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. 2009 Nov 15;47(10):1486-93.
doi: 10.1016/j.freeradbiomed.2009.08.014. Epub 2009 Aug 22.

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK

Tara L Archuleta  1 , Andrew M Lemieux, Vitoon Saengsirisuwan, Mary K Teachey, Katherine A Lindborg, John S Kim, Erik J Henriksen
Affiliations

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK

Tara L Archuleta et al. Free Radic Biol Med. .

Abstract

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60-90 microM, H(2)O(2)) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p<0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser(307) phosphorylation, and decreased phosphorylation of Akt Ser(473) (50%) and GSK-3beta Ser(9) (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 microM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.

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Figures

Fig. 1
Fig. 1
Effects of an oxidant stress on glucose transport activity in mammalian skeletal muscle. Basal and insulin-stimulated (5 mU/ml) rates of 2-deoxyglucose uptake were determined in soleus muscle preparations from lean Zucker rats following 2-hr and 4-hr exposures to an in vitro oxidant stress (60-90 μM H2O2). Values are means ± SE for 6-9 muscles per group. *p<0.05 vs. basal in the absence of H2O2. **p<0.05 vs. insulin-stimulated in the absence of H2O2.
Fig. 2
Fig. 2
Effects of an oxidant stress on IRS-1 and IRS-2 protein expression in mammalian skeletal muscle. The expression of IRS-1 (upper panels) and IRS-2 (lower panels) protein was assessed in soleus muscle preparations from lean Zucker rats following 2-hr (left panels) or 4-hr (right panels) exposures to an oxidant stress (60-90 μM H2O2) in the absence or presence of insulin (5 mU/ml). Values are means ± SE for 4-6 muscles per group. **p<0.05 vs. insulin-stimulated in the absence of H2O2.
Fig. 3
Fig. 3
Effects of an oxidant stress on IRS-1 Ser307 phosphorylation in mammalian skeletal muscle. The phosphorylation of IRS-1 on Ser307 was assessed in soleus muscle preparations from lean Zucker rats following 2-hr (left panels) or 4-hr (right panels) exposures to an oxidant stress (60-90 μM H2O2) in the presence of insulin (5 mU/ml). IRS-1 Ser307 phosphorylation is expressed relative to total IRS-1 protein levels in the samples. Values are means ± SE for 4-5 muscles per group. **p<0.05 vs. insulin-stimulated in the absence of H2O2.
Fig. 4
Fig. 4
Effects of an oxidant stress on protein expression of insulin signaling factors and GLUT-4 in mammalian skeletal muscle. The protein expression of four critical insulin signaling elements (IRß, p85 regulatory subunit of PI3-kinase, Akt, and GSK-3ß) and GLUT-4 was determined in soleus muscle preparations from lean Zucker rats following a 4-hr exposure to an oxidant stress (60-90 μM H2O2) in the absence (upper panel) or presence (lower panel) of insulin (5 mU/ml). Values are means ± SE for 4-6 muscles per group.
Fig. 5
Fig. 5
Effects of an oxidant stress on Akt and GSK-3ß phosphorylation in mammalian skeletal muscle. The phosphorylation of Akt Ser473 and GSK-3ß Ser9 was assessed in soleus muscle preparations from lean Zucker rats following a 4-hr exposure to an oxidant stress (60-90 μM H2O2) in the absence or presence of insulin (5 mU/ml). Values are means ± SE for 4-6 muscles per group. **p<0.05 vs. insulin-stimulated in the absence of H2O2.
Fig. 6
Fig. 6
Effects of an oxidant stress on p38 MAPK phosphorylation in mammalian skeletal muscle. The extent of p38 MAPK Thr180/Tyr182 phosphorylation was measured in soleus muscle preparations from lean Zucker rats following 2-hr or 4-hr exposures to an oxidant stress (60-90 μM H2O2) in the presence of insulin (5 mU/ml). Values are means ± SE for 4-5 muscles per group. *p<0.05 vs. basal in the absence of H2O2. **p<0.05 vs. insulin-stimulated in the absence of H2O2.
Fig. 7
Fig. 7
Effects of selective p38 MAPK inhibition on oxidant-associated loss of IRS-1 and IRS-2 protein and increased IRS-1 Ser307 phosphorylation in mammalian skeletal muscle. The expression of IRS-1 and IRS-2 protein and level of Ser307 phosphorylation on IRS-1 in soleus muscle preparations from lean Zucker rats was determined following a 4-hr exposure to an oxidant stress (60-90 μM H2O2) and insulin (5 mU/ml) in the absence or presence of the selective p38 MAPK inhibitor A304000 (10 μM). IRS-1 Ser307 phosphorylation is expressed relative to total IRS-1 protein levels in the samples. Values are means ± SE for 4-5 muscles per group. **p<0.05 vs. insulin-and H2O2-stimulated in the absence of p38 MAPK inhibitor.
Fig. 8
Fig. 8
Effects of selective p38 MAPK inhibition in the presence of an oxidant stress on glucose transport activity in skeletal muscle. Basal and insulin-stimulated rates of 2-deoxyglucose uptake were determined in soleus muscle preparations from lean Zucker rats following a 4-hr exposure to an in vitro oxidant stress (60-90 μM H2O2) in the absence or presence of the selective p38 MAPK inhibitor A304000 (10 μM). Values are means ± SE for 4-5 muscles per group. *p<0.05 vs. H2O2-stimulated in the absence of p38 MAPK inhibitor. **p<0.05 vs. insulin-stimulated in the absence of p38 MAPK inhibitor.
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