Losartan Prevents Hepatic Steatosis and Macrophage Polarization by Inhibiting HIF-1α in a Murine Model of NAFLD - PubMed (original) (raw)
Losartan Prevents Hepatic Steatosis and Macrophage Polarization by Inhibiting HIF-1α in a Murine Model of NAFLD
Cheng-Hui Wang et al. Int J Mol Sci. 2021.
Abstract
Hypoxia and hepatosteatosis microenvironments are fundamental traits of nonalcoholic fatty liver disease (NAFLD). Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with NAFLD, whereas the route and regulation of lipid droplets (LDs) and macrophage polarization related to systemic inflammation in NAFLD is unknown. Losartan is an angiotensin II receptor antagonist, that approved portal hypertension and related HIF-1α pathways in hepatic injury models. Here, we show that losartan in a murine model of NAFLD significantly decreased hepatic de novo lipogenesis (DNL) as well as suppressed lipid droplets (LDs), LD-associated proteins, perilipins (PLINs), and cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector (CIDE) family in liver and epididymal white adipose tissues (EWAT) of ob/ob mice. Obesity-mediated macrophage M1 activation was also required for HIF-1α expression in the liver and EWAT of ob/ob mice. Administration of losartan significantly diminishes obesity-enhanced macrophage M1 activation and suppresses hepatosteatosis. Moreover, HIF-1α-mediated mitochondrial dysfunction was reversed in ob/ob mice treated with losartan. Together, the regulation of HIF-1α controls LDs protein expression and macrophage polarization, which highlights a potential target for losartan in NAFLD.
Keywords: HIF-1α; lipid droplet; losartan; macrophage polarization; non-alcoholic fatty liver disease.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Losartan attenuated NAFLD development in ob/ob mice. (A) Body weight (g). (B) Liver weight/body weight (%). (C) Plasma ALT (IU/dL). (D) Plasma AST (IU/dL). (E) Plasma TG (mg/dL). (F) Plasma FFA (mmol/L). For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. ALT, alanine transaminase; AST, aspartate aminotransferase; TG, triglyceride; FFA, free fatty acid.
Figure 2
Losartan decreased HIF-1α and LD-associated proteins. (A) Representative HE, Oil Red O, HIF-1α, PLIN1, PLIN2, CIDEA and CIDEC staining of liver from normal mice and losartan-treated ob/ob mice. Green pseudo-color represents visualization of lipofuscin’s autofluorescence at 450–490 nm. Red arrow highlights the positive staining. Scale bar: 100 μm. Quantification of (B) HIF-1α, PLIN1, PLIN2, CIDEA, and CIDEC protein levels by Western blot of liver after losartan treatment. Below graphs indicate quantification relative to Histone or β-actin. (C) Quantification of ATGL, HSL, LPL, and ACO by qRT-PCR. qRT-PCR indicates quantification relative to GAPDH. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. HIF-1α, hypoxia-inducible factor-1α; LDs, lipid droplets; HE, hematoxylin and eosin; PLIN, perilipin; CIDE, cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector; ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; ACO, acyl-CoA oxidase.
Figure 3
Losartan attenuates lipogenesis and improves lipolysis in liver. (A) Representative SREBP-1 and CD36 staining of liver from-treated ob/ob mice and normal mice. Scale bar: 100 μm. (B) Quantification of SREBP-1 and CD36 protein levels by Western blot of liver after losartan treatment. Right-hand graphs indicate quantification relative to Histone (for SREBP-1) and β-actin (for CD36). (C) Quantification of SREBP-1c, FAS and SCD-1, CD36, and FATP by qRT-PCR. qRT-PCR indicate quantification relative to GAPDH. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. SREBP-1, sterol regulatory element binding protein 1; CD36, cluster of differentiation 36; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; FATP, fatty acid transport protein.
Figure 4
Losartan enhanced mitochondrial biogenesis and function in ob/ob mice. (A) The staining for SIRT1, PGC1α, and UCP1 are green, while the staining for mitochondria (Mito Tracker) is red. Staining for DAPI is blue. Magnification of tissue samples is 20× (red box) and for inset, magnification is 40× (white box). Scale bar: 100 μm. (B) Quantification of SIRT1, PGC1α, UCP1, and UCP2 protein levels by Western blot of liver after losartan treatment. Right graphs indicate quantification relative to Histone (for SIRT1 and PGC1α) and β-actin (for UCP1 and UCP2). Quantification of (C) PGC1α, NRF1, NRF2, TFAM, (D) PPARα, CPT-1, CPT-2, LCAD, and MCAD by qRT-PCR. qRT-PCR indicate quantification relative to GAPDH. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. SIRT1, sirtuin-1; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPAR, peroxisome proliferator-activated receptor; UCP1, uncoupling protein 1; NRF, nuclear respiratory factor; TFAM, mitochondrial transcription factor A; CPT, carnitine palmitoyltransferase; LCAD, long-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase.
Figure 5
Losartan related macrophage polarization and angiogenesis in liver. (A) Quantification of IL-1β, MCP-1, TGFβ, TNFα, and IFNγ by qRT-PCR. qRT-PCR indicates quantification relative to GAPDH. (B) Representative CD11b, CD11c, CCR7, CD163 and CD206 staining of liver. Red arrow highlights the positive staining. Scale bar: 100 μm. (C) Quantification of CD11c, CCR7, CD163, and CD206 protein levels by Western blot of liver. Below graphs indicate quantification relative to β-actin. Ratio of CD11c at CD206 and CCR7 at CD163 in liver. (D) Representative TGFβR2, VEGF, and MMP9 staining of liver. Red arrow highlights the positive staining. Scale bar: 100 μm. (E) Quantification of TGFβR2 and VEGF protein levels by Western blot of liver. Below graphs indicate quantification relative to β-actin. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; TGFβ, transforming growth factor beta; TNFα, tumor necrosis factor α; IFNγ, interferon gamma; TGFβR2, transforming growth factor beta receptor 2; VEGF, targets vascular endothelial growth factor; MMP9, matrix metallopeptidase 9.
Figure 6
Losartan attenuated HIF-1α expression and LDs formation in EWAT. (A) Representative HIF-1α staining of EWAT from losartan-treated ob/ob mice and normal mice. Red arrow highlights the positive staining. Scale bar: 100 μm. Quantification of (B) HIF-1α protein level by Western blot. Below graphs indicate quantification relative to Histone. (C) Representative HE, PLIN1, PLIN2, CIDEA and CIDEC staining of EWAT from losartan-treated ob/ob mice and normal mice. Scale bar: 100 μm. Quantification of (D) PLIN1, PLIN2, CIDEA, and CIDEC protein levels by Western blot of EWAT after losartan treatment. Below graphs indicate quantification relative to β-actin. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. HIF-1α, hypoxia-inducible factor-1α; LDs, lipid droplets; EWAT, epididymis white adipose tissue; HE, hematoxylin and eosin; PLIN, perilipin; CIDE, cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector.
Figure 7
Losartan altered macrophage polarization in EWAT. (A) Quantification of IL-1β, MCP1, TGFβ, TNFα, and IFNγ by qRT-PCR. qRT-PCR indicates quantification relative to GAPDH. (B) Representative F4/80, CD11b, CD11c, CCR7, CD206 and CD163 staining of EWAT. Red arrow highlights the positive staining. Scale bar: 100 μm. (C) Quantification of CD11c, CCR7, CD206, and CD163 protein levels by Western blot of EWAT. Below graphs indicate quantification relative to β-actin. The ratio of CD11c at CD206 and CCR7 at CD163 in the EWAT. For each animal group, n = 5. All values represent the mean ± SEM. Data were analyzed by Student’s t test. * p ≤ 0.05; normal vs. ob/ob. # p ≤ 0.05; ob/ob vs. ob/ob + Losartan. EWAT, epididymal white adipose tissue; IL-1β, interleukin-1β; MCP1, monocyte chemoattractant protein-1 (CCL2); TGFβ, transforming growth factor beta; TNFα, tumor necrosis factor α; IFNγ, Interferon gamma.
References
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