Ubiquitin-specific protease 2 regulates hepatic gluconeogenesis and diurnal glucose metabolism through 11β-hydroxysteroid dehydrogenase 1 - PubMed (original) (raw)

. 2012 May;61(5):1025-35.

doi: 10.2337/db11-0970. Epub 2012 Mar 23.

Affiliations

Ubiquitin-specific protease 2 regulates hepatic gluconeogenesis and diurnal glucose metabolism through 11β-hydroxysteroid dehydrogenase 1

Matthew M Molusky et al. Diabetes. 2012 May.

Abstract

Hepatic gluconeogenesis is important for maintaining steady blood glucose levels during starvation and through light/dark cycles. The regulatory network that transduces hormonal and circadian signals serves to integrate these physiological cues and adjust glucose synthesis and secretion by the liver. In this study, we identified ubiquitin-specific protease 2 (USP2) as an inducible regulator of hepatic gluconeogenesis that responds to nutritional status and clock. Adenoviral-mediated expression of USP2 in the liver promotes hepatic glucose production and exacerbates glucose intolerance in diet-induced obese mice. In contrast, in vivo RNA interference (RNAi) knockdown of this factor improves systemic glycemic control. USP2 is a target gene of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a coactivator that integrates clock and energy metabolism, and is required for maintaining diurnal glucose homeostasis during restricted feeding. At the mechanistic level, USP2 regulates hepatic glucose metabolism through its induction of 11β-hydroxysteroid dehydrogenase 1 (HSD1) and glucocorticoid signaling in the liver. Pharmacological inhibition and liver-specific RNAi knockdown of HSD1 significantly impair the stimulation of hepatic gluconeogenesis by USP2. Together, these studies delineate a novel pathway that links hormonal and circadian signals to gluconeogenesis and glucose homeostasis.

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Figures

FIG. 1.

FIG. 1.

Nutritional and circadian regulation of USP2 isoforms. A: Realtime qPCR analysis of USP2–45 and USP2–69 mRNA in liver, white adipose tissue (WAT), and skeletal muscle from fed (open), fasted (filled), and refed (gray) mice. Shown below is a schematic of the USP2 gene locus and the qPCR primers (arrowheads) used to detect USP2–45 and USP2–69 isoforms. Data are means ± SEM (n = 4). *P < 0.05. B: qPCR analysis of liver gene expression at different time points. ZT0 indicates the onset of light phase. C: qPCR analysis of total RNA from hepatocytes in triplicate wells treated with hydrocortisone, glucagon and insulin. Data are means ± SD of samples from one representative experiment. *P < 0.01, hydrocortisone plus glucagon vs. control. #P < 0.01 insulin vs. no insulin. D_–_E: Expression of USP2–45 and USP2–69 in cultured primary hepatocytes (D) or livers from mice (E) transduced with GFP (open, n = 4) or PGC-1α (filled, n = 5) adenovirus. Data are means ± SEM. *P < 0.01, GFP vs. PGC-1α.

FIG. 2.

FIG. 2.

USP2–45 regulates hepatic gluconeogenesis. A: Plasma glucose and insulin concentrations in transduced mice measured under fed condition (ZT5). B: PTT in mice transduced with GFP (open squares, n = 7) or USP2–45 (filled triangles, n = 7) adenovirus. C: qPCR analysis of PEPCK and G6Pase gene expression in livers from mice transduced with GFP (open) or USP2–45 (filled) adenovirus. Data in A-C are means ± SEM. *P < 0.05, GFP vs. USP2–45. D: Blood glucose and insulin levels in mice transduced with control or siUSP2 adenovirus following overnight fast (ZT5). E: PTT in mice transduced with control (open squares, n = 6) or siUSP2 (filled triangles, n = 7) adenovirus. F: qPCR analysis of liver gene expression from mice transduced with control (open) or siUSP (filled) adenovirus. Data in D-F are means ± SEM. *P < 0.05, control vs. siUSP2.

FIG. 3.

FIG. 3.

USP2–45 is downstream of clock and regulates circadian glucose metabolism. A: qPCR analysis of liver gene expression in Bmal1 flox/flox (filled squares) and liver-specific Bmal1 null mice (open diamonds). Data are means ± SD using pooled liver RNA (n = 3–5). B: Regulation of liver Rev-erbα and USP2–45 expression by restricted feeding. Livers were harvested at ZT1 and ZT13 from mice maintained on night feeding (filled, n = 4) or after switching to day feeding for 4 days (open, n = 4). C: Blood glucose levels in mice transduced with control (open squares, n = 11) or siUSP2 (filled triangles, n = 12) adenovirus with feeding restricted to nighttime (upper panel) or 3 days after a switch to daytime feeding (lower panel). Data are double plotted means ± SEM. D: qPCR analysis of liver gene expression from restricted daytime fed mice transduced with control (open) or siUSP2 (filled) harvested at ZT0 or ZT12. Data are means ± SD using pooled RNA assayed in triplicate. *P < 0.05, control vs. siUSP2.

FIG. 4.

FIG. 4.

Hepatic USP2–45 overexpression promotes glucose intolerance in HFD-fed mice. A: qPCR analysis of hepatic USP2 gene expression in mice fed chow (filled, n = 3) or HFD (open, n = 3). Data are means ± SEM. *P < 0.05, chow vs. HFD. B: Plasma glucose concentrations in HFD-fed mice transduced with GFP (open, n = 7) or USP2–45 (filled, n = 7) adenovirus. C_–_D: Plasma insulin concentrations (C) and liver glycogen (D) in ad libitum transduced mice. E: GTT in HFD-fed mice transduced with GFP (open diamonds, n = 8) or USP2–45 (filled squares, n = 8) adenovirus. F: ITT in transduced mice. Data in B_–_F are means ± SEM. *P < 0.05, GFP vs. USP2–45.

FIG. 5.

FIG. 5.

Hepatic gene expression analyses. A: qPCR analysis of liver gene expression in mice transduced with GFP (open) or USP2–45 (filled) adenovirus. Data are means ± SEM. *P < 0.05, GFP vs. USP2–45. B: Immunoblotting analysis of total liver lysates from transduced mice as indicated. C: Quantitation of HSD1 protein expression as normalized to β-actin.

FIG. 6.

FIG. 6.

RNAi knockdown of USP2 in the liver ameliorates glucose intolerance in HFD- fed mice. A: Plasma glucose concentrations in HFD mice transduced with control (open, n = 7) or siUSP2 (filled, n = 7) adenovirus. B: Plasma insulin levels in transduced mice following overnight fasting. C: Liver glycogen content in transduced mice. D and E: GTT (D) and ITT (E) in mice transduced with control (open diamonds) or siUSP2 (filled squares) adenovirus. F: qPCR analysis of liver gene expression in HFD-fed mice transduced with control or siUSP2 adenovirus. Data are means ± SEM. *P < 0.05, control vs. siUSP2. G: Western blot analysis of total liver lysates from transduced mice. H: Quantitation of HSD1 protein expression as normalized to β-actin.

FIG. 7.

FIG. 7.

HSD1 inhibition blocks the effects of USP2–45 on glucose metabolism. A and B: Plasma glucose and insulin concentrations (A) and liver glycogen content (B) in mice transduced with GFP (open, n = 6) or USP2–45 (filled, n = 6) followed by treatments with vehicle or CBX for 3 days. Data are means ± SEM. *P < 0.05 USP2–45 vs. GFP; #P < 0.05 CBX vs. saline. C: qPCR analysis of liver gene expression. Data represents mean ± SD using pooled RNA samples assayed in triplicate. D-E: Plasma glucose (D) and liver gene expression (E) in mice transduced with indicated combinations of adenoviruses. Data are means ± SEM. **P < 0.05 USP2–45+Scrb vs. GFP+Scrb; #P < 0.05 USP2–45+siHSD1 vs. USP2–45+Scrb. Samples were collected at ZT11–12.

FIG. 8.

FIG. 8.

C/EBPα induces HSD1 expression and is a substrate of USP2–45. A and B: Induction of HSD1 mRNA (A) and protein (B) expression by C/EBPα in primary hepatocytes. *P < 0.01, C/EBPα vs. GFP. C: Deubiquitination of C/EBPα by USP2–45 in transiently transfected 293 cells. D: Model depicting the role of USP2–45 in mediating circadian and nutritional regulation of hepatic gluconeogenesis and glucose secretion.

Comment in

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