belong to the nuclear receptor superfamily (65
). PPARα is activated by sterols and is translocated to the nucleus, in which it stimulates the transcription of a variety of enzymes and transporters that promote fatty acid oxidation (23
). Ethanol prevents the activation and nuclear translocation of PPARα (66
), effects abrogated by deletion or blockade of adenosine A2B
receptors. In contrast to PPARα, PPARγ appears to play a direct role in the development of fatty liver (46
). PPARγ is expressed at very low levels in the liver, and overexpression in the liver leads to hepatic steatosis with the expression of several adipogenic genes (26
). Conversely, PPARγ agonists have been used to treat nonalcoholic fatty liver (74
), possibly by increasing expression of the receptor for adiponectin (75
). Ethanol increases Pparg
mRNA expression and expression of lipid synthetic enzymes ACL and FAS in mouse liver, and deletion or blockade of adenosine A1
receptors decreased expression of these genes consistent with the results of the in vitro hepatocyte experiments. These results suggest the hypothesis that adenosine A1
receptors regulate PPARγ and PPARα activation and expression to promote hepatic steatosis (Figure ).
AMPK also plays a key role in the regulation of cellular metabolism. Once activated by phosphorylation of threonine-172 or by AMP, AMPK strongly activates ACC, 3-hydroxy-3-methyl-glutaryl-CoA reductase, and other targets, leading to fatty acid oxidation and diminished cholesterol synthesis (31
). Ethanol inhibits AMPK activation, leading to accumulation of fatty acids within the hepatocyte (30
). Alcohol ingestion diminished AMPK phosphorylation in mouse liver, an effect reversed by deletion or blockade of adenosine A2B
receptors but not A1
receptors. Studies carried out in vitro provided parallel results. Thus, adenosine A2B
receptors are also likely to promote hepatic triglyceride accumulation by diminishing AMPK phosphorylation and activity as well as by diminishing PPARα transcriptional activity (Figure ).
We demonstrated, for the first time to our knowledge, that all 4 adenosine receptors are present in healthy human liver plasma membranes. The number and affinity of adenosine A1
receptors does not change in cirrhotic and fatty livers but the number of A2A
receptors increases in cirrhotic and fatty livers. Prior studies have demonstrated a number of factors that may regulate the expression of adenosine A2A
receptors, including such inflammatory cytokines as IL-1 and TNF (79
) and endotoxin (81
). Interferon-γ, which stimulates increased expression of A2B
), may diminish A2A
receptor signaling (79
The results reported here demonstrate what we believe to be a novel pathogenic mechanism for the development of fatty liver following chronic ethanol ingestion: ethanol-induced adenosine release stimulates hepatic steatosis via activation of A1 and A2B receptors. It is also possible that adenosine and its receptors play a role in the pathogenesis of nonalcoholic fatty liver disease as well. Moreover, these results suggest that adenosine receptor antagonism may provide a novel approach for the development of agents for the treatment and prevention of alcoholic and, possibly, nonalcoholic fatty liver disease.