Hypoxia-inducible factor-2α promotes fibrosis in non-alcoholic fatty liver disease by enhancing glutamine catabolism and inhibiting yes-associated protein phosphorylation in hepatic stellate cells

Abstract

Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence and affects approximately one-third of adults, owing to high-fat dietary habits and a sedentary lifestyle. The role of hypoxia-inducible factor 2α (HIF-2α) in NAFLD progression remains unknown. This study aimed to investigate the effects of chronic hypoxia on NAFLD progression by examining the role of hypoxia-inducible factor 2α (HIF-2α) activation and that of hepatic stellate cell (HSC)-derived myofibroblasts through glutaminolysis. We hypothesised that hypoxia exacerbates NAFLD by promoting HIF-2α upregulation and inhibiting phosphorylated yes-associated protein (YAP), and that increasing YAP expression enhances HSC-derived myofibroblasts. We studied patients with NAFLD living at high altitudes, as well as animal models and cultured cells. The results revealed significant increases in HSC-derived myofibroblasts and collagen accumulation caused by HIF-2α and YAP upregulation, both in patients and in a mouse model for hypoxia and NAFLD. HIF-2α and HIF-2α-dependent YAP downregulation reduced HSC activation and myofibroblast levels in persistent chronic hypoxia. Furthermore, hypoxia-induced HIF-2α upregulation promoted YAP and inhibited YAP phosphorylation, leading to glutaminase 1 (GLS1), SLC38A1, α-SMA, and Collagen-1 overexpression. Additionally, hypoxia restored mitochondrial adenosine triphosphate production and reactive oxygen species (ROS) overproduction. Thus, chronic hypoxia-induced HIF-2α activation enhances fibrosis and NAFLD progression by restoring mitochondrial ROS production and glutaminase-1-induced glutaminolysis, which is mediated through the inhibition of YAP phosphorylation and increased YAP nuclear translocation. In summary, HIF-2α plays a pivotal role in NAFLD progression during chronic hypoxia.

Keywords: HIF-2α; NAFLD/NASH; YAP/p-YAP; glutaminolysis; hepatic stellate cells-derived myofibroblasts.

Figure 1

HIF-2α exacerbates fibrosis in patients with NASH by increasing GLS1 expression during chronic hypoxia. (A) Serum ALT levels were higher in male patients with NASH and hypoxemia (haemoglobin >180 g/L) compared to those with normal haemoglobin levels (120–180 g/L) (p < 0.01). (B) Relative concentration of amino acids in plasma (n=6). (C) H&E staining shows increased fibrosis in NASH patients with hypoxaemia. Immunohistochemistry staining shows increased GLS1 expression in liver tissues of patients with NASH and hypoxaemia. Immunofluorescence staining shows a non-significant increase in the expression of HIF-1α and a significant increase in HIF-2α expression and its co-localisation with α-SMA in liver sections from patients with NASH and hypoxemia. (n = 6; magnification, 200×). (D) GLS1 staining was significantly increased in patients with NASH and hypoxemia compared to those in the control group (n = 6). (E) HIF-2α protein levels were significantly increased in patients with NASH and hypoxemia compared to those in the control group (n = 6). (F) HIF-2α and α-SMA protein levels were significantly increased in patients with NASH and hypoxemia compared to those in the control group (n = 6). Data are presented as means±SD of independent experiments. H&E, Haematoxylin and eosin staining; NASH, non-alcoholic hepatitis; GLS1, Glutaminase 1; HIF-1α, hypoxia-inducible factor-1α; α-SMA, α-smooth muscle actin.

Figure 2

Chronic hypoxia exacerbates fibrosis in mice with NAFLD and NASH, and is associated with HIF-2α and YAP1 overexpression. (A) H&E and Sirius red staining of liver tissue from mouse models for NAFLD and NASH show increased fibrosis in response to hypoxia (n = 5; magnification, 200×). (B) Sirius red staining quantification confirmed increased fibrosis in a mouse model for NASH in response to hypoxia (n = 5). (C) Mice subjected to hypoxia showed impaired glucose tolerance according to the results of the glucose tolerance test. (D) Western blot analysis showed increased expression of α-SMA, YAP1, HIF-2α, and Col1A in mouse models subjected to hypoxia. (E–I) Quantification of western blot results confirmed increased expression of α-SMA, YAP1, HIF-2α, and Col1A, but not HIF-1α, in mouse models subjected to hypoxia (n = 5). (J) Quantification of p-YAP(S127) expression showed decreased phosphorylation of YAP in mouse models in response to hypoxia (n = 5). (K, L) Quantification of mRNA expression levels showed increased expression of SLC38A1 and GLS1 following hypoxia (n = 5). Bars represent mean ± SD of n=5 mice/group. *: p < 0.05 between the two groups under the same conditions, ns: p≥0.05 between the two groups under the same conditions, ^#p < 0.05 vs Control group. NAFLD, Non-alcoholic fatty liver disease; NASH, Nonalcoholic steatohepatitis; YAP1, Yes-associated protein 1; H&E, Haematoxylin and eosin; HIF-2α, Hypoxia-inducible factor-2α; Col1A, Collagen type I alpha 1; α-SMA, α-smooth muscle actin; p-YAP(S127), Phosphorylated yes-associated protein.

Figure 3

Chronic hypoxia impaired hepatic mitochondria and inhibited the oxidative phosphorylation complex in mouse models for NAFLD and NASH. (A) Representative TEM images showing the morphology of mitochondria in liver samples from mouse models for NAFLD and NASH. Samples from animals subjected to hypoxia showed enlarged and swollen mitochondria with disordered cristae. (B) Image of western blot from mouse liver lysates showing bands corresponding to OXPHOS complexes, with β-Tubulin used as the loading control. (C–G) Quantification of band densities for OXPHOS complexes I, II, III, IV, and V. The samples corresponding to animals subjected to hypoxia showed decreased expression for all OXPHOS complexes (n = 5; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs Control group). TEM, Transmission electron microscopy; NAFLD, Non-alcoholic fatty liver disease; NASH, Non-alcoholic hepatitis; OXPHOS, oxidative phosphorylation. ns: No significance.

Figure 4

Chronic hypoxia augmented mitochondrial ROS production by impairing mitochondrial membrane and opening mPTPs in hepatocytes from mouse models for NAFLD and NASH. (A) Flow cytometry images and visualisations of the ΔΨm, ROS, and mPTP opening. Mice subjected to hypoxia showed decreased ΔΨm, increased ROS production, and increased mPTP opening. (B) Quantification of JC-1 levels of ΔΨm. Hypoxic mice showed a significantly decreased ΔΨm. (n = 5; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs Control group). (C) Quantification of mitochondrial ROS levels. Hypoxic mice showed significantly increased ROS production. (n = 5; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs Control group). (D) Quantification of mPTP opening. Mice subjected to hypoxia showed a significant increase in mPTP opening (n = 5; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs Control group). ROS, Reactive oxygen species; mPTP, Mitochondrial permeability transition pore; NAFLD, Non-alcoholic fatty liver disease; NASH, Non-alcoholic hepatitis; ΔΨm, mitochondrial membrane potential.ns: No significance.

Figure 5

HIF-2α overexpression triggered by hypoxia augmented YAP-induced myofibroblast differentiation and inhibited the mitochondrial oxidative phosphorylation complex, and ATP production in LX-2 cells. (A) Western blot results for fibrosis-related proteins from LX-2 cell lysates, with β-actin as loading control. Cell cultures subjected to hypoxia showed increased expression of HIF-2α, α-SMA, YAP1, and Col1A, and decreased phosphorylation of YAP (Ser127). (B–F) Quantification of western blot results confirmed increased expression of HIF-1α, HIF-2α, α-SMA, Col1A, YAP1, and decreased expression of p-YAP (Ser127) in hypoxic cultures. (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs normoxia group). The mRNA expression levels of SLC38A1 (G) and GLS1 (H) were increased in hypoxic cultures. (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs normoxia group). (I) ATP production was decreased in cells subjected to hypoxia. (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs normoxia group). (J) Western blot results for oxidative phosphorylation proteins from LX-2 cell lysates, with VDAC1 as loading control. Cell cultures subjected to hypoxia showed decreased expression of all OXPHOS complexes. (K) Quantification of western blot results confirmed decreased expression of all OXPHOS complexes in hypoxic cultures (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs normoxia group). YAP1, Yes-associated protein; HIF-1α, Hypoxia-inducible factor-1α; HIF-2α, Hypoxia-inducible factor-2α; Col1A, Collagen type I alpha 1; α-SMA, α-smooth muscle actin; p-YAP(S127), Phosphorylated yes-associated protein; GLS1, Glutaminase 1; OXPHOS, oxidative phosphorylation. ns: No significance.

Figure 6

HIF-2α overexpression triggered by hypoxia augmented YAP-induced myofibroblast differentiation in a glutamine-dependent manner, which was abolished by the downregulation of HIF-2α and YAP in LX-2 cells. (A) GLS1 mRNA expression in LX-2 cells under different conditions. Hypoxia increased GLS1 expression, which was abolished by HIF-2α and YAP1 downregulation (n = 3; *p < 0.05 between the two groups under the same conditions, #p < 0.05 vs control group). (B) SLC38A1 mRNA expression in LX-2 cells under different conditions. Hypoxia increased SLC38A1 expression, which was abolished by HIF-2α and YAP1 downregulation (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs control group). (C) GLS1 mRNA expression in LX-2 cells after HIF-2α and YAP1 downregulation. HIF-2α and YAP1 downregulation abolished the hypoxia-induced increase in GLS1 expression (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs control group). (D) SLC38A1 mRNA expression in LX-2 cells after HIF-2α and YAP1 knockdown. HIF-2α and YAP1 knockdown abolished the hypoxia-induced increase in SLC38A1 expression (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs control group). (E) Immunofluorescence staining with Alexa Fluor 488 (green) for α-SMA and Col1A in LX-2 cells, and colocalization with DAPI (blue). (F) Quantification of α-SMA expression. Hypoxia increased α-SMA expression, and this was abolished by HIF-2α and YAP1 downregulation (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs control group). (G) Quantification of Col1a expression. Hypoxia increased Col1A expression, which was abolished by HIF-2α and YAP1 downregulation (n = 3; *p < 0.05 between the two groups under the same conditions, ^#p < 0.05 vs control group). Scale bar: 50 μm. Similar results were obtained from three independent experiments, and representative photographs are shown in each case. Data are presented as means ± SD (n=3). GLS1, Glutaminase 1; YAP1, Yes-associated protein; HIF-2α, Hypoxia-inducible factor-2α; Col1A, Collagen type I alpha 1; α-SMA, α-smooth muscle actin. ns: No significance.

Figure 7

Expression levels and distribution of YAP1 and p-YAP (Ser 127) in LX-2 cells after HIF-2α and YAP1 knockdown. (A) LX-2 cells stained with DAPI to visualise nuclei (blue) and with antibody-conjugated Alexa Fluor 488 or Alexa Fluor 647 to visualise the distribution of HIF-2α (green) and YAP1 (red), respectively. (B) LX-2 cells stained with DAPI to visualise nuclei (blue) and antibody-conjugated Alexa Fluor 488 to visualise the distribution of p-YAP (Ser 127) (green). (C) Quantification of HIF-2α immunofluorescence staining relative to DAPI intensity in LX-2 cells. (D) Quantification of YAP1 immunofluorescence staining relative to DAPI intensity in LX-2 cells. (E) Quantification of p-YAP(S127) immunofluorescence staining relative to DAPI intensity in LX-2 cells. (F) Analysis of immunofluorescence staining intensity ratios between YAP1 and p-YAP (Ser 127). Scale bar: 50 μm. Similar results were obtained from three independent experiments, and representative photographs are shown in each case. Data are presented as means ± SD (n=3). *P<0.05, NS: P>0.05 compared with the same conditions. ^#P<0.05 compared with the control group. YAP1, Yes-associated protein; p-YAP(S127), Phosphorylated yes-associated protein; HIF-2α, Hypoxia-inducible factor-2α.