Activating Adenosine Monophosphate-Activated Protein Kinase Mediates Fibroblast Growth Factor 1 Protection From Nonalcoholic Fatty Liver Disease in Mice

Abstract

Background and aims: Fibroblast growth factor (FGF) 1 demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic and obese mice by an uncertain mechanism. This study investigated the therapeutic activity and mechanism of a nonmitogenic FGF1 variant carrying 3 substitutions of heparin-binding sites (FGF1^△HBS ) against NAFLD.

Approach and results: FGF1^△HBS administration was effective in 9-month-old diabetic mice carrying a homozygous mutation in the leptin receptor gene (db/db) with NAFLD; liver weight, lipid deposition, and inflammation declined and liver injury decreased. FGF1^△HBS reduced oxidative stress by stimulating nuclear translocation of nuclear erythroid 2 p45-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1^△HBS also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1^△HBS . In palmitate-treated hepatic cells, small interfering RNA (siRNA) knockdown of Nrf2 abolished only FGF1^△HBS antioxidative actions but not improvement of lipid metabolism. In contrast, AMPK inhibition by pharmacological agent or siRNA abolished FGF1^△HBS benefits on both oxidative stress and lipid metabolism that were FGF receptor (FGFR) 4 dependent. Further support of these in vitro findings is that liver-specific AMPK knockout abolished therapeutic effects of FGF1^△HBS against high-fat/high-sucrose diet-induced hepatic steatosis. Moreover, FGF1^△HBS improved high-fat/high-cholesterol diet-induced steatohepatitis and fibrosis in apolipoprotein E knockout mice.

Conclusions: These findings indicate that FGF1^△HBS is effective for preventing and reversing liver steatosis and steatohepatitis and acts by activation of AMPK through hepatocyte FGFR4.

Fig. 1.. The therapeutic effects of FGF1^ΔHBS on nonalcoholic fatty liver disease (NAFLD) in late-stage diabetic mice.

Nine-month-old db/db mice were treated with FGF1^ΔHBS (0.5 mg/kg body weight) or phosphate buffered solution vehicle every other day for 3 months. (A) Liver size and (B) weight. (C) Representative images of H&E-stained and Oil Red O stained liver sections. (D) NAFLD activity score (NAS). (E) Triglyceride contents in liver. (F) Non-esterified fatty acid (NEFA) content in liver. (G and H) Plasma ALT and AST activity. (I-L) The mRNA levels of hepatic inflammatory factors (TNFα, PAI-1, MCP-1 and ICAM-1) in liver tissue. (M) MDA contents in liver tissue. Quantitative data are expressed as mean ± SEM, n=6–10. *P < 0.05, ** P < 0.01 vs. vehicle treated mice. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TNFα, tumor necrosis factor α; PAI-1, plasminogen activator inhibitor-1; MCP-1, monocyte chemoattractant protein-1; ICAM-1, intercellular adhesion molecule 1.

Fig. 2.. FGF1^ΔHBS prevents the hepatic oxidative stress and lipid disorder in late-stage diabetic mice.

Nine-month-old db/db mice were treated with FGF1^ΔHBS (0.5 mg/kg body weight) or PBS vehicle every other day for 3 months. (A-D) The protein expressions of Nrf2 anti-oxidative signaling [nuclear (n)-Nrf2, NQO-1, and HO-1], (F-I) the lipogenic genes [mature/precursor (m/pre) SREBP-1, FAS and SCD-1], and (J-L) AMPK signaling [phosphorylated (p)-AMPK/AMPK and p-ACC/ACC] were detected by western blot and quantified by densitometry. (E) The mRNA expression of SREBP-1, FAS and SCD-1 genes was detected by RT-qPCR. GAPDH or Lamin B1 was used as loading controls for all western blot assays. Quantitative data are expressed as mean ± SEM, n=6–10. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle treated mice. Nrf2, nuclear factor erythroid 2-related factor 2; NQO-1, NAD(P)H dehydrogenase (quinone 1); HO-1, heme oxygenase-1; SREBP-1, sterol regulatory element-binding protein 1; FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 3.. FGF1 protects against palmitate (Pal)-induced oxidative stress and lipid disorder in HepG2 cells.

After serum starvation for 24 hours, HepG2 cells were treated with Pal-bovine serum albumin (BSA, 100 μmol/L) or control BSA at the presence or absence of insulin (Ins, 100 nmol/L) in fetal bovine serum (FBS) free medium for 12 hours, followed by incubation with FGF1^WT and FGF1^ΔHBS (1000 ng/ml) for additional 12 hours, and PBS was used as vehicle control. (A) Representative images of dihydroethidium (DHE) stained HepG2 cells. (B) Quantitative analysis of fluorescent intensity of DHE staining. (C) Malondialdehyde (MDA) contents in cell lysate. (D,E) Lipid deposition was determined by Oil Red O staining. (F) Triglyceride contents in cell lysate. Quantitative data are expressed as mean ± SEM, n=3–5. *P < 0.05.

Fig. 4.. The effects of siRNA Knockdown of Nrf2 on the beneficial effects of FGF1 on palmitate (Pal)-induced oxidative stress and lipid disorder in HepG2 cells.

After serum starvation for 24 hours, cells with or without siRNAs against Nrf2 or control (Ctrl) siRNA were treated with Pal-BSA (100 μmol/L) or control BSA at the presence or absence of insulin (Ins, 100 nmol/L) in fetal bovine serum (FBS) free medium for 12 hours, followed by incubation with FGF1^WT and FGF1^ΔHBS (1000 ng/ml) for additional 12 hours, and PBS was used as vehicle control. (A-D) The protein expressions of Nrf2 signaling [n-Nrf2, total (t)-Nrf2, NQO-1 and HO-1] were determined by western blot and quantified by densitometry. (E) MDA contents in cell lysate. (F) Triglyceride contents in cell lysate. (G-J) The protein expressions of lipogenic genes (m/pre SREBP-1, FAS, SCD-1) were determined by western blot and quantified by densitometry. GAPDH or LaminB1 was used as loading controls for all western blot assays. Quantitative data are expressed as mean ± SEM, n=3–5. *P < 0.05. NS, no significant difference.

Fig. 5.. The effects of siRNA knockdown of AMPKα on the protective effects of FGF1 against palmitate (Pal)-induced lipid disorder and oxidative stress in primary hepatocytes.

After serum starvation for 24 hours, cells with or without siRNAs against AMPKα1/2 or control (Ctrl) siRNA were treated with Pal-BSA (100 μmol/L) or control BSA at the presence or absence of insulin (Ins, 100 nmol/L) in FBS free medium for 12 hours, followed by incubation with FGF1^WT and FGF1^ΔHBS (1000 ng/ml) for additional 12 hours, and PBS was used as vehicle control. (A-C) Protein expressions of AMPK signaling pathway (p-AMPK/AMPK and p-ACC/ACC), the (D-G) lipogenic genes (m/pre SREBP-1, FAS, SCD-1), and (I-L) the Nrf2 signaling (n-Nrf2, NQO-1 and HO-1) were determined by western blot and quantified by densitometry. (H) Triglyceride contents in cell lysate. (M) MDA contents in cell lysate. β-actin or LaminB1 was used as loading controls for all western blot assays. Quantitative data are expressed as mean ± SEM, n=3–5. *P < 0.05. NS, no significant difference.

Fig. 6.. The effects of siRNA knockdown of FGFR4 on FGF1 activation of AMPK and protection from palmitate (Pal)-induced lipid accumulation in primary hepatocytes.

(A) The mRNA expressions of FGFRs (R1–4) in primary hepatocytes at 24 hours after isolation (n=3). (B-F) After serum starvation for 24 hours, cells with or without siRNAs against FGFR4 or control (Ctrl) siRNA were treated with Pal-BSA (100 μmol/L) or control BSA at the presence or absence of insulin (Ins, 100 nmol/L) in FBS free medium for 12 hours, followed by incubation with FGF1^WT and FGF1^ΔHBS for additional 12 hours, and PBS was used as vehicle control. (B) Protein expressions of FGFR4 and AMPK signaling pathway (p-AMPK, AMPK, p-ACC and ACC) were determined by western blot. (C-E) The quantitative analysis of FGFR4, p-AMPK/AMPK and p-ACC/ACC western blots by densitometry. (F) Triglyceride contents in cell lysate. Quantitative data are expressed as mean ± SEM, n=4–6. *P < 0.05. NS, no significant difference.

Fig. 7.. The therapeutic effects of FGF1^ΔHBS on chronic NAFLD induced by high-fat/high-sucrose (HFHS) in mice.

At the age of 2 months, male wild type (WT) and liver specific AMPK knockout (AMPK-LKO) mice were fed on a HFHS diet (D12327, Research Diets) for 5 months, and then supplemented with FGF1^ΔHBS (0.5mg/kg body weight) or PBS vehicle every other day for 1 month. (A) The protein expressions of AMPK signaling (p-AMPK/AMPK and p-ACC/ACC) were detected by western blot. (B,C) The quantitative analysis of western blots by densitometry. (D) Representative images of H&E- and (E) Oil Red O-stained liver sections. (F) Triglyceride contents in liver tissues. (G) MDA contents. (H,I) The plasma ALT and AST activity. β-actin was used as loading controls for all western blot assays. Quantitative data are expressed as mean ± SEM, n=6 for A-C, and n=7–11 for D-I. *P < 0.05 vs WT vehicle treated mice; #P < 0.05 vs WT FGF1^ΔHBS treated mice.

Fig. 8.. The preventive effects of FGF1^ΔHBS on nonalcoholic steatohepatitis (NASH) induced by high-fat/high-cholesterol (HFHC) in ApoE-KO mice.

Eight-week-old male ApoE-KO mice were fed on a HFHC diet and treated with FGF1^ΔHBS (0.5 mg/kg body weight) or PBS vehicle every other day for 3 months. Age- and sex-matched ApoE-KO mice fed a normal chow and treated with PBS vehicle were used as controls. (A) Liver size and (B) weight. Representative images of (C) H&E, (E) Oil Red O, (G) Sirius Red, and (I) F4/80 staining of liver sections. (D) NAFLD activity score (NAS). (F) Triglyceride contents in liver tissues. (H) Quantitative analysis of Sirius Red positive-stained area of liver sections, normalized to “Chow+Vehicle” control group. (J-L) Protein expression for AMPK signaling (p-AMPK/AMPK and p-ACC/ACC) was detected by western blot and quantified by densitometry in liver tissues. α-tubulin was used as loading control. Quantitative data are expressed as mean ± SEM, n=6–9 for A-I, n=5 for J-L. (M) A mechanistic illustration of the basis of FGF1 preventection from metabolic disorder (hyperlipidemia and/or hyperglycemia)-induced hepatic oxidative stress and lipid disorder via FGFR4-mediated activation of AMPK signaling pathways. FGF1 enhances hepatic lipid metabolism and anti-oxidative signaling via FGFR4-mediated activation of AMPK to inhibit the lipid accumulation and activate Nrf2-mediated anti-oxidative signaling pathways, thus preventing metabolic syndrome-induced hepatic lipid disorder and oxidative stress, resulting in protection against NAFLD in T2D and obesity.