Hypoxia Induces Dysregulation of Lipid Metabolism in HepG2 Cells via Activation of HIF-2α

Oxygen is crucial to the process of metabolism. A state of hypoxia, or lack of adequate oxygen, activates a variety of complex pathways at both the cellular and organ level to reinstate oxygen homoeostasis. The liver is essential to metabolism, and is extremely sensitive to low-oxygen conditions. Lipid accumulation in the blood vessels and other tissues of the liver leads to an insufficient blood supply to hepatocytes and lipid peroxidation, setting up a hypoxic microenvironment; prolonged hypoxia can promote uncontrolled lipid accumulation [1,2]. The liver's metabolic functions include lipid biosynthesis, uptake, catabolism, and reverse cholesterol transport. Lipid biosynthesis in the liver is regulated by sterol regulatory element-binding proteins (SREBPs), a family of transcription factors that include the isoforms SREBP-1a, SREBP-1c, and SREBP-2 [3]. SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of fatty acids, cholesterol, and triglycerides, whereas SREBP-1c preferentially regulates enzymes of fatty acid synthesis. Fatty acid synthase (FAS) is the principle target of SREBP-1c [4,5]. SREBP-2 regulates cholesterol biosynthesis and uptake, in particular the low-density lipoprotein receptor (LDLR) [6,7]. Another target of SREBP-2 is 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), the rate-limiting step and major control point of de novo cholesterol synthesis [8,9].

Other genes important to lipid metabolism in the liver include adipose differentiation-related protein (ADRP), which participates in fatty acid uptake. Carnitine palmitoyltransferase-1α (CPT-1α), a target of peroxisome proliferator-activated receptor alpha (PPAR-α), controls mitochondrial fatty acid import [5]. Hydroxyacyl-CoA dehydrogenase (HADH) catalyzes the NAD⁺-dependent dehydrogenation step in fatty acid β-oxidation [10].

Cholesterol catabolism involves mainly the biodegradation of cholesterol to bile acids occurring in the hepatocyte endoplasmic reticulum, and begins with the transformation of cholesterol into 7-alpha-hydroxy cholesterol catalyzed by CYP7A1 (cytochrome P450 family 7 subfamily A polypeptide 1) [11,12,13]. Cholesterol clearance pathways are also mediated by members of the ATP-binding cassette (ABC) transporter family. An example is ABCA1 (ABC subfamily A member 1), which participates in transport of cholesterol in the hepatocyte to lipoprotein receptors and the formation of HDL cholesterol [14,15]. ABCG5 and ABCG8 (ABC subfamily G members 5 and 8, respectively) regulate the excretion of liver cellular cholesterol into bile [16,17].

The hypoxia-inducible factor (HIF) family of transcription factors has a central role in the main pathway associated with hypoxic stress [18,19]. They participate in tissue adaptive responses to hypoxia by regulating the expression of hypoxia-induced genes. allowing survival within a large range of reduced oxygen concentrations. The HIF family are heterodimers composed of one α subunit that is unstable in the presence of relatively high levels of oxygen (above 5%), and one constitutively expressed β subunit (or ARNT [aryl hydrocarbon receptor nuclear translocator]). Mammals possess three HIF isoforms, HIF-1, HIF-2, and HIF-3.

Under normoxic conditions, HIF-α subunits are targeted for proteasomal degradation by the Von Hippel-Lindau (VHL) tumor suppressor (Pvhl) catalyzed by oxygen-dependent prolyl hydroxylase domain (PHD) proteins (PHD 1, 2 and 3). Under hypoxic conditions, HIF-α subunits are stabilized and translocate to the nucleus to activate transcription upon binding to hypoxia response elements located within regulatory elements of HIF-target genes.

HIF-2α, also known as EPAS1 (Homo sapiens endothelial PAS domain protein 1), HLF (HIF-like factor), HRF (HIF-related factor), or MOP2 (member of PAS superfamily 2), was first discovered by Tian et al. in 1997 [20]. Recent studies have reported that many cellular processes such as angiogenesis, erythropoiesis, energy metabolism, cell migration, and tumor invasion are influenced by HIF-2α. Since oxygen consumption in mitochondria seriously influences fatty acid oxidation during hypoxia, it is conceivable that lipid metabolism should be regulated through activation of HIFs. However, the effects of HIF-2α and HIF-1α on lipid metabolism have not been definitively determined.

In certain cell types, the expression of PPARα and the DNA-binding affinity of PPARα were reduced during hypoxia, in a HIF-1α-dependent manner [21]. Moreover, HIF-1α has been reported to induce the expression of HIG2, a lipid droplet protein involved in neutral lipid formation [22], and HIF-1α was even considered a protective factor against alcoholic fatty liver [23]. In contrast, Rankin et al. [24] disclosed the importance of HIF-2α rather than HIF-1α in the regulation of lipid droplet formation and continuous lipid accumulation in VHL-deficient mouse liver. This conclusion was further supported by another study indicating that development of fatty liver disease in PHD2/3 knockout mice was under the control of HIF-2α [25]. Considerable controversy remains regarding hepatic steatosis in HIF-2α-deficient mice [26], and lipid accumulation stimulated by forced expression of HIF-1α in mice [27].

Considering the controversies surrounding HIFs in lipid metabolism, it is important to understand all of the mechanisms of hypoxia-induced lipid dysregulation and clarify the roles of HIFs separately. In this study, we focused on whether HIF-2α is associated with lipid metabolism under hypoxic conditions in cells of the human hepatoma cell line HepG2, and investigated the molecular processes activated within the cells.

The Ethics Committee of Nanjing Medical University approved this study. Six male C57BL/6 wild-type mice (age 10 weeks; 22-25 g) were purchased from Model Animal Research Center of Nanjing Medical University, Nanjing, China. The 6 mice were randomly apportioned to a control group or an ischemic-reperfusion (IR) group. Mice were anesthetized by intraperitoneal injection of 60 mg/kg sodium pentobarbital. The skin was disinfected with 1% iodine. The incision was mid-abdominal to allow for full liver exposure. For mice in the IR group, blood vessels were clamped with a non-invasive vascular clamp for one hour as described previously [28], which led to segmental (70%) hepatic ischemia, and then the liver was reperfused for 6 h before tissues were harvested. Mice in the control group underwent the same procedure, but with no clamping of the blood vessels. The mice were euthanized by cutting the diaphragm and heart. Hepatic tissue was obtained and rapidly stored at -70 °C for future analysis.

Cells of the human hepatocellular carcinoma cell line HepG2 were purchased from Shanghai Cell Bank (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Wisent, South America) with 15% fetal bovine serum (FBS; Wisent, South America). Cells were incubated in normoxic (21% O₂, 5% CO₂) or hypoxic (1% O₂, 5% CO₂, 94% N₂) conditions with a humidified chamber at 37 °C.

To silence the expression of HIF-2α, for stable transfection the plasmids pGPU6/GFP/Neo-HIF-2α-homo-1250 and pGPU6/GFP/Neo-shNC were designed and synthesized by GenePharma (Shanghai, China; Table 1). For transient transfection, HIF-2α-silencing small interfering RNA (siRNA; EPAS1-homo-2778) and negative control siRNA (siNC) were also obtained from GenePharma (Shanghai, China; Table 2).

For gene transfection, the cells were cultured in 6-well plates to 50% confluence. The plasmids or siRNAs were transfected into cells using Lipofectamine 2000 in accordance with the manufacturer's instructions (Invitrogen, USA). Cells were transfected for 6 h in medium without FBS. After changing to normal culture medium and culturing for 48 h, siRNA and siNC cells in transient transfection were prepared for further experiments. For stable transfection, 1 mg/mL G418 was incubated with cells for 14 d to obtain stable transfected cell lines (shRNA and shNC cells).

Total RNA from cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the PrimeScript RT Kit (Takara) in accordance with the manufacturer's instructions. Concentrations were measured using a NanoDrop spectrophotometer (ND-1000V3.5.2 software, USA). RT-qPCR was performed with FastStart Universal SYBR Green Master (Rox) (Roche, USA) with an ABI StepOne machine (Applied Biosystsems, Life Technologies, CA, USA). Briefly, qPCR cycling was performed as follows: initial denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 10 s; annealing for 60 s at 60 °C; and a melting curve profile set at 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. Transcript expression levels were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels as an endogenous control. Primers for qPCR were synthesized by Invitrogen (Shanghai, China; Table 3).

Proteins were prepared by homogenizing cells in RIPA buffer (Beyotime, China) containing a protease inhibitor cocktail mix from cOmplete ULTRA Tablets (Roche, Germany) on ice. The protein concentration was determined using a bicinchoninic acid protein assay kit (Keygen, Nanjing, China). Proteins were resolved via SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Temecula, CA, USA), blocked with milk for 2 h, probed with primary antibodies at 4 °C overnight, and incubated with secondary antibodies for 2 h at room temperature. The primary antibodies were: HIF-2α (Abeam, Cambridge, MA, USA), HIF-1α (human; Cell Signaling, USA), HIF-1α (mus; Santa Cruz, USA), and SREBP-1 ( Santa Cruz, USA), each used at a dilution of 1:200; FAS (Cell Signaling, USA) was diluted at 1:1000; β-actin and GAPDH from BioWorld (Irving, TX, USA) were diluted at 1:1000. Secondary antibodies were obtained from ZhongShan JinQiao (China). Protein levels were normalized to β-actin or GAPDH. Electrochemiluminescence was analyzed with a Chemilmager 5500 imaging system (Alpha Innotech, San Leandro, CA, USA).

HepG2 cells were plated in 6-well plates. Cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 30 min and then rinsed twice with PBS. The cells were stained with Nile red (1 µg/mL) in 60% 2-propanol for 5 min and washed three times with PBS. Images were taken with an inverted fluorescence microscope (Olympus, Tokyo, Japan) equipped with an image recorder under a 10× lens.

HepG2 cells at a concentration of 1 × 10⁴/mL were cultured on 22 mm × 22 mm coverslips in 6-well plates overnight. According to group, the corresponding treatment was given. Cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 30 min at 4 °C. They were subsequently treated with 0.1% Triton for 3 min, and washed three times with PBS. Cells were stained with 50 µg/mL Filipin III fluorescent dye at 37 °C for 30 min and washed with PBS three times. Images were obtained with a fluorescent microscope (Olympus) at 10× lens.

HepG2 cells were collected and homogenized in RIPA buffer. Intracellular triglyceride and free cholesterol were measured by using triglyceride and free cholesterol assay kits (Biovision, USA), in accordance with the kit manuals. Triglyceride and cholesterol levels were normalized to total protein concentrations.

HADH activity was measured as described previously [29,30]. Briefly, HepG2 cell pellets were homogenized in 1:10 wt/vol homogenization buffer (50 mM Tris-HCl, 1 mM EDTA, 10% glycerol [wt/vol], 0.1% Triton-100). After homogenization, the cell lysates were centrifuged at 8000 rpm for 30 min at 4 °C. In each well of a 96-well UV plate, 260 µL imidazole (50 mM, pH 7.4), 20 µL reduced nicotinamide adenine dinucleotide (0.15 mM, ε = 6.22 mol· mL^-1· cm^-1), and 10 µL cell lysate were added. The absorbance was detected at 30 °C with a SpectraMax 190 spectrophotometer (Molecular Devices), at 340 nM for 5 min with 30-s intervals both before (baseline) and after adding 10 µL of 3 mM acetoacetyl CoA. HADH activity was normalized to total protein concentrations.

Cholesterol efflux was measured as described previously [31]. Briefly, HepG2 cells were cultured in DMEM media containing 15% FBS, trace labeled with 1 µCi/ml ³H-cholesterol (Perkin-Elmer, USA) overnight. Before treatment, cells were washed with PBS and equilibrated in media for 2 h. Cells were then incubated under normoxia or hypoxia conditions for 24 h, in serum free DMEM containing 10 µg/ml apoA-I and 2 mg/ml fatty acid-free BSA for 24 h. Supernatants were collected, centrifuged to remove cell debris, and radioactivity was quantified by scintillation counting (Microbeta2, Perkin Elmer). Cells were washed with PBS and lysed with 0.1 M NaOH, and cell lysates were analyzed by scintillation counting. Cholesterol efflux was calculated by cpm in media/(cpm in media+cpm in cellular lipid) and expressed in percent.

The equation 2^-∆∆^CT was applied to calculate the fold changes of gene expression. Data are presented as the mean ± standard deviation for at least three separate experiments. Statistical analysis was performed using the paired 2-tailed Student's t test. A P-value of <0.05 was considered statistically significant.

To assess the role of HIF-2α in lipid metabolism, we first determined the HIF-2α mRNA and protein levels in HepG2 cells under normoxia and hypoxia (Fig. 1A, 1C). HIF-2α protein expression increased in hypoxia over time, in vitro reaching a maximum at 24 h of hypoxia. However, there was no change in HIF-2α mRNA levels.

To confirm our in vitro data, we created mouse liver models of ischemic-reperfusion. RNAs and protein was extracted from hepatic tissues as described in Methods. RT-qPCR and western blot analysis both revealed a slight but statistically significant increase in HIF-2α expression in IR mice compared to controls (Fig. 1B, 1D). We also determined quantities of HIF-1α protein both in vitro and in vivo and obtained similar results (Fig. 1E, 1F).

In the present study, we established stable HIF-2α-silenced (shRNA) and negative control (shNC) cell lines by selecting transfected cells with G418. The transfection efficiency was 35% to 40%. The knockdown efficiency was confirmed by western blot analysis (Fig. 2A).

Similarly, the efficiency of knockdown in transient transfection was also confirmed by western blot (Fig. 2B). Western blot analyses showed that cells in which RNA interference (RNAi) had been used had lower levels of HIF-2α protein under hypoxic conditions. However, levels in shNC and siNC cells were comparable to the corresponding controls. As expected, minor efficiency of RNA interference was demonstrated under normoxic conditions.

We detected HIF-1α expression in HepG2 cells to verify the specificity of designed siRNA and shRNA, which was not reversed by HIF-2α shRNA or siRNA (Fig. 2C, 2D). These results indicated that the siRNA/shRNA used in our study specifically targeted HIF-2α mRNA, without an effect on the expression of HIF-1α.

To investigate lipid accumulation in HepG2 cells, we used Nile red staining. Nile red fluorescent dye binds to a wide variety of lipids including triacylglycerol and most fatty acids [32]. Under normoxic conditions, the fluorescence levels of the three groups were not different (Fig. 3A). Twenty-four hours in a hypoxic environment induced marked lipid accumulation in HepG2 cells relative to the control cells that were cultured in normoxic conditions. Comparing siRNA and control cells under hypoxic environments, the results suggested that the accumulation effect was partly inhibited by silencing of HIF-2α, judging from the decreased fluorescence intensity. No significant differences were observed between siNC and the control cells.

We also detected free cholesterol accumulation by means of filipin staining (Fig. 3B). The results were similar to those obtained by Nile red staining. Compared to the control group, blue fluorescence obviously increased under 24-h of hypoxia, evidence of increased cholesterol accumulation. However, under hypoxic conditions the amount of fluorescence increased less in siRNA cells than in the control group, and the degree of fluorescence of siNC cells and that of the control group were very similar. These results suggest that cholesterol accumulation induced by hypoxia can be partly reversed by inhibition of HIF-2α.

Similar results were obtained in the quantitative measurements of lipid levels in HepG2 cells (Fig. 3C, 3D). The increased triglyceride and free cholesterol levels under hypoxic conditions were significantly prevented by silence of HIF-2α expression, compared with the shNC control group.

Expression of ADRP mRNA increased 3.3-fold after 24 h of hypoxia, while CPT-1α mRNA expression was reduced by 45.1% (P < 0.05, both; Fig. 4A, 4B). SREBP-1a and FAS protein levels appeared to be unaffected by hypoxic conditions (Fig. 4C). HADH enzymatic activity, a measure of β-oxidation, was found to be remarkably inhibited by hypoxic conditions (Fig. 4D).

In the case of cholesterol accumulation, genes involved in catabolism (ABCA1, ABCG5, ABCG8, CYP7A1) all low expressed under hypoxia for 24 h, which dropped to 13.2%, 24.0%, 23.4%, 4.8%, respectively (Fig. 5A-D). Unexpectedly, the mRNA expression of genes involved in anabolism (SREBP-2, LDLR, and HMGCR) also were reduced 65.7%, 49.6% and 89.0% under hypoxic conditions (Fig. 5E, 5F, 5G).

To explore the cellular mechanism involved in fatty acid regulation by HIF-2α, we examined via RT-qPCR in shRNA cells the key genes responsible for lipid uptake and β-oxidation. Compared to the shNC group, ADRP mRNA expression was reduced by 33% in the shRNA group under hypoxic conditions (Fig. 6A). There was no difference in CPT-1α mRNA levels between the two groups under hypoxic conditions, nor in HADH enzymatic activity (Fig. 6B, 6C).

To further investigate the possible pathway controlled by HIF-2α in cholesterol metabolism, we also evaluated by RT-qPCR the expression of key genes involved in cholesterol uptake, de novo synthesis, degradation, and reverse cholesterol transportation. Our data indicated that ABCA1 mRNA had a slight but significant increase when HIF-2α was silenced under hypoxia (P< 0.05; Fig. 7A). The cholesterol efflux relevant to hypoxia was also reversed obviously (Fig. 7H). However, between the shRNA and corresponding shNC group there were no significant differences in the mRNA expression of the other genes of interest (SREBP-2, HMGCR, LDLR, CYP7A1, allABCG5/8; Fig. 7B-G).

Dysregulation of lipid metabolism is increasingly recognized as a risk factor for many metabolic diseases, including nonalcoholic fatty liver disease and cardiovascular disease. Currently it is undecided whether obstructive sleep apnea syndrome affects serum lipids [33,34,35,36,37], but studies suggest that hypoxia may influence lipid metabolism. HIFs are important transcription factors with crucial roles in allowing adaption to hypoxic environments in mammals. In the present study, we investigated the effect of HIF-2α on lipid metabolism and the potential underlying molecular mechanisms.

We detected the expressions of HIF-2α in HepG2 cells in vitro and in C57BL/6 male mice. Under hypoxic conditions, HIF-2α mRNA and protein levels were remarkably higher both in vitro and in vivo, except that mRNA expressed in vitro was not affected by 24-h hypoxic conditions. Data obtained from our animal experiments would be better supported by a larger sample than provided at this time. Two studies conducted by Wiesener et al. [38,39] that support our findings also showed that hypoxia has an effect on HIF-2α at the post-transcriptional level rather than the transcriptional level.

In the current study, we observed that lipid accumulation, including fatty acid and cholesterol, obviously increased under hypoxic conditions. Furthermore, we used siRNAs to specifically inhibit HIF-2α production in the human hepatic carcinoma cell line, HepG2. Results from quantitative analysis and staining showed that both triglyceride and free cholesterol accumulation were augmented by HIF-2α knockdown. Therefore, one possible explanation of hypoxia-induced lipid accumulation in hepatocytes is via a HIF-2α transcription factor pathway, consistent with previous studies using mouse models [26,40].

We also investigated the role of transcription factor induction of specific target genes with a variety of functions related to lipid metabolism and the hypoxic response. Our data suggest that the development of steatosis in HepG2 cells is a consequence of increased lipid uptake and reduced lipid β-oxidation. Hu et al. [41] already demonstrated that ADRP coordinates regulation of HIF-1α and HIF-2α in clear cell renal cancer cells. Also, ADRP was found necessary for lipid storage in fibroblasts and a variety of fatty liver disease models [14,15,16].

Unexpectedly, SREBP-1a and FAS, two key fatty acid synthesis relevant proteins induced by hypoxic conditions [42,43] were not changed in our model. With further analysis, we found that HIF-2α significantly upregulated the uptake of fatty acid (ADRP) expression. Finally, we found that reduced β-oxidation was not regulated by HIF-2α, although inhibition of HIF-2α under hypoxia resulted in a slight increase in CPT-1α mRNA expression. As a reflection of β-oxidation efficiency, HADH enzyme activities were significantly decreased under hypoxic conditions, which also were not affected by HIF-2α. Taken together, these data suggest that during hypoxia, ADRP signaling may be regulated by HIF-2α to participate in fatty acid accumulation. This is similar to the conclusions of Rankin et al. [24].

With regard to cholesterol metabolism, in the present study hypoxia was associated with an obvious decline in expression levels of the key genes in HepG2 cells. The data suggest that hypoxia leads to accumulation of cholesterol via a decrease in biodegradation and reverse transportation. Interestingly, we found that expression of SREBP-2 varied with oxygen concentration, but presented a downward trend under hypoxia. LDLR and HMGCR, which are downstream of SREBP-2, also showed similar trends. Our data is in disaccord with that of Li et al. [44,45]who concluded that SREBP-2 signaling as well as the SREBP-2 target LDLR, are not relevant to oxygen concentration in obese mice. With further analysis, we found that only ABCA1 mRNA was significantly elevated by the inhibition of HIF-2α during hypoxic conditions. Furthermore, we validated that the hypoxia related cholesterol excretion was also regulated by HIF-2α. Other pathways affected by hypoxia may be through other mechanisms. One potential pathway mediated by HIF-1α was suggested by Parathath et al. [46] and Manalo et al. [47]. Endoplasmic reticulum stress may also participate in regulation of lipid metabolism [48,49,50,51].

In conclusion, inhibition of HIF-2α protein reversed hypoxia-induced lipid accumulation in HepG2 cells by decreasing the expression of ADRP, involved in fatty acid uptake, increasing ABCA1 expression and relevant cholesterol excretion. These results suggest a possible mechanism for lipid metabolism dysregulation induced by acute hypoxia. These findings indicate that HIF-2α signaling is relevant to oxygen-dependent lipid homeostasis in the liver, and may have implications for the therapeutic treatment of fatty liver and cardiovascular diseases.

All authors report no conflicts of interest.

We would like to express our gratitude to Dr. Junjie Qin and Dr. Jiajie Hou (Nanjing Medical University, China) for technical assistance with the animal study, Dr. Beth Shoshana Zha (Virginia Commonwealth University, USA) and Meijaden Company (Hongkong, China) for help with the native English editing of the manuscript. The study was supported by the National Natural Science Foundation of China (No.81100273; 81100274) and the innovation projects of Jiangsu Province Hospital (No.CX11).

R. Cao and X. Zhao contributed equally to this work.