Glucose Uptake In Muscle Cells Research Articles

LPS-Induced Modifications in Macrophage Transcript and Secretion Profiles Are Linked to Muscle Wasting and Glucose Intolerance.

Macrophages are versatile immune cells that play crucial roles in tissue repair, immune defense, and the regulation of immune responses. In the context of skeletal muscle, they are vital for maintaining muscle homeostasis but macrophage-induced chronic inflammation can lead to muscle dysfunction, resulting in skeletal muscle atrophy characterized by reduced muscle mass and impaired insulin regulation and glucose uptake. Although the involvement of macrophage-secreted factors in inflammation-induced muscle atrophy is well-established, the precise intracellular signaling pathways and secretion factors affecting skeletal muscle homeostasis require further investigation. This study aimed to explore the regulation of macrophage-secreted factors and their impact on muscle atrophy and glucose metabolism. By employing RNA sequencing (RNA-seq) and proteome array, we uncovered that factors secreted by lipopolysaccharide (LPS)-stimulated macrophages upregulated markers of muscle atrophy and pro-inflammatory cytokines, while concurrently reducing glucose uptake in muscle cells. The RNA-seq analysis identified alterations in gene expression patterns associated with immune system pathways and nutrient metabolism. The utilization of gene ontology (GO) analysis and proteome array with macrophage-conditioned media revealed the involvement of macrophage-secreted cytokines and chemokines associated with muscle atrophy. These findings offer valuable insights into the regulatory mechanisms of macrophage-secreted factors and their contributions to muscle-related diseases.

Characterization of Green and Yellow Papaya (Carica papaya) for Anti-Diabetic Activity in Liver and Myoblast Cells and Wound-Healing Activity in Fibroblast Cells.

Obesity and diabetes, often characterized as "metabolic syndrome", have been recognized as two of the most important public health issues worldwide. The objective of the present research was to evaluate green and yellow papaya for anti-oxidation and anti-diabetic properties. Leaves, skin, pulp, and seed samples from papayas were freeze-dried and then extracted in water or 80% methanol. The extracts were used to determine total polyphenolic content and anti-oxidation activities, and to determine biological activities, including glucose uptake, Glut-2 expression, triglyceride reduction, and wound-healing activity. Our data demonstrated that methanol and water extracts of green and yellow papaya have similar concentrations of polyphenols in skin (10-20 mg/g dry powder), leaf (25-30 mg/g dry powder), and pulp (1-3 mg/g dry powder) fractions. However, both methanol and water extracts of seeds from yellow papaya have substantially higher concentrations of polyphenols compared to green papaya. Both water and methanol extracts of yellow papaya exhibited higher anti-oxidation activity compared to green papaya in skin (50-60%), pulp (200-300%), and seeds (10-800%). Old leaves also showed greater anti-oxidation activity (30-40%) compared to new leaves. Pulp extracts from both yellow and green papaya stimulated greater glucose uptake, but only pulp from green papaya stimulated glucose uptake in muscle cells. Similarly, pulp extract stimulated glucose transporter Glut-2 expression in liver cells. The skin, pulp, and seeds of green or yellow papaya showed triglyceride-lowering activity in liver cells by 60-80%, but samples taken from yellow papaya had a more potent effect. Seeds from both green and yellow papaya significantly stimulated the migration of fibroblasts in the wounded area by 2-2.5-fold compared to the untreated control. Consistent with these data, seeds from both green and yellow papaya also significantly stimulated collagen synthesis in fibroblast cells by almost 3-fold. In conclusion, our data indicate that different parts of papaya produce stimulatory effects on glucose uptake, Glut-2 expression, TG reduction, and wound-healing activities. This study concludes that different parts of the papaya can be beneficial for preventing diabetes and diabetes-related wound healing.

Efficacy of the Hibiscus surattensis L. Leaves Active Fraction in Reducing the Levels of HbA1c, AGEs, and Glucose Uptake in Muscle Cells of Diabetic Type 2 Model Rat

Hibiscus surattensis L. is a traditional medicinal plant often used for diabetes treatment in Indonesia, especially in Central Sulawesi. The leaves contain mainly kaempferol, morine, and trifolin which have various pharmacological effects, including high antioxidant properties and the potential to increase insulin secretion. Therefore, this study aims to investigate the efficacy of the Hibiscus surattensis L. leaves (HSL) active fraction in reducing the levels of HbA1c, AGEs, and GLUT-4 expression in the muscle tissue of rat model with type 2 diabetes mellitus using a high-fat and fructose diet of 1.8 g/kg BW (HFD/HF) for 8 weeks. A total of 20 male rats were randomly assigned into four groups namely (1) normal control diet with standard rat chow; (2) negative control with HFD/HF diet; (3) treatment group with metformin 100 mg/kg BW as standard; (4) treatment group given HSL active fraction (FEA) 50 mg/kg BW. All treatments were given orally for 21 days and the data were analyzed by ANOVA and continued with the LSD post hoc test. HFD/HF induction significantly increased the HbA1c and AGEs levels 3.5 times and 1.88 times higher than the normal control group. Moreover, the treatments significantly reduced (p<0.05) HbA1c and AGEs levels compared to the negative control on day 21. Blood HbA1C levels with FEA treatment decreased by 56.4%, while AGEs reduced by 54.7%. GLUT 4 expression in the muscle tissue was significantly different from the negative control with p<0.05. The results also indicate that the administration of FEA had an antidiabetic effect by reducing the levels of HbA1C and AGEs along with other potentially beneficial effects on the treatment of type 2 DM with the probable mechanism of targeting GLUT4 glucose transporter by increasing its translocation and expression.

Pharmacology of α-spinasterol, a phytosterol with nutraceutical values: A review.

α-Spinasterol is a phytosterol found in various edible and non-edible plant sources. The edible plant materials containing α-spinasterol include spinach leaves, cucumber fruits, seeds of pumpkin and watermelon, argan seed oil, cactus pear seed oil and Amaranthus sp. It is a bioavailable nutraceutical, and it can cross the blood-brain barrier. It possesses several important pharmacological properties such as anti-diabetes mellitus, antiinflammation, hypolipidemic, antiulcer, neuroprotection, anti-pain and antitumour activities. For this review, literature search was made focusing on the pharmacological properties of α-spinasterol using PubMed and Google Scholar data bases. Recent studies show the promising antidiabetic properties of α-spinasterol. Its anti-diabetic mechanisms include enhancement of insulin secretion, reduction in insulin resistance, anti-diabetic nephropathy, increase in glucose uptake in muscle cells and inhibition of glucose absorption from intestine. Besides, it is a safe antiinflammatory agent, and its antiinflammatory mechanisms include inhibition of cyclooxygenases, antagonism of TRPV1 receptor and attenuation of proinflammatory cytokines and mediators. It is a promising and safe nutraceutical molecule for human health care. Food supplements, value-added products and nutraceutical formulations can be developed with α-spinasterol for the management of diabetes, chronic inflammatory diseases and improving general health. This review provides all scattered pharmacological studies on α-spinasterol in one place and highlights its immense value for human health care.

Inhibition of Branched‐Chain Ketoacid Dehydrogenase Kinase (BDK) Attenuates BCKA Induced Suppression of Insulin‐Stimulated Glucose Uptake in Muscle Cells

ObjectivePlasma levels of branched‐chain amino acids (BCAAs) and their metabolites, branched‐chain ketoacids (BCKAs) are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus (T2DM). BCAA catabolic enzymes are downregulated in diabetes and obesity in skeletal muscle. We previously showed that leucine and KIC suppressed insulin‐stimulated glucose uptake in L6 myotubes. We have also shown that knocking down branched‐chain ketoacid dehydrogenase (BCKD), an enzyme that decarboxylates BCKAs, suppressed insulin‐stimulated glucose uptake. The objective of this study was to analyze the effect of increasing BCAA catabolism on measures of insulin sensitivity. We hypothesized that upregulating BCAA catabolism increases insulin‐stimulated glucose transport and attenuates BCKA induced insulin resistance.MethodsL6 myotubes were starved of serum‐ and amino acids with vehicle (DMSO) or BDK inhibitor BT2 supplementation for 3 hours. Then myotubes were supplemented with or without BCKAs (200 uM) for 30 minutes. After, cells were incubated with or without insulin (100 nM) for 20 minutes. They were then harvested for immunoblotting or used for glucose transport assay.ResultsBDK inhibition had no effect on insulin‐stimulated glucose uptake. BCKAs suppressed insulin‐stimulated glucose uptake by 31% in control cells (p>0.05; n=3); this suppression was attenuated in cells in which BDK was inhibited. BDK inhibition also reduced KIC‐induced IRS‐1Ser612 and S6K1Thr389 phosphorylation by 28% and 60% respectively but had no effect on AktSer473 phosphorylation.ConclusionsBDK inhibition attenuated BCKA‐induced suppression of insulin‐stimulated glucose uptake, suggesting that BCAA metabolism may be implicated in the pathogenesis of insulin resistance, and is worth further investigation.

CaMKII and Kalirin, a Rac1-GEF, regulate Akt phosphorylation involved in contraction-induced glucose uptake in skeletal muscle cells

Rac1 plays an important role in contraction-stimulated muscle glucose uptake, but the mechanism is not fully elucidated. We previously identified Rac1-dependent activation of Akt played a partial role in contraction-stimulated GLUT4 translocation to the cell surface of C2C12 myotubes. Recognizing that contraction activates CaMKII in muscle and CaMKII is known to regulate Rac1 activity in other systems, here we investigated the relationship between CaMKII, Akt and contraction-stimulated glucose uptake. Expression of a constitutively-active mutant of CaMKIIδ stimulated Akt phosphorylation that was inhibited by Rac1 inhibitor II. C2C12 myotubes were contracted by electrical pulse stimulation (EPS). We observed the CaMKII inhibitor, KN-93 and CaMKIIδ siRNA-mediated knockdown, reduced EPS-induced Akt phosphorylation in C2C12 myotubes. ITX3, an inhibitor of the Rac-GTPase Kalirin and Kalirin siRNA-mediated knockdown reduced EPS-stimulated Akt phosphorylation in myotubes. In addition, the Akt inhibitor MK2206 partly reduced EPS-stimulated glucose uptake without simultaneously affecting CaMKII phosphorylation and Kalirin protein abundance. Our findings demonstrate EPS leads to Akt activation through a CaMKII-Kalirin-Rac1 signaling pathway and partly regulates contraction-stimulated glucose uptake in muscle cells.

Depletion of Branched-Chain Ketoacid Dehydrogenase Kinase (BDK) Upregulates Insulin-Stimulated Glucose Uptake in Muscle Cells

Abstract Objectives Plasma levels of branched-chain amino acids (BCAAs) and their metabolites, branched-chain ketoacids (BCKAs) are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus (T2DM). BCAA catabolic enzymes are downregulated in diabetes and obesity. We previously showed that leucine and KIC suppressed insulin-stimulated glucose uptake in L6 myotubes. We have also shown that knocking down branched-chain ketoacid dehydrogenase (BCKD), an enzyme that decarboxylates BCKAs, suppressed insulin-stimulated glucose uptake. The objective of this study is to analyze how stimulating BCAA catabolic flux, by depleting branched-chain ketoacid dehydrogenase kinase (BDK), a negative regulator of BCKD, affects insulin sensitivity. We hypothesize that upregulating BCAA catabolism will increase insulin-stimulated glucose transport and attenuate insulin resistance. Methods L6 myoblasts were cultured in differentiation media for 4 days. On day 4 of differentiation, cells were transfected with control (SCR) or branched-chain ketoacid dehydrogenase kinase (BDK) siRNA oligonucleotides. Forty-eight hours later, myotubes were starved of serum- and amino acids for 3 hours then supplemented with or without KIC (200 mM) for 30 minutes. After, cells were incubated with or without insulin (100 nM) for 20 minutes. They were then harvested for immunoblotting or used for glucose transport assay. Results There was a 32% increase in insulin-stimulated glucose uptake with BDK depletion. KIC suppressed insulin-stimulated glucose uptake by 25% in control (SCR) cells; this suppression was attenuated in cells depleted of BDK. BDK depletion also reduced KIC-induced IRS-1Ser612 phosphorylation by 64% but had no effect on AktSer473 phosphorylation. Conclusions BDK depletion increased insulin-stimulated glucose transport, and attenuated KIC-induced suppression of insulin-stimulated glucose uptake, suggesting that increasing BCKD activity can be a therapeutic strategy against insulin resistance. Funding Sources Natural Science and Research Council (NSERC)

Tmod3 Phosphorylation Mediates AMPK-Dependent GLUT4 Plasma Membrane Insertion in Myoblasts.

Insulin and muscle contractions mediate glucose transporter 4 (GLUT4) translocation and insertion into the plasma membrane (PM) for glucose uptake in skeletal muscles. Muscle contraction results in AMPK activation, which promotes GLUT4 translocation and PM insertion. However, little is known regarding AMPK effectors that directly regulate GLUT4 translocation. We aim to identify novel AMPK effectors in the regulation of GLUT4 translocation. We performed biochemical, molecular biology and fluorescent microscopy imaging experiments using gain- and loss-of-function mutants of tropomodulin 3 (Tmod3). Here we report Tmod3, an actin filament capping protein, as a novel AMPK substrate and an essential mediator of AMPK-dependent GLUT4 translocation and glucose uptake in myoblasts. Furthermore, Tmod3 plays a key role in AMPK-induced F-actin remodeling and GLUT4 insertion into the PM. Our study defines Tmod3 as a key AMPK effector in the regulation of GLUT4 insertion into the PM and glucose uptake in muscle cells, and offers new mechanistic insights into the regulation of glucose homeostasis.

ATM inhibitor KU-55933 induces apoptosis and inhibits motility by blocking GLUT1-mediated glucose uptake in aggressive cancer cells with sustained activation of Akt.

Enhanced glucose uptake is coupled with elevated aerobic glycolysis (the Warburg effect) in cancer cells and is closely correlated with increased tumor aggressiveness and poor prognosis. We previously discovered that ATM, a protein kinase deficient in Ataxia-telangiectasia (A-T) disease, is an insulin-responsive protein that participates in insulin-mediated glucose uptake in muscle cells by stimulating glucose transporter 4 (GLUT4) translocation. However, the role of ATM in glucose uptake and tumorigenesis of cancer cells is unclear. In the present study, we found that aggressive breast and prostate cancer cell lines with overactivated Akt activity exhibit enhanced glucose uptake and GLUT1 translocation upon insulin treatment, and KU-55933, a specific inhibitor of ATM, inhibits insulin-mediated glucose uptake by blocking translocation of GLUT1 to the cell surface. KU-55933 also inhibits aerobic glycolysis and ATP production in these cells. Moreover, KU-55933 induces apoptosis and inhibits motility of cancer cells by inhibiting glucose uptake. Our results showed that while high concentration of glucose and insulin promote the expression of a mesenchymal biomarker (vimentin) in these cancer cells, KU-55933 strongly inhibits its expression as well as epithelial to mesenchymal transition. The roles of ATM in stimulating glucose uptake, glycolysis, motility, and proliferation of cancer cells were demonstrated by knocking-down ATM in these cells. KU-55933 treatment also inhibits tumor growth and metastasis in vivo in mouse mammary tumors through inhibition of GLUT1 translocation and vimentin expression. These results suggest that ATM acts as a promoter of tumorigenesis in cancer cells with overactivated Akt, and KU-55933 induces apoptosis and inhibits motility by blocking GLUT1-mediated glucose uptake and glycolysis in these cancer cells, which may lead to the use of KU-55933 and its analogs as new preventive or therapeutic agents against cancer.

Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation.

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.

Evaluation of the antidiabetic potential of Psidium guajava L. (Myrtaceae) using assays for α-glucosidase, α-amylase, muscle glucose uptake, liver glucose production, and triglyceride accumulation in adipocytes

Ethnopharmacological relevancePsidium guajava L. (Myrtaceae) leaves are used as an herbal antidiabetic remedy in several parts of the world. On Madagascar, both the bark and leaves are used for treatment of diabetes. Materials and methodsDilution series of ethanolic extracts of P. guajava leaves and bark were used for determining inhibitory activities against yeast α-glucosidase and porcine α-amylase. Skeletal muscle glucose uptake was measured using 2-deoxy-D-(1–3H)-glucose in murine C2C12 skeletal muscle cells. Hepatic glucose-6-phosphatase activity in rat hepatoma H4IIE cells and triglyceride accumulation in murine 3T3-L1 adipocyte-like cells were assessed using Wako AutoKit Glucose assays and AdipoRed reagent, respectively. Cells were incubated for 18 h with the maximal non-toxic concentrations of the plant extracts determined by the lactate dehydrogenase cytotoxicity assay. ResultsEthanolic extracts of P. guajava leaf and bark inhibited α-glucosidase with IC50 values of 1.0 ± 0.3 and 0.5 ± 0.01 μg/mL, respectively. In the α-amylase inhibition assay, the ethanolic extract of bark of P. guajava showed an IC50 value of 10.6 ± 0.4 μg/mL. None of the extracts were able to reduce glucose-6-phosphatase activity in rat hepatoma H4IIE cells. In contrast, P. guajava leaf extract significantly increased 2-deoxy-D-[1–3H]-glucose uptake in C2C12 muscle cells (161.4 ± 10.1%, p = 0.0015) in comparison to the dimethyl sulfoxide (DMSO) vehicle control, as did the reference compounds metformin (144.0 ± 7.7%, p = 0.0345) and insulin (141.5 ± 13.8%, p = 0.0495). Furthermore, P. guajava leaf and bark extracts, as well as the reference compound rosiglitazone, significantly enhanced triglyceride accumulation in 3T3-L1 cells (252.6 ± 14.2%, p < 0.0001, 211.1 ± 12.7%, p < 0.0001, and 201.1 ± 9.2%, p < 0.0001, respectively) to levels higher than the DMSO vehicle control. Moreover, P. guajava leaf extract significantly enhanced the triglyceride accumulation in 3T3-L1 cells compared to rosiglitazone. ConclusionThe results demonstrated that P. guajava leaf and bark extracts can be used as a natural source of α-glucosidase inhibitors. In addition, the bark extract of P. guajava was an effective α-amylase inhibitor. Moreover, P. guajava leaf extract improved glucose uptake in muscle cells, while both leaf and bark extracts enhanced the triglyceride content in adipocytes in culture. P. guajava leaf and bark extracts may thus hypothetically have future applications in the treatment of type 2 diabetes.

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0–0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

BackgroundLowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines. PurposeOur goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4′-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells. MethodsTo determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [14C] 2-deoxy-D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin. ResultsAcacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells. ConclusionTaken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.

Attenuation of FFA‐Induced Skeletal Muscle Insulin Resistance by Carnosic and Rosmarinic Acid

Impaired insulin action in muscle leads to insulin resistance and Type 2 diabetes mellitus, a disease on the rise. Elevated blood free fatty acids (FFAs), as seen in obesity, are associated with insulin resistance and studies have shown increased serine phosphorylation of the insulin receptor substrate (IRS‐1) and reduced insulin‐stimulated glucose uptake in muscle cells exposed to FFA palmitate. A number of serine/threonine kinases including JNK, IKKβ, mTOR and p70 6SK have been implicated in serine phosphorylation of IRS‐1 and insulin resistance. On the other hand, activation of the energy sensor AMP‐activated protein kinase (AMPK) increases glucose uptake and has become an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased skeletal muscle cell glucose uptake and activated AMPK. The polyphenols carnosic acid (CA) and rosmarinic acid (RA) are found in high concentration in RE and in the present study we investigated their effect on palmitate‐induced insulin resistant L6 muscle cells. Glucose uptake was measured using [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting.Exposure of L6 myotubes to the FFA palmitate (P) (0.2 mM, 16 hours) resulted in significant reduction of insulin‐stimulated glucose uptake (I: 201±1.21% , P+I: 117±15.6% of control, P&lt;0.001) indicating insulin resistance. Importantly, in the presence of 2 mM CA or 5 mM RA, the insulin‐stimulated glucose uptake was restored (CA+P+I: 185±7.8%, RA+P+I: 181±14.4% of control, P&lt;0.001). Treatment with palmitate increased serine phosphorylation of IRS‐1, increased the phosphorylation/activation of JNK, mTOR and p70 6SK and significantly decreased the insulin‐stimulated phosphorylation of Akt. The effects of CA and RA on these signaling molecules are under investigation. Our data indicate that treatment with CA or RA attenuates the FFA‐induced muscle insulin resistance. These polyphenols may have potent effects against insulin resistance and deserve further study.Support or Funding InformationThe research was funded by the Natural Sciences and Engineering Research Council (NSERC) grant to ET.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SELENON (SEPN1) protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to a congenital myopathy associated with insulin resistance (SEPN1-related myopathy). The exact cause of the insulin resistance in patients with SELENON loss of function is not known. Skeletal muscle is the main contributor to insulin-mediated glucose uptake, and a defect in this muscle-related mechanism triggers insulin resistance and glucose intolerance. We have studied the chain of events that connect the loss of SELENON with defects in insulin-mediated glucose uptake in muscle cells and the effects of this on muscle performance. Here, we show that saturated fatty acids are more lipotoxic in SELENON-devoid cells, and blunt the insulin-mediated glucose uptake of SELENON-devoid myotubes by increasing ER stress and mounting a maladaptive ER stress response. Furthermore, the hind limb skeletal muscles of SELENON KO mice fed a high-fat diet mirrors the features of saturated fatty acid-treated myotubes, and show signs of myopathy with a compromised force production. These findings suggest that the absence of SELENON together with a high-fat dietary regimen increases susceptibility to insulin resistance by triggering a chronic ER stress in skeletal muscle and muscle weakness.Importantly, our findings suggest that environmental cues eliciting ER stress in skeletal muscle (such as a high-fat diet) affect the pathological phenotype of SEPN1-related myopathy and can therefore contribute to the assessment of prognosis beyond simple genotype-phenotype correlations.

TAZ couples Hippo/Wnt signalling and insulin sensitivity through Irs1 expression

Insulin regulates blood glucose levels by binding its receptor and stimulating downstream proteins through the insulin receptor substrate (IRS). Impaired insulin signalling leads to metabolic syndrome, but the regulation of this process is not well understood. Here, we describe a novel insulin signalling regulatory pathway involving TAZ. TAZ upregulates IRS1 and stimulates Akt- and Glut4-mediated glucose uptake in muscle cells. Muscle-specific TAZ-knockout mice shows significantly decreased Irs1 expression and insulin sensitivity. Furthermore, TAZ is required for Wnt signalling-induced Irs1 expression, as observed by decreased Irs1 expression and insulin sensitivity in muscle-specific APC- and TAZ-double-knockout mice. TAZ physically interacts with c-Jun and Tead4 to induce Irs1 transcription. Finally, statin administration decreases TAZ, IRS1 level and insulin sensitivity. However, in myoblasts, the statin-mediated decrease in insulin sensitivity is counteracted by the expression of a constitutively active TAZ mutant. These results suggest that TAZ is a novel insulin signalling activator that increases insulin sensitivity and couples Hippo/Wnt signalling and insulin sensitivity.

Effect of sub-toxic exposure to Malathion on glucose uptake and insulin signaling in L6 myoblast derived myotubes

Biochemical basis of Malathion exposure-induced diabetes mellitus is not known. Hence, effects of its sub-toxic exposure on redox sensitive kinases (RSKs), insulin signaling and insulin-induced glucose uptake were assessed in rat muscle cell line. In this in vitro study, rat myoblast (L6) cells were differentiated to myotubes and were exposed to sub-toxic concentrations (10 mg/l and 20 mg/l) of Malathion for 18 hours. Total antioxidant level and insulin-stimulated glucose uptake by myotubes were assayed. Activation of JNK, NFκB, p38MAPK and insulin signaling from tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Akt were assessed in myotubes after Malathion exposure by western blot and was compared with those in controls. Paraoxonase (PON) activity was measured in cell lysate using p-nitrophenyl acetate as substrate. PON1 and PON2 expression in myotubes were assessed by PCR. The glucose uptake and total antioxidant level in L6-derived myotubes after sub-toxic exposure to Malathion were decreased in a dose-dependent manner. Phosphorylation levels of RSKs (JNK, p38MAPK and IκBα component of NFκB) were increased and that of IRS-1 and Akt on insulin stimulation was decreased following Malathion exposure as compared to those in controls. PON1 and PON2 genes were expressed in myotubes with and without Malathion exposure. Significant PON activity was present in cell lysate. We conclude that sub-toxic Malathion exposure induces oxidative stress in muscle cells activating RSKs that impairs insulin signaling and thereby insulin-stimulated glucose uptake in muscle cells. This probably explains the biochemical basis of Malathion-induced insulin resistance state and diabetes mellitus.

Effect of sub-toxic chlorpyrifos on redox sensitive kinases and insulin signaling in rat L6 myotubes.

Sub-chronic exposures to chlorpyrifos, an organophosphorus pesticide is associated with incidence of diabetes mellitus. Biochemical basis of chlorpyrifos-induced diabetes mellitus is not known. Hence, effect of its sub-toxic exposure on redox sensitive kinases, insulin signaling and insulin-induced glucose uptake were assessed in rat muscle cell line. In an in vitro study, rat myoblasts (L6) cell line were differentiated to myotubes and then were exposed to sub-toxic concentrations (6mg/L and 12mg/L) of chlorpyrifos for 18h. Then total anti-oxidant level in myotubes was measured and insulin-stimulated glucose uptake was assayed. Assessment of activation of NFκB & p38MAPK and insulin signaling following insulin stimulation from tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Akt were done in myotubes after chlorpyrifos exposure by western blot (WB) and compared with those in vehicle-treated controls. The glucose uptake and total antioxidant level in L6-derived myotubes after sub-toxic exposure to chlorpyrifos were decreased in a dose-dependent manner. As measured from band density of WB, phosphorylation levels increased for redo-sensitive kinases (p38MAPK and IκBα component of NFκB) and decreased for IRS-1 (at tyrosine 1222) and Akt (at serine 473) on insulin stimulation following chlorpyrifos exposure as compared to those in controls. We conclude that sub-toxic chlorpyrifos exposure induces oxidative stress in muscle cells activating redox sensitive kinases that impairs insulin signaling and thereby insulin-stimulated glucose uptake in muscle cells. This probably explains the biochemical basis of chlorpyrifos-induced insulin resistance state and diabetes mellitus.

Attenuation of Free Fatty Acid-Induced Muscle Insulin Resistance by Rosemary Extract

Elevated blood free fatty acids (FFAs), as seen in obesity, impair muscle insulin action leading to insulin resistance and Type 2 diabetes mellitus. Serine phosphorylation of the insulin receptor substrate (IRS) is linked to insulin resistance and a number of serine/threonine kinases including JNK, mTOR and p70 S6K have been implicated in this process. Activation of the energy sensor AMP-activated protein kinase (AMPK) increases muscle glucose uptake, and in recent years AMPK has been viewed as an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased muscle cell glucose uptake and activated AMPK. However, the effect of RE on FFA-induced muscle insulin resistance has never been examined. In the current study, we investigated the effect of RE in palmitate-induced insulin resistant L6 myotubes. Exposure of myotubes to palmitate reduced the insulin-stimulated glucose uptake, increased serine phosphorylation of IRS-1, and decreased the insulin-stimulated phosphorylation of Akt. Importantly, exposure to RE abolished these effects and the insulin-stimulated glucose uptake was restored. Treatment with palmitate increased the phosphorylation/activation of JNK, mTOR and p70 S6K whereas RE completely abolished these effects. RE increased the phosphorylation of AMPK even in the presence of palmitate. Our data indicate that rosemary extract has the potential to counteract the palmitate-induced muscle cell insulin resistance and further studies are required to explore its antidiabetic properties.

Isoform-specific AMPK association with TBC1D1 is reduced by a mutation associated with severe obesity.

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237. Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation.