Implication of phosphorylation of the myosin II regulatory light chain in insulin-stimulated GLUT4 translocation in 3T3-F442A adipocytes

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca(2+)/calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca(2+) in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3-F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca(2+)-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin-stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca(2+)/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.     

Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach

Type-2 diabetes mellitus (T2DM), the leading global health burden of this century majorly develops due to obesity and hyperglycemia-induced oxidative stress in skeletal muscles. Hence, developing novel drugs that ameliorate these pathological events is an immediate priority. The study was designed to analyze the possible role of Stevioside, a characteristic sugar from leaves of Stevia rebaudiana (Bertoni) on insulin signaling molecules in gastrocnemius muscle of obesity and hyperglycemia-induced T2DM rats. Adult male Wistar rats rendered diabetic by administration of high fat diet (HFD) and sucrose for 60 days were orally administered with SIT (20 mg/kg/day) for 45 days. Various parameters were estimated including fasting blood glucose (FBG), serum lipid profile, oxidative stress markers, antioxidant enzymes and expression of insulin signaling molecules in diabetic gastrocnemius muscle. Stevioside treatment improved glucose and insulin tolerances in diabetic rats and restored their elevated levels of FBG, serum insulin and lipid profile to normalcy. In diabetic gastrocnemius muscles, Setvioside normalized the altered levels of lipid peroxidase (LPO), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH*), antioxidant enzymes (CAT, SOD, GPx and GSH) and molecules of insulin signaling including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt mRNA levels. Furthermore, Stevioside enhanced glucose uptake (GU) and oxidation in diabetic muscles by augmenting glucose transporter 4 (GLUT 4) synthesis very effectively in a similar way to metformin. Results of molecular docking analysis evidenced the higher binding affinity with IRS-1 and GLUT 4. Stevioside effectively inhibits oxidative stress and promotes glucose uptake in diabetic gastrocnemius muscles by activating IR/IRS-1/Akt/GLUT 4 pathway. The results of the in silico investigation matched those of the in vivo study. Hence, Stevioside could be considered as a promising phytomedicine to treat T2DM.     

S-Adenosyl-L-methionine ameliorates TNF��-induced insulin resistance in 3T3-L1 adipocytes

An association between inflammatory processes and the pathogenesis of insulin resistance has been increasingly suggested. The IκB kinase-β (IKK-β)/ nuclear factor-κB (NF-κB) pathway is a molecular mediator of insulin resistance. S-Adenosyl-L-methionine (SAM) has both antioxidative and anti-inflammatory properties. We investigated the effects of SAM on the glucose transport and insulin signaling impaired by the tumor necrosis factor α (TNFα) in 3T3-L1 adipocytes. SAM partially reversed the basal and insulin stimulated glucose transport, which was impaired by TNFα. The TNFα-induced suppression of the tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1) and Akt in 3T3-L1 adipocytes was also reversed by SAM. In addition, SAM significantly attenuated the TNFα-induced degradation of IκB-α and NF-κB activation. Interestingly, SAM directly inhibited the kinase activity of IKK-β in vitro. These results suggest that SAM can alleviate TNFα mediated-insulin resistance by inhibiting the IKK-β/NF-κB pathway and thus can have a beneficial role in the treatment of type 2 diabetes mellitus.     

Astragalus polysaccharide improves palmitate-induced insulin resistance by inhibiting PTP1B and NF-κB in C2C12 myotubes

We investigated the effects of Astragalus polysaccharide (APS) on palmitate-induced insulin resistance in C2C12 skeletal muscle myotubes. Palmitate-reduced glucose uptake was restored by APS. APS prevented palmitate-induced C2C12 myotubes from impaired insulin signaling by inhibiting Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and increasing Ser473 phosphorylation of Akt. Moreover, the increases in protein-tyrosine phosphatase-1B (PTP1B) protein level and NF-κB activation associated with palmitate treatment were also prevented by APS. However the treatment with APS didn't change AMP-activated protein kinase (AMPK) activation in palmitate-induced myotubes. The results of the present study suggest that Astragalus polysaccharide inhibits palmitate-induced insulin resistance in C2C12 myotubes by inhibiting expression of PTP1B and regulating NF-κB but not AMPK pathway.     

Modulation of the caveolin-3 and Akt status in caveolae by insulin resistance in H9c2 cardiomyoblasts

We investigated glucose uptake and the translocation of Akt and caveolin-3 in response to insulin in H9c2 cardiomyoblasts exposed to an experimental insulin resistance condition of 100 nM insulin in a 25 mM glucose containing media for 24 h. The cells under the insulin resistance condition exhibited a decrease in insulin-stimulated 2-deoxy[(3)H]glucose uptake as compared to control cells grown in 5 mM glucose media. In addition to a reduction in insulin-induced Akt translocation to membranes, we observed a significant decrease in insulin-stimulated membrane association of phosphorylated Akt with a consequent increase of the cytosolic pool. Actin remodeling in response to insulin was also greatly retarded in the cells. When translocation of Akt and caveolin-3 to caveolae was examined, the insulin resistance condition attenuated localization of Akt and caveolin-3 to caveolae from cytosol. As a result, insulin-stimulated Akt activation in caveolae was significantly decreased. Taken together, our data indicate that the decrease of glucose uptake into the cells is related to their reduced levels of caveolin-3, Akt and phosphorylated Akt in caveolae. We conclude that the insulin resistance condition induced the retardation of their translocation to caveolae and in turn caused an attenuation in insulin signaling, namely activation of Akt in caveolae for glucose uptake into H9c2 cardiomyoblasts.     

Label-Free Proteomic Identification of Endogenous,Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

Protein–protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein–protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein–protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.     

GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels

Many phosphorylation signal transduction pathways in the eukaryotic cell are modulated by posttranslational modification of specific serines/threonines with N-acetylglucosamine (O-GlcNAc). Levels of O-GlcNAc on key proteins regulate biological processes as diverse as the cell cycle, insulin signaling, and protein degradation. The two enzymes involved in this dynamic and abundant modification are the O-GlcNAc transferase and O-GlcNAcase. Structural data have recently revealed that the O-GlcNAcase possesses an active site with significant structural similarity to that of the human lysosomal hexosaminidases HexA/HexB. PUGNAc, an O-GlcNAcase inhibitor widely used to raise levels of O-GlcNAc in human cell lines, also inhibits these hexosaminidases. Here, we have exploited recent structural information of an O-GlcNAcase-PUGNAc complex to design and synthesize a glucoimidazole-based inhibitor, GlcNAcstatin, which is a 5 pM competitive inhibitor of enzymes of the O-GlcNAcase family, shows 100000-fold selectivity over HexA/B, and binds to the O-GlcNAcase active site by mimicking the transition state as revealed by X-ray crystallography. This compound is able to raise O-GlcNAc levels in human HEK 293 and SH-SY5Y neuroblastoma cell lines and thus provides a novel, potent tool for the study of the role of O-GlcNAc in intracellular signal transduction pathways.     

Study on glucose transport in muscle cells by extracts from Mitragyna speciosa (Korth) and mitragynine

The leaves of Mitragyna speciosa Korth (Rubiaceae) have been used in folk medicine for its unique medicinal properties. This study examined the water, methanolic and crude alkaloidal extracts from M. speciosa leaves and its major constituent mitragynine for the enhancement of glucose transport. Cellular uptake of radioactive 2-deoxyglucose was determined in rat L8 myotubes. Involving signalling pathway was determined with the specific inhibitors. Cell cytotoxicity was monitored by lactate dehydrogenase assay. Protein levels of glucose transporters (GLUTs) were measured by Western blotting. The results show that test samples significantly increased the rate of glucose uptake. The uptake was associated with increase in GLUT1 protein content. Co-incubation with insulin had no additional effect, but the cellular uptake was decreased by wortmannin and SB 203580, specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK), respectively. It is concluded that the increased glucose transport activity of M. speciosa is associated with increases in activities of the key enzymes dependent to the insulin-stimulated glucose transport for its acute action, and increases in the GLUT1 content for its long-term effect. This study demonstrated the effect of M. speciosa in stimulating glucose transport in muscle cells, implicating the folkloric use of M. speciosa leaves for treating diabetes.     

Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.     

Vitamin K2 as a New Modulator of the Ceramide De Novo Synthesis Pathway

The aim of the study was to evaluate the influence of vitamin K2 (VK2) supplementation on the sphingolipid metabolism pathway in palmitate-induced insulin resistant hepatocytes. The study was carried out on human hepatocellular carcinoma cells (HepG2) incubated with VK2 and/or palmitic acid (PA). The concentrations of sphingolipids were measured by high-performance liquid chromatography. The expression of enzymes from the sphingolipid pathway was assessed by Western blotting. The same technique was used in order to determine changes in the expression of the proteins from the insulin signaling pathway in the cells. Simultaneous incubation of HepG2 cells with palmitate and VK2 elevated accumulation of sphinganine and ceramide with increased expression of enzymes from the ceramide de novo synthesis pathway. HepG2 treatment with palmitate and VK2 significantly decreased the insulin-stimulated expression ratio of insulin signaling proteins. Moreover, we observed that the presence of PA w VK2 increased fatty acid transport protein 2 expression. Our study showed that VK2 activated the ceramide de novo synthesis pathway, which was confirmed by the increase in enzymes expression. VK2 also intensified fatty acid uptake, ensuring substrates for sphingolipid synthesis through the de novo pathway. Furthermore, increased concentration of sphingolipids, mainly sphinganine, inhibited insulin pathway proteins phosphorylation, increasing insulin resistance development.     

Bioactive Compounds from Lemon (Citrus limon) Extract Overcome TNF-α-Induced Insulin Resistance in Cultured Adipocytes

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.     

Phloridzin Acts as an Inhibitor of Protein-Tyrosine Phosphatase MEG2 Relevant to Insulin Resistance

Inhibition of the megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2, also named PTPN9) activity has been shown to be a potential therapeutic strategy for the treatment of type 2 diabetes. Previously, we reported that PTP-MEG2 knockdown enhances adenosine monophosphate activated protein kinase (AMPK) phosphorylation, suggesting that PTP-MEG2 may be a potential antidiabetic target. In this study, we found that phloridzin, isolated from Ulmus davidiana var. japonica, inhibits the catalytic activity of PTP-MEG2 (half-inhibitory concentration, IC₅₀ = 32 ± 1.06 μM) in vitro, indicating that it could be a potential antidiabetic drug candidate. Importantly, phloridzin stimulated glucose uptake by differentiated 3T3-L1 adipocytes and C2C12 muscle cells compared to that by the control cells. Moreover, phloridzin led to the enhanced phosphorylation of AMPK and Akt relevant to increased insulin sensitivity. Importantly, phloridzin attenuated palmitate-induced insulin resistance in C2C12 muscle cells. We also found that phloridzin did not accelerate adipocyte differentiation, suggesting that phloridzin improves insulin sensitivity without significant lipid accumulation. Taken together, our results demonstrate that phloridzin, an inhibitor of PTP-MEG2, stimulates glucose uptake through the activation of both AMPK and Akt signaling pathways. These results strongly suggest that phloridzin could be used as a potential therapeutic candidate for the treatment of type 2 diabetes.     

Novel Triterpenoids from Cassia fistula Stem Bark Depreciates STZ-Induced Detrimental Changes in IRS-1/Akt-Mediated Insulin Signaling Mechanisms in Type-1 Diabetic Rats

Here, we identified the mechanisms of action of antidiabetic activity of novel compounds isolated from Cassia fistula stem bark in STZ-diabetic animals. Novel triterpenoid compounds (C1, C2 and C3) were treated to STZ-administered diabetic animals at a concentration of 20mg/kg body weight orally for 60 days to assess their effects on plasma glucose, plasma insulin/C-peptide, serum lipid markers and the enzymes of carbohydrate metabolism, glucose oxidation and insulin signaling molecules. Oral administration of novel triterpenoid compounds to STZ-diabetic animals significantly decreased (p < 0.05) the plasma glucose concentration on the 7th, 15th, 30th, 45th and 60th daysin a duration-dependent manner (p < 0.05). Plasma insulin (p < 0.0001)/C-peptide (p < 0.0006), tissue glycogen (p < 0.0034), glycogen phosphorylase (p < 0.005), glucose 6-phosphatase (p < 0.0001) and lipid markers were significantly increased (p < 0.0001) in diabetic rats, whereas glucokinase (p < 0.0047), glycogen synthase (p < 0.003), glucose oxidation (p < 0.001), GLUT4 mRNA (p < 0.0463), GLUT4 protein (p < 0.0475) and the insulin-signaling molecules IR mRNA (p < 0.0195), IR protein (p < 0.0001), IRS-1 mRNA (p < 0.0478), p-IRS-1^Tyr612 (p < 0.0185), Akt mRNA (p < 0.0394), p–Akt^Ser473 (p < 0.0162), GLUT4 mRNA (p < 0.0463) and GLUT4 (p < 0.0475) were decreased in the gastrocnemius muscle. In silico analysis of C1–C3 with IRK and PPAR-γ protein coincided with in vivo findings. C1–C3 possessed promising antidiabetic activity by regulating insulin signaling mechanisms and carbohydrate metabolic enzymes.     

A quantum mechanics/molecular mechanics study of the protein-ligand interaction of two potent inhibitors of human O-GlcNAcase: PUGNAc and NAG-thiazoline

O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.     

Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.     

Biochemical and molecular basis of insulin resistance

Insulin-resistance is a major problem associated with diabetes and that is increasing rapidly worldwide. Insulin is a peptide hormone secreted by the beta-cells of the pancreatic islets of Langerhans in response to increased circulating levels of glucose and amino acids and it is essential for appropriate tissue development, growth, and maintenance of whole-body glucose homeostasis by regulating carbohydrate, lipid and protein metabolism. Insulin resistance is a defect in signal transduction. The signaling mechanisms involved in the various biologic responses to insulin remain somewhat elusive. This review focuses on the structure and activity of insulin receptor, inheritance of insulin resistance, insulin receptor and alleles, enzyme activity in insulin resistance, insulin receptor in phosphorylation and relating substrate. We have discussed insulin receptor substrate-family (IRS) related to insulin resistance, detail downstream signaling effects, GLUT4 vesicle translocation and related events, cytokine-mediated insulin resistance, and feedback control mechanisms. This review also focuses on insulin resistance in obesity-linked metabolic syndrome, insulin resistance related to plasma membrane disturbances and insulin resistance for exercise and cellular integrity. Finally, we can conclude that insulin resistance is really a complex phenomenon in which several genetic defects combine with environmental stresses.     

Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise

O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2'-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.     

A divergent synthesis of 2-acyl derivatives of PUGNAc yields selective inhibitors of O-GlcNAcase

A divergent route facilitating the rapid synthesis of a series of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate (PUGNAc)-based inhibitors, bearing different N-acyl groups has been developed. All compounds of this series are inhibitors of both human O-GlcNAcase and human beta-hexosaminidase, yet some effectively exploit differences between the active site architectures of these two human enzymes which render them selective for O-GlcNAcase. Such inhibitors may be valuable tools in dissecting the role of the O-GlcNAc post-translational modification at the cellular and organismal level since these compounds may have different pharmacokinetic properties when compared to other inhibitors of beta-N-acetyl-glucosaminidases.     

Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4

Background  

Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4.     

β-Sitosterol Circumvents Obesity Induced Inflammation and Insulin Resistance by down-Regulating IKKβ/NF-κB and JNK Signaling Pathway in Adipocytes of Type 2 Diabetic Rats

β-sitosterol (SIT), the most abundant bioactive component of vegetable oil and other plants, is a highly potent antidiabetic drug. Our previous studies show that SIT controls hyperglycemia and insulin resistance by activating insulin receptor and glucose transporter 4 (GLUT-4) in the adipocytes of obesity induced type 2 diabetic rats. The current research was undertaken to investigate if SIT could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of SIT (20 mg/kg b.wt) was administered orally for 30 days to high fat diet and sucrose induced type-2 diabetic rats. Metformin, the conventionally used antidiabetic drug was used as a positive control. Interestingly, SIT treatment restores the elevated serum levels of proinflammatory cytokines including leptin, resistin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to normalcy and increases anti-inflammatory adipocytokines including adiponectin in type 2 diabetic rats. Furthermore, SIT decreases sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator–activated receptor-γ (PPAR-γ) gene expression in adipocytes of diabetic rats. The gene and protein expression of c-Jun-N-terminal kinase-1 (JNK1), inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) and nuclear factor kappa B (NF-κB) were also significantly attenuated in SIT treated groups. More importantly, SIT acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that SIT inhibits obesity induced insulin resistance by ameliorating the inflammatory events in the adipose tissue through the downregulation of IKKβ/NF-κB and c-Jun-N-terminal kinase (JNK) signaling pathway.