Requirement of JIP1-mediated c-Jun N-terminal kinase activation for obesity-induced insulin resistance

Abstract

The c-Jun NH(2)-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr(103) replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.

FIG. 1.

Creation of mice with a germ line knock-in mutation in the Jip1 gene. (A) Schematic illustration of the gene targeting strategy. A floxed Neo^R cassette was inserted into intron 3 of the Jip1 gene by homologous recombination. Point mutations were introduced into exon 3. The Neo^R cassette was excised using Cre recombinase. HindIII restriction sites are indicated (H). (B) The DNA sequence of the region of exon 3 of the Jip1 gene that surrounds codon 103 is presented. The mutated allele replaces codon 103, which encodes Thr (ACT) with Ala (GCC). Translationally silent mutations were also introduced to create an XmaI restriction site in the mutated Jip1 allele (Jip1^TA). (C) Genomic DNA was prepared from wild-type (Jip1^+/+) mice, heterozygous (Jip1^+/TA), and homozygous (Jip1^TA/TA) mice. Genotype analysis was performed by PCR. (D) Extracts prepared from the cerebellum of Jip1^+/+, Jip1^+/TA, and Jip1^TA/TA mice were examined by immunoblot analysis by probing with antibodies to JIP1, JIP2, and β-tubulin. (E) The expression of Jip1 mRNA in white adipose tissue (WAT), brown adipose tissue (BAT), quadriceps muscle, and liver of C57BL/6J mice was measured by quantitative RT-PCR analysis of mRNA. The relative mRNA expression was calculated by normalization of the data to the amount of Gapdh mRNA in each sample (mean ± SE; n = 8).

FIG. 2.

Comparison of HFD-induced obesity in JIP^WT and JIP^TA mice. (A) Mice (8 to 10 weeks old) were fed either a chow diet (ND) or a high-fat diet (HFD) for 16 weeks. The body weight of the mice was measured (mean ± SE; n = 10 to 15). No significant difference between the weights of JIP^WT and JIP^TA mice was detected (P > 0.05). (B) Fat and lean body mass of mice fed (3 weeks) a chow diet (ND) or an HFD (HF) was measured by ¹H-MRS. The data presented are the means ± SE (n = 8). No significant difference between JIP^WT and JIP^TA mice was detected (P > 0.05). (C) Chow-fed (ND) and HFD-fed (HF) mice (16 weeks) were fasted overnight. JNK1 activity in epididymal fat was measured in a protein kinase (KA) assay using c-Jun and [γ-³²P]ATP as substrates. The cell extracts used for the protein kinase assay were also examined by immunoblot analysis by probing with an antibody to JNK. Representative data are presented (upper panel) and quantitated (mean ± SE [n = 5]) (lower panel). Statistically significant differences are indicated (**, P < 0.01). (D) Chow-fed (ND) and HFD-fed (HF) mice (16 weeks) were fasted overnight. The blood glucose concentration was measured (mean ± SE; n = 10 to 15). Statistically significant differences are indicated (**, P < 0.01). (E) Chow-fed (ND) and HFD-fed (HF) mice (16 weeks) were fasted overnight. Epididymal fat was examined by immunoblot analysis by probing with antibodies to phospho-Ser³⁰⁷ IRS1, IRS1, and tubulin. (F) Chow-fed (ND) and HFD-fed (HF) mice (16 weeks) were fasted overnight. The blood insulin concentration was measured (mean ± SE; n = 10 to 15). Statistically significant differences are indicated (*, P < 0.05).

FIG. 3.

Comparison of the responses of JIP^WT and JIP^TA mice to glucose and insulin tolerance tests. (A) Glucose tolerance tests (GTT) with chow-fed (ND) and HFD-fed JIP^WT and JIP^TA mice (16 weeks) were performed by measurement of blood glucose concentrations in animals following intraperitoneal injection of glucose (1 g/kg). The data presented represent the means ± SE (n = 10 to 15). No statistically significant differences between JIP^WT and JIP^TA mice were detected (P > 0.05). (B) Glucose-induced insulin release. The effect of administration of glucose (2 g/kg body mass) by intraperitoneal injection on the blood insulin concentration was examined (means ± SE; n = 13 to 15). Statistically significant differences between JIP^WT and JIPT^TA mice are indicated (*, P < 0.05). (C) Insulin tolerance tests (ITT) with JIP^WT and JIP^TA mice fed either a chow diet (ND) or an HFD for 16 weeks were performed by intraperitoneal injection of insulin (1.5 U/kg body mass). The concentration of blood glucose was measured (mean ± SE; n = 10). Statistically significant differences between JIP^WT and JIPT^TA mice are indicated (*, P < 0.05; **, P < 0.01).

FIG. 4.

Effect of the Thr103Ala JIP1 mutation on insulin sensitivity. Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp in conscious chow-fed (ND) and HFD-fed (HF) JIP1^WT and JIP1^TA mice. The data presented are the means ± SE for 8 or 9 experiments. Statistically significant differences between JIP1^WT and JIP1^TA mice are indicated (*, P < 0.05). (A) Steady-state glucose infusion rate during the clamp. (B) Hepatic glucose production (HGP) during the clamp. (C) Hepatic insulin action, expressed as insulin-mediated percent suppression of basal HGP. (D) Insulin-stimulated whole-body glucose turnover. (E) Whole-body glycogen plus lipid synthesis. (F) Whole-body glycolysis.

FIG. 5.

Increased insulin sensitivity of HFD-fed JIP1^TA mice compared with that of HFD-fed JIP1^TA mice. (A) Chow-fed (ND) and HFD-fed (HF) JIP1^WT and JIP1^TA mice (16 weeks) were fasted overnight and then treated by intraperitoneal injection of 1.5 U/kg insulin. Extracts prepared from epididymal fat pads at 10 min postinjection were examined by immunoblot analysis using antibodies to phospho-AKT (Thr³⁰⁸ and Ser⁴⁷³), AKT, and GAPDH. (B) Quantitative analysis of AKT phosphorylation (Ser⁴⁷³) was performed by ELISA (Luminex 200 machine). The relative AKT phosphorylation is presented (mean ± SE; n = 5 to 8). Ins, insulin.

FIG. 6.

The Thr103Ala JIP1 mutation reduces HFD-induced hepatic steatosis. (A) Sections prepared from chow-fed (ND) and HFD-fed (HF) JIP1^WT and JIP1^TA mice (16 weeks) were stained with hematoxylin and eosin (H&E) or with Sudan Black B. Scale bar = 100 μm. (B) The liver mass at the time of necropsy (16 weeks) is presented (mean ± SE; n = 10). Statistically significant differences between JIP1^WT and JIP1^TA mice are indicated (*, P < 0.05). (C and D) The amount of hepatic or blood triglycerides was measured (mean ± SE; n = 10). Statistically significant differences between JIP1^WT and JIP1^TA mice are indicated (*, P < 0.05; **, P < 0.01). (E to G) The amount of C/ebpα, C/ebpβ, or Srebp1c mRNA in the liver of JIP1^WT and JIP1^TA mice was measured by quantitative RT-PCR and was normalized to the amount of Gapdh mRNA in each sample (mean ± SE; n = 8). Statistically significant differences between JIP1^WT and JIP1^TA mice are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001)).

FIG. 7.

Comparison of energy balance of JIP^WT and JIP^TA mice using metabolic cages. (A to G) Groups of 6 mice were examined during a 3-day period to measure the mean food and water consumption, gas exchange (V_O2 and V_CO2), respiratory exchange quotient [V_CO2]/[V_O2], energy expenditure, and physical activity. Statistically significant differences between JIP^WT and JIP^TA mice are indicated (*, P < 0.05; **, P < 0.01).

FIG. 8.

Downregulation of the hepatic IL-6 pathway in JIP1^TA mice. (A and B) Chow-fed (ND) and HFD-fed (HF) JIP1^WT and JIP1^TA mice (16 weeks) were fasted overnight. The amounts of IL-6 and TNF-α in the blood of chow-fed and HFD-fed JIP^WT and JIPT^TA mice were examined by ELISA (mean ± SE; n = 10). Statistically significant differences between JIP^WT and JIPT^TA mice are indicated (**, P < 0.01). (C and D) The expression of Socs3 and Vldlr mRNA in the liver was measured by quantitative RT-PCR analysis of mRNA. The relative mRNA expression was calculated by normalization of the data to the amount of Gapdh RNA in each sample (mean ± SE; n = 8). Statistically significant differences between JIP^WT and JIPT^TA mice are indicated (**, P < 0.01; ***, P < 0.001). (E) The expression of SOCS3 and α-tubulin in the livers of chow-fed and HFD-fed JIP^WT and JIPT^TA mice was measured by immunoblot analysis.

FIG. 9.

Adipokines in JIP1^WT and JIP1^TA mice. (A and B) The blood concentration of leptin or resistin from chow-fed (ND) or HFD-fed (HF) JIP1^WT and JIP1^TA mice (16 weeks.) was measured by ELISA (mean ± SE; n = 10). (C) The amount of adiponectin mRNA in epididymal fat was measured by quantitative RT-PCR and was normalized to the amount of Gapdh mRNA in each sample (mean ± SE; n = 8). Statistically significant differences between JIP1^WT and JIP1^TA mice are indicated (*, P < 0.05; **, P < 0.01).