Insulin-stimulated Akt Ser473 Phosphorylation Research Articles

148-OR: Partial Knockout of Skeletal Muscle Dynamin-Related Protein 1 Improves Insulin Sensitivity in Diet-Induced Insulin Resistant Mice, but Not in Normal Control Mice

Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported excessive Drp1 activity in skeletal muscle from both animals and humans with insulin resistance, which is strongly correlated to impaired skeletal muscle insulin sensitivity. To examine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis, we generated tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 partial knockout mice (mDrp1+/-) . Male mDrp1+/- and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, received tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. Improvements in whole-body glucose tolerance and insulin sensitivity were observed in HFD-fed mDrp1+/- mice (P<0.05) , but not in LFD-fed mDrp1+/- mice, when compared to their respective WT controls. Further, HFD-induced impairment in skeletal muscle insulin-stimulated Akt Ser473 phosphorylation (P<0.05) was mitigated in HFD-fed mDrp1+/- mice. In contrast, insulin-stimulated Akt Ser473 phosphorylation was reduced in LFD-fed mDrp1+/- mice compared to LFD-fed WT mice (P<0.05) . mDrp1+/- mice exhibited more fused and interconnected mitochondrial networks in skeletal muscle than WT mice. The examination of mitochondrial function revealed no change in mitochondrial respiration but reductions in mitochondrial Complex I and II and pyruvate dehydrogenase derived H2O2 production in HFD-fed mDrp1+/- mice compared to HFD-fed WT mice (P<0.05) . In conclusion, our data suggest that partial knockout of skeletal muscle Drp1 improves skeletal muscle insulin sensitivity and whole-body glucose homeostasis in diet-induced insulin-resistant mice but not in normal control mice. These improvements are, at least in part, due to reduced mitochondrial-derived H2O2 production. Disclosure B.A.Kugler: None. N.Lin: None. P.D.Nguyen: None. A.Ali: None. A.Sesay: None. B.Kalemba: None. H.Sesaki: None. K.Zou: None. Funding National Institutes of Health (R15DK131512) Diabetes Action Research and Education Foundation Grant (#505)

Inhibiting Drp1-Mediated Mitochondrial Fission Attenuates High-Fat Diet Induced Skeletal Muscle Insulin Resistance

Dynamin-related protein 1 (Drp1) is a critical mediator of mitochondrial fission. Recent studies have reported increased Drp1 activation in obese skeletal muscle, which is associated with reduced mitochondrial function and insulin sensitivity. PURPOSE: To determine if inhibiting Drp1-mediated mitochondrial fission by a pharmacologic inhibitor attenuates skeletal muscle insulin resistance induced by a high-fat diet (HFD) in mice. METHODS: 6-week old male C57BL/6J mice (n=9/group) were assigned to either a HFD (45% Fat) or low-fat diet (LFD, 10% Fat) group for a total of 5-weeks. A subgroup of HFD-fed mice received intraperitoneal injections of Mitochondrial Division Inhibitor 1 (MDIVI-1) (20 mg/kg) while the other mice received saline every other day for the last week of diet intervention. A glucose tolerance test was performed after 4 hours of fasting. Twenty-four hours after the final injection, quadriceps and gastrocnemius muscles were collected for further analysis. H2O2 levels were detected using Amplex Red Hydrogen Peroxide kit. Insulin signaling and protein markers of mitochondrial dynamics were measured by immunoblot analysis. RESULTS: HFD significantly increased glucose area under the curve (AUC) than LFD mice (19074 ± 2137 vs. 10726 ± 1254; p < 0.05), but MDIVI-1 treatment attenuated glucose AUC in HFD-fed mice (15354 ± 1278). MDIVI-1 treatment reduced Drp1 expression in mitochondrial fraction from HFD-fed mice when compared to the saline-treated counterparts (0.003 ± 0.001 vs. 0.012 ± 0.003; p < 0.05). The mitochondrial fraction Drp1 expression was positively associated with glucose AUC (r = 0.497, p = 0.042). Insulin-stimulated Aktser473 phosphorylation was reduced in insulin-stimulated skeletal muscle from HFD-fed mice compared to mice fed with LFD (0.70 ± 0.22 vs. 2.21 ± 0.39; p < 0.05), but no difference was found between HFD-fed mice treated with MDIVI-1 and LFD-fed mice. HFD had a significant increase in H2O2 than LFD-fed mice (0.351 ± 0.046 ± 0.186 ± 0.043; p < 0.05), while MDIVI-1 treatment reduced H2O2 production.in HFD-fed mice (0.351 ± 0.046 vs. 0.215 ± 0.364; p < 0.05) CONCLUSION: These data suggest that inhibiting Drp1-mediated mitochondrial fission attenuates skeletal muscle insulin resistance and improves whole-body glucose homeostasis in mice fed by HFD.

Palmitic acid but not palmitoleic acid induces insulin resistance in a human endothelial cell line by decreasing SERCA pump expression

Palmitic acid is a negative regulator of insulin activity. At the molecular level, palmitic acid reduces insulin stimulated Akt Ser473 phosphorylation. Interestingly, we have found that incubation with palmitic acid of human umbilical vein endothelial cells induced a biphasic effect, an initial transient elevation followed by a sustained reduction of SERCA pump protein levels. However, palmitic acid produced a sustained inhibition of SERCA pump ATPase activity. Insulin resistance state appeared before there was a significant reduction of SERCA2 expression. The mechanism by which palmitic acid impairs insulin signaling may involve endoplasmic reticulum stress, because this fatty acid induced activation of both PERK, an ER stress marker, and JNK, a kinase associated with insulin resistance. None of these effects were observed by incubating HUVEC-CS cells with palmitoleic acid. Importantly, SERCA2 overexpression decreased the palmitic acid-induced insulin resistance state. All these results suggest that SERCA pump might be the target of palmitic acid to induce the insulin resistance state in a human vascular endothelial cell line. Importantly, these data suggest that HUVEC-CS cells respond to palmitic acid-exposure with a compensatory overexpression of SERCA pump within the first hour, which eventually fades out and insulin resistance prevails.

Ceramide inhibits insulin-stimulated Akt phosphorylation through activation of Rheb/mTORC1/S6K signaling in skeletal muscle

Ceramide is a negative regulator of insulin activity. At the molecular level, it causes a decrease in insulin-stimulated Akt Ser473 phosphorylation in C2C12 myotubes. Interestingly, we found that the phosphorylation of S6K at Thr389 was increased under the same conditions. Utilizing both rapamycin to inhibit mTORC1 activity and shRNA to knock down Rheb, we demonstrated that the decrease in Akt Ser473 phosphorylation stimulated by insulin after C2-ceramide incubation can be prevented. The mechanism by which C2-ceramide impairs signaling would seem to involve a negative feedback of activated S6K via phosphorylation of insulin receptor substrate-1 at Ser636/639, since S6K inhibitor can block this phenomenon. Finally, rapamycin treatment was found not to affect C2-ceramide-induced PKCζ activation, suggesting that the pathway revealed in this study is parallel to the one involving PKCζ activation. We proposed a novel pathway/mechanism involving Rheb/mTORC1/S6K signaling to explain how C2-ceramide impairs insulin signaling via Akt phosphorylation. The existence of multiple pathways involved in insulin signaling impairment by C2-ceramide treatment implies that different strategies might be needed to ameliorate insulin resistance caused by C2-ceramide.

Cafeteria diet-induced insulin resistance is not associated with decreased insulin signaling or AMPK activity and is alleviated by physical training in rats

Excess energy intake via a palatable low-fat diet (cafeteria diet) is known to induce obesity and glucose intolerance in rats. However, the molecular mechanisms behind this adaptation are not known, and it is also not known whether exercise training can reverse it. Male Wistar rats were assigned to 12-wk intervention groups: chow-fed controls (CON), cafeteria diet (CAF), and cafeteria diet plus swimming exercise during the last 4 wk (CAF(TR)). CAF feeding led to increased body weight (16%, P < 0.01) and increased plasma glucose (P < 0.05) and insulin levels (P < 0.01) during an IVGTT, which was counteracted by training. In the perfused hindlimb, insulin-stimulated glucose transport in red gastrocnemius muscle was completely abolished in CAF and rescued by exercise training. Apart from a tendency toward an approximately 20% reduction in both basal and insulin-stimulated Akt Ser(473) phosphorylation (P = 0.051) in the CAF group, there were no differences in insulin signaling (IR Tyr(1150/1151), PI 3-kinase activity, Akt Thr(308), TBC1D4 Thr(642), GSK3-alpha/beta Ser(21/9)) or changes in AMPKalpha1 or -alpha2, GLUT4, Munc18c, or syntaxin 4 protein expression or in phosphorylation of AMPK Thr(172) among the groups. In conclusion, surplus energy intake of a palatable but low-fat cafeteria diet resulted in obesity and insulin resistance that was rescued by exercise training. Interestingly, insulin resistance was not accompanied by major defects in the insulin-signaling cascade or in altered AMPK expression or phosphorylation. Thus, compared with previous studies of high-fat feeding, where insulin signaling is significantly impaired, the mechanism by which CAF diet induces insulin resistance seems different.

Abstract 5447: Thromboxane A 2 Receptor Activation Attenuates Insulin Signaling and Angiogenesis by Upregulating Phosphatase PTEN in Cultured Endothelial Cells

Increasing evidence indicates that elevated levels of endogenous thromboxane receptor (TPr) agonists including thromboxane (Tx) A 2 , prostaglandin H 2 , and 8-iso-prostaglandin F 2a contribute to insulin resistance, endothelial dysfunction, and accelerated atherosclerosis in diabetes. However, the mechanisms by which TPr triggers insulin resistance in endothelial cells remaine poorly defined. Here we show how TPr activation impairs insulin signaling and angiogenesis in endothelial cells. Exposure of cultured human umbilical vein endothelial cells (HUVEC) to a structurally related TxA 2 mimetic (IBOP, U46619, or carboxy-TxA 2 ) for 3 or 16h significantly inhibited both basal- and insulin-stimulated Akt-Ser473 phosphorylation, Akt activity, the serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS-Ser1177), and angiogenesis. Concomitantly, IBOP significantly increased the levels of Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and PTEN phosphorylation at Ser380/Thr382/383. Further, Y27632, a Rho kinase (ROCK) inhibitor, abolished the effects of IBOP on the phosphorylation of PTEN, Akt and eNOS, and the PTEN level. Similarly, inhibition of PTEN by small interference RNA (siRNA), like the selective TPr antagonist, SQ29548, or knockdown of TPr by siRNA, abolished IBOP-induced inhibition on both the phosphorylation of the Akt-eNOS axis and angiogenesis. Furthermore, IBOP significantly increased the Ser428 phosphorylation of LKB1, a known upstream kinase for PTEN. Finally, suppression of LKB1 abolished IBOP-induced PTEN phosphorylation and PTEN accumulation in HUVEC. We conclude that TPr suppresses insulin signaling and angiogenesis by upregulating RhoA mediated but LKB1-dependent PTEN phosphorylation in endothelial cells. This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma &amp; Texas).

Effects of fasting on insulin action and glucose kinetics in lean and obese men and women

The development of insulin resistance in the obese individual could impair the ability to appropriately adjust metabolism to perturbations in energy balance. We investigated a 12- vs. 48-h fast on hepatic glucose production (R(a)), peripheral glucose uptake (R(d)), and skeletal muscle insulin signaling in lean and obese subjects. Healthy lean [n = 14; age = 28.0 +/- 1.4 yr; body mass index (BMI) = 22.8 +/- 0.42] and nondiabetic obese (n = 11; age = 34.6 +/- 2.3 yr; BMI = 36.1 +/- 1.5) subjects were studied following a 12- and 48-h fast during 2 h of rest and a 3-h 40 mUxm(-2)xmin(-1) hyperinsulinemic-euglycemic clamp (HEC). Basal glucose R(a) decreased significantly from the 12- to 48-h fast (lean 1.96 +/- 0.23 to 1.63 +/- 0.15; obese 1.23 +/- 0.07 to 1.07 +/- 0.07 mgxkg(-1)xmin(-1); P = 0.004) and was equally suppressed during the HEC after both fasts. The increase in glucose R(d) during the HEC after the 12-h fast was significantly decreased in lean and obese subjects after the 48-h fast (lean 9.03 +/- 1.17 to 4.16 +/- 0.34, obese 6.10 +/- 0.77 to 3.56 +/- 0.30 mgxkg FFM(-1)xmin(-1); P < 0.001). After the 12- but not the 48-h fast, insulin-stimulated AKT Ser(473) phosphorylation was greater in lean than obese subjects. We conclude that 1) 48 h of fasting produces a marked decline in peripheral insulin action, while suppression of hepatic glucose production is maintained in lean and obese men and women; and 2) the magnitude of this decline is greater in lean vs. obese subjects.