Combination of metformin and thiazolidindiones restore insulin signalling in insulin-resistant cultured myotubes

We examined the effect of combination of thiazolidinediones (TZDs) and metformin on insulin-resistant skeletal muscle cells. The combined use of TZDs and metformin resulted in maximum tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) at 12.5 microM of TZDs and 100 microM of metformin as compared to the maximum tyrosine phosphorylation of IR and IRS-1 achieved at 50 microM of TZDs or 400 microM of metformin. The glucose uptake was significantly high at the combination of lower concentration (12.5 microM of TZDs and 100 microM of metformin) as compared to the combination of higher concentration (50 microM of TZDs and 400 microM of metformin). Results demonstrated that (1) Additive effect on insulin sensitization can be achieved by a combination of TZDs and metformin at lower concentration; (2) combination of TZDs and metformin act on insulin signaling molecules in insulin resistance; (3) in vitro system has the potentiality to determine possible target molecule(s) and mechanism of action of drugs.     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.     

Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.     

Interleukin-15 increases glucose uptake in skeletal muscle An antidiabetogenic effect of the cytokine

Previous studies have demonstrated that interleukin-15 (IL-15) has important anabolic effects on muscle protein metabolism. In the present investigation we have analysed the effects of IL-15 on glucose metabolism in skeletal muscle. Administration of a single dose of the cytokine (100 μg/kg body weight) resulted in a 32% increase on glucose uptake (as measured by the uptake of 2-deoxyglucose) in skeletal muscle. The effects observed on glucose uptake were direct since in vitro incubations of rat EDL muscles in the presence of the cytokine resulted in a 30% increase in glucose uptake. Similarly, IL-15 increased glucose uptake in C2C12 cell cultures, this being related with an increase in both glucose oxidation to CO₂ and the incorporation into muscle lipid. The effects of the cytokine were associated with an increase in GLUT-4 mRNA, suggesting a higher effect in insulin sensitivity. In conclusion, the data presented here indicate that IL-15 facilitates glucose metabolism in skeletal muscle and, therefore, a possible role of the cytokine as an antidiabetogenic drug merits future investigations.     

Mechanotransduction pathways in skeletal muscle hypertrophy

In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.     

Proteomic analysis reveals cellular pathways regulating carbohydrate metabolism that are modulated in primary human skeletal muscle culture due to treatment with bioactives from Artemisia dracunculus L

Insulin resistance is a major pathophysiologic abnormality that characterizes metabolic syndrome and type 2 diabetes. A well characterized ethanolic extract of Artemisia dracunculus L., termed PMI 5011, has been shown to improve insulin action in vitro and in vivo, but the cellular mechanisms remain elusive. Using differential proteomics, we have studied mechanisms by which PMI 5011 enhances insulin action in primary human skeletal muscle culture obtained by biopsy from obese, insulin-resistant individuals. Using iTRAQ™ labeling and LC–MS/MS, we have identified over 200 differentially regulated proteins due to treatment with PMI 5011 and insulin stimulation. Bioinformatics analyses determined that several metabolic pathways related to glycolysis, glucose transport and cell signaling were highly represented and differentially regulated in the presence of PMI 5011 indicating that this extract affects several pathways modulating carbohydrate metabolism, including translocation of GLUT4 to the plasma membrane. These findings provide a molecular mechanism by which a botanical extract improves insulin stimulated glucose uptake, transport and metabolism at the cellular level resulting in enhanced whole body insulin sensitivity.     

Hydroxylamine enhances glucose uptake in C2C12 skeletal muscle cells through the activation of insulin receptor substrate 1

Diabetes mellitus is a global disease, and the number of patients with it is increasing. Of various agents for treatment, those that directly act on muscle are currently attracting attention because muscle is one of the main tissues in the human body, and its metabolism is decreased in type II diabetes. In this study, we found that hydroxylamine (HA) enhances glucose uptake in C2C12 myotubes. Analysis of HA’s mechanism revealed the involvement of IRS1, PI3 K and Akt that is related to the insulin signaling pathway. Further investigation about the activation mechanism of insulin receptor or IRS1 by HA may provide a way to develop a novel anti-diabetic agent alternating to insulin.     

Chronic effects of ethanol on muscle metabolism in the rat

Chronic ethanol feeding in the rat is associated with a skeletal myopathy involving primarily type-II muscle fibers, which is recognised to be mediated via a specific impairment in protein turnover. This paper investigates whether the cause of this myopathy may be related to abnormalities in carbohydrate and lipid metabolism in different muscles. [U-¹⁴C]Glucose metabolism was examined in two muscles with different fibre compsitions, the extensor digitorum longus (EDL) muscle, which contains predominantly type-II muscle fibres, and the soleus muscle, which is composed primarily of type-I muscle fibres. Feeding on the ethanol-supplemented Lieber-DeCarli liquid diet for 2 or 6 weeks was associated with profound distubances in glucose metabolism in both EDL and soleus muscles, particularly in relation to rates of glycogen and alanine formation. We discuss the importance of these metabolic changes in relation to the genesis of chronic alcoholic skeletal myopathy.     

An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes

An enzymatic assay adapted to photometric analysis with 96-well microplates was evaluated for the measurement of 2-deoxyglucose (2DG) uptake in insulin-responsive tissues and differentiated 3T3-L1 adipocytes. For in vivo measurements, a small amount of nonradiolabeled 2DG was injected into mice without affecting glucose metabolism. For photometric quantification of the small amount of 2-deoxyglucose 6-phosphate (2DG6P) that accumulates in cells, we introduced glucose-6-phosphate dehydrogenase, glutathione reductase, and 5,5′-dithiobis(2-nitrobenzoic acid) to the recycling amplification reaction of NADPH. We optimized the enzyme reaction for complete oxidation of endogenous glucose 6-phosphate (G6P) and glucose in mouse tissues in vivo and serum as well as in 3T3-L1 adipocytes in vitro. All reactions are performed in one 96-well microplate by consecutive addition of reagents, and the assay is able to quantify 2DG and 2DG6P in the range of 5–80 pmol. The results obtained with the assay for 2DG uptake in vitro and in vivo in the absence or presence of insulin stimulation was similar to those obtained with the standard radioisotopic method. Thus, the enzymatic assay should prove to be useful for measurement of 2DG uptake in insulin-responsive tissues in vivo as well as in cultured cells.     

Critical role of the transient activation of p38 MAPK in the etiology of skeletal muscle insulin resistance induced by low-level in vitro oxidant stress

Increased cellular exposure to oxidants may contribute to the development of insulin resistance and type 2 diabetes. Skeletal muscle is the primary site of insulin-dependent glucose disposal in the body; however, the effects of oxidative stress on insulin signaling and glucose transport activity in mammalian skeletal muscle are not well understood. We therefore studied the effects of a low-level in vitro oxidant stress (30–40 μM H₂O₂) on basal and insulin-stimulated (5 mU/ml) glucose transport activity and insulin signaling at 2, 4, and 6 h in isolated rat soleus muscle. H₂O₂ increased basal glucose transport activity at 2 and 4 h, but not at 6 h. This low-level oxidant stress significantly impaired insulin-stimulated glucose transport activity at all time points, and was associated with inhibition of insulin-stimulated phosphorylation of Akt Ser⁴⁷³ and GSK-3β Ser⁹. In the presence of insulin, H₂O₂ decreased total protein expression of IRS-1 at 6 h and IRS-2 at 4 and 6 h. Phosphorylation of p38 MAPK Thr¹⁸⁰/Tyr¹⁸² was transiently increased by H₂O₂ in the presence and absence of insulin at 2 and 4 h, but not at 6 h. Selective inhibition of p38 MAPK with A304000 partially rescued the H₂O₂-induced reduction in insulin-stimulated glucose transport activity. These results indicate that direct in vitro exposure of isolated mammalian skeletal muscle to a low-level oxidant stress impairs distal insulin signaling and insulin-stimulated glucose transport activity, at least in part, due to a p38 MAPK-dependent mechanism.     

ANG-(1-7) reduces ANG II-induced insulin resistance by enhancing Akt phosphorylation via a Mas receptor-dependent mechanism in rat skeletal muscle

The nonapeptide angiotensin II (ANG II) induces vasoconstriction via the ANG II type I receptor, while its splice product ANG-(1-7) elicits an antihypertensive effect via the Mas receptor. Although a critical role of ANG II in the etiology of skeletal muscle insulin resistance is well documented, the role of the ANG-(1-7)/Mas receptor axis in this context is poorly understood. Therefore, we determined whether ANG-(1-7) is effective in ameliorating the negative effects of ANG II on insulin-stimulated insulin signaling and glucose transport activity in isolated soleus muscle from normotensive lean Zucker rats. ANG II alone (500 nM for 2 h) decreased insulin-stimulated glucose transport activity by 45% (P < 0.05). In the presence of 500-1000 nM ANG-(1-7), insulin-stimulated glucose transport activity in muscle exposed to ANG II improved by ∼30% (P < 0.05). Moreover, ANG-(1-7) treatment increased Akt Ser⁴⁷³ phosphorylation (47%, P < 0.05) without an effect on glycogen synthase kinase-3β Ser⁹ phosphorylation. The dependence of ANG-(1-7) action on the Mas receptor was assessed using A779 peptide, a selective Mas receptor antagonist. The positive effects of ANG-(1-7) on insulin-stimulated glucose transport activity and Akt Ser⁴⁷³ phosphorylation in soleus muscle were completely prevented in presence of 1000 nM A779. In conclusion, the present study demonstrates that ANG-(1-7), via a Mas receptor-dependent mechanism, can ameliorate the inhibitory effect of ANG II on glucose transport activity in mammalian skeletal muscle, associated with enhanced Akt phosphorylation. These results provide further evidence supporting the targeting of the renin-angiotensin system for interventions designed to reduce insulin resistance in skeletal muscle tissue.     

Estradiol stimulates Akt, AMP-activated protein kinase (AMPK) and TBC1D1/4, but not glucose uptake in rat soleus

Post-menopausal women exhibit decreases in circulating estrogen levels and whole body insulin sensitivity, suggesting that estrogen regulates skeletal muscle glucose disposal. Thus, we assessed whether estrogen stimulates glucose uptake or enhances insulin sensitivity in skeletal muscle. Ex vivo muscle stimulation with 17β-estradiol (10 nM) resulted in a rapid (?10 min) increase in the phosphorylation of Akt, AMP-activated protein kinase (AMPK), and TBC1D1/4, key signaling proteins that regulate glucose uptake in muscle. Treatment with the estrogen receptor antagonist, ICI 182,780, only partly inhibited signaling, suggesting both an estrogen receptor-dependent and independent mechanism of estradiol action. 17β-Estradiol did not stimulate ex vivo muscle [³H]-2-deoxyglucose uptake or enhance insulin-induced glucose uptake, demonstrating discordance between the estradiol-induced stimulation of signaling proteins and muscle glucose uptake. This study is the first to demonstrate that estradiol stimulates Akt, AMPK, and TBC1D1/4 in intact skeletal muscle, but surprisingly, estradiol does not stimulate muscle glucose uptake.     

A newly synthetic chromium complex--chromium(phenylalanine)3 improves insulin responsiveness and reduces whole body glucose tolerance

Low-molecular-weight organic chromium complexes such as chromium picolinate are often used as dietary supplements to improve insulin sensitivity and to correct dyslipidemia. However, toxicity associated with such chromium compounds has compromised their therapeutic value. The aim of this study was to evaluate the impact of a newly synthesized complex of chromium with phenylalanine, Cr(pa)3 on insulin-signaling and glucose tolerance. Cr(pa)3 was synthesized by chelating chromium(III) with D-phenylalanine ligand in aqueous solution. In mouse 3T3-adipocytes, Cr(pa)3 augmented insulin-stimulated glucose-uptake as assessed by a radioactive-glucose uptake assay. At the molecular level, Cr(pa)3 enhanced insulin-stimulated phosphorylation of Akt in a time- and concentration-dependent manner without altering the phosphorylation of insulin receptor. Oral treatment with Cr(pa)3 (150 microg/kg/d, for six weeks) in ob/ob+/+ obese mice significantly alleviated glucose tolerance compared with untreated obese mice. Unlike chromium picolinate, Cr(pa)3 does not cleave DNA under physiological reducing conditions. Collectively, these data suggest that Cr(pa)3 may represent a novel, less-toxic chromium supplement with potential therapeutic value to improve insulin sensitivity and glycemic control in type II diabetes.     

滇产植物乌墨中胰岛素增敏活性成分

从乌墨（Syzygium cumini）根的甲醇提取物中分离鉴定8个已知化合物,并对其进行了经高糖、高胰岛素处理后胰岛素抵抗L6肌管细胞糖摄取活性的筛选。初步研究结果表明,其中7个化合物在有无胰岛素刺激下均能明显促进葡萄糖的利用。在无胰岛素刺激状态下,浓度为10μgml^-1时,无羁萜（1）的葡萄糖消耗量比抵抗细胞增加了17．35％;在有胰岛素刺激状态下,浓度为0．1μgml^-1,5,7,3’,4’,5＇-五羟基黄酮（8）葡萄糖消耗量比抵抗细胞增加了51．11％。     

Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice

Early diabetic nephropathy is characterized by renal hypertrophy that is mainly due to proximal tubular hypertrophy. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, and its signaling has been reported to regulate protein synthesis and cellular growth, specifically, hypertrophy. Therefore, we examined the effect of mTOR signaling on diabetic renal hypertrophy by using the specific inhibitor for mTOR, rapamycin. Ten days after streptozotocin-induced diabetes, mice showed kidney hypertrophy with increases in the phosphorylation of p70S6kinase and the expression of cyclin kinase inhibitors, p21(Cip1) and p27(Kip1), in the kidneys. The intraperitoneal injection of rapamycin (2 mg/kg/day) markedly attenuated the enhanced phosphorylation of p70S6kinase, the increment of cyclin-dependent kinase inhibitors, and renal enlargement without any changes of clinical parameters, including blood glucose, blood pressure, and food intake. Overexpression of a constitutive active form of p70S6kinase resulted in increased cell size of cultured mouse proximal tubule cells; thus, activation of p70S6kinase causes hypertrophy of proximal tubular cells. Our findings suggest that activation of mTOR signaling causes renal hypertrophy at the early stage of diabetes.     

ESAT6通过mTOR抑制自噬并促进BCG增殖

目的 研究mTOR在结核杆菌毒力因子ESAT6诱导的自噬抑制以及促进BCG增殖中的作用。方法 PCMV-HA-ESAT6质粒转染Raw264.7细胞,用蛋白免疫印迹检测LC3、P62、P-mTOR和P-70S6K表达水平;用mTOR阻断剂Torin1联合ESAT6转染以及分别作用于Raw264.7细胞后,免疫印迹检测P62和P-mTOR表达水平,LysoTracker Red染色观察溶酶体变化,BCG增殖实验计数各组菌落数。结果 ESAT6转染细胞后,细胞P62、P-mTOR和P-70S6K表达水平显著增高,LC3I完成向LC3II的转化;联合Torin1的ESAT6转染组和Torin1处理组的P-mTOR和P62无显著变化,溶酶体无变化,BCG菌落数减少。结论 ESAT6诱导的自噬抑制和BCG的增殖依赖于mTOR的活化。