Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism

Abstract

Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver.

FIG 1

Glucose metabolism in mice with SkM-specific Nrf2 induction. (A) Blood glucose levels and AUCs measured by ipGTT in Keap1FB/FB and Keap1MuKO-B mice (n = 8 or 9). Glucose (2 g/kg of body weight) was administered intraperitoneally to 9-week-old male mice after 16 h of fasting. (B) Blood glucose levels of Keap1FB/FB and Keap1MuKO-B mice either fed ad libitum or under fasting conditions (n = 9). (C) Plasma insulin levels of 9-week-old male Keap1FB/FB and Keap1MuKO-B mice (n = 8 or 9) fed ad libitum. (D) Body weights of 9-week-old male Keap1FB/FB and Keap1MuKO-B mice (n = 9). (E) Keap1 mRNA expression in SkM and liver tissues from Keap1FB/FB and Keap1MuKO-B mice (n = 9). The data were normalized to Hprt, and the expression levels in Keap1FB/FB mice were set as 1. (F) Blood glucose levels and AUCs measured by ipGTT in Nrf2F/F::Keap1FB/FB and Nrf2F^del/F^del::Keap1MuKO-B mice (n = 7). Glucose (2 g/kg of body weight) was intraperitoneally administered to 9-week-old male mice after 16 h of fasting. (G) Blood glucose levels of Nrf2F/F::Keap1FB/FB and Nrf2F^del/F^del::Keap1MuKO-B mice either fed ad libitum or under fasting conditions (n = 7). (H) Body weights of 9-week-old male Nrf2F/F::Keap1FB/FB and Nrf2F^del/F^del::Keap1MuKO-B mice (n = 7). (I) Keap1 and Nrf2 mRNA expression in SkM from Nrf2F/F::Keap1FB/FB and Nrf2F^del/F^del::Keap1MuKO-B mice (n = 7). The data were normalized to Hprt, and the expression levels in Nrf2F/F::Keap1FB/FB mice were set as 1. Error bars show the mean ± SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (versus the control). All of the data were collected from ICR background mice.

FIG 2

Energy consumption in mice with SkM-specific Nrf2 induction. (A) CT images of 28-week-old male Keap1FB/FB and Keap1MuKO-B mice. Note that the epididymal fat masses of these mice were almost comparable. (B) Estimated whole-body lean and fat mass volumes by HU in the continuous-slice CT images (n = 5). (C to E) Tissue weights (C), oxygen consumption (D), and mitochondrial DNA content (E) in dissected SkM (n = 5). Oxygen consumption levels were normalized to tissue weight (D). The mitochondrial DNA content data were normalized to genomic DNA content, and the average content of Keap1FB/FB mice was set as 1 (E). Error bars show the mean ± SEM. *, P < 0.05 (versus the control); n. s., not significant. All of the data were collected from ICR background mice.

FIG 3

Glucose metabolism in mice with SkM-specific Nrf2 deletion. (A) Blood glucose levels and AUCs measured by ipGTT in Nrf2F/F and Nrf2MuKO mice (n = 6). Glucose (2 g/kg of body weight) was administered intraperitoneally to 8-week-old male mice after 16 h of fasting. (B) Blood glucose levels of Nrf2F/F and Nrf2MuKO mice either fed ad libitum or under fasting conditions (n = 6). (C) Body weights of Nrf2F/F and Nrf2MuKO mice (n = 6). Error bars show the mean ± SEM. **, P < 0.01; *, P < 0.05 (versus Nrf2F/F mice). All of the data were collected from C57BL/6J background mice.

FIG 4

Nrf2 regulates the expression of glycogen metabolism-related enzymes. (A) Gbe1, Phka1, and Nqo1 mRNA expression levels in SkM from 9-week-old male Keap1FB/FB, Keap1MuKO-B, Nrf2F/F::Keap1FB/FB, and Nrf2F^del/F^del::Keap1MuKO-B mice (n = 6 to 9). (B) Keap1, Gbe1, Phka1, and Nqo1 mRNA expression levels in SkM from 9-week-old male Keap1FA/+, Keap1FA/FA, and Keap1MuKO-A mice (n = 6). (C) Nrf2, Gbe1, Phka1, and Nqo1 mRNA expression levels in SkM from 6-week-old male Nrf2F/F and Nrf2MuKO mice (n = 4). The data were normalized to Hprt, and the expression level in each control was set as 1. (D, E) Immunoblot analysis of GBE, PhKα1, NQO1, and α-tubulin (TUB) in SkM from 9-week-old male Keap1FB/FB and Keap1MuKO-B mice (n = 6) (D) and 6-week-old Nrf2F/F and Nrf2MuKO mice (n = 6 or 7) (E). Protein expression was quantified and normalized to TUB. The expression level in each control was set as 1. Error bars show the mean ± SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (versus the control). All of the data were collected from ICR background mice.

FIG 5

Nrf2 regulates SkM glycogen metabolism. (A) Protocol used to evaluate SkM glycogen metabolism. (B) Glycogen contents of SkM from Keap1FB/FB mice under fasting (n = 4) and F/R conditions (n = 6) and Keap1MuKO-B mice under F/R conditions (n = 6); the data are normalized to tissue weight. (C) Glycogen phosphorylase-mediated G1P release from SkM glycogen from Keap1FB/FB and Keap1MuKO-B mice under F/R conditions (n = 6). Oyster glycogen served as a positive control; the data are presented as arbitrary units (AU) of A₃₄₀ (left side), and the rate of glycogen phosphorylase release of G1P was calculated according to the change in A₃₄₀ per milligram of glycogen in 30 min (right side). Error bars show the mean ± SEM. ***, P < 0.001 (versus the control). All of the data were collected from ICR background mice.

FIG 6

Nrf2 and glycogen metabolism in the liver. (A and B) Keap1, Nqo1, and Gbe1 mRNA expression profiles in the livers of 15-week-old male Keap1FB/FB and Keap1LKO-B mice (A; n = 6) and Keap1FA/+, Keap1FA/FA, and Keap1LKO-A mice (B; n = 6). The data were normalized to Gapdh, and the expression levels in the controls were set as 1. (C) Protocol for evaluation of liver glycogen metabolism. (D and E) Liver glycogen content (D) and blood glucose levels (E) of Keap1FB/FB mice under fasting (n = 3) and F/R conditions (n = 8), in Keap1MuKO-B mice under F/R conditions (n = 8), in Keap1FA/+ mice under fasting and F/R conditions, and in Keap1MuKO-A mice under F/R conditions (n = 6). Glycogen content was normalized to tissue weight. (F) Blood glucose levels of Keap1LKO-B and Keap1FB/FB mice (n = 10 or 11) as measured by ipGTT. Glucose (2 g/kg of body weight) was intraperitoneally administered to 9-week-old male mice after 16 h of fasting. Error bars show the mean ± SEM. ***, P < 0.001; ***, P < 0.01; *, P < 0.05. n. s., not significant. All of the data were collected from ICR background mice.

FIG 7

Effects of chemical Nrf2 induction on Gbe1 and Phka1 expression. (A) Effects of Nrf2-inducing chemicals on Gbe1, Phka1, and Nqo1 gene expression. C2C12 myotubes were treated with 0.1% DMSO (vehicle [Veh]), 100 μmol/liter DEM, 50 μmol/liter tBHQ, 10 μmol/liter SFN, or 100 nmol/liter CDDO-Im for 24 h. The data were normalized to Hprt, and the expression levels in vehicle-treated cells were set as 1 (n = 4 each). (B) Dose-response analysis. C2C12 myotubes were treated with either 0.1% DMSO (vehicle in dose 0) or CDDO-Im for 24 h. The data were normalized to Hprt, and the expression levels in the vehicle-treated groups were set as 1 (n = 4 each). (C) Time course analysis. C2C12 myotubes were treated with either 0.1% DMSO or 100 nmol/liter CDDO-Im for the times indicated. The data were normalized to Hprt, and the expression levels in the time zero groups were set as 1 (n = 4 each). Error bars show the mean ± SEM. ***, P < 0.001; *, P < 0.05 (versus the control).

FIG 8

CDDO-Im effects on Gbe1 and Phka1 in vivo. (A, B) Time course analysis of the effects of CDDO-Im administration in mouse SkM (A) and liver (B). Wild-type (WT) and Nrf2 knockout mice received either CDDO-Im (30 μmol/kg of body weight) or the vehicle orally, and SkM and livers were collected at the times indicated (n = 3 each for 3 and 6 h, n = 4 for 12 h). CDDO-Im (30 μmol/kg of body weight) and the vehicle were readministered to the 12-h groups 6 h after the first administration. The data were normalized to Hprt, and the expression levels in the vehicle-treated wild-type mice were set as 1. Error bars show the mean ± SEM. ***, P < 0.001; **, P < 0.01 (versus the vehicle-treated wild type). All of the data were collected from C57BL/6J background mice.

FIG 9

Effect of Nrf2 induction on exercise capacity. (A and B) Protocols for CDDO-Im administration (A) and the treadmill test (B) to estimate exercise capacity. (C) Treadmill test results of 8-week-old male mice after treatment with CDDO-Im or the vehicle (n = 6 or 7). Maximum running speed and distance were determined. (D) Blood glucose levels after the treadmill test (n = 6 or 7). (E to G) SkM tissue weights (E); glycogen contents in SkM and liver (F); and expression levels of Gbe1, Phka1, and Nqo1 in SkM (G) before and 1 day after treadmill exercise (n = 6 or 7). The glycogen contents were normalized to tissue weights (F). The mRNA data were normalized to Hprt, and the expression levels in the vehicle-treated pretreadmill mice were set as 1. Error bars show the mean ± SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (versus vehicle-treated mice). All of the data were collected from C57BL/6J background mice.

FIG 10

Nrf2 transcriptional regulation of Gbe1 and Phka1 expression. (A) Screen shots of ChIP-seq profiles around the Gbe1 and Nqo1 TSSs from two independent experiments (Exp.) with CDDO-Im (100 nmol/liter, 3 h)-treated C2C12 myotubes and anti-Nrf2 antibody. (B) Manual ChIP analysis of Gbe1 (kb −0.4 and kb +15 from the TSS) and Nqo1 (a positive locus). Nrf2-DNA complexes were immunoprecipitated with either anti-Nrf2 antibody (n = 4) or control IgG (n = 3) from nuclear extracts of C2C12 myotubes cultured with either 0.1% DMSO or 100 nmol/liter CDDO-Im for 3 h. The data are percentages of the input DNA. (C) Immunoblot analysis of Nrf2 and lamin B from nuclear extracts of C2C12 myotubes cultured in 0.1% DMSO or 100 nmol/liter CDDO-Im for 3 h. (D) Schematic of the chimeric reporter containing the 5′-flanking region of the mouse Gbe1 gene and luciferase cDNA. SV40, simian virus 40. (E and F) Luciferase reporter analysis in C2C12 myoblasts. C2C12 myoblasts were transiently transfected with reporter vectors and cultured for 24 h with the concentrations of CDDO-Im indicated. Analysis of luciferase reporter expression in response to various doses of CDDO-Im (E) and mutational analysis of Gbe1 AREs (F). The transcriptional activities at dose 0 (E, 0.1% DMSO vehicle) and of the vehicle-treated wild-type reporter (F) were set as 1 (n = 4). Data are relative firefly luciferase activity (test) normalized to Renilla luciferase activity (control). (G) Manual ChIP analysis of Phka1 (kb −1.6 from the TSS) in nuclear extracts from C2C12 myotubes cultured with 0.1% DMSO or 100 nmol/liter CDDO-Im for 3 h with anti-Nrf2 antibody (n = 4) or control IgG (n = 3). The data are the percentages of the input DNA. Error bars show the mean ± SEM. ***, P < 0.001 (versus the vehicle).

FIG 11

Nrf2 enhances glucose uptake in SkM. (A) 2-DG uptake in SkM of 8-week-old male Keap1FA/+ and Keap1FA/– mice for 4 h. After 16 h of fasting, 2-DG (1 g/kg of body weight) was intraperitoneally administered. The data are intracellular 2-DG–6-phosphate levels normalized to tissue weight (n = 7), and the level in the control was set as 1. (B) Profiles of Keap1FA/–::Nrf2MuKO mice. (C) Blood glucose levels and AUCs in ipGTT of Keap1FA/+::Nrf2F/F, Keap1FA/–::Nrf2F/F, and Keap1FA/–::Nrf2MuKO mice (n = 5 or 6). Glucose (2 g/kg of body weight) was administered intraperitoneally to 16-week-old male mice after 16 h of fasting. (D) Gbe1 and Phka1 mRNA expression levels in SkM (n = 5 or 6). The data were normalized to Hprt, and the expression levels in the controls were set as 1. Error bars show the mean ± SEM. ***, P < 0.001; *, P < 0.05 (versus the control). †, P < 0.001 (versus Keap1FA/–::Nrf2F/F mice). All of the data were collected from ICR background mice.

FIG 12

SkM-specific induction of Nrf2 improves diabetes mellitus. Blood glucose levels (A) and body weights (B) of 8- to 9-week-old control db/db::Keap1FA/FA (n = 9; all females) and db/db::Keap1MuKO-A (n = 5; 4 females and 1 male) mice fed ad libitum are shown. Error bars show the mean ± SEM. *, P < 0.05 (versus the control). All of the data were collected from ICR background mice.

FIG 13

Schematic representation of Nrf2 regulation of liver and SkM glycogen and glucose metabolism. Nrf2-mediated GBE and PhKα1 induction accelerates SkM glycogen branching and breakdown. In SkM, Nrf2 enhances glycogen release of G1P and promotes glucose uptake, exercise capacity, and locomotive activity. In contrast, Nrf2 enhances expression of GBE but not PhKα1 in the liver, promoting glycogen storage and contributing to the maintenance of blood glucose levels during fasting.