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. 2011 Sep 20;108(38):15852-7.
doi: 10.1073/pnas.1107394108. Epub 2011 Sep 12.

PKA phosphorylation couples hepatic inositol-requiring enzyme 1alpha to glucagon signaling in glucose metabolism

Ting Mao  1 , Mengle Shao, Yifu Qiu, Jialiang Huang, Yongliang Zhang, Bo Song, Qiong Wang, Lei Jiang, Yi Liu, Jing-Dong J Han, Pengrong Cao, Jia Li, Xiang Gao, Liangyou Rui, Ling Qi, Wenjun Li, Yong Liu
Affiliations

PKA phosphorylation couples hepatic inositol-requiring enzyme 1alpha to glucagon signaling in glucose metabolism

Ting Mao et al. Proc Natl Acad Sci U S A. .

Abstract

The endoplasmic reticulum (ER)-resident protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) is activated through transautophosphorylation in response to protein folding overload in the ER lumen and maintains ER homeostasis by triggering a key branch of the unfolded protein response. Here we show that mammalian IRE1α in liver cells is also phosphorylated by a kinase other than itself in response to metabolic stimuli. Glucagon-stimulated protein kinase PKA, which in turn phosphorylated IRE1α at Ser(724), a highly conserved site within the kinase activation domain. Blocking Ser(724) phosphorylation impaired the ability of IRE1α to augment the up-regulation by glucagon signaling of the expression of gluconeogenic genes. Moreover, hepatic IRE1α was highly phosphorylated at Ser(724) by PKA in mice with obesity, and silencing hepatic IRE1α markedly reduced hyperglycemia and glucose intolerance. Hence, these results suggest that IRE1α integrates signals from both the ER lumen and the cytoplasm in the liver and is coupled to the glucagon signaling in the regulation of glucose metabolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Glucagon stimulates the phosphorylation of hepatic IRE1α. (A) Fasting induced hepatic IRE1α phosphorylation. Male C57BL/6 mice were ad libitum fed or subjected to a 6-h fast. Liver extracts were analyzed by immunoblotting using the indicated antibodies. Results are shown for three individual mice in the fed or fasted state, representative of two independent experiments. (B and C) Glucagon stimulated IRE1α phosphorylation in vivo and in primary hepatocytes. (B) Mice were treated for the indicated time intervals (n = 3 per group) by intraperitoneal injection of glucagon (100 μg/kg body weight). (C) Mouse primary hepatocytes were treated with 100 nM glucagon as indicated, or with dimethyl sulfoxide (DMSO) or thapsigargin (Tg, 1 μM) for 1 h. Protein extracts were analyzed by immunoblotting, and spliced (s) and unspliced (u) Xbp1 mRNA transcripts were measured by RT-PCR. Results are representative of at least three independent experiments. (D) Glucagon induced a distinct phosphorylation state of IRE1α from that caused by ER stress. Primary hepatocytes were treated with PBS, 100 nM glucagon, or 1 μM Tg for 1 h. Immunoblotting was performed for band-shift analysis using the IRE1α antibody. Results are representative of three independent experiments.
Fig. 2.
Fig. 2.
IRE1α mediates glucagon's metabolic effects in liver cells. (A) IRE1α knockdown reduced glucagon-induced expression of gluconeogenic genes. Primary hepatocytes, infected for 72 h with the indicated adenoviruses, were incubated with 100 nM glucagon for 4 h. Immunoblotting was performed for IRE1α expression analysis. Expression of Pepck and G6pase was analyzed by real-time RT-PCR using actin as an internal control, shown as averaged fold-induction by glucagon ± SEM (n = 3 independent experiments). **P < 0.01 compared with Ad-CON by one-way ANOVA. (B) Impact of IRE1α knockdown on glucagon-induced transcriptomic changes in hepatocytes. Microarray analysis was performed as described in SI Appendix, Fig. S3. Heat maps represent averaged fold changes of gene expression from three independent experiments, showing differentially expressed genes caused by glucagon induction (GI) as aligned with changes of these genes as a result of IRE1α knockdown in the presence of glucagon stimulation (GI/shIRE1α_shCON). PCC value was calculated between the fold-changes in the two heat maps. The pie charts indicate the percentages of glucagon-up-regulated or -down-regulated genes that were oppositely changed or unaltered by IRE1α knockdown. (C and D) IRE1α deficiency reduced glucose production from liver cells and attenuated the hyperglycemic response to glucagon in vivo. Mice were infected with Ad-shCON or Ad-shIRE1α-#2 through tail vein injection. (C) Primary hepatocytes were isolated after 5 d of infection and glucose production was measured in the absence or presence of 10 μM forskolin (Fsk). Data are presented as the mean ± SEM (n = 3 independent experiments). **P < 0.01, ##P < 0.01 by two-way ANOVA. (D) Glucagon challenge test. Mice infected for 10 d were injected intraperitoneally with glucagon (150 μg/kg) after a 15-h fast. Blood glucose was measured at the indicated time points after glucagon injection. The areas under the curve (AUC) were shown as the mean ± SEM (n = 10–11/group). *P < 0.05, **P < 0.01 by two-way ANOVA or t test.
Fig. 3.
Fig. 3.
PKA is directly responsible for phosphorylating IRE1α. (A and B) Inhibition of PKA decreased glucagon- or epinephrine-induced phosphorylation of IRE1α. (A) Primary hepatocytes precultured for 30 min with PKA inhibitor H89 (at 5 and 10 μM) or PLC inhibitor U73122 (at 5 and 15 μM) were treated with 100 nM glucagon or PBS/DMSO (Veh) for 1 h. (B) Hepatocytes precultured with 5 and 10 μM H89 were stimulated with 10 μM epinephrine for 10 min. Immunoblotting was performed with the indicated antibodies. (C) PKA knockdown blunted glucagon-stimulated IRE1α phosphorylation. Hepatocytes infected for 72 h with adenoviruses Ad-shCON or Ad-shPKA were subsequently incubated for 1 h with 100 nM glucagon or 1 μM Tg. (D) Glucagon-induced phosphorylation of IRE1α was independent of its capability of autophosphorylation. Hepatocytes were infected with adenoviruses expressing EGFP, Flag-tagged human wild-type (WT) IRE1α or IRE1α-K599A and IRE1α-S724A mutants. Cells with or without preincubation for 30 min in 10 μM H89 were then treated with 100 nM glucagon for 1 h. Phosphorylation of IRE1α and splicing of Xbp1 mRNA were analyzed. (E and F) PKA directly phosphorylated IRE1α in vitro. (E) Hepatocytes were infected for 48 h with adenoviruses expressing the indicated forms of Flag-tagged IRE1α. IRE1α proteins were immunoprecipitated with Flag antibody and subsequently incubated with purified mouse PKA or human PKCε at 30 °C for 1 h. (F) Purified recombinant protein of the cytoplasmic portion of human IRE1α was likewise incubated with PKA or PKC. Phosphorylation of IRE1α was analyzed by immunoblotting. All results are shown as representative of three (A–E) or two (F) independent experiments.
Fig. 4.
Fig. 4.
Phosphorylation at Ser724 dictates the functional output of IRE1α. (A) Blocking Ser724 phosphorylation resulted in altered autophosphorylation status of IRE1α. Primary hepatocytes infected with adenoviruses expressing the indicated forms of Flag-tagged human IRE1α were treated with PBS or 100 nM glucagon for 1 h. Immunoblotting was performed using IRE1α antibody for band-shift analysis. (B) Impact of disruption of Ser724 phosphorylation on IRE1α-evoked transcriptomic changes in hepatocytes. Microarray analysis was performed as described in SI Appendix, Fig. S7. Heat maps represent averaged fold-changes of gene expression from three independent experiments, showing differentially expressed genes upon IRE1α-WT overexpression (WT versus EGFP control) as aligned with changes of these genes caused by S724A mutation (S724A versus WT). PCC value was calculated, and the pie charts indicate the percentages of IRE1α up-regulated or down-regulated genes, which were oppositely changed or unaltered by S724A mutation. (C) S724A mutation impaired IRE1α-WT up-regulation of gluconeogenic genes. Hepatocytes infected with the indicated adenoviruses were treated with PBS or 100 nM glucagon for 1 h. The mRNA abundance of Pepck and G6pase was determined by real-time RT-PCR using actin as an internal control. Data are shown as the mean ± SEM after normalization to the Ad-EGFP control. *P or #P < 0.05, **P or ##P < 0.01 by one-way ANOVA.
Fig. 5.
Fig. 5.
PKA-dependent hyperactivation of hepatic IRE1α contributes to obesity-associated disruption of glucose metabolism. (A–C) Highly increased IRE1α phosphorylation in the livers of db/db mice is PKA-dependent and distinct from that induced by ER stress. Male db/db mice were treated for 2 h with PBS or H89 (5 mg/kg body weight) through intraperitoneal injection. For ER stress control, primary hepatocytes were treated with 1 μM Tg or DMSO for 1 h. (A) Liver extracts from db/db mice and their wild-type littermates (+/+) were analyzed by immunoblotting with the indicated antibodies. Shown are representative results for three individual mice from each group (n = 4–5 per group). (B) Abundance of the spliced (s) and total (t) Xbp1 mRNA was determined by real time RT-PCR. (C) Band-shift immunoblot analysis of hepatic IRE1α from wild-type or db/db mice and from Tg-treated hepatocytes. (D–G) Knockdown of hepatic IRE1α improved glucose metabolism in db/db mice. Male db/db mice were infected with Ad-shCON or Ad-shIRE1α-#2 (n = 5/group). (D) At 21 d after infection, the mRNA of liver G6pase and Pepck was analyzed by real-time RT-PCR after a 6-h fast, using actin as an internal control. (E) Glucose was measured after a 6-h fast from mice infected for 18 d. (F and G) Pyruvate tolerance test and glucose tolerance test. Mice infected for 15 or 18 d were fasted for 6 h before intraperitoneal injection with 2 g/kg pyruvate (F) or 1.5 g/kg glucose (G). Blood glucose was measured at the indicated time points, and the AUCs are shown as the mean ± SEM (n = 5/group). *P < 0.05, **P < 0.01 by two-way ANOVA or t test.
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References

    1. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007;8:519–529. - PubMed
    1. Patil C, Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus: The unfolded protein response in yeast and mammals. Curr Opin Cell Biol. 2001;13:349–355. - PubMed
    1. Mori K, Ma W, Gething MJ, Sambrook J. A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 1993;74:743–756. - PubMed
    1. Sidrauski C, Walter P. The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell. 1997;90:1031–1039. - PubMed
    1. Cox JS, Shamu CE, Walter P. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 1993;73:1197–1206. - PubMed

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