1 Nature 2006 Vol: 440(7086):944-948. DOI: 10.1038/nature04634

Reactive oxygen species have a causal role in multiple forms of insulin resistance

Insulin resistance is a cardinal feature of type 2 diabetes and is characteristic of a wide range of other clinical and experimental settings. Little is known about why insulin resistance occurs in so many contexts. Do the various insults that trigger insulin resistance act through a common mechanism? Or, as has been suggested1, do they use distinct cellular pathways? Here we report a genomic analysis of two cellular models of insulin resistance, one induced by treatment with the cytokine tumour-necrosis factor-α and the other with the glucocorticoid dexamethasone. Gene expression analysis suggests that reactive oxygen species (ROS) levels are increased in both models, and we confirmed this through measures of cellular redox state. ROS have previously been proposed to be involved in insulin resistance, although evidence for a causal role has been scant. We tested this hypothesis in cell culture using six treatments designed to alter ROS levels, including two small molecules and four transgenes; all ameliorated insulin resistance to varying degrees. One of these treatments was tested in obese, insulin-resistant mice and was shown to improve insulin sensitivity and glucose homeostasis. Together, our findings suggest that increased ROS levels are an important trigger for insulin resistance in numerous settings.

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Figures
Figure 1: Characterization of the insulin-resistant state.a, Rates of glucose transport in TNF-treated cells. Basal glucose transport (grey) and insulin-stimulated glucose transport (white) are shown. Cells were untreated, treated with TNF alone, TNF plus pioglitazone (Pio), or TNF followed by medium (TNF washout). Basal rate refers to the rate of glucose transport in the absence of insulin. Insulin-stimulated rate was calculated as the rate of transport in the presence of insulin minus the basal rate. All values are normalized to the insulin-stimulated rate from untreated cells. Asterisk indicates a significant difference (P < 0.05, t-test) compared to TNF treatment alone. b, Rates of glucose transport in dexamethasone (Dex)-treated cells. Data are analogous to a. c, Measurement of cellular redox status, showing rates of dichlorofluorescein (DCF) oxidation. Asterisk, P < 0.05 versus untreated; plus sign, P < 0.05 versus TNF or dexamethasone alone (t-tests). Results in a–c are mean s.e.m. d, Measurement of chronic oxidative stress. Immunoblots show total protein carbonylation. In the left panel, lanes 1–3 were loaded with an equal amount of protein from cells that were untreated, treated with TNF alone or treated with TNF plus pioglitazone. The right panel is analogous, with dexamethasone replacing TNF. Figure 2: Effect of antioxidants on insulin resistance.a, Partial restoration of TNF- or dexamethasone-induced insulin resistance by MnTBAP. Bars show insulin-dependent glucose transport. Results are mean s.e.m. Asterisks indicate P < 0.05 versus TNF or dexamethasone (Dex) treatment alone (t-test). b, Partial restoration of TNF- or dexamethasone-induced insulin resistance by NAC. Data are analogous to a. c–e, Effect of 250 M MnTBAP on measures of insulin signal transduction in cells with dexamethasone-induced (top) or TNF-induced (bottom) insulin resistance. c, Immunoblots show phospho-Akt (P-Akt) levels on acute stimulation of cells with insulin (left) or in the absence of insulin (right). Total Akt protein levels are shown in the panel immediately below the phosphorylated counterpart. Signals were quantified by densitometry and normalized to total protein levels. The level of insulin-stimulated phospho-Akt decreases by 40% and 60%, respectively, in TNF- and dexamethasone-treated cells. MnTBAP treatment recovers phospho-Akt levels to within 5% (TNF) and 30% (dexamethasone) of that found in untreated cells. d, Stimulation of p70S6K phosphorylation (P-S6K) by insulin was assayed, with data arranged and analysed as in c. The level of insulin-stimulated phospho-S6K decreases by 35% and 65%, respectively, in TNF- and dexamethasone-treated cells. MnTBAP treatment recovers phospho-S6K levels to within 25% (both TNF and dexamethasone) of that found in untreated cells. e, Immunoblot showing JNK phosphorylation (P-JNK) levels on treatment with dexamethasone, dexamethasone and MnTBAP, TNF, or TNF and MnTBAP. Total JNK levels are shown in the panel immediately below phospho-JNK. The level of phospho-JNK increases by 25% and 80%, respectively, in TNF- and dexamethasone-treated cells. MnTBAP treatment restores phospho-JNK to untreated levels. Figure 3: Effect of transgenes on insulin resistance.a–d, Effect of cytocatalase (a), mitocatalase (b), CuZnSOD (c) or MnSOD (d) transgene expression on TNF- and dexamethasone-induced insulin resistance. Bars show insulin-dependent rates of glucose transport in untreated cells with or without transgene induction (white), or in TNF- or dexamethasone-treated cells with or without transgene induction (grey). Results are mean s.e.m. Asterisks indicate P < 0.05 versus TNF or dexamethasone alone (t-test). Figure 4: Effects of chronic treatment with MnTBAP or rosiglitazone on obese mice.a, Effect on body weight. Bars show weights of animals after 12 weeks of treatment with daily subcutaneous injection of MnTBAP or rosiglitazone (Rosiglit.). Animals were treated with 2.5 mg kg-1 (n = 7), 5 mg kg-1 (n = 8) or 10 mg kg-1 (n = 8) MnTBAP, 3 mg kg-1 rosiglitazone (n = 8) or vehicle (phosphate-buffered saline) (n = 8). b, Effect on fed glucose levels. Bars indicate mean fed glucose levels averaged over 18 days during the last eight weeks of treatment. Glucose levels were determined at random times during the day and on random days during this period. c, Effect on insulin sensitivity. Insulin tolerance tests were performed on mice from each treatment group (vehicle, 5 mg kg-1 MnTBAP or rosiglitazone). Lines indicate the time course of glucose levels after subcutaneous injection of human insulin (2 U kg-1). d, Effect on glucose tolerance. Glucose tolerance tests were performed, with lines indicating the time course of glucose excursion following subcutaneous injection of glucose (1 g kg-1) in vehicle-treated, 10 mg kg-1 MnTBAP and rosiglitazone treatment groups. Results are mean s.e.m. Astersisks indicate P < 0.05 versus vehicle-treated animals (t-test). Glucose and insulin tolerance test data using different dose levels of MnTBAP are provided in Supplementary Information.
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References
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