Steatohepatitis/Metabolic Liver Disease
Article first published online: 22 APR 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 53, Issue 5, pages 1526–1537, May 2011
How to Cite
Nath, B., Levin, I., Csak, T., Petrasek, J., Mueller, C., Kodys, K., Catalano, D., Mandrekar, P. and Szabo, G. (2011), Hepatocyte-specific hypoxia-inducible factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice. Hepatology, 53: 1526–1537. doi: 10.1002/hep.24256
Potential conflict of interest: G. S. is a member of the University of Massachusetts Diabetes Endocrinology Research Center.
Supported by grants R21 AA017544 (to G. S.) and F30 AA017030 (to B. N.) from the National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health. The core resources supported by the Diabetes Endocrinology Research Center grant DK32520 were also used.
- Issue published online: 22 APR 2011
- Article first published online: 22 APR 2011
- Accepted manuscript online: 3 MAR 2011 08:58AM EST
- Manuscript Accepted: 10 FEB 2011
- Manuscript Received: 7 OCT 2010
Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia-regulated hypoxia-inducible factor 1-α, (HIF-1α) may regulate liporegulatory genes, but the relationship of HIF-1 to steatosis remains unknown. We investigated HIF-1α in alcohol-induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels, and increased serum alanine aminotransferase (ALT) levels, suggesting liver injury in wild-type (WT) mice. There was increased hepatic HIF-1α messenger RNA (mRNA), protein, and DNA-binding activity in alcohol-fed mice compared with controls. Mice engineered with hepatocyte-specific HIF-1 activation (HIF1dPA) had increased HIF-1α mRNA, protein, and DNA-binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared with WT mice. In contrast, hepatocyte-specific deletion of HIF-1α [HIF-1α(Hep−/−)], protected mice from alcohol- and lipopolysaccharide (LPS)-induced liver damage, serum ALT elevation, hepatomegaly, and lipid accumulation. HIF-1α(Hep−/−), WT, and HIF1dPA mice had equally suppressed levels of peroxisome proliferator-activated receptor α mRNA after chronic ethanol, whereas the HIF target, adipocyte differentiation-related protein, was up-regulated in WT mice but not HIF-1α(Hep−/−) ethanol-fed/LPS-challenged mice. The chemokine monocyte chemoattractant protein-1 (MCP-1) was cooperatively induced by alcohol feeding and LPS in WT but not HIF-1α(Hep−/−) mice. Using Huh7 hepatoma cells in vitro, we found that MCP-1 treatment induced lipid accumulation and increased HIF-1α protein expression as well as DNA-binding activity. Small interfering RNA inhibition of HIF-1α prevented MCP-1–induced lipid accumulation, suggesting a mechanistic role for HIF-1α in hepatocyte lipid accumulation. Conclusion: Alcohol feeding results in lipid accumulation in hepatocytes involving HIF-1α activation. The alcohol-induced chemokine MCP-1 triggers lipid accumulation in hepatocytes via HIF-1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease. (HEPATOLOGY 2011;)
Alcoholic liver disease (ALD) is a spectrum of disorders ranging from mild and reversible steatosis to life-threatening and irreversible cirrhosis. The cellular and molecular mechanisms that contribute to ALD continue to be elucidated, and over past decades numerous paradigms have been proposed, including the pivotal inflammatory role of tumor necrosis factor α signaling downstream of Toll-like receptor 4 stimulation by gut-derived endotoxin.1 However, no unifying mechanism for hepatic lipid accumulation has emerged thus far, with various lines of evidence suggesting roles for nuclear regulatory factors such as the family of peroxisome-proliferator activated receptors, sterol-regulatory element binding proteins, metabolic enzymes such as cytochrome P4502E1, or hormonal factors such as adiponectin.2-7 Increasing evidence suggests that inflammation and hepatic lipid accumulation are linked processes, because knockout of several genes involved in the inflammatory response, such as those of the Toll-like receptor 4 pathway or nuclear factor κB pathway, also prevent lipid accumulation in response to chronic alcohol feeding.1 A recent report demonstrated that treatment of the hepatoma cell line, Huh7, with the chemokine MCP-1 directly induced lipid accumulation in vitro, raising the possibility that a relationship between proinflammatory mediators and hepatic lipid accumulation may exist.8
The hypoxia-inducible factors (HIFs) are a family of heterodimeric transcription factors that promote a homeostatic transcriptional response to low oxygen tension. Mature HIF is composed of one of three isoforms of an alpha subunit (HIF-1α, HIF-2α, or HIF3α) and a beta subunit, the major isoform of which is termed HIF-1β or the aryl-hydrocarbon receptor nuclear translocator (ARNT). Under conditions of normal oxygen tension, the alpha subunits of HIF are rapidly scaffolded on the Von-Hippel Lindau tumor suppressor protein, where they are hydroxylated and subsequently ubiquitinated and degraded. Under conditions of low oxygen tension, HIF alpha subunits escape hydroxylation and dimerize with HIF-1β/ARNT, translocate to the nucleus and activate hypoxia response elements (HREs) in the genome.9 HIFs are named by their alpha subunit, with HIF-1 and HIF-2 having a wide, overlapping but nonidentical set of transcriptional targets.10 In a recently described model, HIF1dPA, a mutant of HIF-1α construct in which the proline that is normally targeted for hydroxylation is mutated to alanine, enables tissue-specific constitutive activation of HIF.10 In the HIF-1α(Hep−/−) model, floxed exons of the native HIF-1α gene enable tissue-specific ablation of HIF activity.11 Recent investigation with the HIF1dPA model demonstrated that whereas activation of HIF-1 alone resulted in minimal lipid accumulation, and activation of HIF-2 alone resulted in gross vascular changes without any appreciable increase in hepatic lipid, simultaneous activation of HIF-1 and HIF-2 results in a phenotype of hepatomegaly with macrovesicular lipid accumulation.10 However, the relationship of this phenotype to human diseases characterized by steatosis (e.g., alcoholic steatosis or nonalcoholic fatty liver disease) remains to be elucidated.
The role of HIFs in ALD is yet to be fully explored. Liver hypoxia has been documented in rats on a continuous ethanol diet, and some investigators suggest that a process analogous to ischemia-reperfusion injury may be implicated.12-15 Others have postulated an increase in HIF-1α messenger RNA (mRNA) as a mechanism of ethanol-induced liver injury.16 However, the direct contribution of HIF-1 to alcoholic liver injury is unknown.
We hypothesized that HIF-1α protein, mRNA, and downstream gene activation would be up-regulated in the livers of mice after chronic ethanol feeding, and that modifying HIF expression in hepatocytes might affect the progression of ALD. In order to dissect the contribution of HIF-1α to ALD, we used cre-lox mouse models of hepatocyte-specific HIF-1α activation (HIF1dPA) as well as hepatocyte-specific HIF-1α deletion (HIF-1α(Hep−/−)).
Materials and Methods
All animals received care in compliance with protocols approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. Mice were gradually habituated to a Lieber-DeCarli liquid diet with 5% ethanol (vol/vol) over a period of 2 weeks, then maintained on the 5% diet for 4 weeks. Consumption was recorded daily throughout and isocaloric amounts of a non–alcohol-containing diet (in which dextran-maltose replaced calories from ethanol) were dispensed to pair-fed animals. Weights were recorded weekly. Wild-type (WT) mice (C57/Bl6), Alb-Cre, and HIF-1flox/flox mice were purchased from Jackson Laboratories (Bar Harbor, ME). LSL-HIF1dPA mice were a kind gift of William Kim (University of North Carolina, Chapel Hill, NC). The HIF1dPA allele was engineered by Kim et al.10 Briefly, a stop codon is flanked by loxP sites upstream of a HIF-1α transgene in which a proline-to-alanine substitution enables the transgene to escape recognition by proline hydroxylases and subsequent proteasomal degradation. Coexpression of the albumin-cre transgene excises the stop codon, and subsequently enables expression of the transgene in hepatocytes. LSL-HIF1dPA and HIF-1flox/flox mice were bred against Cre mice as described,10, 11 tagged by ear notching, and housed in separate cages.10, 11 Prior to the conclusion of the study, some mice were randomly assigned to receive lipopolysaccharide (LPS) (Sigma) injection (500 μg/kg) or saline injection. Mice were sacrificed 18 hours after LPS injection. At the conclusion of the feeding, mice were weighed and euthanized. Livers were excised and weighed, and portions were snap-frozen in liquid nitrogen for protein and biochemical assays, preserved in 10% neutral-buffered formalin for histopathological analysis, or soaked in RNALater (Qiagen GmbH, Hilden, Germany) for RNA extraction. Blood was collected and serum was separated for biochemical analysis. Tail snips were collected for genotyping. Nuclear extracts were prepared via sucrose gradient centrifugation and two-step purification as described.17
Sections of formalin-fixed livers were stained with hematoxylin/eosin and analyzed via microscopy. Frozen sections were prepared from liver tissue frozen in OCT media and stained with Oil Red O. Photomicrographs were analyzed with Metamorph software.
Multiplex Cytokine Bead Array.
Multiplex cytokine bead array was performed using the BioRad Precision Pro multiplex cytokine bead array kit (BioRad) according to the instructions of the manufacturer. Briefly, serum aliquots stored at −80°C were diluted at a 1:4 ratio using dilution buffer provided in the kit. Serum was allowed to mix with beads coated with antibodies to one of eight different cytokines and subsequently incubated with a second antibody that detects conjugated bead-cytokine pairs.
Cell Culture Studies.
All studies were performed using the human hepatocellular carcinoma cell line Huh7. Cells were maintained in complete growth medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2% penicillin/streptomycin, and 1% 100× amino acid supplement mixture.) For in vitro assays, cells were plated on 10-cm plates and transfected with plasmid DNA or small interfering RNA (siRNA) and/or treated with recombinant monocyte chemoattractant protein-1 (MCP-1) (R&D Systems).
HIF1dPA and HIF2dPA plasmids were a kind gift of William Kim. Plasmid DNA was transfected into Huh7 cells using Fugene transfection reagent according to the manufacturer's instructions. Briefly, Huh7 cells on 10-cm plates at 50%-60% confluence were transfected with 5 μg plasmid DNA (HIF1dPA or HIF2dPA transgenes encoded into pcDNA3.1) with 15 μL Fugene 6 and 160 μL serum-free media. For verification of plasmid transfection, pcDNA3.1 encoding green fluorescent protein was used and cells imaged 24 hours posttransfection with a fluorescent microscope.
HIF-1α siRNA, HIF-2α siRNA, or scrambled siRNA were purchased from Santa Cruz Biotechnology. Transfection was achieved using siPORT Amine transfection agent (Applied Biosystems) according to the manufacturer's protocol. Briefly, for a 10-cm plate, 17 μL siPORT Amine reagent at room temperature was added to 333 μL Opti-MEM serum-free medium. 7.5 μL of 10 μm HIF-1α, HIF-2α, or scrambled siRNA was diluted in 142 μL Opti-MEM. Subsequently, both the transfection reagent and the siRNA mixture were mixed and transfection complexes were formed at room temperature. The mixture (500 μL) was dispersed on a 10-cm plate, and overlaid with 7 × 105 cells in a final volume of 7 mL. After 24 hours, medium was aspirated and replaced with 10 mL complete culture medium for subsequent treatment.
Real-Time Polymerase Chain Reaction.
RNA was purified using the RNeasy Mini kit (Qiagen, Gaithersburg, MD) with on-column DNA digestion (Qiagen). Complementary DNA was prepared using random hexamer primers and a Reverse Transcription System kit (Promega, Madison, WI). Real-time quantitative polymerase chain reaction (PCR) was performed using an iCycler (Bio-Rad Laboratories Inc., Hercules, CA), using specific primers. Primer sequences available on request. Fold change in gene expression was determined by normalizing to 18S mRNA.
Oil Red O Staining.
Cultured and treated cells were washed with 2 mL phosphate-buffered saline. Plates were incubated in 2 mL 10% formalin for 10 minutes, the formalin solution was replaced, and the plates were incubated overnight, then washed twice with ddH2O and once with 60% isopropanol. Dried plates were incubated for 10 minutes in 1 mL of Oil Red O working solution. Plates were immediately destained with four washes of ddH2O and photographed.
Electrophoretic Mobility Shift Assay.
A total of 30-50 μg nuclear extract was resolved on 10% polyacrylamide gels and transferred overnight to nitrocellulose. Membranes were blocked overnight with blocking buffer (5% bovine serum albumin in Tris-Borate-SDS with 0.01% Tween 20) with refrigeration, and subsequently probed overnight with anti–HIF-1α (R&D Biosciences) mouse monoclonal antibodies. Detection was performed using anti-mouse horseradish-peroxidase–conjugated secondary antibody and chemiluminescent substrates.
Quantification and Statistics.
Band density was quantified using Labworks 4.0 image analysis. Statistical analysis was performed with Microsoft Excel using a two-tailed Student t test. P < 0.05 was considered significant.
Chronic Alcohol Administration Induces Liver Steatosis and HIF-1α Activation in Mice.
As has been reported elsewhere, ethanol feeding increased liver weight to body weight ratio, liver triglyceride, and serum ALT values and resulted in liver steatosis in WT mice compared with isocaloric diet-fed controls (Fig. 1A-E). To test our hypothesis that alcohol may increase the expression and activity of hypoxia-inducible factor-1, nuclear extracts from liver tissue were evaluated for HIF-1 expression. We found that HIF-1α mRNA was up-regulated by ethanol feeding in WT mice (Fig. 1D). HIF-1α protein was also more abundant in alcohol-fed than in pair-fed livers (Fig. 2A,B). HIFs are primarily degraded by posttranslational hydroxylation and subsequent degradation of the alpha subunits by the ubiquitin/proteasomal system. To confirm that HIF-1α was transcriptionally active, we performed an EMSA using a commercially available HRE oligonucleotide. Our results showed a significant up-regulation of HIF DNA-binding activity in ethanol-fed animals versus pair-fed animals, suggesting HIF-1 activation (Fig. 2C,D).
Increased Liver Steatosis in Alcohol-Fed HIF1dPA Mice.
In order to determine the contribution of HIF-1α to ethanol-induced liver pathology, we used a mouse model of hepatocyte-specific HIF-1α activation (HIF1dPA) described by Kim et al.10 Due to a mixed genetic background, Alb-Cre littermates that did not harbor the HIF1dPA transgene were selected as controls. To confirm the activation of HIF-1α in HIF1dPA mice, HIF-1α DNA-binding activity was examined in liver nuclear extracts from HIF1dPA and Alb-Cre control mice, and a significant up-regulation of HIF-1α DNA-binding activity was observed (P < 0.01; HIF1dPA pair-fed versus Alb-Cre pair-fed) (Supporting Fig. 1.) We found increased liver weight/body weight (LW/BW) ratios in HIF1dPA mice versus Alb-Cre controls even without alcohol feeding (Fig 3A). Specifically, both HIF1dPA pair-fed mice and control ethanol-fed mice had a statistically significant up-regulation of LW/BW ratio versus pair-fed controls after 4 weeks of ethanol feeding (Fig. 3A). Ethanol-fed HIF1dPA mice had the highest LW/BW ratios (P < 0.05 versus HIF1dPA pair-fed mice).
Examination of liver triglycerides in whole-liver extracts revealed that alcohol caused an up-regulation of triglyceride in hepatic extracts in control mice at 4 weeks (Fig. 3B). Triglyceride levels were higher in pair-fed HIF1dPA mice versus pair-fed control mice (P < 0.05, HIF1dPA pair-fed versus Alb-Cre pair-fed) indicating an effect of constitutive HIF-1α on lipid accumulation in the absence of any other stimulus. Alcohol-fed HIF1dPA mice had the highest average hepatic triglyceride content (P < 0.05 versus all other groups). The presence of HIF1dPA transgene also led to serum ALT levels comparable to Alb-Cre ethanol-fed mice (Fig. 3C). Histopathology analysis also confirmed that ethanol-fed HIF1dPA mice had more lipid vacuolization than ethanol-fed Alb-Cre mice (Fig. 3D). These results suggested that constitutive HIF1 activation in hepatocytes (HIF1dPA mice) results in liver abnormalities reminiscent of ALD and that alcohol feeding and constitutive HIF-1 activation cooperatively up-regulated hepatic steatosis.
Deletion of HIF-1 in Hepatocytes Protects from Alcohol-Induced Liver Damage.
Because our findings suggested an effect of hepatocyte-specific HIF-1α expression on lipid accumulation, we sought to test whether elimination of HIF-1α activity in hepatocytes could ameliorate the pathology associated with chronic ethanol feeding. We used a mouse engineered by Johnson and coworkers11 where native HIF-1α is flanked by LoxP sites, and coexpression of Cre recombinase results in tissue-specific deletion of HIF-1α. Analysis of mice with hepatocyte-specific deletion of HIF-1α and controls maintained on the ethanol diet revealed increased LW/BW ratios in WT ethanol-fed mice versus control mice at 4 weeks. In contrast, HIF-1α(Hep−/−) mice showed no significant difference in LW/BW ratio between pair-fed and ethanol-fed groups (Fig. 4A). Consistent with the role of HIF-1α in hepatocyte steatosis, HIF-1α(Hep−/−) mice were protected from the increase in liver triglyceride content observed in WT mice after alcohol feeding (Fig. 4B). WT mice showed a robust cooperative up-regulation of serum ALT with chronic ethanol and LPS challenge (P < 0.02, WT ethanol/LPS versus WT pair-fed). In contrast, HIF1α(Hep−/−) mice were protected against serum ALT increase, even in the presence of chronic ethanol and LPS (Fig. 4C). Next, we performed immunoblotting on nuclear extracts from WT and HIF-1α(Hep−/−) mice. Ethanol feeding resulted in a significant increase in HIF-1α expression in nuclear extracts prepared from WT mice (Fig. 4D). In contrast, nuclear extracts from HIF-1α(Hep−/−) mice had very low levels of HIF-1α expression, and no further up-regulation with ethanol feeding was observed, confirming suppression of HIF-1α signaling in our mouse model (Fig. 4D,E). On histology examination, livers from WT mice exhibited significant steatosis after chronic alcohol feeding, but no significant difference was observed between livers from pair-fed and ethanol-fed HIF1α(Hep−/−) mice. (Fig. 5).
Because peroxisome proliferator-associated receptor α (PPARα) is associated with lipid accumulation, we examined PPARα mRNA levels in ethanol- and pair-fed control and HIF-1α(Hep−/−) mice. To amplify the effect of ethanol feeding, we also applied an LPS challenge. LPS has been identified in the portal circulation after chronic alcohol intake in mice and men, and it contributes to the development of ALD.20 To our surprise, we found that PPARα was similarly suppressed by ethanol feeding in each of these experimental groups, indicating that the HIF-1α effect on lipid accumulation was independent of PPARα (Fig. 6A). Next, we examined adipocyte differentiation-related protein (ADRP), which has been associated with HIF-1α expression.21 We found that ADRP mRNA was significantly up-regulated with ethanol feeding alone (P < 0.05) (Fig. 6B). Although no cooperative effect of LPS injection and chronic ethanol was observed in ADRP mRNA expression 2 hours after LPS injection (Fig. 6B), by 18 hours there was a robust cooperative up-regulation of ADRP mRNA with chronic ethanol and LPS injection (P < 0.05) (Supporting Fig. 2). HIF-1α(Hep−/−) mice were protected from any up-regulation of ADRP with chronic ethanol alone or with chronic ethanol and LPS challenge (Fig. 6B; Supporting Fig. 2). These results indicated that ADRP may be implicated in the differential effect of HIF-1α on lipid accumulation. Thus, we examined the effect of constitutive HIF activation on the expression of ADRP in HIF1dPA and in control Alb-Cre mice (Fig. 6C). We found a significant increase in ADRP expression with ethanol feeding in Alb-Cre mice, similar to that observed in WT mice (P < 0.02). Furthermore, we found that the presence of the HIF1dPA transgene up-regulated hepatic ADRP protein expression to a similar extent as ethanol feeding (P < 0.01) (Fig. 6D,E).
MCP-1 Induces Steatosis via HIF-1α Expression in Hepatocytes.
In order to further dissect the mechanism of HIF-1α regulation in hepatic lipid accumulation, we supplemented our in vivo work with an in vitro model of hepatic lipid accumulation. The chemokine MCP-1 has recently been demonstrated to result in lipid accumulation in the hepatocyte cell line Huh7.8 First, we examined MCP-1 expression levels in ethanol-fed control and HIF-1α(Hep−/−) mice. We found that alcohol feeding alone resulted in a small, but significant up-regulation in MCP-1 serum levels (Fig. 7A). This corresponded to increased MCP-1 hepatic mRNA with chronic ethanol (Fig. 7B). LPS stimulation and ethanol cooperatively up-regulated MCP-1 in WT mice (Fig. 7C). LPS induced MCP-1 in HIF1α(Hep−/−) mice to an extent comparable to WT, but there was no further increase in HIF-1α(Hep−/−) with alcohol feeding (Fig. 7C). To evaluate mechanistic events, we next treated Huh7 cells with recombinant MCP-1 or with a plasmid containing the degradation-resistant HIF1dPA mutant. MCP-1 treatment resulted in increased HIF-1α protein in nuclear extracts from Huh7 cells (Fig. 8A). We also determined that HIF1dPA overexpression resulted in increased HIF-1α mRNA (Fig. 8B), and this was associated with increased triglyceride levels compared with control cells (Fig. 8C). Furthermore, we found that either MCP-1 treatment or HIF1dPA plasmid treatment resulted in increased lipid accumulation in Huh7 cells (Fig. 8E).
To establish a further mechanistic insight into the role of HIF1 in hepatocyte lipid accumulation, we sought to determine whether we could block lipid accumulation in MCP-1 treated cells by silencing HIF-1α. When Huh7 cells were treated with HIF-1α siRNA, we found that expression of HIF-1α mRNA was significantly suppressed at 24 and 36 hours (Supporting Fig. 3). Next, Huh7 cells that had been pretreated with HIF-1α siRNA were challenged with MCP-1 stimulation. We found increased triglyceride in scrambled siRNA control but not in HIF-1α–siRNA pretreated cells after the MCP-1 challenge (Fig. 8E). Using Oil Red O staining we also confirmed that HIF-1α siRNA pretreatment could prevent MCP-1 treatment–induced lipid accumulation (Fig. 8F). These results suggest a link between alcohol-induced increases in HIF-1, MCP-1, and lipid accumulation in hepatocytes.
In this study, we provide evidence for an effect of HIF-1α on hepatic lipid accumulation in ALD. Although the relationship between alcohol and hypoxia in the liver has been described, our novel observations ascribe a specific pathophysiological role to the dysregulation of a hypoxia-responsive transcription factor in ALD. We found that chronic alcohol feeding results in increased HIF-1α levels and activation in the liver. We further demonstrated that constitutive activation of HIF-1α in hepatocytes accelerates lipid accumulation with chronic ethanol feeding, and report that HIF1dPA mice have higher steatosis on histology evaluation and increased hepatic triglyceride levels compared with control mice. We report for the first time that alcohol-induced lipid accumulation can be prevented in mice with hepatocyte-specific deletion of HIF-1α. Using an in vitro system, we found that inhibition of HIF-1α prevents lipid accumulation. We also demonstrated that the protective effect of HIF-1α deletion may be independent of PPARα, and may depend upon regulation of other genes involved in lipid homeostasis, including the adipocyte differentiation related protein. Our data further suggested that the up-regulation of MCP-1 observed in LPS-injected, ethanol-fed mice may be an upstream mediator of HIF-1α expression, as MCP-1 treatment resulted in increased HIF-1α expression in vitro. Finally we present data to show that inhibition of HIF-1α prevents lipid accumulation in vitro in response to MCP-1 treatment.
Our novel observations link alcohol-induced induction of HIF-1α and alcohol-induced steatosis in a mechanistic way. Previous studies have suggested a role for HIFs in hepatic lipid accumulation, and our findings offer a novel perspective on the role of HIFs in alcohol-induced steatosis. We found induction of HIF-1α after alcohol feeding and demonstrated that hepatocyte-specific inhibition of HIF-1 prevented the alcohol-induced steatosis, suggesting that HIF-1α alone can mediate alcohol-induced steatosis. This observation is somewhat different from the results of Rankin et al.,22 who recently described a dominant role for the HIF-2α isoform in hepatic lipid regulation using a scheme of cre-lox–mediated activation of HIF-1α or HIF-2α in hepatocytes; in that model, disruption of either HIF isoform in combination with pVHL knockout resulted in activation of the remaining isoform. Their findings, however, were in sharp contrast to work by Scortegagna et al.23 that demonstrated that adult HIF-2 knockout mice developed severe hepatic steatosis that could be reversed by treatment with a superoxide dismutase inhibitor. Kim et al., as well, found no significant contribution to hepatic lipid accumulation with a constitutively active mutant of HIF-2, despite finding a robust effect on angiogenesis. On the other hand, they demonstrated a mild HIF-1α–dependent effect on lipid accumulation.10 The different genetic techniques used to create specific gene expression or knockout in each of these studies may offer some explanation of the different results each describes.
Many of the genes involved in lipid homeostasis are regulated by HIFs.24, 25 However, it is yet to be dissected whether significant differences exist in the contribution of HIF-1α and HIF-2α in a given cell type and/or cell-specific effects. Our data suggest that in hepatocytes both in vivo and in vitro (in mice as well as in human cells), HIF-1α activation alone is sufficient to induce lipid accumulation. We explored the contribution of ADRP, a lipid droplet-associated surface protein that is regulated by HIF.21 ADRP has been shown to be up-regulated in human steatosis as well as in mice developing steatosis after a high-fat diet.26, 27 Here we report the novel observation that ADRP is up-regulated with chronic ethanol alone. We found further cooperative up-regulation of ADRP in WT mice after alcohol feeding and LPS injection that correlated with HIF-1α induction. ADRP was up-regulated with constitutive HIF-1α expression but conversely, ADRP up-regulation with chronic ethanol and/or LPS injection was prevented in mice with hepatocyte-specific HIF-1α deletion. This suggested a mechanistic role for HIF-1α in ADRP induction and liver steatosis.
Increasing evidence suggests that lipid accumulation is affected by proinflammatory stimuli. In support of this notion, the chemokine MCP-1 was recently shown to cause lipid accumulation in human hepatoma cells.8 We found a synergistic up-regulation of MCP-1 in the serum of chronic alcohol-fed, LPS-challenged mice suggesting that increased gut-derived LPS could amplify MCP-1 induction in ALD. Consistent with the role of HIF-1α in induction of MCP-1 by LPS, we found lower increase in MCP-1 serum levels in mice with hepatocyte-specific HIF-1 deletion compared with controls after an in vivo LPS challenge. These data imply a role of HIF-1α in MCP-1 induction in ALD. We speculate that gut-derived LPS could be an inducer of HIF-1α in vivo as serum LPS levels are increased after chronic alcohol feeding in mice and reportedly in humans as well.28 More important, we found that MCP-1 can induce HIF-1α mRNA, and protein in hepatoma cells in vitro and this was associated with induction of lipid accumulation in hepatocytes. Together, these results suggest a cross-regulation between HIF-1α activation and MCP-1 in promoting hepatocyte lipid accumulation where MCP-1 can induce HIF-1α activation and in turn, HIF-1α can contribute to MCP-1 induction.
The clinically relevant question is the implication of these findings for the development of clinical strategies for the treatment of alcoholic fatty liver disease. Whereas cessation of alcohol use tends to result in a rapid reversal of alcoholic fatty liver, the increasingly recognized epidemic of the related entity, nonalcoholic fatty liver disease, suggests that therapies that modify hepatic lipid accumulation are likely to find clinical use.
Additional Supporting Information may be found in the online version of this article.
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