Urea-induced ROS generation causes insulin resistance in mice with chronic renal failure

Abstract

Although supraphysiological concentrations of urea are known to increase oxidative stress in cultured cells, it is generally thought that the elevated levels of urea in chronic renal failure patients have negligible toxicity. We previously demonstrated that ROS increase intracellular protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc), and others showed that increased modification of insulin signaling molecules by O-GlcNAc reduces insulin signal transduction. Because both oxidative stress and insulin resistance have been observed in patients with end-stage renal disease, we sought to determine the role of urea in these phenotypes. Treatment of 3T3-L1 adipocytes with urea at disease-relevant concentrations induced ROS production, caused insulin resistance, increased expression of adipokines retinol binding protein 4 (RBP4) and resistin, and increased O-GlcNAc-modified insulin signaling molecules. Investigation of a mouse model of surgically induced renal failure (uremic mice) revealed increased ROS production, modification of insulin signaling molecules by O-GlcNAc, and increased expression of RBP4 and resistin in visceral adipose tissue. Uremic mice also displayed insulin resistance and glucose intolerance, and treatment with an antioxidant SOD/catalase mimetic normalized these defects. The SOD/catalase mimetic treatment also prevented the development of insulin resistance in normal mice after urea infusion. These data suggest that therapeutic targeting of urea-induced ROS may help reduce the high morbidity and mortality caused by end-stage renal disease.

Figure 1. Uremic mice are insulin resistant and glucose intolerant.

(A) Insulin tolerance test. Mice were injected i.p. with insulin (0.75 U/kg), and blood glucose was measured at 0, 30, and 60 minutes. (B) i.p. glucose tolerance test. Mice were injected i.p. with glucose (1 mg/g body weight), and blood glucose was measured at 0, 15, 30, 60, and 120 minutes. (C) Insulin resistance in uremic mice. Insulin values from the IPGTT animals shown in B were determined by ELISA. (D) Glucose intolerance in uremic mice. Glucose values from B at 0 and 120 minutes. All results represent mean ± SEM of 9 animals per group. *P < 0.01 compared with controls.

Figure 2. Uremic mice have elevated plasma levels of insulin resistance–associated adipokines.

Plasma RBP4 (A) and resistin (B) concentrations were measured in the plasma of control and uremic mice by semiquantitative Western blot (A) or ELISA (B). Each bar represents the mean ± SEM of 9 mice per group. *P < 0.01 compared with controls.

Figure 3. Urea causes decreased insulin sensitivity in differentiated 3T3L1 adipocytes.

(A) Effect of urea on insulin-stimulated glucose uptake in differentiated 3T3L1 cells. (B) Immunoblot analysis of insulin-induced phosphorylation of IRS-1 tyrosine and Ser636 in urea-treated 3T3L1 cells and controls. (C) Immunoblot analysis of insulin-induced AKT phosphorylation in urea-treated 3T3L1 cells and their controls. Maximum levels of IRS-1 and AKT phosphorylation are shown as 100% in bar graphs.IP, immunoprecipitation. n = 5; *P < 0.01 compared with controls. Data represent mean ± SEM.

Figure 4. Urea-induced decrease in insulin-stimulated glucose transport is mediated by mitochondrial ROS.

Cells were infected with adenoviral vectors expressing catalase, UCP1, or MnSOD. After 48 hours incubation with urea, both intracellular ROS generation (A) and insulin-stimulated glucose uptake (B) were measured. Each bar represents the mean ± SEM of 5 separate experiments, each with n = 8. *P < 0.01 compared respectively with cells not treated with urea (A) or with insulin (B).

Figure 5. Urea-induced ROS increase IRS-1 modification by O-GlcNAc in 3T3L1 adipocytes.

(A) IP Western blot analysis of IRS-1 modification by O-GlcNAc in urea-treated 3T3L1 cells (48 hours) and their controls. (B) IP Western blot analysis of IRS-1 modification by O-GlcNAc in urea-treated 3T3-L1 cells alone and after overexpression of catalase, UCP1, and MnSOD. Cells were infected with adenoviral vectors expressing catalase, UCP1, or MnSOD. After incubation with urea for 48 hours, IRS-1 was immunoprecipitated and the amount of O-GlcNAc modification was analyzed by immunoblotting. Levels of modification in cells infected with empty vector are shown as 100% in bar graphs. n = 5; *P < 0.01 compared with controls. Data represent mean ± SEM.

Figure 6. Urea-induced ROS increase expression and secretion of insulin resistance–associated adipokines in 3T3L1 adipocytes.

(A) Effect of urea on RBP4 and resistin mRNA. Adipokine expression was evaluated by quantitative PCR (qPCR) in cells exposed to urea for 48 hours and in control cells. (B) Effect of urea on RBP4 and resistin protein secreted in the medium by 3T3L1 adipocytes. RBP4 and resistin concentration were measured by ELISA. (C and D) Effect of catalase, MnSOD, and UCP1 overexpression on urea-induced increases in RBP4 (C) and resistin (D) expression. Cells were infected with adenoviral vectors as described above and adipokine expression measured by qPCR. Results are expressed as fold changes versus the controls. Each bar represents the mean ± SEM of 5 different experiments. *P < 0.01 compared with controls.

Figure 7. Treatment of uremic mice with a SOD/catalase mimetic normalizes hyperinsulinemia.

(A) Effect of MnTBAP on uremia. After 2 weeks of treatment with MnTBAP, plasma urea concentration was measured in untreated and MnTBAP-treated control mice and in untreated and MnTBAP-treated uremic mice. Results represent the mean ± SEM of 7 animals per group. *P < 0.01 compared with controls. (B) Effect of MnTBAP on plasma glucose in uremic mice. After 2 weeks of treatment with MnTBAP, plasma glucose was measured in the indicated groups of mice. Results represent mean ± SEM of 7 animals per group. (C) Effect of MnTBAP on uremia-induced hyperinsulinemia in uremic mice. After 2 weeks of treatment with MnTBAP, plasma insulin concentration was measured by ELISA in untreated and MnTBAP-treated control mice and in untreated and MnTBAP-treated uremic mice. The results represent mean ± SEM of 7 animals per group. *P < 0.01 compared with controls.

Figure 8. Treatment of uremic mice with a SOD/catalase mimetic normalizes insulin resistance and glucose intolerance.

Treated uremic mice and treated controls received MnTBAP i.p. for 2 weeks. (A) Insulin tolerance test. Mice were injected i.p. with insulin (0.75 U/kg), and blood glucose was measured at 0, 30, and 60 minutes. (B) i.p. glucose tolerance test. Mice were injected i.p. with glucose (1 mg/g body weight), and blood glucose was measured at 0, 15, 30, 60, and 120 minutes. (C) Insulin resistance in uremic mice. Insulin values from the IPGTT animals shown in B were determined by ELISA. (D) Glucose intolerance in uremic mice. Glucose values from B at 0 and 120 minutes. All results represent mean ± SEM of 7 animals per group. *P < 0.01 compared with controls.

Figure 9. Treatment of uremic mice with a SOD/catalase mimetic normalizes oxidative stress, IRS-1 O-GlcNAc modification, and IRS-1 pTyr in epididymal fat.

Treated uremic mice and treated controls received MnTBAP i.p. for 2 weeks. (A) Oxidative stress was quantified by immunoblotting of epididymal adipose tissue protein using anti–3-nitrotyrosine antibody and normalized for total protein by blotting with anti–β-actin antibody. (B) IP: Western blot analysis of uremia-induced O-GlcNAc modification of IRS-1 and (C) IRS-1 Tyr phosphorylation in epididymal adipose tissue. Levels of IRS-1 modification in epididymal adipose tissue from untreated control mice are shown as 100% in bar graphs. n = 5, *P < 0.01 compared with controls. Data represent mean + SEM.

Figure 10. Treatment of uremic mice with a SOD/catalase mimetic normalizes insulin resistance–associated adipokine levels in adipose tissue and in plasma.

Treated uremic mice and treated controls received MnTBAP i.p. for 2 weeks. (A) Relative expression of RBP4 and resistin in epididymal adipose tissue of untreated and MnTBAP-treated control and uremic mice measured by qPCR. The control values are shown as 1.0. (B) Plasma levels of RBP4 and resistin measured in the plasma of the untreated and MnTBAP-treated control mice and in the plasma of untreated and MnTBAP-treated CRF mice by semiquantitative Western blot or ELISA, respectively. Each bar represents the mean ± SEM of 7 mice per group. *P < 0.01 compared with controls.

Figure 11. Urea infusion induces insulin resistance and elevated insulin resistance–associated adipokines in normal rats.

(A) GIR necessary to maintain euglycemia at 120 minutes of EU clamps of rats infused with PBS or urea with or without MnTBAP treatment. (B) Plasma glucose levels at 120 minutes of EU clamps of rats infused with PBS or urea with or without MnTBAP treatment. (C) Plasma levels of RBP4 at 120 minutes of euglycemic-hyperglycemic clamps in rats infused with PBS or urea with or without MnTBAP treatment. (D) Plasma levels of resistin at 120 minutes of euglycemic-hyperglycemic clamps in rats infused with PBS or urea with or without MnTBAP treatment. All results represent mean ± SEM of 5 animals per group. *P < 0.01 compared with controls.