Complexity of NAC Action as an Antidiabetic Agent: Opposing Effects of Oxidative and Reductive Stress on Insulin Secretion and Insulin Signaling

Abstract

Dysregulated redox balance is involved in the pathogenesis of type 2 diabetes. While the benefit of antioxidants in neutralizing oxidative stress is well characterized, the potential harm of antioxidant-induced reductive stress is unclear. The aim of this study was to investigate the dose-dependent effects of the antioxidant N-acetylcysteine (NAC) on various tissues involved in the regulation of blood glucose and the mechanisms underlying its functions. H₂O₂ was used as an oxidizing agent in order to compare the outcomes of oxidative and reductive stress on cellular function. Cellular death in pancreatic islets and diminished insulin secretion were facilitated by H₂O₂-induced oxidative stress but not by NAC. On the other hand, myotubes and adipocytes were negatively affected by NAC-induced reductive stress, as demonstrated by the impaired transmission of insulin signaling and glucose transport, as opposed to H₂O₂-stimulatory action. This was accompanied by redox balance alteration and thiol modifications of proteins. The NAC-induced deterioration of insulin signaling was also observed in healthy mice, while both insulin secretion and insulin signaling were improved in diabetic mice. This study establishes the tissue-specific effects of NAC and the importance of the delicate maintenance of redox balance, emphasizing the challenge of implementing antioxidant therapy in the clinic.

Keywords: N-acetylcysteine; glucose uptake; glutathionylation; insulin signaling; oxidative stress; redox balance; type 2 diabetes.

Figure 1

Effect of NAC on viability and functionality of pancreatic islets. Pancreatic islets were isolated from ICR mice and treated with NAC or H₂O₂ for 24 h. Cytotoxicity was detected using PI staining. Hoechst staining indicates viable cells. Two representative micrographs are presented for each treatment (A). (B,C): Basal and glucose-induced insulin secretion were measured as described in Methods. The data represent the mean ± SEM of 5 independent experiments. * p < 0.05, ** p < 0.005 and *** p < 0.0005, compared to control, or as indicated, analyzed by two-way ANOVA, followed by Bonferroni’s post hoc testing.

Figure 2

Cytotoxic effects of H₂O₂ and NAC. L6 myotubes (A,B) and 3T3-L1 adipocytes (C,D) were treated with H₂O₂ or NAC for 24 h. LDH release was measured as a biomarker of cytotoxicity, as described in Methods. The data represent the mean ± SEM of 4 independent experiments. *** p < 0.0005, compared to control, analyzed by one-way ANOVA, followed by Bonferroni’s post hoc testing.

Figure 3

Short-term NAC treatment did not affect, while H₂O₂ increased glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with H₂O₂ (A) or NAC (B) for 30 min. Insulin (100 nM) was given as a positive control. In a second set of experiments, cells were treated with H₂O₂ (C) or NAC (D) for 10 min before the administration of insulin (100 nM) for an additional 30 min. Deoxy-D-glucose in cells was determined as described in Methods. Data are expressed as percent of basal uptake in control cells. The data represent the mean ± SEM of 5 independent experiments. * p < 0.05, ** p < 0.005 and *** p < 0.0005, compared to control or as indicated, analyzed by one-way ANOVA, followed by Bonferroni’s post hoc testing.

Figure 4

Long-term NAC treatment reduced glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with H₂O₂ (A) or NAC (B) for 24 h. In a second set of experiments, cells treated with H₂O₂ (C) or NAC (D) for 24 h were incubated with insulin (100 nM) for an additional 30 min. Deoxy-D-glucose in cells was determined as described in Methods. Data are expressed as percent of basal uptake in control cells. The data represent the mean ± SEM of 5 independent experiments. * p < 0.05, ** p < 0.005 and *** p < 0.0005, compared to control or as indicated, analyzed by one-way ANOVA, followed by Bonferroni’s post hoc testing.

Figure 5

NAC reduced insulin-induced phosphorylation of signaling proteins in 3T3-L1 adipocytes and L6 myotubes. 3T3-L1 (A) and L6 (B) were treated with H₂O₂ or NAC for 24 h (B) followed by incubation with insulin (100 nM) for an additional 10 min. Western blot analysis of proteins involved in insulin signaling pathway was performed. Representative blot of 3 independent experiments is shown.

Figure 6

NAC increased glutathione level in 3T3-L1 adipocyte and L6 myotubes. 3T3-L1 (A–C) and L6 myotubes (D–F) were treated with H₂O₂ or NAC for 24 h. Levels of GSH, GSSG and total glutathione (tGSH) were measured as described in Methods. The data represent the mean ± SEM of 5 independent experiments. * p < 0.05, ** p < 0.005 and *** p < 0.0005, compared to control, analyzed by one-way ANOVA, followed by Dunnett’s post hoc testing.

Figure 7

NAC increased the level of free thiol residues in 3T3-L1 and L6 cells. 3T3-L1 adipocytes (A) and L6 myotubes (B) were treated with H₂O₂ or NAC for 24 h. Free thiol (SH) level was measured according to Elmann’s assay, as described in Methods. The data represent the mean ± SEM of 5 independent experiments. * p < 0.05, ** p < 0.005 and *** p < 0.0005, compared to control, analyzed by one-way ANOVA, followed by Dunnett’s post hoc testing.

Figure 8

Protein glutathionylation is reduced by NAC in L6 cells. L6 myotubes were treated with H₂O₂ or NAC for 24 h. (A) Western blot analysis was performed under non-reducing conditions using anti-GSH antibody. Representative blot of 3 independent experiments is shown. (B) Number and classification of proteins with glutathionylation level that was increased or decreased by NAC. Level of glutathionylation was detected in L6 myotubes by mass spectrometry as described in Methods. Function of affected proteins was determined using Uniprot.

Figure 9

NAC attenuated insulin signaling in normoglycemic mice. C57BL/6 mice (n = 8, 6 weeks old) were given NAC (1200 mg/kg/day) for 6 weeks, as described in Methods. (A) Body weight was measured every week. (B) Glucose tolerance test (GTT) was performed at age of 12 weeks as described in Methods. Western blot analysis and densitometry analysis of proteins involved in insulin signaling pathway was performed in muscle (C,D) and liver (E,F). Optical density was normalized to the non-phosphorylated form or to housekeeping protein. The results are presented as the mean ± SEM. * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001 by one-way Anova (A) followed by Bonferroni’s post hoc test.

Figure 10

NAC improved glycemic control in diabetic KK-Ay mice. Mice (n = 10, 6 weeks old) were given NAC (1200 mg/kg/day) for 9 weeks, as described in Methods. (A) GTT was performed at the age of 15 weeks as described in Methods (n = 7). Western blot analysis and densitometry analysis of proteins involved in insulin signaling pathway was performed in muscle (B,C) and liver (D,E). Optical density was normalized to the non-phosphorylated form or to housekeeping protein. (F) Pancreatic islets were isolated, and GSIS was performed as described in Methods. The results are presented as the mean ± SEM. * p < 0.05, ** p < 0.005 and *** p < 0.0005 by one-way Anova followed by Bonferroni’s post hoc test.