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. 2013 Jan;123(1):455-68.
doi: 10.1172/JCI62819. Epub 2012 Dec 21.

The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism

Yingfeng Deng  1 , Zhao V Wang, Caroline Tao, Ningguo Gao, William L Holland, Anwarul Ferdous, Joyce J Repa, Guosheng Liang, Jin Ye, Mark A Lehrman, Joseph A Hill, Jay D Horton, Philipp E Scherer
Affiliations

The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism

Yingfeng Deng et al. J Clin Invest. 2013 Jan.

Abstract

Postprandially, the liver experiences an extensive metabolic reprogramming that is required for the switch from glucose production to glucose assimilation. Upon refeeding, the unfolded protein response (UPR) is rapidly, though only transiently, activated. Activation of the UPR results in a cessation of protein translation, increased chaperone expression, and increased ER-mediated protein degradation, but it is not clear how the UPR is involved in the postprandial switch to alternate fuel sources. Activation of the inositol-requiring enzyme 1 (IRE1) branch of the UPR signaling pathway triggers expression of the transcription factor Xbp1s. Using a mouse model with liver-specific inducible Xbp1s expression, we demonstrate that Xbp1s is sufficient to provoke a metabolic switch characteristic of the postprandial state, even in the absence of caloric influx. Mechanistically, we identified UDP-galactose-4-epimerase (GalE) as a direct transcriptional target of Xbp1s and as the key mediator of this effect. Our results provide evidence that the Xbp1s/GalE pathway functions as a novel regulatory nexus connecting the UPR to the characteristic postprandial metabolic changes in hepatocytes.

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Figures

Figure 1
Figure 1. Postprandial activation of hepatic Xbp1s in WT mice and Xbp1s induction in LIXs mice.
(A and B) WT FVB mice (n = 3 per group) were fasted for 24 hours and refed up to 15 hours. The experiments in A and B were repeated twice. (A) A representative image of Xbp1 RT-PCR products from liver with GAPDH as a loading control. U, unspliced Xbp1; S, spliced Xbp1. (B) Xbp1s levels as determined by qPCR. (C) Xbp1s protein levels during fasting-refeeding in WT livers and by induction in LIXs mice with lamin as loading control for nuclear fraction. (D) RT-PCR analysis of liver Xbp1 from mice fed ad libitum with Dox chow diet. (E) The comparison of Xbp1s protein expression in WT by refeeding and LIXs mice by induction. *P < 0.05.
Figure 2
Figure 2. Hepatic Xbp1s overexpression mimics the metabolic effects of refeeding.
RER (A) and food intake (B) of WT mice (n = 5 per group) during refeeding after a 24-hour fast. Time 0 represents 12 p.m. RER (C) and food intake (D) of WT and LIXs mice after Dox induction (n = 4 per group). Mice were switched to a Dox chow diet at 2 p.m. on day 3 in the metabolic cage. Time 0 represents 12 p.m. of day 4 (22 hours after induction). *P < 0.05.
Figure 3
Figure 3. Hepatic Xbp1s overexpression triggers hypoglycemia.
(A) Hepatic glycogen staining from mice on Dox chow diet for 48 hours. Scale bars: 50 μm. (B) GLUT2 immunofluorescence staining. Scale bars: 50 μm. (C–E) Mice (n = 3 per group) were fed Dox chow diet for 24–96 hours. Serum glucose levels (C), liver TG content (D), and serum free fatty acid levels (E) under fed or 6-hour fast conditions. *P < 0.05. (F) Serum glucose levels during a pyruvate tolerance test (n = 3 per group). *P < 0.05.
Figure 4
Figure 4. Hepatic Xbp1s overexpression induces GalE.
Gene transcription of GalE (A) and GalK (C) and immunoblotting of GalE (B) from mice (n = 3 per group) fed Dox food for 24–72 hours. GAPDH served as a loading control. *P < 0.05.
Figure 5
Figure 5. Hepatic GalE expression associates with Xbp1s induction in vivo.
(A and B) WT mice were fasted for different durations and refed for 2 hours. Expression of Xbp1s, GalE, and GalK (B) was examined by qPCR (n = 3 per group). *P < 0.05. (C and D) WT and LIXs mice were fasted for 6 or 24 hours before a 2-hour refeeding. Induction was performed once by Dox gavage 3 hours prior to refeeding. Sac, sacrifice. Expression of Xbp1s, GalE, and GalK (D) is shown (n = 3 per group). *P < 0.05. (E) Xbp1s expression in genetically (ob/ob) and diet-induced (HFD) obese mice (n = 3 per group) with actin as loading control. U, unspliced Xbp1; S, spliced Xbp1. Expression of GalE was determined by qPCR (F). *P < 0.05.
Figure 6
Figure 6. GalE expression is upregulated by Xbp1s in vitro.
(A) Gene expression in mouse primary hepatocytes (n = 5–6 per group) infected with lentivirus overexpressing LacZ or Xbp1s. *P < 0.05. (B) Gene expression in Huh7 cells after treatment with tunicamycin (TM) and insulin (n = 3 per group). *P < 0.05. (C) Serum insulin levels in mice from Figure 5C (n = 3 per group). *P < 0.05.
Figure 7
Figure 7. GalE is a target gene of Xbp1s.
(A) A consensus binding site of Xbp1s is indicated on alignment of GalE promoters from mouse, rat, and human. (B) Luciferase assays for GalE promoter regulated by Xbp1s in HEK293T cells (n = 3–6 per group). (C) Deletion or mutation of the Xbp1s binding site reduces luciferase activity (n = 3 per group). *P < 0.05. (D) Xbp1s protein association with Gale promoter examined by ChIP assay using WT livers under condition of fasting, 2 hours refeeding, and 6 hours refeeding. Ctrl, control.
Figure 8
Figure 8. Regulation of GalE by fasting-refeeding.
(A) Expression of GalE in WT mice (n = 3 per group) during fasting-refeeding. Experiments were repeated twice. (B) Model for the role of GalE in glucose assimilation in WT mice by refeeding (left) and LIXs mice by induction (right). See text for details.
Figure 9
Figure 9. Hepatic GalE overexpression improves insulin sensitivity.
(A and B) Serum glucose levels during an oral glucose tolerance test (A) and a pyruvate tolerance test (B) in ob/ob mice (n = 4–5 per group) assayed 7 days after adenoviral infection. *P < 0.05. (C) Fasting (6 hours) serum glucose levels from WT mice (n = 3–4 per group) assayed 7 days after adenoviral infection. The mice were fed a HFD for 10 weeks before infection. *P < 0.05. (D and E) Serum glucose and insulin levels during an oral glucose tolerance test (D) and serum glucose levels during an insulin tolerance test (E) in WT mice (n = 5–6 per group) assayed 7 days after infection. *P < 0.05.
Figure 10
Figure 10. GalE overexpression enhances biosynthesis of nucleotide sugars and protein glycosylation.
(A–C) Representative FACE images of total hepatic N-glycan with G4-G7 glucose oligomer standards (A) and nucleotide-sugar pools that have been hydrolyzed to allow detection of the corresponding free monosaccharides (B), and quantification of cellular UDP sugars (C) from mice (n = 4–5 per group) after 48 hours of induction. *P < 0.05. Man., mannose. (D) Immunofluorescence staining of hepatic LDL receptor 24 hours after induction. Scale bars: 50 μm.
Figure 11
Figure 11. GalE regulates protein synthesis and cellular response to insulin.
(A) Measurement of protein synthesis stimulated by Xbp1s in primary hepatocytes using 3H-leucine incorporation. *P < 0.05. (B) Knockdown of GalE reduces protein synthesis rate in both WT and LIXs hepatocytes. *P < 0.05. (C) Knockdown of GalE impairs insulin signaling in WT hepatocytes. (D) Overexpression of GalE enhances the insulin response in WT rat hepatocytes. WB, Western blot. (E) Adiponectin levels were examined by immunoblotting in HEK293T cells when co-transfected with a GalE expression plasmid (1 μg) or the vehicle plasmid (1 μg). Actin served as loading control. APN, adiponectin. GDI, guanosine nucleotide dissociation inhibitor. NA, no virus infection.
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