Manganese supplementation protects against diet-induced diabetes in wild type mice by enhancing insulin secretion

Abstract

Mitochondrial dysfunction is both a contributing mechanism and complication of diabetes, and oxidative stress contributes to that dysfunction. Mitochondrial manganese-superoxide dismutase (MnSOD) is a metalloenzyme that provides antioxidant protection. We have previously shown in a mouse model of hereditary iron overload that cytosolic iron levels affected mitochondrial manganese availability, MnSOD activity, and insulin secretion. We therefore sought to determine the metallation status of MnSOD in wild-type mice and whether altering that status affected β-cell function. 129/SvEVTac mice given supplemental manganese exhibited a 73% increase in hepatic MnSOD activity and increased metallation of MnSOD. To determine whether manganese supplementation offered glucose homeostasis under a situation of β-cell stress, we challenged C57BL/6J mice, which are more susceptible to diet-induced diabetes, with a high-fat diet for 12 weeks. Manganese was supplemented or not for the final 8 weeks on that diet, after which we examined glucose tolerance and the function of isolated islets. Liver mitochondria from manganese-injected C57BL/6J mice had similar increases in MnSOD activity (81%) and metallation as were seen in 129/SvEVTac mice. The manganese-treated group fed high fat had improved glucose tolerance (24% decrease in fasting glucose and 41% decrease in area under the glucose curve), comparable with mice on normal chow and increased serum insulin levels. Isolated islets from the manganese-treated group exhibited improved insulin secretion, decreased lipid peroxidation, and improved mitochondrial function. In conclusion, MnSOD metallation and activity can be augmented with manganese supplementation in normal mice on normal chow, and manganese treatment can increase insulin secretion to improve glucose tolerance under conditions of dietary stress.

Figure 1.

Manganese supplementation increases MnSOD activity in liver. A, Activity of mitochondrial MnSOD in liver was measured after ip injection of 4 mg/kg MnCl₂ for 7 days in wild-type 129/SvEvTac mice on normal chow (n = 3 mice/group). B, Protein levels of MnSOD were measured by immunoblotting. Data were analyzed by the Student t test (2 tail). *P < .05. ANT, adenine nucleotide translocase.

Figure 2.

Metallation of MnSOD protein increases with Mn supplementation in mitochondria from livers of 129/SvEvTac mice. Equal amounts of crude mitochondrial protein extracts of liver from saline-injected (A) and 4 mg/kg Mn-injected mice (B) were fractionated by size exclusion column to isolate SOD protein. Manganese and iron content in each fraction was measured by ICP-OES (PerkinElmer). Immunoblotting was used to determine MnSOD protein content in each fraction. C, MnSOD activity was measured in each fraction from saline- or Mn-injected mouse liver mitochondria. D, Quantitative analysis of triplicate experiments of A and B. E, Quantitative analysis of triplicate experiments of C. Data were analyzed by the Student t test (2 tail), comparing the difference between saline-IP vs MN-IP groups. **P < .01.

Figure 3.

Manganese supplementation does not improve glucose tolerance but increases β-cell function in 129/SvEvTac mice on normal chow. Saline or Mn was administered to the mice for 7 days consecutively. A, Glucose (1 g/kg body weight) was injected ip and tail blood glucose levels were determined at the indicated times. B, Serum insulin levels were determined before and 30 minutes after glucose challenge. C, HOMA-IR. D, HOMA-B. A and B were analyzed by the bivariate ANOVA (2 way ANOVA). Different letters show statistical difference (P < .05). C and D were analyzed by the Student t test (2 tail), comparing difference between saline-IP vs MN-IP groups. * P < .01 (n = 15–18 mice/group).

Figure 4.

Mn supplementation improves glucose tolerance and insulin secretion in a high-fat diet-induced diabetic model. High-fat or normal chow diets were given to age-matched C57BL/6J (3–4 months old) male mice for a month. Maintaining the same diet, saline or Mn was then administered ip for 8 weeks. A, Tail glucose levels were determined at the indicated times after the glucose challenge. B, Area under the glucose curve (AUC) of the glucose tolerance test was calculated. C, Body weights were determined of the mice on normal chow or the high-fat diet, saline or Mn treated. D, HOMA-IR. E, Serum insulin levels were determined before and 30 minutes after a glucose challenge. F, HOMA-B. A, C, and E were analyzed by the bivariate ANOVA (2 way ANOVA). Different letters show statistical differences (P < .05). B, D, and F were analyzed by the Student t test (2 tail), comparing the difference between saline-IP vs MN-IP groups. ***P < .001, *P < .05, 8–11 mice/group (n = 3–11 mice/group for insulin measurement). Values are represented as the mean ± SEM.

Figure 5.

Improved glucose tolerance with Mn supplementation is not due to increased insulin content in pancreas but increased glucose-stimulated insulin secretion. Whole pancreas tissue was homogenized and insulin levels measured, normalized to protein content (3 mice/group) (A). Isolated pancreatic islets were incubated with the indicated glucose concentrations for 1 hour and insulin content of the media measured (B). Panel A was analyzed by the Student t test (2 tail), comparing difference between saline-IP vs MN-IP groups. Panel B was analyzed by the bivariate ANOVA (2 way ANOVA). Differences in letters indicate statistically significant differences (P < .05) (n = 5 mice/group). Values are represented as the mean ± SEM.

Figure 6.

Increased Mn content and mitochondrial SOD activity in liver from Mn-treated mice. Liver cytosolic and mitochondrial preparations were isolated from saline- and Mn-treated mice on high-fat diets. Copper, iron, manganese, and zinc contents were measured in both cytosolic (A) and mitochondrial fractions (B). Manganese supplementation increases activity of mitochondrial MnSOD in Mn-treated mice compared with control mice without a change in cytosolic Cu/Zn SOD activity (C). Data were analyzed by the Student t test (2 tail), comparing the difference between saline-IP vs MN-IP groups. ***P < .001, *P < .05 (n = 4-5 mice/group). Values are represented as the mean ± SEM.

Figure 7.

Mn supplementation increases SOD activity, reduces lipid peroxidation, and increases oxygen consumption rate in pancreatic islets. Pancreatic islets were isolated and SOD activity was measured in whole-islet extracts (A) and purified mitochondria (B). C, Lipid peroxidation levels were assessed in extracts of whole islets. D, Oxygen consumption rates were measured (n = 3/group). Data in panels A, C, and D were obtained from 3 mice/normal chow diet group and 5 mice/high-fat diet group. Data in panel B were from 10 mice per group but to facilitate mitochondrial isolation islets from 2–3 mice were pooled prior to isolation, with each pool being counted as 1 assay, hence 3-4 SOD assays per group. Mice in panel B were left on the high-fat diet for 5 weeks rather than the 8 weeks in the other panels. Panels A–C were analyzed by the Student t test (2 tail), comparing the difference between saline-IP vs MN-IP groups. ***P < .001; **P < .01; *P < .05. Panel D was analyzed by the bivariate ANOVA (2 way ANOVA). Values are represented as the mean ± SEM.