Insulin is a stronger inducer of insulin resistance than hyperglycemia in mice with type 1 diabetes mellitus (T1DM)

Abstract

Subjects with type 1 diabetes mellitus (T1DM) eventually develop insulin resistance and other features of T2DM such as cardiovascular disorders. The exact mechanism has been not been completely understood. In this study, we tested the hypothesis that excessive or inappropriate exposure to insulin is a primary mediator of insulin resistance in T1DM. We found that continuous exposure of mice with non-obese diabetes to insulin detemir, which is similar to some current conventional treatment of human T1DM, induced severe insulin resistance, whereas untreated hyperglycemia for the same amount of time (2 weeks) did not cause obvious insulin resistance. Insulin resistance was accompanied by decreased mitochondrial production as evaluated by mitochondrial DNA and levels of transcripts and proteins of mitochondrion-associated genes, increased ectopic fat accumulation in liver and skeletal muscle (gastrocnemius) evaluated by measurements of triglyceride content, and elevated oxidative stress detected by the GSH/GSSG ratio. Prolonged exposure of cultured hepatocytes to insulin induced significant insulin resistance, whereas the same length of exposure to a high level of glucose (33 mm) did not cause obvious insulin resistance. Furthermore, our results showed that prolonged exposure to insulin caused oxidative stress, and blockade of mitochondrion-derived oxidative stress by overexpression of manganese-superoxide dismutase prevented insulin resistance induced by the prolonged exposure to insulin. Together, our results show that excessive exposure to insulin is a primary inducer of insulin resistance in T1DM in mice.

Figures

FIGURE 1.

Continuous exposure to insulin induces insulin resistance while hyperglycemia for the same amount of time does not cause obvious insulin resistance in NOD mice with T1DM. NOD mice with fasting blood glucose levels at ∼300 mg/dl were treated with either the vehicle solution (saline, n = 6) or detemir (n = 5) for 2 weeks as detailed under “Materials and Methods.” NOD mice of the same age without diabetes were used as controls (n = 5). A, doses of detemir used. B, blood glucose levels. ***, p < 0.001 versus vehicle and control. C and D, ITT was performed 14 h after the last dose of detemir at the end of a 2-week treatment. *, p < 0.05; ***, p < 0.01 versus basal (time point 0). E, body weights. F, food consumption. G, water consumption. BW, body weight.

FIGURE 2.

Continuous exposure to insulin increases the basal level of insulin signaling. Protein levels of insulin signaling components from mice described in Fig. 1 were evaluated by immunoblotting. A, PI 3-kinase ((PI3K) p55 subunit) level in liver. B, Akt level in liver. C, PI 3-kinase-α purified from liver lysates was used to convert PI(4,5)P2 into PI(3,4,5)P3. The level of PI 3-kinase activity needed to produce PI(3,4,5)P3 was measured and presented as the mean ± S.E. D, levels of PI 3-kinase (p55 subunit) and Akt in gastrocnemius. E, PI 3-kinase-α purified from gastrocnemius lysates was used to convert PI(4,5)P2 into PI(3,4,5)P3. The level of PI 3-kinase activity needed to produce PI(3,4,5)P3 was measured and presented as the mean ± S.E.

FIGURE 3.

Continuous exposure to insulin increases the basal level of IRS-1 serine phosphorylation. IRS-1 serine phosphorylation in liver (A) and gastrocnemius (B) from the mice described in Fig. 1 were evaluated by immunoblotting with specific antibodies.

FIGURE 4.

Continuous exposure to insulin suppresses mitochondrial production while increasing ectopic fat accumulation and oxidative stress. Liver and gastrocnemius samples from NOD mice described in Fig. 1 were collected for measurement of mitochondrial DNA (A), TFAM protein levels (B and C), triglyceride (TG) content (D), and the ratio between reduced GSH and oxidized GSSG (E) as detailed under “Materials and Methods.” *, p < 0.05 versus control; #, p < 0.05 versus vehicle; ##, p < 0.01 versus vehicle.

FIGURE 5.

Chronic exposure to insulin causes insulin resistance, whereas the same length of exposure to a high level of glucose does not cause insulin resistance in cultured hepatocytes. Hepa1c1c7 cells were precultured in 5.5 mm glucose for 2 days followed by prolonged exposure to insulin (10 nm) in the presence of either a normal level of glucose (5.5 mm) or a high level of glucose (33 mm) for 72 h as noted. After extensive washings with warm phosphate-buffered saline to remove residual insulin, cells were acutely stimulated with 1 or 10 nm insulin for 5 min. Phosphorylation of insulin signaling components was then evaluated by immunoblotting using specific antibodies. The results are representative of three of independent experiments. *, p < 0.05; **, p < 0.01, lane 5 versus lane 2; #, p < 0.05, lane 6 versus lane 3; ¶, p < 0.05; ¶¶, p < 0.01, lane 11 versus lane 8; †, p < 0.05, lane 12 versus lane 9.

FIGURE 6.

Prolonged exposure to insulin induces oxidative stress in isolated hepatocytes. Isolated mouse hepatocytes were incubated with insulin (100 nm) for 24 h followed by measurement of the GSH/GSSG ratio as detailed under “Materials and Methods” (A) or for 6 h followed by evaluation of transcript levels of oxidation responsive genes by real-time PCR (B). H2O2 (0.1 mm) was used as a control. Results represent the mean ± S.E. of two independent experiments. *, p < 0.05 versus vehicle. Gclc, glutamine-cysteine ligase.

FIGURE 7.

Mitochondrion-derived oxidative stress is required for the development of insulin resistance that is induced by prolonged exposure to insulin in isolated hepatocytes. Isolated mouse hepatocytes were infected by recombinant adenoviruses that encoded either manganese-superoxide dismutase (Mn-SOD/SOD2) or no target protein for 36 h. Cells were subsequently preincubated with insulin (100 nm) for 24 h as noted, washed thoroughly with warm media, and then stimulated by fresh insulin (10 nm) for 1 min as noted. Akt phosphorylation and the Mn-SOD expression then were detected by immunoblotting (A). Some of the similarly treated cells were stimulated with cAMP (10 μm)/dexamethasome ((Dex) 50 nm)) for 2.5 h to activate gluconeogenesis in the presence or absence of fresh media with insulin followed by measurement of the gluconeogenic genes glucose-6-phosphatase (G6Pase) and phosphoenoylpyruvate carboxykinase (PEPCK) (B). Results represent the mean ± S.E. of two independent experiments. **, p < 0.01, comparing lanes 9 and 4.