Stimulation of glucose uptake by theasinensins through the AMP-activated protein kinase pathway in rat skeletal muscle cells

Theasinensins, dimeric catechins, have been reported to possess anti-hyperglycemic activity, but the underlying mechanism for this activity remains unknown. In this study, the effect of theasinensins A and B on glucose uptake into rat skeletal muscle cells (L6 myotubes) was investigated. A glucose uptake study using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) indicated that both theasinensins A and B stimulated glucose uptake in a concentration-dependent manner and translocation of glucose transporter 4 (GLUT4) to the plasma membrane. In addition, inhibition studies measuring 2-NBDG uptake in L6 cells revealed that compound C (AMP-activated protein kinase inhibitor) suppressed theasinensin-stimulated glucose uptake, whereas genistein (insulin receptor tyrosine kinase inhibitor) and wortmannin (phosphatidylinositol 3-kinase inhibitor) were inactive. Subsequent experiments on GLUT4-related signaling pathways in L6 cells demonstrated that theasinensins promoted the phosphorylation of AMPK, but not that of Akt, and that the theasinensin-promoted glucose uptake was blocked in the presence of a CaMKK inhibitor. The promotion of AMPK phosphorylation by theasinensins was not blocked in LKB1-knockdown cells. Consequently, it was concluded that theasinensins A and B did in fact promote GLUT4 translocation to the plasma membrane in L6 myotubes through the CaMKK/AMPK signaling pathway, but not through the PI3K/Akt pathway.     

Prolonged exposure to resistin inhibits glucose uptake in rat skeletal muscles

AIM: To assess the effects and mechanisms of the action of resistin on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells. METHODS: Rat myoblasts (L6) were cultured and differentiated into myotubes followed by stimulation with single commercial resistin (130 ng/mL, 0-24 h) or cultured supernatant from 293-T cells transfected with resistin-expressing vectors (130 ng/mL, 0-24 h). Liquid scintillation counting was used to quantitate [3H] 2-deoxyglucose uptake. The translocation of insulin-sensitive glucose transporters GLUT4 and GLUT1, synaptosomal-associated protein 23 (SNAP23) and GLUT protein content, as well as the tyrosine phosphorylation status and protein content of insulin receptor substrate (IRS)-1, were assessed by Western blotting. RESULTS: Treatment of L6 myotubes with single resistin or cultured supernatant containing recombinant resistin reduced basal and insulin-stimulated 2-deoxyglucose uptake and impaired insulin-stimulated GLUT4 translocation. While SNAP23 protein content was decreased, no effects were noted in GLUT4 or GLUT1 protein content. Resistin also diminished insulin-stimulated IRS-1 tyrosine phosphorylation levels without affecting its protein content. The effects of recombinant resistin from 293-T cells transfected with resistin-expressing vectors were greater than that of single resistin treatment. CONCLUSION: Resistin regulated IRS-1 function and decreased GLUT4 translocation and glucose uptake in response to insulin. The downregulated expression of SNAP23 may have been partly attributed to the decrease of glucose uptake by resistin treatment. These observations highlight the potential role of resistin in the pathophysiology of type 2 diabetes related to obesity.     

Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells

Skeletal muscle accounts for ∼80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1^−/− knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell.     

Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation

Compounds that increase the activity of the energy sensor AMP‐activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [³H]‐2‐deoxy‐d ‐glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post‐hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0 μmol/L CA a response (226 ± 9.62% of control, P=.001), similar to maximum insulin (201 ± 7.86% of control, P=.001) and metformin (213 ± 10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA‐stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.     

Rubiscolin-6 activates opioid receptors to enhance glucose uptake in skeletal muscle

Rubiscolin-6 is an opioid peptide derived from plant ribulose bisphosphate carboxylase/oxygenase (Rubisco). It has been demonstrated that opioid receptors could control glucose homeostasis in skeletal muscle independent of insulin action. Therefore, Rubiscolin-6 may be involved in the control of glucose metabolism. In the present study, we investigated the effect of rubiscolin-6 on glucose uptake in skeletal muscle. Rubiscolin-6-induced glucose uptake was measured using the fluorescent indicator 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG) in L6 and C2C12 cell lines. The protein expressions of glucose transporter 4 (GLUT4) and AMP-activated protein kinase (AMPK) in L6 cells were observed by Western blotting. The in vivo effects of rubiscolin-6 were characterized in streptozotocin (STZ)-induced diabetic rats. Rubiscolin-6 induced a concentration-dependent increase in glucose uptake levels. The increase of phospho-AMPK (pAMPK) and GLUT4 expressions were also observed in L6 and C2C12 cells. Effects of rubiscolin-6 were blocked by opioid receptor antagonists and/or associated signals inhibitors. Moreover, Rubiscolin-6 produced a dose-dependent reduction of blood glucose and increased GLUT4 expression in STZ-induced diabetic rats. In conclusion, rubiscolin-6 increases glucose uptake, potentially via an activation of AMPK to enhance GLUT4 translocation after binding to opioid receptors in skeletal muscle.     

Fargesin, a component of Flos Magnoliae, stimulates glucose uptake in L6 myotubes

Flos Magnoliae (FM) is a commonly used Chinese medicinal herb for symptomatic relief of allergic rhinitis, sinusitis and headache. Although several FM species have been used as substitutes or adulterants for clinical use, possible differences in their pharmacological actions have not been reported. To confirm the effects of FM on skeletal muscle glucose metabolism, we tested the effects of several compounds isolated from FM on glucose uptake by L6 myotubes. We found that fargesin, a component of FM, dose-dependently stimulated glucose consumption in L6 myotubes, which was accompanied by enhanced glucose transporter (GLUT)-4 translocation to the cell surface. Fargesin-stimulated glucose uptake was blocked by wortmannin, a phosphatidylinositol-3 kinase (PI3 K) inhibitor. Fargesin stimulated Akt phosphorylation, a key component in the insulin signaling pathway, which was completely inhibited by wortmannin. Here, we demonstrated that fargesin, a bioactive component of Flos Magnoliae, increases basal glucose uptake and GLUT4 translocation in L6 myotubes by activating the PI3 K–Akt pathway.     

Corosolic acid induces GLUT4 translocation in genetically type 2 diabetic mice

The effect of corosolic acid (CA) on blood glucose was studied in KK-Ay mice, an animal model of type 2 diabetes. CA (10 mg/kg) reduced the blood glucose (p<0.05) of KK-Ay mice 4 h after single oral administration when compared with the control group. However, CA did not change the plasma insulin. The muscle facilitative glucose transporter isoform 4 (GLUT4) translocation from low-density microsomal membrane to plasma membrane was significantly increased in the orally CA-treated mice when compared with that of the controls (p<0.05). These results suggest that the hypoglycemic effect of CA is derived, at least in part, from an increase in GLUT4 translocation in muscle. Therefore, it may be that CA has beneficial effects on hyperglycemia in type 2 diabetes.     

Role of SNARE's in the GLUT4 translocation response to insulin in adipose cells and muscle

Insulin stimulates glucose transport in skeletal muscle, heart, and adipose tissue by promoting the appearance of GLUT4, the major glucose transporter isoform present in these tissues, on the cell surface. This is achieved by differentially modulating GLUT4 exocytosis and endocytosis, between a specialized intracellular compartment and the plasma membrane. Ligands which activate the heterotrimeric GTP-binding proteins Gs and Gi appear to modulate insulin-stimulated glucose transport through effects on the fusion of docked GLUT4-containing vesicles with the plasma membrane. In insulin resistance states, reduced cellular GLUT4 levels in adipose cells fully account for the decreased glucose transport response to insulin in these cells. In contrast, although insulin-stimulated GLUT4 translocation is also impaired in muscle, total cellular levels of GLUT4 are not altered. The defect in muscle has been attributed to a GLUT4 trafficking problem and thus studies of this mechanism could provide clues as to the nature of the impairment. The movement of GLUT4-containing vesicles from an intracellular storage site to the plasma membrane and the fusion of docked GLUT4-containing vesicles with the plasma membrane are conceptually similar to some secretory processes. A general hypothesis called the SNARE hypothesis (soluble NSF attachment protein receptors where NSF stands for N-ethylmaleimide-sensitive fusion protein) postulates that the specificity of secretory vesicle targeting is generated by complexes that form between membrane proteins on the transport vesicle (v-SNARE's) and membrane proteins located on the target membrane (t-SNARE's). Several v- and t-SNARE's have been identified in adipose cells and muscle. VAMP2 and VAMP3/cellubrevin (v-SNARE's) have been shown to interact with the t-SNARE's syntaxin 4 and SNAP-23. The cytosolic protein NSF has the characteristic of binding to the v-/t-SNARE complex through its interaction with alpha-SNAP, another soluble factor. Furthermore, recent studies have demonstrated that VAMP2/3, syntaxin 4, SNAP-23, and NSF are functionally involved in insulin-stimulated GLUT4 translocation in adipose cells and thus are likely to be involved in the Gs- and Gi-mediated modulation of the glucose transport response to insulin as well. This review summarizes recent advances on the normal mechanism of GLUT4 translocation and discusses how this process could be affected in insulin resistant states such as type II diabetes.     

Synthetic (+)-antroquinonol exhibits dual actions against insulin resistance by triggering AMP kinase and inhibiting dipeptidyl peptidase IV activities

BACKGROUND AND PURPOSE

The fungal product (+)-antroquinonol activates AMP kinase (AMPK) activity in cancer cell lines. The present study was conducted to examine whether chemically synthesized (+)-antroquinonol exhibited beneficial metabolic effects in insulin-resistant states by activating AMPK and inhibiting dipeptidyl peptidase IV (DPP IV) activity.

EXPERIMENTAL APPROACH

Effects of (+)-antroquinonol on DPP IV activity were measured with a DPPIV Assay Kit and effects on GLP-1-induced PKA were measured in AR42J cells. Translocation of the glucose transporter 4, GLUT4, induced either by insulin-dependent PI3K/AKT signalling or by insulin-independent AMPK activation, was assayed in differentiated myotubes. Glucose uptake and GLUT4 translocation were assayed in L6 myocytes. Mice with diet-induced obesity were used to assess effects of acute and chronic treatment with (+)-antroquinonol on glycaemic control in vivo.

KEY RESULTS

The results showed that of (+)-antroquinonol (100 μM ) inhibited the DPP IV activity as effectively as the clinically used inhibitor, sitagliptin. The phosphorylation of AMPK Thr¹⁷² in differentiated myotubes was significantly increased by (+)-antroquinonol. In cells simultaneously treated with S961 (insulin receptor antagonist), insulin and (+)-antroquinonol, the combination of (+)-antroquinonol plus insulin still increased both GLUT4 translocation and glucose uptake. Further, (+)-antroquinonol and sitagliptin reduced blood glucose, when given acutely or chronically to DIO mice.

CONCLUSIONS AND IMPLICATIONS

Chemically synthesized (+)-antroquinonol exhibits dual effects to ameliorate insulin resistance, by increasing AMPK activity and GLUT4 translocation, along with inhibiting DPP IV activity.     

Prenylflavonoids from fruit of Macaranga tanarius promote glucose uptake via AMPK activation in L6 myotubes

Skeletal muscle is a major tissue of glucose consumption and plays an important role in glucose homeostasis. Prenylflavonoids, a component of Macaranga tanarius fruits, have been reported to have antioxidant, antibacterial, and anticancer effects. However, the effects of these compounds on skeletal muscle glucose metabolism are unclear. Here, we isolated five prenylflavonoids from M. tanarius fruits, and investigated the mechanism of action of these compounds on skeletal muscle cells using L6 myotubes. We found that isonymphaeol B and 3′-geranyl naringenin increased glucose uptake in a dose-dependent manner. Furthermore, both isonymphaeol B and 3′-geranyl naringenin increased AMPK phosphorylation but did not affect PI3K-Akt phosphorylation. Isonymphaeol B and 3′-geranyl naringenin also increased Glut1 mRNA expression and plasma membrane GLUT1 protein levels. These results suggest that isonymphaeol B and 3′-geranyl naringenin have beneficial effects on glucose metabolism through AMPK and GLUT1 pathway. Isonymphaeol B and 3′-geranyl naringenin may be potential lead candidates for antidiabetic drug development.

Graphical abstract

     

Effects of metformin treatment on glucose transporter proteins in subcellular fractions of skeletal muscle in (fa/fa) Zucker rats.

1. The present study was designed to clarify the cellular mechanism through which the antihyperglycaemic drug, metformin, exerts its effects. For this purpose the contents of glucose transporter protein isoforms GLUT1 and GLUT4 were measured in plasma membrane and intracellular membrane fractions of skeletal muscle obtained from genetically obese, insulin-resistant Zucker rats. 2. Hindlimb muscles were dissected from metformin-treated (300 mg kg-1 day-1, p.o., for 12 days) and control rats in basal treatment state, and after acute stimulation with insulin (22 u kg-1, i.p.). Since metformin treatment reduces food intake, we also used a pair-fed control group to investigate the effects of altered insulinaemia per se. Glucose transporter levels were analysed by Western blot and slot blot-techniques. In addition, 2-deoxy-[14C]-glucose uptake in isolated muscle strips was evaluated. 3. No changes were noted in the contents of GLUT1 proteins in any of the subcellular fractions after metformin treatment. The contents of GLUT4 in subcellular fractions were not altered in the basal treatment state. After acute insulin exposure the content of GLUT4 in the intracellular membrane fraction declined significantly in the metformin-treated group, while no significant effect was seen in the plasma membrane fraction. In agreement with these results, metformin treatment did not alter 2-deoxyglucose uptake into isolated muscle strips. 4. In conclusion, the present study does not support the concept that metformin would enhance translocation of glucose transporter proteins from the intracellular compartment to the plasma membrane in skeletal muscle in vivo.     

Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling

Insulin induces a translocation of the glucose transporter GLUT4 from intracellular storage compartments towards the cell surface in adipocytes and skeletal muscle cells, allowing the cells to take up glucose. In type 2 diabetes-associated insulin resistance, the efficiency of this process is reduced. The thiazolidinediones, widely prescribed as anti-diabetic therapy, are generally regarded as insulin-sensitizers. The aim of this study was to evaluate the effect of the thiazolidinedione rosiglitazone (BRL 49653) on GLUT4 in adipocytes. When applied during differentiation, rosiglitazone dose dependently augmented GLUT4 expression along with the formation of lipid droplets. Intriguingly, its presence during differentiation led to increases in both cell surface GLUT4 levels and insulin sensitivity of GLUT4 translocation in mature adipocytes. Treatment of fully differentiated adipocytes with rosiglitazone also led to increases in GLUT4 at the plasma membrane. Rosiglitazone similarly affected cell surface levels of the endosomal transferrin receptor, but did not alter the GLUT4 internalization rate. The augmentation in cell surface GLUT4 levels was maintained in adipocytes that were rendered insulin-resistant in vitro by a 24 h insulin treatment and moreover in these cells rosiglitazone also fully restored insulin-induced GLUT4 translocation. We conclude that in adipocytes, rosiglitazone increases cell surface GLUT4 levels by increasing its endosomal recycling and restores insulin-induced GLUT4 translocation in insulin resistance. These results implicate novel modes of action on GLUT4 that are all likely to contribute to the insulin-sensitizing effect of rosiglitazone in type 2 diabetes.     

Regulation of microvascular flow and metabolism: An overview

Skeletal muscle is an important site for insulin to regulate blood glucose levels. It is estimated that skeletal muscle is responsible for ~80% of insulin‐mediated glucose disposal in the post‐prandial period. The classical action of insulin to increase muscle glucose uptake involves insulin binding to insulin receptors on myocytes to stimulate glucose transporter 4 (GLUT 4) translocation to the cell surface membrane, enhancing glucose uptake. However, an additional role of insulin that is often under‐appreciated is its action to increase muscle perfusion thereby improving insulin and glucose delivery to myocytes. Either of these responses (myocyte and/or vascular) may be impaired in insulin resistance, and both impairments are apparent in type 2 diabetes, resulting in diminished glucose disposal by muscle. The aim of this review is to report on the growing body of literature suggesting that insulin‐mediated control of skeletal muscle perfusion is an important regulator of muscle glucose uptake and that impairment of microvascular insulin action has important physiological consequences early in the pathogenesis of insulin resistance. This work was discussed at the 2015 Australian Physiological Society Symposium “Physiological mechanisms controlling microvascular flow and muscle metabolism”.     

Exercise-induced galanin release facilitated GLUT4 translocation in adipocytes of type 2 diabetic rats

Although galanin has been shown to increase insulin sensitivity in skeletal muscle of rats, there is no literature available about the effect of galanin on Glucose Transporter 4 (GLUT4) translocation from intracellular membrane pools to plasma membranes in adipocytes of type 2 diabetic rats. In the present study M35, a galanin antagonist was used to elucidate whether exercise-induced galanin release increased GLUT4 translocation in adipocytes of streptozotocin-induced diabetic rats. The present findings showed that plasma galanin levels after swimming training in all four trained groups were higher compared with each sedentary control. M35 treatment had an inhibitory effect on glucose infusion rates in the euglycemic-hyperinsulinemic clamp test and GLUT4 mRNA expression levels in adipocytes. Moreover, M35 treatment reduced GLUT4 concentration in both plasma membranes and total cell membranes. The ratios of GLUT4 contents in plasma membranes to total cell membranes in four drug groups were lower compared with each control. These data demonstrate a beneficial role of endogenous galanin to transfer GLUT4 from internal stores to plasma membranes in adipocytes of type 2 diabetic rats. Galanin plays a significant role in regulation of glucose metabolic homeostasis and is an important hormone relative to diabetes.     

Evaluation of fish oil-rich in MUFAs for anti-diabetic and anti-inflammation potential in experimental type 2 diabetic rats

The advantages of monounsaturated fatty acids (MUFAs) on insulin resistance and type 2 diabetes mellitus (T2DM) have been well established. However, the molecular mechanisms of the anti-diabetic action of MUFAs remain unclear. This study examined the anti-hyperglycemic effect and explored the molecular mechanisms involved in the actions of fish oil- rich in MUFAs that had been acquired from hybrid catfish (Pangasius larnaudii×Pangasianodon hypophthalmus) among experimental type 2 diabetic rats. Diabetic rats that were fed with fish oil (500 and 1,000 mg/kg BW) for 12 weeks significantly reduced the fasting plasma glucose levels without increasing the plasma insulin levels. The diminishing levels of plasma lipids and the muscle triglyceride accumulation as well as the plasma leptin levels were identified in T2DM rats, which had been administrated with fish oil. Notably, the plasma adiponectin levels increased among these rats. The fish oil supplementation also improved glucose tolerance, insulin sensitivity and pancreatic histological changes. Moreover, the supplementation of fish oil improved insulin signaling (p-Akt^Ser473 and p-PKC-ζ/λ^Thr410/403), p-AMPK^Thr172 and membrane GLUT4 protein expressions, whereas the protein expressions of pro-inflammatory cytokines (TNF-α and nuclear NF-κB) as well as p-PKC-θ^Thr538 were down regulated in the skeletal muscle. These data indicate that the effects of fish oil-rich in MUFAs in these T2DM rats were partly due to the attenuation of insulin resistance and an improvement in the adipokine imbalance. The mechanisms of the anti-hyperglycemic effect are involved in the improvement of insulin signaling, AMPK activation, GLUT4 translocation and suppression of pro-inflammatory cytokine protein expressions.     

Antidiabetic effect of plumbagin isolated from Plumbago zeylanica L. root and its effect on GLUT4 translocation in streptozotocin-induced diabetic rats

Plumbago zeylanica L. root is widely used in Indian medicine to treat diabetes mellitus. The aim of the present investigation was to evaluate the antidiabetic effects of plumbagin isolated from P. zeylanica L. root and its effect on GLUT4 translocation in STZ-induced diabetic rats. Plumbagin (15 and 30 mg/kg b wt) was orally administered to STZ-induced diabetic rats for 28 days. An oral glucose tolerance test was performed on 21st day. The effect of plumbagin on body weight, blood glucose, plasma insulin, total protein, urea, creatinine, liver glycogen, plasma enzymes (SGOT, SGPT and ALP) and carbohydrate metabolism enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase and hexokinase) were investigated. GLUT4 mRNA and protein expression in skeletal muscles were also studied. Plumbagin significantly reduced the blood glucose and significantly altered all other biochemical parameters to near normal. Further, plumbagin increased the activity of hexokinase and decreased the activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly in treated diabetic rats. Enhanced GLUT4 mRNA and protein expression were observed in diabetic rats after treatment with plumbagin. The results indicated that plumbagin enhanced GLUT4 translocation and contributed to glucose homeostasis. It could be further probed for use as a drug to treat diabetes.     

Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells differentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of differentiation of the C2C12 myotubes. Insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to significant levels (P<0.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were unaffected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin- and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the effect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.     

Differential activation of glucose transport in cultured muscle cells by polyphenolic compounds from Canna indica L. Root

Effects of extracts of a plant, which has been used as a traditional medicine for treating diabetes on glucose transport activity was evaluated in cultured L8 muscle cells. The aqueous extract of Canna indica root (CI) at doses of 0.1-0.5 mg/ml, which contains total phenolic compounds equivalent to 6-30 microg of catechin caused a dose- and time-dependent induction of 2-deoxy-[3H]glucose (2-DG) uptake activity. The induced 2-DG uptake was significantly increased within 8 h and reached a maximum by 16 h. The CI extract increased the amount of glucose transporter isoforms 1 (GLUT1) and 4 (GLUT4) at the cell surface and enhanced expression of GLUT1 protein. Cycloheximide treatment almost completely reversed CI-induced 2-DG uptake to the basal level. Exposure of muscle cells to wortmannin and SB203580 diminished CI-mediated glucose uptake by 38 and 14%, respectively. The effect of CI and insulin was partially additive. Phytochemical analysis detected the presence of flavonoids and catechol in the CI. Taken together, these data provide evidence for differential effects of CI on regulated-glucose transport in muscle cells. Our findings suggest that GLUT1 protein synthesis and the activation of phosphatidylinositol 3-kinase (PI3K) are critical for the increase in glucose transporter activity at the plasma membrane and essential for the maximal induction of glucose transport by CI in L8 muscle cells.     

GLUT4 associated proteins as therapeutic targets for diabetes

Type 2 diabetes is a complex, multifactorial disease resulting from insulin resistance in target tissues and the impairment of insulin secretion from the pancreas. One of the key metabolic actions of insulin is to control blood sugar levels by promoting glucose uptake into adipocyte and muscle cells. This is achieved by activation of a complex signal transduction cascade that stimulates the trafficking of the insulin responsive glucose transporter protein, GLUT4, from specific intracellular sites to the plasma membrane. This review is divided into two major sections. The first section gives an overview of GLUT4 trafficking and the second section focuses on the patents that have been acquired for GLUT4 associated proteins and which demonstrate potential as therapeutic targets for the treatment of diabetes. Inventions in this area include methods and agents to translocate GLUT4 to the plasma membrane independent of insulin and methods to increase the level of GLUT4 in insulin responsive cells.     

脂联素影响骨骼肌胰岛素抵抗模型中葡萄糖转运蛋白4表达的研究

目的：通过建立骨骼肌L6细胞胰岛素抵抗模型,探讨骨骼肌L6细胞中脂联素（ APN）的分泌,以及脂联素对骨骼肌胰岛素抵抗模型中葡萄糖转运蛋白4（GLUT4）表达的影响。方法（1）体外培养大鼠L6成肌细胞,诱导分化后,给予不同浓度的棕榈酸（PA）,测定不同时间细胞培养上清液葡萄糖浓度,观察PA对L6细胞摄取葡萄糖的影响,建立胰岛素抵抗模型;（2）根据实验条件的不同分为三组：NC组（正常对照组,即骨骼肌L6细胞组）;IR组（胰岛素抵抗模型组）;IR＋PIO组（胰岛素抵抗模型＋吡格列酮组）。应用Western blot方法分别测定上述三组APN和GLUT4表达水平。结果（1）0．4 mmol· L^-1的棕榈酸在作用12、24、36 h以及0．6～0．8 mmol· L^-1棕榈酸作用8～36 h后,细胞培养上清液中葡萄糖含量,明显高于对照组。胰岛素抵抗模型建立;（2）Western blot结果显示：①与NC组比较,IR组APN和GLUT4表达均减少,差异有统计学意义;②与IR组比较,IR＋PIO组其APN和GLUT4表达均增加,差异有统计学意义。结论（1）大鼠L6成肌细胞培养并诱导分化后,经过一定条件下PA刺激,可以建立胰岛素抵抗模型;（2）大鼠L6细胞可分泌和表达脂联素,骨骼肌源性脂联素上调L6细胞GLUT4的表达;（3）吡格列酮作为PPAR-γ激动剂,可增加大鼠L6细胞脂联素的分泌,进而改善胰岛素敏感性。