Dissociation of inositol-requiring enzyme (IRE1α)-mediated c-Jun N-terminal kinase activation from hepatic insulin resistance in conditional X-box-binding protein-1 (XBP1) knock-out mice - PubMed (original) (raw)

Dissociation of inositol-requiring enzyme (IRE1α)-mediated c-Jun N-terminal kinase activation from hepatic insulin resistance in conditional X-box-binding protein-1 (XBP1) knock-out mice

Michael J Jurczak et al. J Biol Chem. 2012.

Abstract

Hepatic insulin resistance has been attributed to both increased endoplasmic reticulum (ER) stress and accumulation of intracellular lipids, specifically diacylglycerol (DAG). The ER stress response protein, X-box-binding protein-1 (XBP1), was recently shown to regulate hepatic lipogenesis, suggesting that hepatic insulin resistance in models of ER stress may result from defective lipid storage, as opposed to ER-specific stress signals. Studies were designed to dissociate liver lipid accumulation and activation of ER stress signaling pathways, which would allow us to delineate the individual contributions of ER stress and hepatic lipid content to the pathogenesis of hepatic insulin resistance. Conditional XBP1 knock-out (XBP1Δ) and control mice were fed fructose chow for 1 week. Determinants of whole-body energy balance, weight, and composition were determined. Hepatic lipids including triglyceride, DAGs, and ceramide were measured, alongside markers of ER stress. Whole-body and tissue-specific insulin sensitivity were determined by hyperinsulinemic-euglycemic clamp studies. Hepatic ER stress signaling was increased in fructose chow-fed XBP1Δ mice as reflected by increased phosphorylated eIF2α, HSPA5 mRNA, and a 2-fold increase in hepatic JNK activity. Despite JNK activation, XBP1Δ displayed increased hepatic insulin sensitivity during hyperinsulinemic-euglycemic clamp studies, which was associated with increased insulin-stimulated IRS2 tyrosine phosphorylation, reduced hepatic DAG content, and reduced PKCε activity. These studies demonstrate that ER stress and IRE1α-mediated JNK activation can be disassociated from hepatic insulin resistance and support the hypothesis that hepatic insulin resistance in models of ER stress may be secondary to ER stress modulation of hepatic lipogenesis.

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Figures

FIGURE 1.

FIGURE 1.

XBP1Δ mice fed fructose chow have reduced hepatic lipid levels and expression of lipogenic genes. a, 24-h energy expenditure (EE) during fructose chow feeding. b, 24-h feeding during fructose feeding. c, body weight and fat mass after 1 week of fructose feeding. d, plasma triglyceride levels. e, hepatic triglyceride levels. f, hepatic DAG levels in cytosol (Cyto) and membrane (Memb) compartments, and ceramide and LCCoA levels. g, quantitative PCR of cDNA from liver of genes involved in lipid metabolism. The data are expressed as fold change relative to WT. (n = 6–8 for both genotypes aged 14–16 weeks for each panel). *, p < 0.05; **, p < 0.001; ***, p < 0.001.

FIGURE 2.

FIGURE 2.

Markers of ER stress are elevated in fructose chow-fed XBP1Δ mice. a, Western blots of liver lysates for ER stress markers IRE1α, phosphorylated and total eIF2α, and GAPDH loading control. b, quantitative PCR of cDNA isolated from liver for genes involved in the ER stress response. The data are reported as fold change relative to WT. c, Western blot of liver lysate for the ER chaperone GRP78 and GAPDH loading control. d, hepatic JNK activity from whole cell liver lysates (WC), cytoplasmic (Cyto), and nuclear (Nuc) fractions obtained by differential centrifugation. e, plasma levels of the cytokines IL1-β, IL-6, IL-10, and IL-12. TNFα and INFγ were below the level of detection (2.5 pg/ml) (n = 6–8 for both genotypes for each panel). The blots shown are representative of six to eight mice per genotype. *, p < 0.05; **, p < 0.001; ***, p < 0.001.

FIGURE 3.

FIGURE 3.

Hepatic insulin sensitivity is improved in fructose chow-fed XBP1Δ mice. a, plasma glucose levels after a 14-h overnight fast. b, plasma insulin levels after a 14-h overnight fast. c, basal and insulin-stimulated (clamp) hepatic glucose production. d, plasma glucose levels (upper panel) and glucose infusion rate (lower panel) during hyperinsulinemic euglycemic clamps. e, glucose infusion rate required to maintain euglycemia during the final 40 min of the clamp. f, whole-body glucose uptake measured over the final 40 min of the clamp. g, hepatic glycogen levels measured following hyperinsulinemic euglycemic infusion (n = 6–9 for both genotypes for each panel). *, p < 0.05.

FIGURE 4.

FIGURE 4.

Decreased PKCϵ activity is associated with improved insulin signaling in fructose chow-fed XBP1Δ mice. a, immunoprecipitation of liver lysates from clamped mice for the indicated antibodies, followed by Western blotting. IP, immunoprecipitation; IB, immunoblot. b, quantification of Western blotting data in A. c, total FoxO1 protein levels in insulin-treated (post-clamp) liver. d, hepatic G6PC and PEPCK mRNA levels in insulin-treated (post-clamp) liver determined by QPCR. e, PKCϵ activity in post-clamp liver determined as the ratio of membrane to cytosolic levels of PKCϵ. *, p < 0.05 (n = 6–9 for both genotypes for each panel). The blots shown are representative of six to nine mice per genotype. *, p < 0.05.

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