Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Abstract

Calorie restriction [CR; ∼40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.

Fig. 1.

ERK1/2^{Thr202/Tyr204} phosphorylation in epitrochlearis (left) and soleus muscles (right) with 0, 1.2, or 30 nM insulin. Phosphorylation of ERK1 for each muscle are shown (top), and phosphorylation of ERK2 are shown (bottom). Main effects of diet, insulin (Ins), and diet × insulin interactions from 2-way ANOVA are shown. AL, ad libitum fed group; CR, calorie-restricted group (∼40% below AL group intake); NS, not significant. Data are means ± SE; n = 8 muscles per diet group and insulin concentration.

Fig. 2.

JNK^{Thr183/Tyr185} phosphorylation in epitrochlearis (left) and soleus muscles (right) with 0, 1.2, or 30 nM insulin. Phosphorylation of JNK1 for each muscle is shown (top), and phosphorylation of JNK2 is shown (bottom). Main effects of diet, insulin, and diet × insulin interactions from 2-way ANOVA are shown. Additionally, there were significant increases (*P < 0.05; CR > AL) in JNK1 phosphorylation in the epitrochlearis with 1.2 nM insulin and in the soleus with 30 nM insulin as revealed by post hoc analysis. Data are means ± SE; n = 8 muscles per diet group and insulin concentration.

Fig. 3.

p38^{Thr180/Tyr182} phosphorylation in epitrochlearis (left) and soleus muscles (right) with 0, 1.2, or 30 nM insulin. Main effects of diet, insulin, and diet × insulin interactions from 2-way ANOVA are shown. Additionally, there was a significant increase (*P < 0.05; CR > AL) in p38 phosphorylation in the epitrochlearis with 1.2 nM insulin as revealed by post hoc analysis. Data are means ± SE; n = 8 muscles per diet group and insulin concentration.

Fig. 4.

Phosphorylated ERK1, ERK2, JNK1, JNK2, and p38 in the gastrocnemius. *P < 0.05, CR vs. AL. Data are means ± SE; n = 8 rats per diet group.

Fig. 5.

Phosphorylated mammalian target of rapamycin (mTOR), GSK3α, and GSK3β in the gastrocnemius; n = 8 rats per diet group.

Fig. 6.

Detection of protein carbonyls in the gastrocnemius. Aliquots of each muscle are derivatized with 2–4-dinitrophenylhydrazine (DNPH) (+) or treated with a control solution [without DNPH (−)], and then proteins were subsequently separated by SDS-PAGE and blotted with a DNP-specific antibody (left). Bands at 25, 28, 43, and 67 kDa were quantified (right). MW, molecular weight. Data are means ± SE; n = 8 muscles per diet group.

Fig. 7.

Detection of 4-hydroxynonenal protein conjugates in the gastrocnemius. Protein lysates were subsequently separated by SDS-PAGE and blotted with 4-hydroxynonenal antibody (left). Bands at 38, 48, 63, and 92 kDa were quantified (right). Data are means ± SE; n = 8 muscles per diet group.