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. 2011 Mar;17(3):356-65.
doi: 10.1038/nm.2293. Epub 2011 Feb 13.

Regulation of glucose homeostasis through a XBP-1-FoxO1 interaction

Yingjiang Zhou  1 , Justin Lee, Candace M Reno, Cheng Sun, Sang Won Park, Jason Chung, Jaemin Lee, Simon J Fisher, Morris F White, Sudha B Biddinger, Umut Ozcan
Affiliations

Regulation of glucose homeostasis through a XBP-1-FoxO1 interaction

Yingjiang Zhou et al. Nat Med. 2011 Mar.

Abstract

To date, the only known role of the spliced form of X-box-binding protein-1 (XBP-1s) in metabolic processes has been its ability to act as a transcription factor that regulates the expression of genes that increase the endoplasmic reticulum (ER) folding capacity, thereby improving insulin sensitivity. Here we show that XBP-1s interacts with the Forkhead box O1 (FoxO1) transcription factor and directs it toward proteasome-mediated degradation. Given this new insight, we tested modest hepatic overexpression of XBP-1s in vivo in mouse models of insulin deficiency or insulin resistance and found it improved serum glucose concentrations, even without improving insulin signaling or ER folding capacity. The notion that XBP-1s can act independently of its role in the ER stress response is further supported by our finding that in the severely insulin resistant ob/ob mouse strain a DNA-binding-defective mutant of XBP-1s, which does not have the ability to increase ER folding capacity, is still capable of reducing serum glucose concentrations and increasing glucose tolerance. Our results thus provide the first evidence to our knowledge that XBP-1s, through its interaction with FoxO1, can bypass hepatic insulin resistance independent of its effects on ER folding capacity, suggesting a new therapeutic approach for the treatment of type 2 diabetes.

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Conflict of interest statement

Authors do not have any conflict of interest.

Figures

Figure 1
Figure 1
XBP1s binds to FoxO1 and promotes its degradation. (a) XBP1s and FoxO1 protein levels in MEFs expressing FoxO1 and increasing levels of XBP1s. Foxo1 mRNA levels were analyzed by qPCR. (b) Endogenous FoxO3a and FoxO1 protein levels in MEFs expressing increasing levels of XBP1s. (c) Nuclear FoxO1 and XBP1s protein levels in MEFs treated with DMSO or MG132. (d) Pulse-chase analysis of FoxO1 stability in MEFs overexpressing XBP1s. (e) Ubiquitinylated FoxO1 levels in MEFs expressing ubiquitin and XBP1s after DMSO or MG132 treatment. (f) Immunoblotting of FoxO1 and XBP1s in XBP1s–immunoprecipitates from MG132-treated MEFs, expressing FoxO1 and XBP1s. Total FoxO1 protein level were determined in whole cell lysates (WCL). (g) Mammalian two-hybrid assay with pM-XBP1s and pVP16-FoxO1 or pM-FoxO1 and pVP16-XBP1s. (h) Nuclear FoxO1 and XBP1s protein levels in MEFs treated with 1 µM BEZ235 for 4 h. Phospho-AktSer473 and total Akt levels were determined in cell lysates. (i) FoxO1, pAktSer473, total Akt and XBP1s levels in WT and Akt1/2 double knockout (Akt1/2-DKO) MEFs treated with DMSO or 20 µM Akt inhibitor for 30 min. (j) Phosphorylation resistant mutant FoxO1(ADA) protein levels in MEFs expressing FoxO1(ADA) and increasing levels of XBP1s. Each experiment was independently reproduced three times. Error bars are ± s.e.m.; P values were determined by Student’s t-test (** P < 0.01, *** P < 0.001).
Figure 2
Figure 2
Medium-level expression of XBP1s in ob/ob mice improves glucose homeostasis without altering the insulin receptor signaling. Seven-week-old, male, ob/ob mice were injected with Ad-LacZ (n = 6) or Ad-XBP1s (n = 6) (4 × 107 PFU g−1) through tail vein. (a) XBP1s protein levels in the liver of ob/ob mice injected with low or medium dose of adenovirus. (b) Relative mRNA levels of XBP1s target genes, Dnajb9, Pida3 and Hspa5 in the liver of Ad-LacZ- or Ad-XBP1s-injected mice. (c) Blood glucose levels in fed, 6h and 14h fasting on indicated days after injections. (d) Plasma insulin levels on day 9, (e) GTT on day 5, and (f) ITT on day 7 after the injections. (g) IR and IRS1 tyrosine and AktSer473 phosphorylations with or without insulin stimulation in the liver on post-injection day 9. (h) Total and nuclear levels of FoxO1 protein in the liver of Ad-LacZ- or Ad-XBP1s-injected ob/ob mice. Graph depicts total FoxO1/tubulin and nuclear FoxO1/lamin A/C ratios. Relative mRNA levels of (i) Foxo1, (j) Igfbp1, G6pc, Pck1, and Ppargc1a in the liver of adenovirus-injected mice. Experiments were repeated in five independent cohorts. Error bars are ± s.e.m.; P values were determined by Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).
Figure 3
Figure 3
High-level expression of XBP1s in the liver of ob/ob mice increases insulin sensitivity. Seven-week-old, male, ob/ob mice were injected with Ad-LacZ (n = 6) or Ad-XBP1s (n = 6) (1.8 × 108 PFU g−1) through tail vein. (a) XBP1s protein levels in the liver lysates on day 6 after injection. (b) Dnajb9, Pida3 and Hspa5 mRNA levels in the liver of Ad-LacZ- or Ad-XBP1s-injected mice. (c) Fed blood glucose levels on day 5, (d) plasma insulin levels on day 7, and (e) GTT on day 3 after the adenovirus injections. (f) In vivo insulin receptor signaling in the liver of Ad-LacZ- or Ad-XBP1s-injected ob/ob mice on post injection day 6. Graphs depicts the (phospho/total)/pIR ratios. (g) Relative mRNA levels of Insr in the liver of adenovirus-injected mice on day 6 after injection. (h) Total and nuclear FoxO1 levels in the liver of Ad-LacZ- or Ad-XBP1s-injected ob/ob mice. Graph depicts total FoxO1/tubulin and nuclear FoxO1/lamin A/C ratios. Relative mRNA levels of (i) Foxo1 and (j) G6pc, Pck1, and Ppargc1a in the liver of Ad-LacZ- or Ad-XBP1s-injected ob/ob mice. Experiments were repeated in three independent cohorts. N.S: non-specific. Error bars are ± s.e.m.; P values were determined by Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).
Figure 4
Figure 4
A DNA binding defective mutant XBP1s (ΔDBD) improves glucose homeostasis in ob/ob mice. (a) Nuclear protein levels of XBP1s and ΔDBD in MEFs infected with the same dose of Ad-XBP1s or Ad-ΔDBD. (b) ERSE-luciferase activity and Hspa5 mRNA levels in MEFs infected with Ad-XBP1s or Ad-ΔDBD. (B) Immunoblotting of FoxO1 and ΔDBD in ΔDBD–immunoprecipitates from MEFs expressing FoxO1 and ΔDBD. Total FoxO1 protein levels were determined in whole cell lysates (WCL). (d) FoxO1 protein levels and (e) Foxo1 mRNA levels in MEFs expressing FoxO1 and increasing levels of ΔDBD. (f–j) Seven-week-old, male, ob/ob mice were injected with Ad-LacZ (n = 6), Ad-XBP1s (n = 6) or Ad-ΔDBD (n = 6) (4 × 107 PFU g−1) through tail vein. (f) 6-h fasted blood glucose levels on day 3 and (g) GTT on day 5 after the adenovirus injections. (h) Phospho-AktSer473, total Akt, FoxO1, XBP1s and ΔDBD protein levels in the liver of Ad-LacZ- or Ad-XBP1s- or Ad-ΔDBD-injected ob/ob mice on day 7 after the injections. Graph depicts total FoxO1/tubulin protein ratio. Relative mRNA levels of (i) Hspa5, (j) Igfbp1, G6pc, and Pck1 in the liver of the liver of Ad-LacZ- or Ad-XBP1s- or Ad-ΔDBD-injected ob/ob mice. Error bars are ± s.e.m.; P values were determined by Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).
Figure 5
Figure 5
XBP1s can also improve glucose homeostasis in insulin independent manner. (a–d) Eight-week-old, male, STZ-treated mice were injected with Ad-XBP1s (n = 9) or Ad-LacZ (n = 9) (1.5 × 108 PFU g−1) though tail vein. (a) Plasma insulin levels before and after STZ treatment. (b) Fed and 12-h fasted blood glucose levels on indicated days. (c) Total and nuclear FoxO1 protein levels and (d) Liver Igfbp1, G6pc, and Pck1 mRNA levels 10 days after adenovirus injections. (e–g) Eight-week-old, male, LIRKO mice were injected with Ad-LacZ (n = 8) or Ad-XBP1s (n = 8) (4 × 107 PFU g–1) through tail vein. (e) Liver IR protein levels and GTT on day 4 after adenovirus injections. (f) FoxO1, XBP1s, pAktSer473, pAktThr308 and total Akt levels and (g) Igfbp1, G6pc, and Pck1 mRNA levels in the liver of Ad-XBP1s- or Ad-LacZ-injected LIRKO mice 8 days after adenovirus injection. (h–j) Eight-week-old, male DKO mice were injected with Ad-LacZ (n = 8) or Ad-XBP1s (n = 8) (7.5 × 107 PFU g–1) through tail vein. (h) Liver IRS1 and IRS2 protein levels in Irs1Flox/Flox;Irs2Flox/Flox (DF) and DKO mice. GTT was performed on post injection day 4. (i) FoxO1 and XBP1s protein levels and (j) Igfbp1, G6pc, and Pck1 mRNA levels in the liver of adenovirus-injected DKO mice on day 8 after the injections. Error bars are ± s.e.m.; P values were determined by Student’s t-test. (* P < 0.05, ** P < 0.01, *** P < 0.001).
Figure 6
Figure 6
Enhanced glucose tolerance after medium-level expression of XBP1s is FoxO1-dependent. Seven-week-old, male, ob/ob mice were injected with Ad-shGFP + Ad-LacZ (n = 5), Ad-shGFP + Ad-XBP1s (n = 5), Ad-shFoxO1 + Ad-LacZ (n = 5) or Ad-shFoxO1 + Ad-LacZ (n = 5) through tail vein. (a) Blood glucose levels on day 7, and (b) GTT on day 5 after the adenovirus injections. (c) FoxO1 and XBP1s protein levels in the liver of adenovirus-injected ob/ob mice on day 7 after injections. Relative mRNA levels of (d) Foxo1, (e) Igfbp1 and Ppargc1a, (f) Dnajb9 and Hspa5 on day 7 after adenovirus injections. Error bars are ± s.e.m.; P values were determined by Student’s t-test. (*p<0.05, **p<0.01, ***p<0.001).
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Comment in

  • The other sweet face of XBP-1.
    Ueki K, Kadowaki T. Ueki K, et al. Nat Med. 2011 Mar;17(3):246-8. doi: 10.1038/nm0311-246. Nat Med. 2011. PMID: 21383716 No abstract available.

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