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Pur? promotes hepatic glucose production by increasing Adcy6 transcription.

ABSTRACT: OBJECTIVE:Enhanced glucagon signaling and hepatic glucose production (HGP) can account for hyperglycemia in patients with obesity and type 2 diabetes. However, the detailed molecular mechanisms underlying the enhanced HGP in these patients are not fully understood. Here, we identify Pur? as a positive regulator of HGP and study its molecular mechanisms in the regulation of HGP both in vivo and in vitro. METHODS:Adenovirus-mediated knockdown or overexpression of Pur? was performed in either primary hepatocytes or the livers of db/db mice. Glucose metabolism, insulin sensitivity, and HGP were determined by glucose, insulin, and lactate tolerance tests, respectively. Pur?/ADCY6 protein levels, glucagon signaling (p-CREB/CREB), and insulin signaling (p-Akt/Akt) were measured by immunoblotting. Gene expression was measured by RNA-seq and real-time quantitative polymerase chain reaction. Luciferase reporter and chromatin immunoprecipitation assays were used to study the interaction between Pur? and the Adcy6 promoter. RESULTS:Pur? was abnormally elevated in obese mice and was also increased under fasting conditions or via the glucagon signaling pathway, which promoted HGP by increasing Adcy6 expression. Liver-specific knockdown of Pur? in db/db mice significantly ameliorated hyperglycemia and glucose intolerance by suppressing the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway. Consistent with this observation, the knockdown of Pur? also inhibited glucose production in isolated primary hepatocytes by inhibiting the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway, whereas the overexpression of Pur? promoted glucose production by activating this signaling pathway. Mechanistically, Pur? directly binds to the promoter of the Adcy6 gene and thereby promotes its transcription. CONCLUSIONS:Taken together, these results illustrate a new model in which Pur? functions to regulate the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to help maintain glucose homeostasis.

SUBMITTER: Jia L

PROVIDER: S-EPMC6920194 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Purβ promotes hepatic glucose production by increasing Adcy6 transcription.

Jia Linna L Jiang Yunfeng Y Li Xinzhi X Chen Zheng Z

Molecular metabolism 20191116

<h4>Objective</h4>Enhanced glucagon signaling and hepatic glucose production (HGP) can account for hyperglycemia in patients with obesity and type 2 diabetes. However, the detailed molecular mechanisms underlying the enhanced HGP in these patients are not fully understood. Here, we identify Purβ as a positive regulator of HGP and study its molecular mechanisms in the regulation of HGP both in vivo and in vitro.<h4>Methods</h4>Adenovirus-mediated knockdown or overexpression of Purβ was performed ...[more]

PMID: 31918924

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Project description:ObjectiveDeregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway.MethodsGlucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane.ResultsHepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells.ConclusionsOur data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

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Pur? promotes hepatic glucose production by increasing Adcy6 transcription.

Publications

Purβ promotes hepatic glucose production by increasing Adcy6 transcription.

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