Heterozygous SOD2 deletion impairs glucose-stimulated insulin secretion, but not insulin action, in high-fat-fed mice

Abstract

Elevated reactive oxygen species (ROS) are linked to insulin resistance and islet dysfunction. Manganese superoxide dismutase (SOD2) is a primary defense against mitochondrial oxidative stress. To test the hypothesis that heterozygous SOD2 deletion impairs glucose-stimulated insulin secretion (GSIS) and insulin action, wild-type (sod2(+/+)) and heterozygous knockout mice (sod2(+/-)) were fed a chow or high-fat (HF) diet, which accelerates ROS production. Hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI) clamps were performed to assess GSIS and insulin action in vivo. GSIS during HG clamps was equal in chow-fed sod2(+/-) and sod2(+/+) but was markedly decreased in HF-fed sod2(+/-). Remarkably, this impairment was not paralleled by reduced HG glucose infusion rate (GIR). Decreased GSIS in HF-fed sod2(+/-) was associated with increased ROS, such as superoxide ion. Surprisingly, insulin action determined by HI clamps did not differ between sod2(+/-) and sod2(+/+) of either diet. Since insulin action was unaffected, we hypothesized that the unchanged HG GIR in HF-fed sod2(+/-) was due to increased glucose effectiveness. Increased GLUT-1, hexokinase II, and phospho-AMPK protein in muscle of HF-fed sod2(+/-) support this hypothesis. We conclude that heterozygous SOD2 deletion in mice, a model that mimics SOD2 changes observed in diabetic humans, impairs GSIS in HF-fed mice without affecting insulin action.

Figure 1

SOD2 expression and activity in islets and muscle. A: Western blotting of SOD2 in isolated islets. GAPDH was used as a loading control. B: Western blotting of SOD2 in muscle mitochondria. V-DAC was used as a loading control. Representative bands were displayed. Data are normalized to chow-fed sod2^+/+ mice and represented as mean ± SEM. C: SOD2 activity was measured in mitochondria isolated from gastrocnemius muscle. n = 7–8. *P < 0.05 compared with sod2^+/+ within a diet.

Figure 2

In vivo GSIS as assessed by the HG clamp. (A) Arterial glucose, (B) GIR, (C and E) arterial insulin, and (D and F) arterial C-peptide were measured throughout the HG clamp. Data are represented as mean ± SEM. n = 13 for chow-fed sod2^+/+ (7 females/6 males); n = 11 for chow-fed sod2^+/− (5 females/6 males); n = 12 for HF-fed sod2^+/+ (5 females/7 males); n = 13 for HF-fed sod2^+/− (6 females/7 males). *P < 0.05 compared with sod2^+/+.

Figure 3

Ex vivo GSIS as assessed by islet perifusion and static incubation. A and B: Isolated islets were perifused with 16.7 mmol/L glucose and 16.7 mmol/L glucose plus IBMX. C and D: Areas under the insulin peaks in response to glucose and IBMX stimulation were quantified. E: Isolated islets were cultured and incubated with either 3 or 20 mmol/L glucose, and insulin was measured in the culture medium. Data are represented as mean ± SEM. n = 4–8 males for all groups. *P < 0.05 compared with sod2^+/+ with the same diet/treatment. IEQs, islet equivalents.

Figure 4

Redox status, gene expression, and areas of islets. A: ROS production was assessed by the ROS indicator dichlorofluorescein diacetate in islets isolated from the HF-fed mice. B and C: Protein carbonyl groups and TBARS were measured in isolated islet extracts. D: Superoxide level was measured by DHE staining in frozen pancreatic sections. Representative images are displayed and fluorescence was quantified in 10–30 islets per genotype for chow-fed mice and 60–70 islets per genotype for HF-fed mice. E: mRNA levels were determined by quantitative real-time PCR and normalized to HF-fed sod2^+/+ mice. F: Islet size was quantified and representative images are displayed. Data are represented as mean ± SEM. n = 4–8 males for all groups. *P < 0.05 compared with sod2^+/+ with the same diet; #P < 0.05 compared with chow-fed mice with the same genotype. IEQs, islet equivalents; Ins, insulin.

Figure 5

In vivo insulin action as determined by the HI clamp. (A) Arterial glucose, (B) GIR, (C) endoRa, (D) Rd, and (E) Rg, an index of muscle glucose uptake, were determined during the HI clamp. F: Western blotting of p-Akt and total Akt in gastrocnemius muscle post the HI clamp. Representative bands are displayed. GAPDH was used as a loading control. The integrated intensities of bands are normalized to chow-fed sod2^+/+ and represented as mean ± SEM. n = 9 for chow-fed sod2^+/+ (4 females/5 males); n = 10 for chow-fed sod2^+/− (5 females/5 males); n = 12 for HF-fed sod2^+/+ (5 females/7 males); n = 13 for HF-fed sod2^+/− (6 females/7 males). f, P < 0.05 for the main diet effect; +P < 0.05 compared with basal with the same genotype; #P < 0.05 compared with chow-fed mice with the same genotype; *P < 0.05 compared with sod2^+/+ with the same diet. SVL, superficial vastus lateralis.

Figure 6

Glucose effectiveness during the HG clamp. (A) Western blotting of GLUT-1, (B) hexokinase II, and (C) phosphorylation of AMPK and total AMPK was performed in gastrocnemius muscle extracts post HG clamp. GAPDH was used as loading control. Representative bands are displayed. The integrated intensities of bands are normalized to chow-fed sod2^+/+ and are represented as mean ± SEM. n = 4–5 (males). D and E: Muscle and liver glycogen content was determined in tissues post HG clamp. n = 6–7 (males). *P < 0.05 compared with sod2^+/+ HF. HK II, hexokinase II.

Figure 7

Redox status and mitochondrial H₂O₂ level in muscle. A: GSH and GSSG concentrations in gastrocnemius muscle were measured. B and C: Protein carbonyl groups and TBARS were measured in gastrocnemius homogenates. D: Mitochondrial H₂O₂ level was measured during succinate titration at state-4 respiration (10 µg/mL oligomycin) in permeabilized muscle fiber bundles prepared from chow- and HF-fed sod2^+/+ and sod2^+/− mice. E: Mitochondrial H₂O₂ level was measured during palmitoylcarnitine-supported state-4 respiration. *P < 0.05 compared with sod2^+/+ with the same diet; #P < 0.05 compared with chow with same genotype. n = 3–8.