The PPARα-FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway - PubMed (original) (raw)

. 2014 Sep 2;20(3):512-25.

doi: 10.1016/j.cmet.2014.06.010. Epub 2014 Jul 17.

Julie Cavanagh-Kyros 2, Luisa Garcia-Haro 1, Guadalupe Sabio 3, Tamera Barrett 2, Dae Young Jung 1, Jason K Kim 4, Jia Xu 5, Hennady P Shulha 5, Manuel Garber 6, Guangping Gao 7, Roger J Davis 8

Affiliations

The PPARα-FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway

Santiago Vernia et al. Cell Metab. 2014.

Abstract

The cJun NH2-terminal kinase (JNK) stress signaling pathway is implicated in the metabolic response to the consumption of a high-fat diet, including the development of obesity and insulin resistance. These metabolic adaptations involve altered liver function. Here, we demonstrate that hepatic JNK potently represses the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). Therefore, JNK causes decreased expression of PPARα target genes that increase fatty acid oxidation and ketogenesis and promote the development of insulin resistance. We show that the PPARα target gene fibroblast growth factor 21 (Fgf21) plays a key role in this response because disruption of the hepatic PPARα-FGF21 hormone axis suppresses the metabolic effects of JNK deficiency. This analysis identifies the hepatokine FGF21 as a critical mediator of JNK signaling in the liver.

Copyright © 2014 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Hepatic JNK contributes to diet-induced obesity and insulin resistance

(A) The liver, adipose tissue (epididymal), and skeletal muscle (gastrocnemius) of LWT, LΔ1, LΔ2 and LΔ1,2 mice were examined by immunoblot analysis by probing with antibodies to JNK and GAPDH. (B) Genomic DNA isolated from the liver of LWT, LΔ1, LΔ2 and LΔ1,2 mice was examined by PCR analysis to detect Jnk and Δ_Jnk_ alleles. (C) Liver extracts prepared from mice fed a chow diet or a HFD (16 wk) and starved overnight were examined by immunoblot (IB) analysis by probing with antibodies to αTubulin and JNK. In vitro protein kinase assays (KA) with the substrates GST-cJun and [γ-32P]ATP were performed to measure JNK activity. The amount of cJun and phosphorylated cJun (p-cJun) were detected by staining with Coomassie blue and Phosphorimager (Applied Biosystems) analysis, respectively. (D) The fat and lean mass of chow-fed and HFD-fed (16 weeks) mice were measured by 1H-MRS analysis (mean ± SEM; n = 8 ~ 10). Significant differences between LWT and LΔ1,2 mice were detected (**, p < 0.01, ***, p < 0.001). (E,F) Glucose (GTT) and insulin (ITT) tolerance tests were performed on mice fed (16 wk) a HFD (mean ± SEM; n = 35 ~ 50; **, p < 0.01; ***, p < 0.001). (G) Pyruvate (PTT) tolerance tests were performed on mice fed (16 wk) a HFD (mean ± SEM; n = 20 ~ 30; *, ***, p < 0.001). (H–K) Insulin sensitivity was measured using hyperinsulinemic-euglycemic clamps with conscious LWT and LΔ1,2 mice fed a chow diet or HFD. The hepatic glucose production (HGP) during the clamp, hepatic insulin action (expressed as insulin-mediated percent suppression of basal HGP), glucose infusion rate, and glycogen plus lipid synthesis (mean ± SEM for 6 ~ 8 experiments) are presented. Statistically significant differences between LWT and LΔ1,2 mice are indicated (* p < 0.05). (L–N) Chow-fed or HFD-fed LWT and LΔ1,2 mice were starved overnight and then administered insulin (1.5U/kg body mass) by intraperitoneal injection. Liver, epididymal adipose tissue, and gastrocnemius muscle extracts (prepared 15 mins post-injection) were examined (upper panels) by multiplexed ELISA for pS473-AKT and AKT (mean ± SEM; n = 5 ~ 6; *, p < 0.05). Representative extracts were also examined by immunoblot analysis using antibodies to AKT, phospho-AKT (pSer473 and pThr308) and GAPDH (lower panels). See also Figures S1 & S2.

Figure 2

Figure 2. Effect of liver-specific JNK-deficiency on hyperinsulinemia

(A) Mice were fed a chow diet or a HFD (16 wk). Sections of the pancreas were stained with an antibody to insulin. Bar, 100μm. (B) Relative islet size was measured using Image J64 software (mean ± SEM; n = 30; *, p < 0.05). (C) The mice were fasted overnight and the blood concentration of insulin was measured (mean ± SEM; n = 16; *, p < 0.05). (D) Glucose-induced insulin secretion was examined using overnight fasted mice by intraperitoneal injection of glucose and measurement of blood insulin concentration (mean ± SEM; n = 8 ~ 10; **, p < 0.01). (E,F) Mice were fed a chow diet or a HFD (16 wk). Blood glucose concentration in mice fasted overnight or fed ad libitum was measured (mean ± SEM; n = 35 ~ 50; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3

Figure 3. Hepatic JNK suppresses the PPARα pathway and fatty acid oxidation

(A) Fgf21 mRNA expression by primary hepatocytes obtained from LWT, LΔ1, LΔ2 and LΔ1,2 mice was measured by quantitative RT-PCR assays (mean ± SEM; n = 6; **, p < 0.01; ***, p < 0.001). (B, C) Heatmap representation of RNA-seq analysis of hepatic genes in overnight fasted HFD-fed mice with the expression profile LΔ1 < LWT < LΔ2 < LΔ1,2. The genes are displayed with lowest expression (top) to highest expression (bottom) in LΔ1 liver. Gene ontology analysis of these genes is presented (C). (D–F) Seahorse XF24 analysis was performed using primary hepatocytes isolated from LWT and LΔ1,2 mice. Mitochondrial oxygen consumption rate (OCR) in the presence of 200μM palmitate/BSA, 15 mM glucose, or 1 mM pyruvate / 10 mM lactate (D). Glucose production rate per μg protein in the presence of 1 mM pyruvate / 10 mM lactate (E). Lactate production by LWT and LΔ1,2 mice hepatocytes (incubated with 15 mM glucose) and the effect of treatment of LWT hepatocytes with 15 mM 2-deoxyglucose (2DG) or 100 nM rotenone (Rot). (F). The data presented are the mean ± SEM; n=10–15; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (G) Differentially expressed glycolysis, tricarboxylic acid cycle, and electron transport chain genes identified by RNA-seq analysis in the liver of HFD-fed LΔ1,2 mice compared with HFD-fed LWT are illustrated (padj < 0.05). (H) LWT and LΔ1,2 mice were fed a chow diet or a HFD (16 weeks). Sections of the liver were stained with hematoxylin and eosin. Bar, 25 μm. (I–K) Gene expression in the liver of overnight-fasted LWT and LΔ1,2 mice was measured by quantitative RT-PCR assays of mRNA (mean ± SEM; n = 8; **, p < 0.01; ***, p < 0.001). See also Figures S3 & S4.

Figure 4

Figure 4. Hepatic JNK-deficiency increases peroxisome number and mitochondrial size

(A, B) LWT and LΔ1,2 mice were fed a HFD (16 wks). Sections of the liver were examined by transmission electron microscopy. Representative images are presented. Key: nucleus (N); lipid droplet (LD). Arrow heads indicate peroxisomes (A) and mitochondria (B). Bar, 2 μm (A) and 0.5 μm (B). (C, D) The number of peroxisomes (C) and mitochondria (D) per field was measured (mean ± SEM; n = 30; *, p < 0.05). (E) Relative mitochondrial DNA copy number was measured (mean ± SEM; n = 3).

Figure 5

Figure 5. Hepatic JNK inhibits PPARα-dependent gene expression

(A) Primary hepatocytes were obtained from LWT and LΔ1,2 mice and incubated with solvent (DMSO, Control), PPARα agonists (50 μM WY14043 or 100 μM Fenofibrate), or PPARα antagonists (10 μM GW6471 or 20 μM MK886) for 16h. PPARα target gene (Acox1, Ehhadh, and Pdk4) expression was examined by measurement of mRNA by quantitative RT-PCR analysis (mean ± SEM; n = 6; ***, p < 0.001). (B) The expression of _Ppar_α, _Rxr_α, Ncor1, Ncor2, and Nrip1 mRNA by LWT and LΔ1,2 primary hepatocytes was measured by quantitative RT-PCR analysis (mean ± SEM; n = 6; ***, p < 0.001). (C) LWT and LΔ1,2 primary hepatocytes were examined by immunoblot analysis by probing with antibodies to PPARα, NCoR1, NCoR2, NRIP1, JNK, and GAPDH. (D) Primary wild-type hepatocytes were treated (12 h) with DMSO (vehicle) or 1μM JNK-in-8 (a small molecule JNK inhibitor) prior to measurement of mRNA expression by quantitative RT-PCR analysis (mean ± SEM; n = 6; *, p < 0.05; **, p < 0.01; ***, p < 0.001). The effect of treatment (8 h) of the hepatocytes with DMSO (NT) or 100 μM Fenofibrate (Fibrate) was examined. (E) Recombinant AAV8 vectors were employed to express Control shRNA or Ncor1 shRNA in the liver of HFD-fed (4 wks.) of LWT and LΔ1,2 mice. Hepatic mRNA expression at 10 ~ 16 days post-infection was examined by quantitative RT-PCR analysis (mean ± SEM; n = 14 ~ 15; *, p < 0.05; **, p < 0.01). (F, G) NCoR1 or GFP (Control) were expressed in the liver of HFD-fed (4 wks.) of LWT and LΔ1,2 mice using recombinant adenovirus vectors (10 ~ 16 days). The hepatic expression of Acox1, Ehhadh & Pdk4 mRNA was measured by quantitative RT-PCR analysis (F). The mice were examined by glucose tolerance tests (G). The data presented are the mean ± SEM (n = 6; *, p < 0.05; ***, p < 0.001). See also Figures S5 & S6.

Figure 6

Figure 6. The FGF21 pathway is repressed by JNK

(A–D) Chromatin immunoprecipitation (ChIP) assays were performed using liver isolated from overnight fasted LWT and LΔ1,2 mice with antibodies to non-immune immunoglobulin (Control), PPARα, NCoR1, NRIP1, and acetyl-lysine16 histone H4 (H4K16ac). The fold enrichment of a fragment of the Fgf21 promoter with a PPRE site was measured by quantitative PCR analysis (mean ± SEM; n = 6; p < 0.05). (E) The hepatic expression of Fgf21 mRNA in overnight fasted mice was examined by quantitative RT-PCR analysis (mean ± SEM; n = 8; ***, p < 0.001). (F) The concentration of FGF21 in the blood of chow-fed and HFD-fed LWT and LΔ1,2 mice was measured by ELISA. Blood was collected from mice fed ad libitum or fasted overnight (mean ± SEM; n=6 *, p < 0.05; **, p < 0.01). (G) Ketone body production was measured in overnight-fasted LWT and LΔ1,2 mice challenged (6 h) without (Control) and with Octanoate (mean ± SEM; n = 5; *, p < 0.05). (H–J) NCoR1 or GFP (Control) were expressed in the liver of HFD-fed (4 wks.) LWT and LΔ1,2 mice using recombinant adenovirus vectors (10 ~ 16 days). The mice were fasted overnight and hepatic expression of NCoR1 and GAPDH were examined by immunoblot analysis (H). The amount of Fgf21 mRNA was measured by quantitative RT-PCR analysis (I) and the concentration of FGF21 in the blood was measured by ELISA (J). The data presented are the mean ± SEM; n = 6; *, p < 0.05; **, p < 0.01. See also Figure S7.

Figure 7

Figure 7. FGF21 mediates metabolic effects of hepatic JNK-deficiency

(A) AAV8 vectors were employed to express Control shRNA and Fgf21 shRNA in the liver. The mice were fed a HFD (4 wk). FGF21 in the blood of mice fed ad-libitum or fasted overnight was measured by ELISA (mean ± SEM; n = 6; *, p < 0.05). (B) Ketogenesis was examined in HFD-fed mice that were fasted overnight and then challenged with octanoate (6 h) by measurement of blood β-hydroxybutyrate (mean ± SEM; n = 15 ~ 30; p < 0.05). The data are normalized to blood β-hydroxybutyrate in control mice at 0 h (100%). (C) Insulin tolerance tests (ITT) were performed on HFD-fed mice. The time course of blood glucose clearance and the area under the curve (AUC) are presented (mean ± SEM; n = 15 ~ 30; *, p < 0.05; **, p < 0.01).

References

    1. Aguirre V, Uchida T, Yenush L, Davis R, White MF. The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307) J Biol Chem. 2000;275:9047–9054. - PubMed
    1. Ahmed SS, Li J, Godwin J, Gao G, Zhong L. Gene transfer in the liver using recombinant adeno-associated virus. Curr Prot Micro. 2013;Chapter 14(Unit14D):16. - PMC - PubMed
    1. Astapova I, Lee LJ, Morales C, Tauber S, Bilban M, Hollenberg AN. The nuclear corepressor, NCoR, regulates thyroid hormone action in vivo. Proc Natl Acad Sci USA. 2008;105:19544–19549. - PMC - PubMed
    1. Badman MK, Koester A, Flier JS, Kharitonenkov A, Maratos-Flier E. Fibroblast growth factor 21-deficient mice demonstrate impaired adaptation to ketosis. Endocrinol. 2009;150:4931–4940. - PMC - PubMed
    1. Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos-Flier E. Hepatic fibroblast growth factor 21 is regulated by PPARalphα and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab. 2007;5:426–437. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources