Prevention of steatosis by hepatic JNK1

Abstract

Nonalcoholic steatosis (fatty liver) is a major cause of liver dysfunction that is associated with insulin resistance and metabolic syndrome. The cJun NH(2)-terminal kinase 1 (JNK1) signaling pathway is implicated in the pathogenesis of hepatic steatosis and drugs that target JNK1 may be useful for treatment of this disease. Indeed, mice with defects in JNK1 expression in adipose tissue are protected against hepatic steatosis. Here we report that mice with specific ablation of Jnk1 in hepatocytes exhibit glucose intolerance, insulin resistance, and hepatic steatosis. JNK1 therefore serves opposing actions in liver and adipose tissue to both promote and prevent hepatic steatosis. This finding has potential implications for the design of JNK1-selective drugs for the treatment of metabolic syndrome.

Figure 1. Mice with hepatocyte-specific deficiency of JNK1 are glucose intolerant

(A) The liver and quadriceps muscle of Alb-cre Jnk1^+/+ (L^WT) mice, Alb-cre Jnk1^LoxP/LoxP (L^KO) mice, and Jnk1^−/− (KO) mice were examined by immunoblot analysis by probing with antibodies to JNK1 and GAPDH. (B) L^WT and L^KO mice were fed a chow diet (ND) or a high fat diet (HFD) for 16 wks. JNK activity in epididymal (white) fat and liver was measured in a protein kinase (KA) assay using cJun and ATP[γ-³²P] as substrates. The cell extracts used for the protein kinase assay were also examined by immunoblot analysis by probing with an antibody to GAPDH. (C) The phosphorylation of IRS1 on Ser-307 in the liver was examined using ND- and HFD- fed L^WT and L^KO mice by immunoblot analysis by probing with antibodies to IRS1 and phosphoSer-307 IRS1. (D) Chow- fed L^WT and L^KO mice were fasted overnight and administered glucose (2g/kg) by intraperitoneal injection. The activation of AKT in the liver was examined by immunoblot analysis by probing with antibodies to AKT and phosphoAKT. (E) Glucose tolerance tests of chow-fed L^WT and L^KO mice were performed by measurement of blood glucose concentration in animals following intraperitoneal injection of glucose (1g/kg). The data presented represent the mean ± SD (n =10 ~ 15). Statistically significant differences are indicated (*, P < 0.05; **, P < 0.01). (F) Chow- fed L^WT and L^KO mice were fasted overnight or fed ad libitum and the blood glucose concentration was measured (mean ± SD; n = 10 ~ 15). Statistically significant differences are indicated (***, P < 0.001).

Figure 2. JNK1-deficiency in hepatocytes increases insulin clearance

Chow-fed L^WT and L^KO mice were fasted overnight. (A) The concentration of blood insulin was measured (mean ± SD; n = 10). No statistically significant difference between L^KO and L^WT mice was detected. (B) The effect of administration of glucose (2 g/ kg body mass) by intraperitoneal injection on blood insulin concentration was examined (mean ± SD; n = 13~15). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.01). (C) The concentration of insulin C-peptide in the blood was measured (mean ± SD; n = 10~15). Statistically significant differences between L^KO and L^WT are indicated (****, P < 0.0001). (D) The effect of administration of glucose (2 g/kg body mass) on insulin C-peptide concentration in the blood was examined (mean ± SD; n = 15). No statistically significant difference between L^KO and L^WT was detected). (E) Mice were injected with human insulin (1.5U/kg body mass). The concentration of human insulin in the blood was measured (mean ± SD; n = 15). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.05). (F) The expression of the insulin receptor, Ceacam-1, and Gapdh mRNA in the liver was measured by quantitative RT-PCR (Taqman^©) assays. The expression of insulin receptor (InsR) and Caecam-1 mRNA was normalized to the amount of 18S RNA in each sample (mean ± SD; n = 7). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.05; ***, P < 0.001).

Figure 3. Mice with JNK1-deficient hepatocytes exhibit hepatic insulin resistance

(A-F) Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp in conscious chow-fed L^KO and L^WT mice. (A) Basal hepatic glucose production (HGP). (B) Insulin-stimulated rate of HGP. (C) Hepatic insulin action, expressed as insulin-mediated percent suppression of basal HGP. (D) Insulin-stimulated whole body glucose turnover. (E) Whole body fat mass measured using ¹H-MRS. (F) Whole body lean mass. The data presented are the mean ± SE for 6 ~ 8 experiments. Statistically significant differences between L^KO mice and L^WT mice are indicated (*, P < 0.05). (G,H) Chow-fed L^KO and L^WT mice were administered insulin (0.3U/kg body mass) by intravenous injection (5 mins). The activation of AKT in the liver was examined by immunoblot analysis by probing with antibodies to AKT and phosphoAKT. The relative amount of phosphoAKT is presented as the mean ± SD (n = 3). Statistically significant differences between L^KO and L^WT mice are indicated (*, P < 0.05). (I) Chow-fed L^KO and L^WT mice were administered insulin (0.3U/kg body mass) by intravenous injection (5 mins). The phosphorylation of IRS1 on Ser-307 in the liver was examined by immunoblot analysis by probing with antibodies to IRS1 and phosphoSer-307 IRS1.

Figure 4. JNK1-deficiency in hepatocytes causes steatosis

(A) Chow-fed L^WT and L^KO mice were fasted overnight. Representative sections of the liver stained with Oil Red-O are presented. (B) The amount of hepatic triglyceride was measured in mice fasted over night (mean ± SD; n = 10). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.05). (C) The amount of hepatic lipogenesis was measured in mice fasted 6 hours (mean ± SD; n =8 ~ 9). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.05). (D) The expression of genes that encode lipogenic transcription factors and co-activators in the liver of chow-fed L^KO and L^WT mice that were fasted overnight (C/ebpα, C/ebpβ, Pgc-1β,Pparγ, and Srebp1) was measured by quantitative RT-PCR assays of the amount of mRNA and was normalized to the amount of 18S RNA in each sample (mean ± SD; n = 7). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.05; **, P < 0.01). (E) The expression of genes that encode enzymes that promote lipogenesis in the liver of chow-fed L^KO and L^WT mice that were fasted overnight (Fas, fatty acid synthase; Acsl1/4, acetyl-CoA synthetase long chain family member 1/4; Acacα/β, acetyl-CoA carboxylaseα/β, Acot3, Acetyl-CoA thioesterase; Dgat1, Diacylglycerol O-acyltransferase homolog 1; Glyk, Glycerol kinase; Mttp, microsomal triglyceride transfer protein) was measured by quantitative RT-PCR assays of the amount of mRNA and was normalized to the amount of 18S RNA in each sample (mean ± SD; n = 7). Statistically significant differences between L^KO and L^WT are indicated (*, P < 0.05; **, P < 0.01).