Diabetic downregulation of Nrf2 activity via ERK contributes to oxidative stress-induced insulin resistance in cardiac cells in vitro and in vivo - PubMed (original) (raw)

Diabetic downregulation of Nrf2 activity via ERK contributes to oxidative stress-induced insulin resistance in cardiac cells in vitro and in vivo

Yi Tan et al. Diabetes. 2011 Feb.

Abstract

Objective: Oxidative stress is implicated in cardiac insulin resistance, a critical risk factor for cardiac failure, but the direct evidence remains missing. This study explored a causal link between oxidative stress and insulin resistance with a focus on a regulatory role of redox sensitive transcription factor NF-E2-related factor 2 (Nrf2) in the cardiac cells in vitro and in vivo.

Research design and methods: Chronic treatment of HL-1 adult cardiomyocyte with hydrogen peroxide led to insulin resistance, reflected by a significant suppression of the insulin-induced glucose uptake. This was associated with an exaggerated phosphorylation of extracellular signal-related kinase (ERK). Although U0126, an ERK inhibitor, enhanced insulin sensitivity and attenuated oxidative stress-induced insulin resistance, LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), worsened the insulin resistance. Moreover, insulin increased Nrf2 transcriptional activity, which was blocked by LY294002 but enhanced by U0126. Forced activation of Nrf2 by adenoviral over-expression of Nrf2 inhibited the increased ERK activity and recovered the blunted insulin sensitivity on glucose uptake in cardiomyocytes that were chronically treated with H(2)O(2). In the hearts of streptozotocin-induced diabetic mice and diabetic patients Nrf2 expression significantly decreased along with significant increases in 3-nitrotyrosine accumulation and ERK phosphorylation, whereas these pathogenic changes were not observed in the heart of diabetic mice with cardiac-specific overexpression of a potent antioxidant metallothionein. Upregulation of Nrf2 by its activator, Dh404, in cardiomyocytes in vitro and in vivo prevented hydrogen peroxide- and diabetes-induced ERK activation and insulin-signaling downregulation.

Conclusions: ERK-mediated suppression of Nrf2 activity leads to the oxidative stress-induced insulin resistance in adult cardiomyocytes and downregulated glucose utilization in the diabetic heart.

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Figures

FIG. 1.

H2O2-induced insulin resistance in HL-1 cells. Cells were pretreated with H2O2 (100 μmol/L) with or without NAC (1 mmol/L) in 0.1% FBS medium in the absence of norepinephrine for 16 h and then subjected to basal or insulin (100 nmol/L for 10 min)-stimulated 2-DG uptake assay (A), followed by Western blotting of phosphorylated and total ERK (B). Results are representatives of 3 separated experiments (n = 4). *P < 0.05 vs. control (H2O2− and insulin−), #P < 0.05 vs. H2O2 (+) without NAC; otherwise statistical difference was indicated. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Effect of inhibitors of mitogen-activated protein kinase kinase (MEK) and PI3K on basal or insulin-induced glucose uptake in HL-1 cells with or without oxidative stress. Cells were pretreated with or without H2O2 (100 μmol/L) as in Fig. 1, with absence (A) and presence (B) of 100 nmol/L insulin pretreatment for 10 min. Cells were treated with or without U0126 (10 μmol/L) or LY294002 (10 μmol/L) as indicated for 30 min and then subjected to 2-DG uptake assay. *P < 0.05 vs. control (−), #P < 0.05 vs. insulin (+) plus H2O2 (+); n = 4.

FIG. 3.

A negative regulation of Nrf2 by ERK worsens insulin sensitivity in HL-1 cells. A: Insulin activates Nrf2 via a negative cross-talk between PI3K and ERK. Cells were transfected with ARE-luc and pRL-TK-luc in Opti-MEM (Invitrogen) for 6 h and then changed with 0.1% FBS medium in the absence of norepinephrine for 48 h. The cells were next stimulated with 100 nmol/L of insulin, 10 μmol/L of U0126, and 10 μmol/L of LY294002 as indicated for 12 h. Nrf2 transcriptional activity was measured by a dual luciferase assay kit (Promega). *P < 0.05 vs. control (−), #P < 0.05 vs. insulin (+); n = 4. B: Effect of overexpression of Nrf2 on insulin sensitivity in HL-1 cells. Cells infected with Ad-βGal (20 multiplicity of infection [MOI]) and Ad-Nrf2 (20 MOI) were pretreated with H2O2 (100 μmol/L) as in Fig. 1 and subjected to 2-DG uptake assay. *P < 0.05 vs. control (−), n = 4. C: Infected cells were pretreated as in Fig. 1 and stimulated with or without insulin (100 nmol/L) for 10 min. *P < 0.05 vs. control (H2O2− and insulin−); otherwise statistical difference was indicated.

FIG. 4.

Cardiac Nrf2 expression in the mice with and without cardiac-specific MT overexpression. Diabetes was induced by multiple low doses of STZ in mice with cardiac-specific MT overexpression (MT-TG) and littermate WT mice. A: Cardiac Nrf2 expression at 2 months after the onset of diabetes was detected by Western blot (WT, n = 6 for control [Ctrl], n = 8 for diabetes mellitus [DM]; MT-TG, n = 7 for control, n = 8 for DM). At 5 months after the onset of diabetes, cardiac Nrf2 expression (B); nitrosative damage, measured by 3-NT (C); and ERK phosphorylation (D) were detected by Western blot, respectively (WT, n = 7 for control, n = 8 for DM; MT-TG, n = 6 for control, n = 7 for DM). β-Actin or total ERK was used as loading control. *P < 0.05 vs. control.

FIG. 5.

Downregulation of Nrf2 expression in human diabetic hearts. A: Representatives of Nrf2 staining on left ventricular tissue sections of normal (males, n = 5; females, n = 5) and diabetic (males, n = 4; females, n = 2) human hearts. Red is Nrf2, and green is α-myosin heavy chain. Blue is nuclei. The semiquantification of Nrf2 protein levels by measuring mean integrated optical density (IOD) of eight randomly chosen fields of each tissue section for males (B) and females (C) are presented, respectively. Two sections of each heart specimen have been analyzed. *P < 0.05 vs. normal group. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

A therapeutic effect of Dh404 on cardiomyocyte insulin resistance. A: Effect of Dh404 on insulin-induced Nrf2 transcriptional activity in HL-1 cells. Cells were transfected as in Fig. 3. The cells were stimulated with 100 nmol/L of insulin and 200 nmol/L of Dh404 as indicated for 12 h. Nrf2 transcriptional activity was measured as in Fig. 3. Effect of Dh404 on oxidative stress–induced ERK phosphorylation (B) and glucose uptake in HL-1 cells (C) is shown. Cells were pretreated with H2O2 (100 μmol/L) with or without Dh404 (200 nmol/L) in 0.1% FBS medium in the absence of norephineprine for 16 h and then stimulated with or without insulin (100 nmol/L for 10 min) as indicated. 2-DG uptake assay and Western blotting of phosphorylated and total ERK were performed as described in

research design and methods

. *P < 0.05 vs. normal group; #P < 0.05 vs. insulin alone (A) or H2O2 alone (C); †P < 0.05 vs. H2O2 plus insulin (C).

FIG. 7.

Dh404 cardiac prevention of diabetic oxidative stress and inhibition of glucose metabolism. Diabetes was induced by multiple low doses of STZ as used in Fig. 4. Diabetic and age-matched nondiabetic mice were treated with Dh404 at 10 mg/kg body wt every other day from the onset of diabetes for 2 weeks. Cardiac Nrf2 expression (A); nitrosative damage, measured by 3-NT (B); ERK phosphorylation (C); Akt phosphorylation (D); and GSK-3β phosphorylation (E) were detected by Western blot, respectively (n = 5 for control [Ctrl], n = 7 for DM). β-Actin, total ERK, total Akt, or total GSK-3β was used as loading control. *P < 0.05 vs. control; #P < 0.05 vs. DM; $P < 0.05 vs. Dh404. F: A working hypothesis of Nrf2-mediated regulation of insulin sensitivity in the cardiomyocytes. (A high-quality color representation of this figure is available in the online issue.)

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Diabetic downregulation of Nrf2 activity via ERK contributes to oxidative stress-induced insulin resistance in cardiac cells in vitro and in vivo - PubMed (original) (raw)