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Hepatic p38α MAPK controls gluconeogenesis via FOXO1 phosphorylation at S273 during glucagon signalling in mice

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Abstract

Aims/hypothesis

Hyperglucagonaemia-stimulated hepatic glucose production (HGP) contributes to hyperglycaemia during type 2 diabetes. A better understanding of glucagon action is important to enable efficient therapies to be developed for the treatment of diabetes. Here, we aimed to investigate the role of p38 MAPK family members in glucagon-induced HGP and determine the underlying mechanisms by which p38 MAPK regulates glucagon action.

Methods

p38α, β, γ and δ MAPK siRNAs were transfected into primary hepatocytes, followed by measurement of glucagon-induced HGP. Adeno-associated virus serotype 8 carrying p38α MAPK short hairpin RNA (shRNA) was injected into liver-specific Foxo1 knockout, liver-specific Irs1/Irs2 double knockout and Foxo1S273D knockin mice. Foxo1S273A knockin mice were fed a high-fat diet for 10 weeks. Pyruvate tolerance tests, glucose tolerance tests, glucagon tolerance tests and insulin tolerance tests were carried out in mice, liver gene expression profiles were analysed and serum triglyceride, insulin and cholesterol levels were measured. Phosphorylation of forkhead box protein O1 (FOXO1) by p38α MAPK in vitro was analysed by LC–MS.

Results

We found that p38α MAPK, but not the other p38 isoforms, stimulates FOXO1-S273 phosphorylation and increases FOXO1 protein stability, promoting HGP in response to glucagon stimulation. In hepatocytes and mouse models, inhibition of p38α MAPK blocked FOXO1-S273 phosphorylation, decreased FOXO1 levels and significantly impaired glucagon- and fasting-induced HGP. However, the effect of p38α MAPK inhibition on HGP was abolished by FOXO1 deficiency or a Foxo1 point mutation at position 273 from serine to aspartic acid (Foxo1S273D) in both hepatocytes and mice. Moreover, an alanine mutation at position 273 (Foxo1S273A) decreased glucose production, improved glucose tolerance and increased insulin sensitivity in diet-induced obese mice. Finally, we found that glucagon activates p38α through exchange protein activated by cAMP 2 (EPAC2) signalling in hepatocytes.

Conclusions/interpretation

This study found that p38α MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis in both health and disease. The glucagon-induced EPAC2–p38α MAPK–pFOXO1-S273 signalling pathway is a potential therapeutic target for the treatment of type 2 diabetes.

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Abbreviations

AAV8:

Adeno-associated virus serotype 8

AMPK:

AMP-activated protein kinase

ATGL:

Adipose triglyceride lipase

CaMKII:

Calcium calmodulin-dependent kinase II

CREB:

cAMP-responsive element-binding protein 

DIO:

Diet-induced obese

DKO:

Liver-specific Irs1 and Irs2 double knockout

EPAC2:

Exchange protein activated by cAMP 2

FAO:

Fatty acid oxidation

FOXO1:

Forkhead box protein O1

G6PC:

Glucose-6-phosphatase

HFD:

High-fat diet

HGP:

Hepatic glucose production

HMOX1:

Heme oxygenase 1

INSP3R1:

Inositol triphosphate receptor 1

PCK:

Phosphoenolpyruvate carboxykinase

PKA:

Protein kinase A

pp38:

p38 phosphorylated at T180/Y182

shRNA:

Short hairpin RNA

t-FOXO1:

Total FOXO1

TKO:

Liver-specific Irs1, Irs2 and Foxo1 triple knockout

WT:

Wild-type

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Authors and Affiliations

  1. Department of Nutrition, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX, USA

    Wanbao Yang, Wang Liao, Xiaopeng Li, Weiqi Ai, Quan Pan, Zheng Shen, Wen Jiang & Shaodong Guo

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  1. Wanbao Yang
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Correspondence to Shaodong Guo.

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Acknowledgements

The authors thank L. Li (Proteomics Service Center, University of Texas Health Science Center, Houston. TX, USA) for help in the analysis of the LC–MS/MS data.

Data availability

All data from this article are available from the corresponding authors on request.

Funding

This work was supported by National Institutes of Health grants (R01 DK095118, R01 DK120968, and R01 DK124588), an ADA Career Development Award (1-15-CD-09), faculty start-up funds from Texas A&M University Health Science Center and AgriLife Research, and an USDA National Institute of Food and Agriculture grant (Hatch 1010958) to SG (PI). SG is the recipient of the 2015 ADA Research Excellence Thomas R. Lee Award and is a 2021 Presidential Impact Fellow at Texas A&M University.

Authors’ relationships and activities

The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work.

Contribution statement

SG conceived the hypothesis, designed the experiments, performed the data analyses and wrote the manuscript. WY designed and conducted the experiments, performed the data analyses and wrote the manuscript. WL conducted the experiments. QP and ZS helped design the study, performed the animal studies, analysed the data and assisted in the preparation of the manuscript. XL, WA and WJ performed the animal studies and assisted in the preparation of the manuscript.. WL, XL, WA, QP, ZS and WJ critically reviewed the manuscript. All authors approved the final version of the manuscript. SG is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.

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Yang, W., Liao, W., Li, X. et al. Hepatic p38α MAPK controls gluconeogenesis via FOXO1 phosphorylation at S273 during glucagon signalling in mice. Diabetologia 66, 1322–1339 (2023). https://doi.org/10.1007/s00125-023-05916-5

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