Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis - PubMed (original) (raw)

Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis

Haibo Sha et al. Diabetes. 2014 Mar.

Abstract

The physiological role of the spliced form of X-box-binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A-like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia.

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Figures

Figure 1

Figure 1

Generation of adipocyte-specific XBP1s TG mice. A: qPCR analysis in WAT of 20-week-old lean (+/+) and obese (ob/ob) female mice (n = 6). B: qPCR analysis in primary adipocytes (Adipo) and SVCs isolated from WAT of 18-week-old lean (+/+) and obese (ob/ob) male mice (representative of three independent experiments). C: qPCR analysis in Adipo and SVCs isolated from WAT of 25-week-old females fed LFD or high-fat diet (HFD) (n = 3 mice). D: The diagram of the transgene construct in which HA-XBP1s is driven by adipocyte-specific aP2 promoter flanked by the H19 chromatin insulators. E: RT-PCR analysis of Xbp1 transcripts in WAT. F: qPCR analysis in WAT of WT (n = 6), TGm (n = 3), and TG (n = 6) females. G: RT-PCR analysis of WAT and liver using transgene-specific primers to demonstrate tissue specificity. H: Western blot analysis of HA-XBP1s protein in WAT following immunoprecipitation (IP) with HA-agarose. I: qPCR analysis of XBP1s target genes Erdj4, P58ipk, and Edem and nontarget gene Chop in WAT of WT and TG mice (n = 6). J: Western blot analysis of phospho-IRE1α (p-IRE1α) using Phos-tag gel in WAT of 17-week-old males (n = 6) with quantitation shown at top. 0, nonphosphorylated IRE1α. K: Transmission electron microscopy images of WAT of WT and TG mice. Arrows point to the ER. Gene expression levels in qPCR analysis were normalized to ribosomal gene L32, which is also used as a loading control in the RT-PCR assay. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t test. a.u., arbitrary unit; L, lipid droplet.

Figure 2

Figure 2

Improved glucose homeostasis in TG mice on LFD. BW (A) and epididymal WAT weight (B) of 16-week-old WT and TG male mice (n = 6). C: Representative hematoxylin and eosin images of WAT sections. Right: Quantitation of adipocyte cell size (n = 300 cells each). D: GTT in 16-week-old males (n = 6) with 1.5 g glucose/kg BW. Right: Area under the curve (AUC) analysis. E: Linear regression analysis of AUC in GTT (D) plotted to Xbp1s expression levels in WAT determined by qPCR (n = 6 each). F: ITT in 15-week-old WT and TG males (n = 6) with 1.5 U insulin/kg BW. Right: AUC analysis. Western blot analysis of phospho-AKT (p-AKT) (S473) in the liver (G), WAT (H), and skeletal muscle (I) of mice with or without intraperitoneal (i.p.) insulin (1 units/kg BW) for 30 min. HSP90 is a loading control. G–I, right: Quantitation of p-AKT/AKT with insulin injection (n = 4). J: Serum levels of pancreatic hormones (insulin and glucagon) and gut hormones (ghrelin, gastric inhibitory polypeptide [GIP], and glucagon-like peptide 1 [GLP-1]) determined by the multiplex assay (n = 4–5). Data are mean ± SEM. *P < 0.05; **P < 0.01 by Student t test. a.u., arbitrary unit.

Figure 3

Figure 3

Elevated adiponectin multimerization in TG lean mice. A: Serum levels of adipokines leptin, resistin, and PAI-1. Leptin was measured in 12-week-old mice (n = 11–12), while resistin and PAI-1 levels were measured in 20-week-old mice (n = 4–5 mice). B: Serum adiponectin levels of 17-week-old males (n = 12 each). C: Adipoq mRNA levels in WAT of 17-week-old WT and TG males determined by qPCR (n = 11–12). D: Western blot analysis of XBP1s in 3T3-L1 adipocytes acutely overexpressing green fluorescent protein (CON) or XBP1s. To visualize XBP1s, cells were treated with thapsigargin (Tg) for 6 h. Right: qPCR analysis of Adipoq mRNA levels in 3T3-L1 adipocytes (without Tg treatment). E: Nonreducing Western blot analysis (left) and quantitation (right) of adiponectin (ADPN) complexes in the serum. Serum IgG levels were used as loading controls for normalization. F: Western blot analysis of adiponectin complexes in the serum following gel-filtration chromatography. Relative abundance of different forms of adiponectin shown at bottom. G: Nonreducing Western blots of adiponectin complexes in the serum following velocity-based sucrose-gradient fractionation (n = 5). Quantitation shown on the right. H: Representative nonreducing Western blots of adiponectin complexes in the WAT following velocity-based sucrose-gradient fractionation (n = 2). Data are shown as mean ± SEM. Gene expression levels in qPCR analysis were normalized to L32. *P < 0.05; **P < 0.01 by Student t test. a.u., arbitrary unit; CREB, cAMP-responsive element–binding protein.

Figure 4

Figure 4

Improved glucose homeostasis and elevated HMW adiponectin in TG ob/ob mice. A: Western blot analysis of HA-XBP1s protein in whole-cell lysates (Total), cytosol (Cyt), and nucleus (Nuc) of WAT from 13-week-old TG and TG-ob males following immunoprecipitation (IP) with HA-agarose. Quantitation shown below with total and cytosol HA-XBP1s normalized to HSP90 and nuclear HA-XBP1s normalized to CREB. * indicates a nonspecific band. B: qPCR analysis in primary adipocytes (Adipo), SVCs, and macrophages (MΦ) isolated from WAT of 12-week-old females as determined by qPCR (n = 3). C: BWs of 9-week-old males (n = 5–8). D: GTT of mice in C with 1 g glucose/kg BW. Right: AUC analysis. E: BWs of 12-week-old females (n = 4). F: ITT of mice in E with 4 U insulin/kg BW and AUC analysis (right). G: Adipoq mRNA levels in WAT of 14-week-old females (n = 4–6 each). H: Nonreducing Western blots of adiponectin (ADPN) complexes in the serum following velocity-based sucrose-gradient fractionation with quantitation shown on the right (n = 3). Fractions were pooled together as indicated. I: Regression analysis of growth curves of males (left) and females (right) of WT-ob and TG-ob mice. J: Representative picture of mice at the age of 40 weeks. K: Daily food intake of 35-week-old WT-ob (n = 5) and TG-ob (n = 7) males. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t test. a.u., arbitrary unit; l.e., longer exposure; LMW, low–molecular weight; wk, week.

Figure 5

Figure 5

Adipocyte XBP1s affects systemic glucose tolerance via adiponectin. A: qPCR analysis of Xbp1s in WAT of 14-week-old males (n = 4). B: BWs of 14-week-old males (n = 6). C: GTT of mice in B with 1 g glucose/kg BW (left) and AUC analysis (right). Data are shown as mean ± SEM. ***P < 0.001; N.S., not significant by Student t test.

Figure 6

Figure 6

XBP1s-mediated global transcriptional regulation in WAT. A: Gene ontology analysis of top 10 pathways that are upregulated by XBP1s in WAT of 16-week-old WT and TG males on LFD under ad libitum. B: Heat map showing changes in UPR genes organized by the ratio of signals in TG to signals in WT in each functional category with average level of WT signals set as 1. Genes increased over 1.2-fold in TG mice are shown. n = 4 mice/cohort. ERAD, ER-associated protein degradation.

Figure 7

Figure 7

XBP1s regulates the expression of genes involved in adiponectin maturation. qPCR (A) and Western blot (B) analyses in WAT of 18-week-old WT and TG males (n = 6). B: Quantitation after normalization to HSP90 shown on right. C: qPCR analysis in WAT of 14-week-old females (n = 4–6). #P < 0.05 comparing WT-ob vs. WT-lean; *P < 0.05 comparing TG-ob vs. WT-ob. D: qPCR analysis in primary adipocytes (Adipo), SVCs, and macrophages (MΦ) purified from WAT of 12-week-old WT-ob and TG-ob females (n = 3). E: Linear regression analyses of mRNA levels plotted to Xbp1s expression levels in WAT as determined by qPCR. n = 6 for WT and 12 for TG mice. F: Chromatin immunoprecipitation–PCR analysis of XBP1s targets in WAT of TG mice using the anti-XBP1 antibody. Nonspecific IgG and 3′ untranslated region (UTR) were included as negative controls. G: Luciferase assays showing XBP1s-mediated transactivation of target gene promoters (p) in human embryonic kidney 293T cells. Gene expression levels in qPCR analysis were normalized to L32. Data are shown as mean ± SEM. H: The working model for XBP1s-mediated adiponectin multimerization and glucose homeostasis. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t test. a.u., arbitrary unit; CON, control; N.D., not detected.

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