Insulin Resistance In C2C12 Cells Research Articles

Ursonic acid from Artemisia montana exerts anti-diabetic effects through anti-glycating properties, and by inhibiting PTP1B and activating the PI3K/Akt signaling pathway in insulin-resistant C2C12 cells

Artemisia is one of the largest genera in the plant family Asteraceae and has long been used in traditional medicine for its antitussive, analgesic, antihypertensive, antitoxic, antiviral, antimalarial, and anti-inflammatory properties. However, the anti-diabetic activity of Artemisia montana has not been broadly studied. The goal of this study was to determine whether extracts of the aerial parts of A. montana and its main constituents inhibit protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase activities. We isolated nine compounds from A. montana including ursonic acid (UNA) and ursolic acid (ULA), which significantly inhibited PTP1B with IC50 values of 11.68 and 8.73 μM, respectively. In addition, UNA showed potent inhibitory activity against α-glucosidase (IC50 = 61.85 μM). Kinetic analysis of PTP1B and α-glucosidase inhibition revealed that UNA was a non-competitive inhibitor of both enzymes. Docking simulations of UNA demonstrated negative binding energies and close proximity to residues in the binding pockets of PTP1B and α-glucosidase. Molecular docking simulations between UNA and human serum albumin (HSA) revealed that UNA binds tightly to all three domains of HSA. Furthermore, UNA significantly inhibited fluorescent AGE formation (IC50 = 4.16 μM) in a glucose-fructose-induced HSA glycation model over the course of four weeks. Additionally, we investigated the molecular mechanisms underlying the anti-diabetic effects of UNA in insulin-resistant C2C12 skeletal muscle cells and discovered that UNA significantly increased glucose uptake and decreased PTP1B expression. Further, UNA increased GLUT-4 expression level by activating the IRS-1/PI3K/Akt/GSK-3 signaling pathway. These findings clearly demonstrate that UNA from A. montana shows great potential for treatment of diabetes and its complications.

PDIA4, a new endoplasmic reticulum stress protein, modulates insulin resistance and inflammation in skeletal muscle.

Endoplasmic reticulum (ER) stress has emerged as a key player in insulin resistance (IR) progression in skeletal muscle. Recent reports revealed that ER stress-induced the expression of protein disulfide isomerase family a member 4 (PDIA4), which may be involved in IR-related diseases. A previous study showed that metformin modulated ER stress-induced IR. However, it remained unclear whether metformin alleviated IR by regulating PDIA4 expression in skeletal muscle. Herein, we used palmitate-induced IR in C2C12 cells and a high-fat diet-induced IR mouse model to document the relations between metformin, IR, and PDIA4. In C2C12 cells, palmitate-induced IR increased inflammatory cytokines and PDIA4 expression. Besides, knocking down PDIA4 decreased palmitate-induced IR and inflammation in C2C12 cells. Furthermore, metformin modulated PDIA4 expression and alleviated IR both in vitro and in vivo. In addition, serum PDIA4 concentrations are associated with IR and inflammatory cytokines levels in human subjects. Thus, this study is the first to demonstrate that PDIA4 participates in the metformin-induced effects on skeletal muscle IR and indicates that PDIA4 is a potential novel therapeutic target for directly alleviating IR.

Mineralocorticoid Receptors Mediate Diet-Induced Lipid Infiltration of Skeletal Muscle and Insulin Resistance.

Excess circulating lipids increase total intramyocellular (IMC) lipid content and ectopic fat storage, resulting in lipotoxicity and insulin resistance in skeletal muscle. Consumption of a diet high in fat and refined sugars-a Western diet (WD)-has been shown to activate mineralocorticoid receptors (MRs) and promote insulin resistance. However, our understanding of the precise mechanisms by which enhanced MR activation promotes skeletal muscle insulin resistance remains unclear. In this study, we investigated the mechanisms by which enhanced MR signaling in soleus muscle promotes ectopic skeletal muscle lipid accumulation and related insulin resistance. Six-week-old C57BL/6J mice were fed either a mouse chow diet or a WD with or without spironolactone (1 mg/kg/day) for 16 weeks. Spironolactone attenuated 16 weeks of WD-induced in vivo glucose intolerance and insulin resistance, and improved soleus insulin metabolic signaling. Improved insulin sensitivity was accompanied by increased glucose transporter 4 (Glut4) expression in conjunction with decreased soleus free fatty acid and IMC lipid content, as well as CD36 expression. Additionally, spironolactone prevented WD-induced soleus mitochondria dysfunction. Furthermore, MR signaling also mediated WD/aldosterone-induced reductions in soleus microRNA (miR)-99a, which was identified to negatively target CD36 and prevented palmitic acid-induced increases in CD36 expression, lipid droplet formation, mitochondria dysfunction, and insulin resistance in C2C12 cells. These data indicate that inhibition of MR activation with spironolactone prevented diet-induced abnormal expression of miR-99a, which had the capacity to reduce CD36, leading to reduced IMC lipid content and improved soleus mitochondria function and insulin sensitivity.

Butyrate oxidation attenuates the butyrate-induced improvement of insulin sensitivity in myotubes

Skeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin resistance, butyrate, a short-chain fatty acid (SCFA) produced from dietary fibre fermentation, prevents it. This preventive role of butyrate has been attributed to histone deacetylase (HDAC)-mediated transcription regulation and activation of mitochondrial fatty-acid oxidation. Here we address the interplay between butyrate and the long-chain fatty acid palmitate and investigate how transcription, signalling and metabolism are integrated to result in the butyrate-induced skeletal muscle metabolism remodelling. Butyrate enhanced insulin sensitivity in palmitate-treated, insulin-resistant C2C12 cells, as shown by elevated insulin receptor 1 (IRS1) and pAKT protein levels and Slc2a4 (GLUT4) mRNA, which led to a higher glycolytic capacity. Long-chain fatty-acid oxidation capacity and other functional respiration parameters were not affected. Butyrate did upregulate mitochondrial proteins involved in its own oxidation, as well as concentrations of butyrylcarnitine and hydroyxybutyrylcarnitine. By knocking down the gene encoding medium-chain 3-ketoacyl-CoA thiolase (MCKAT, Acaa2), butyrate oxidation was inhibited, which amplified the effects of the SCFA on insulin sensitivity and glycolysis. This response was associated with enhanced HDAC inhibition, based on histone 3 acetylation levels. Butyrate enhances insulin sensitivity and induces glycolysis, without the requirement of upregulated long-chain fatty acid oxidation. Butyrate catabolism functions as an escape valve that attenuates HDAC inhibition. Thus, inhibition of butyrate oxidation indirectly prevents insulin resistance and stimulates glycolytic flux in myotubes treated with butyrate, most likely via an HDAC-dependent mechanism.

Terpenoid-Rich Extract of Dillenia indica L. Bark Displays Antidiabetic Action in Insulin-Resistant C2C12 Cells and STZ-Induced Diabetic Mice by Attenuation of Oxidative Stress.

Insulin resistance (IR) plays a key role in the pathogenesis and clinical outcome of patients with multiple diseases and diabetes. In this study, we examined the antidiabetic effects of a terpenoid-rich extract from Dillenia indica L. bark (TRDI) in palmitic acid-induced insulin resistance (PA-IR) in C2C12 myotube and a streptozotocin (STZ)-induced diabetic mice model and explored the possible underlying mechanism. TRDI showed potential DPPH- and ABTS-radical scavenging effects with a half-maximal inhibitory concentration (IC50) value of 9.76 ± 0.50 µg/mL and 17.47 ± 1.31 µg/mL, respectively. Furthermore, TRDI strongly mitigated α-glucosidase activity with an IC50 value of 3.03 ± 1.01 µg/mL, which was 92-fold higher than the positive control, acarbose (IC50 = 279.49 ± µg/mL). TRDI stimulated the insulin receptor substrarte-1 (INS-1), downregulated phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (Akt) in both normal and PA-IR C2C12 cells as well as in STZ-induced diabetic mice, enhanced glucose transporter 4 (GLUT4) translocation to the plasma membrane (PM), and increased glucose absorption. Furthermore, TRDI administration significantly reduced PA-induced reactive oxygen species (ROS) formation in C2C12 cells and increased the protein level of numerous antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase-1 (GPx-1) and thioredoxin reductase (TrxR) both in vitro and in vivo. Furthermore, TRDI facilitated nuclear factor erythroid 2 related factor 2 (Nrf2) nuclear translocation and increased HO-1 expression in PA-IR C2C12 cells and STZ-induced diabetic mice. However, for the inhibition of Nrf2, TRDI failed to resist the effects of IR. Thus, this study provides new evidence to support the use of TRDI for diabetes treatment.

Structure-Activity Relationship of Synthetic Ginkgolic Acid Analogs for Treating Type 2 Diabetes by PTPN9 Inhibition.

Ginkgolic acid (C13:0) (GA), isolated from Ginkgo biloba, is a potential therapeutic agent for type 2 diabetes. A series of GA analogs were designed and synthesized for the evaluation of their structure–activity relationship with respect to their antidiabetic effects. Unlike GA, the synthetic analog 1e exhibited improved inhibitory activity against PTPN9 and significantly stimulated glucose uptake via AMPK phosphorylation in differentiated 3T3-L1 adipocytes and C2C12 myotubes; it also induced insulin-dependent AKT activation in C2C12 myotubes in a concentration-dependent manner. Docking simulation results showed that 1e had a better binding affinity through a unique hydrophobic interaction with a PTPN9 hydrophobic groove. Moreover, 1e ameliorated palmitate-induced insulin resistance in C2C12 cells. This study showed that 1e increases glucose uptake and suppresses palmitate-induced insulin resistance in C2C12 myotubes via PTPN9 inhibition; thus, it is a promising therapeutic candidate for treating type 2 diabetes.

Bredemolic acid restores glucose utilization and attenuates oxidative stress in palmitic acid-induced insulin-resistant C2C12 cells.

Objective. Due to insulin resistance and oxidative stress that are associated with type 2 diabetes mellitus (T2DM), T2DM has become a prevalent metabolic disorder that presents various side effects. However, alternative antidiabetic treatment has commonly been used in treating diabetes mellitus in diabetic patients. In our previous studies, bredemolic acid has been reported as an antidiabetic agent that improves glucose uptake, ameliorates insulin resistance, and oxidative stress in the liver, heart, kidney, and skeletal muscle of prediabetic rats. However, these effects have not been validated in vitro. Therefore, this study was aimed to investigate the effects of bredemolic acid on insulin-mediated glucose utilization, lipid peroxidation, and the total antioxidant capacity (TOAC) in palmitic acid-induced insulin-resistant C2C12 skeletal muscle cells in vitro. Methods. Insulin resistance was induced in the skeletal muscle cells after 4 h of exposure to palmitic acid (0.5 mmol/l). Different cell groups were incubated in culture media DMEM supplemented with fetal calf serum (10%), penicillin/streptomycin (1%), and L-glutamine (1%) and then treated with either insulin (4 µg/ml) or bredemolic acid (12.5 mmol/l) or with both. Thereafter, the cells were seeded in 24- or 96-well plates for determination of the cell viability, glucose utilization, glycogen formation, and antioxidant capacity. Results. The results showed that bredemolic acid significantly improved TOAC and promoted glucose utilization via attenuation of lipid peroxidation and increased glycogen formation in the insulin-resistant cells, respectively. Conclusion. This study showed that bredemolic acid restored the insulin resistance through improved glucose utilization, glycogen formation, and TOAC in the skeletal muscle cells.

PEG-BHD1028 Peptide Regulates Insulin Resistance and Fatty Acid β-Oxidation, and Mitochondrial Biogenesis by Binding to Two Heterogeneous Binding Sites of Adiponectin Receptors, AdipoR1 and AdipoR2.

Adiponectin plays multiple critical roles in modulating various physiological processes by binding to its receptors. The functions of PEG-BHD1028, a potent novel peptide agonist to AdipoRs, was evaluated using in vitro and in vivo models based on the reported action spectrum of adiponectin. To confirm the design concept of PEG-BHD1028, the binding sites and their affinities were analyzed using the SPR (Surface Plasmon Resonance) assay. The results revealed that PEG-BHD1028 was bound to two heterogeneous binding sites of AdipoR1 and AdipoR2 with a relatively high affinity. In C2C12 cells, PEG-BHD1028 significantly activated AMPK and subsequent pathways and enhanced fatty acid β-oxidation and mitochondrial biogenesis. Furthermore, it also facilitated glucose uptake by lowering insulin resistance in insulin-resistant C2C12 cells. PEG-BHD1028 significantly reduced the fasting plasma glucose level in db/db mice following a single s.c. injection of 50, 100, and 200 μg/Kg and glucose tolerance at a dose of 50 μg/Kg with significantly decreased insulin production. The animals received 5, 25, and 50 μg/Kg of PEG-BHD1028 for 21 days significantly lost their weight after 18 days in a range of 5–7%. These results imply the development of PEG-BHD1028 as a potential adiponectin replacement therapeutic agent.

Ca2+/calmodulin-dependent protein kinase IV attenuates inflammation and mitochondrial dysfunction under insulin resistance in C2C12 cells

CaMKIV has been reported involved in the improvement of whole-body insulin sensitivity and mitochondrial biogenesis of skeletal muscle. Here, we first investigate the effects of CaMKIV on glucose metabolism, cell viability, inflammatory function, and mitochondrial function in palmitate-induced C2C12 cells of insulin resistance. Then we explored the potential mechanism of these effects. Differentiated C2C12 cells were treated with or without 100 ng/ml of CaMKIV under palmitate-induced insulin resistance. The results suggest palmitate induced insulin sensitivity, reduced glucose uptake, decreased cell viability, increased inflammatory factors, and caused mitochondrial dysfunction in C2C12 cells. Of note, CaMKIV reversed palmitate-induced insulin resistance, increased the reduction of glucose uptake, inhibited inflammatory response, and mitochondrial dysfunction, despite of no change in cells viabilities. However, these beneficial effects of CaMKIV were blocked by the downregulation of CREB1. Taken together, our data demonstrated CaMKIV prevents palmitate-induced insulin resistance, inflammatory response, and mitochondrial dysfunction through phosphorylated CREB1 in differentiated C2C12 cells.

Investigation of C-glycosylated apigenin and luteolin derivatives’ effects on protein tyrosine phosphatase 1B inhibition with molecular and cellular approaches

In our previous study, we investigated C-glycosylated apigenin and luteolin derivatives’ antidiabetic potential via protein tyrosine phosphatase 1B (PTP1B) inhibition in vitro, and found that they exhibited varying degrees of effect on PTP1B (IC50 = 17.76, 24.76, 7.62, 24.54, 6.70, and 57.11 µM for isovitexin, apigenin, vitexin, isoorientin, luteolin, and orientin, respectively). However, the precise molecular mechanisms in apigenin and luteolin derivatives’ PTP1B inhibition remain unclear. Therefore, in this study, we examined C-glycosylated apigenin and luteolin derivatives’ PTP1B inhibition via molecular docking simulation and PTP1B expression at the cellular level in insulin-resistant C2C12 cells. Kinetic studies revealed that apigenin, luteolin, and isoorientin were competitive, vitexin and isovitexin mixed-type, and orientin noncompetitive inhibitors of PTP1B. Docking simulations of these flavonoids demonstrated different negative binding energies and close proximity to residues in PTP1B’s binding pocket. The number of hydrogen bonds and other interactions determined the strength of the protein-inhibitor interaction. Further, we also found that apigenin and luteolin derivatives stimulate glucose uptake in insulin-resistant C2C12 cells. Therefore, we speculate that the C-glycosylation apigenin and luteolin derivatives at the C-6 and C-8 positions affect PTP1B inhibition greatly depending upon the type of mode of inhibition and binding affinity with the PTP1B expression level. Thus, the study contributes to our knowledge of the mechanisms by which C-glycosylated flavonoids exert varying degrees of PTP1B inhibition activity.

Poncirin, an orally active flavonoid exerts antidiabetic complications and improves glucose uptake activating PI3K/Akt signaling pathway in insulin resistant C2C12 cells with anti-glycation capacities

Poncirin, a natural flavanone glycoside present abundantly in many citrus fruits, contains an extensive range of biological activities. However, the antidiabetic mechanism of poncirin is unexplored yet. In this study, we examined the anti-diabetic prospective of poncirin by evaluating its ability to inhibit protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, human recombinant AR (HRAR), rat lens aldose reductase (RLAR), and advanced glycation end-product (AGE) formation (IC50 = 7.76 ± 0.21, 21.31 ± 1.26, 3.56 ± 0.33, 11.91 ± 0.21, and 3.23 ± 0.09 µM, respectively). Kinetics data and docking studies showed the lowest binding energy and highestaffinityforthemixed and competitivetypeof inhibitorsof poncirin. Moreover, the molecular mechanisms underlying the antidiabetic outcomes of poncirin in insulin resistant C2C12 skeletal muscle cells were explored, which significantly increased glucose uptake and decreased the expression of PTP1B in C2C12 cells. Consequently, poncirin increased GLUT-4 expression level by activating the IRS-1/PI3K/Akt/GSK-3 signaling pathway. Moreover, poncirin (0.5–50 µM) remarkably inhibited the formation of fluorescent AGE, nonfluorescent CML, fructosamine, and β-cross amyloid structures in glucose-fructose-induced BSA glycation during 4 weeks of study. Poncirin also notably prevented protein oxidation demonstrated with decreasing the protein carbonyl and the consumption of protein thiol in the dose-dependent manner. The results clearly expressed the promising activity of poncirin for the therapy of diabetes and its related complications.

Reducing effect of insulin resistance on alpha-synuclein gene expression in skeletal muscle

BackgroundAlpha-synuclein (SNCA) as the presynaptic protein is expressed in different tissues and prevents insulin-resistance (IR) through increasing glucose-uptake by adipocytes and muscles. However, the effect of insulin metabolism on SNCA expression has scarcely elucidated. In present study we assessed the probable effect of insulin resistance on SNCA expression in muscle C2C12 cells and also skeletal muscle tissues of type 2 diabetic mice.Materials and methodsSixteen male C57BL/6 mice were divided into two experimental groups, including control and type 2 diabetic mice with IR (induced by high-fat diet + low-dose streptozotocin). The animals of the study involved the measurements of fasting blood glucose, oral-glucose-tolerance-test, as well as fasting plasma insulin. Moreover, insulin-resistant and insulin-sensitive muscle C2C12 cells were prepared. The insulin-resistance was confirmed by the glucose-uptake assay. Comparative quantitative real time PCR was used to assess the SNCA expression.ResultsThe obtained results have showed a significant ~ 27% decrease in SNCA expression level in muscle tissue of diabetic mice (P = 0.022). Moreover, there was a significant change of SNCA expression in insulin-resistant C2C12 cells (P < 0.001).ConclusionType 2 diabetes due to insulin-resistance can decrease SNCA gene expression in muscles. In addition to the role of SNCA in cell susceptibility to insulin and glucose uptake, the SNCA expression can also be affected by insulin metabolism.

The Phosphorylation of IRS1S307 and AktS473 Molecules in Insulin-Resistant C2C12 Cells Induced with Palmitate Is Influenced by Epigallocatechin Gallate from Green Tea.

In the current investigation, the effect of epigallocatechin gallate (EGCG) on the phosphorylation of IRS1S307 and AktS473 molecules in insulin-resistant C2C12 muscle cells induced with palmitate was studied and compared with the effect of the antidiabetic drug, rosiglitazone. C2C12 myoblasts were cultured in Dulbecco's modified Eagle's medium and differentiated into myotubes using horse serum and the creatine kinase test was used to confirm their differentiation. The treatment of C2C12 myotubes was carried out with palmitate, where albumin was used as the conjugator. The Western blot technique was used to check the useful phosphorylation of IRS1S307 and AktS473 in C2C12 myotubes, in the presence or absence of palmitate. There was a significant (p < 0.00) and linear increase in the activity of creatine kinase over time (0 to 96 h after differentiation) with everyday myoblast formation. While neither EGCG nor rosiglitazone showed a significant (p > 0.05) effect on palmitate content during 96 h of incubation of IRS1S307 , EGCG alone or combined with rosiglitazone increased the phosphorylation of AktS473 , leading to the increase of glucose uptake into C2C12 cells. Thus, it can be concluded that EGCG alone or in combination with rosiglitazone may show some therapeutic effects for the prevention or treatment of Type 2 diabetes owing to its substantial effect on increasing the phosphorylation of AktS473 and the subsequent glucose uptake into the cells.

Aqueous-Methanol Extracts of Orange-Fleshed Sweet Potato (Ipomoeabatatas) Ameliorate Oxidative Stress and Modulate Type 2 Diabetes Associated Genes in Insulin Resistant C2C12 Cells.

Edible plants such as sweet potato are sources of natural antioxidants that can be exploited in the management and treatment of insulin resistance. This present study investigated the effects of the extracts of an orange-fleshed sweet potato on oxidative stress biomarkers (glutathione status and lipid peroxidation) and activities of antioxidant enzymes (catalase, CAT and glutathione peroxidase, GPx) in palmitate-induced insulin resistant C2C12 cells. The intracellular antioxidant status of the cells was also measured using Ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. Furthermore, this study determined the effect of the extracts on the regulation of some type 2 diabetes associated genes; glucose transporter 4 (glut4), Nuclear respiratory factor 1 (nrf1), Myocyte enhanced factor 2A (mef2a), Carnitine palmitoyltransferase 1 (cpt1) and Acetyl-CoA carboxylase 2 (acc2). The results showed a significant (p < 0.05) increase in intracellular GSH level, a significant reduction in the level of malonaldehyde and a significant improvement in the intracellular antioxidant status upon treatment of the insulin resistant cells with the extracts. The extracts were also able to positively modulate the expression levels of the type 2 diabetes associated genes. On the other hand, HPLC-MS analysis of the extracts showed the presence of polyphenols which could have contributed to the bioactivity of the extracts through their antioxidant effects.

A novel mechanism of pre-transplant insulin resistance contributing to post-transplant complications: Cyclosporin A-induced O-GlcNAcylation

Pre-transplant insulin resistance has been proved to be an important risk factor for organ transplantation, predicting increased post-transplant complications and worse survival outcomes. However, the underlying mechanism is still unclear. Cyclosporin A (CsA) is widely used as an immunosuppressant after organ transplantation, while emerging evidence has shown that CsA increases the risk of post-transplant complications. Thus, in this study, using a cellular model of palmitate-induced insulin resistance, we evaluate the effect of CsA on apoptosis in skeletal muscle C2C12 cells with palmitate-induced insulin resistance. Western blot and flow cytometric analysis showed that CsA induced apoptosis in insulin-resistant C2C12 cells. Mechanistically, a sustained increase of global protein O-GlcNAcylation was observed after CsA treatment, and suppression of protein O-GlcNAcylation with its inhibitors (alloxan or 5-oxo-6-diazo-norleucine) resulted in decreased O-GlcNAcylation levels and apoptosis. Furthermore, CsA increased mitochondrial membrane potential and intracellular ROS production in insulin-resistant C2C12 cells, and inhibition of ROS production with SS-31 suppressed CsA-induced O-GlcNAcylation. In summary, our results suggest that CsA treatment induced apoptosis in insulin-resistant C2C12 cells, partly via CsA-induced ROS production and resultant O-GlcNAcylation, indicating that O-GlcNAcylation serves as a potent therapeutical target for organ transplantation.

Metformin ameliorates high uric acid-induced insulin resistance in skeletal muscle cells

Hyperuricemia occurs together with abnormal glucose metabolism and insulin resistance. Skeletal muscle is an important organ of glucose uptake, disposal, and storage. Metformin activates adenosine monophosphate-activated protein kinase (AMPK) to regulate insulin signaling and promote the translocation of glucose transporter type 4 (GLUT4), thereby stimulating glucose uptake to maintain energy balance. Our previous study showed that high uric acid (HUA) induced insulin resistance in skeletal muscle tissue. However, the mechanism of metformin ameliorating UA-induced insulin resistance in muscle cells is unknown and we aimed to determine it. In this study, differentiated C2C12 cells were exposed to UA (15 mg/dl), then reactive oxygen species (ROS) was detected with DCFH-DA and glucose uptake with 2-NBDG. The levels of phospho-insulin receptor substrate 1 (IRS1; Ser307), phospho-AKT (Ser473) and membrane GLUT4 were examined by western blot analysis. The impact of metformin on UA-induced insulin resistance was monitored by adding Compound C, an AMPK inhibitor, and LY294002, a PI3K/AKT inhibitor. Our data indicate that UA can increase ROS production, inhibit IRS1-AKT signaling and insulin-stimulated glucose uptake, and induce insulin resistance in C2C12 cells. Metformin can reverse this process by increasing intracellular glucose uptake and ameliorating UA-induced insulin resistance.

Rosiglitazone, but not epigallocatechin-3-gallate, attenuates the decrease in PGC-1α protein levels in palmitate-induced insulin-resistant C2C12 cells.

Alteration of lipid metabolism is an important mechanism for the treatment of insulin resistance. PGC-1α, a key regulator of mitochondrial biogenesis and function, plays an important role in the improvement of insulin sensitivity by increasing fatty acids β-oxidation. In the present study, the effects of epigallocatechin-3-gallate (EGCG), an anti-obesity agent and enhancer of lipid catabolism, on PGC-1α protein expression was examined and compared with anti-diabetic drug rosiglitazone (RGZ). After differentiation of C2C12 myoblasts to myotubes, insulin resistance was induced by palmitate treatment. Then the expression of the PGC-1a gene and glucose uptake were evaluated before and after treatment with RGZ and EGCG. Palmitate treatment significantly decreased PGC-1α protein expression in C2C12 cells (P<0.05). RGZ could restore the expression of PGC-1α in palmitate treated cells (P>0.05), while EGCG had no significant effect on the expression of this gene (P<0.05). RGZ and EGCG significantly improved glucose uptake (by 2- and 1.54-fold, respectively) in myotubes treated with palmitate. These data suggest that RGZ and EGCG both exert their anti-diabetic activity by increasing insulin sensitivity, but with different molecular mechanisms. This effect of RGZ, unlike EGCG, is mediated, at least partly, by increasing PGC-1α protein expression.

Docosahexaenoic acid attenuated palmitate-induced insulin resistance in C2C12 cells

To explore the effects of docosahexaenoic acid (DHA) on palmitate-induced insulin resistance in C2C12 cells. Differentiated C2C12 myotubes were used. The gene expression of inflammatory cytokine and insulin signaling pathway were evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. And glucose uptake was measured by [(3)H]-2DG uptake. The levels of reactive oxygen species (ROS) were evaluated by DCF fluorescence. And the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured with a glutathione assay kit. Palmitate-induced cellular insulin resistance was clarified by reduced [(3)H]-2DG uptake and impaired gene expression of insulin signaling pathway (GLUT4, p-IRβ and p-IRS-1). DHA decreased the expression of palmitate-caused pro-inflammatory cytokines (MCP-1, IL-6 and iNOS) and oxidative stress and increased the gene expression of insulin signaling pathway (Glut4 and p-IRβ). Palmitate-induced insulin resistance is accompanied by elevated pro-inflammatory cytokines, oxidative stress and impaired gene expression of insulin signaling pathway. And DHA decreases inflammation and oxidative stress and increases glucose uptake in C2C12 cells. Thus DHA attenuates palmitate-induced insulin resistance.

Amelioration of palmitate-induced insulin resistance in C2C12 muscle cells by rooibos (Aspalathus linearis)

Increased levels of free fatty acids (FFAs), specifically saturated free fatty acids such as palmitate are associated with insulin resistance of muscle, fat and liver. Skeletal muscle, responsible for up to 80% of the glucose disposal from the peripheral circulation, is particularly vulnerable to increased levels of saturated FFAs. Rooibos (Aspalathus linearis) and its unique dihydrochalcone C-glucoside, aspalathin, shown to reduce hyperglycemia in diabetic rats, could play a role in preventing or ameliorating the development of insulin resistance. This study aims to establish whether rooibos can ameliorate experimentally-induced insulin-resistance in C2C12 skeletal muscle cells. Palmitate-induced insulin resistant C2C12 cells were treated with an aspalathin-enriched green (unfermented) rooibos extract (GRE), previously shown for its blood glucose lowering effect in vitro and in vivo or an aqueous extract of fermented rooibos (FRE). Glucose uptake and mitochondrial activity were measured using 2-deoxy-[3H]-d-glucose, MTT and ATP assays, respectively. Expression of proteins relevant to glucose metabolism was analysed by Western blot. GRE contained higher levels of all compounds, except the enolic phenylpyruvic acid-2-O-glucoside and luteolin-7-O-glucoside. Both rooibos extracts increased glucose uptake, mitochondrial activity and ATP production. Compared to FRE, GRE was more effective at increasing glucose uptake and ATP production. At a mechanistic level both extracts down-regulated PKC θ activation, which is associated with palmitate-induced insulin resistance. Furthermore, the extracts increased activation of key regulatory proteins (AKT and AMPK) involved in insulin-dependent and non-insulin regulated signalling pathways. Protein levels of the glucose transporter (GLUT4) involved in glucose transport via these two pathways were also increased. This in vitro study therefore confirms that rooibos can ameliorate palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Inhibition of PKC θ activation and increased activation of AMPK and AKT offer a plausible mechanistic explanation for this ameliorative effect.

Leukocyte antigen-related inhibition attenuates palmitate-induced insulin resistance in muscle cells

Palmitate has been shown to induce insulin resistance in skeletal muscle cells. The aim of this study was to investigate the role of the leukocyte common antigen-related (LAR) gene in palmitate-induced insulin resistance in C2C12 cells. A stable C2C12 cell line was generated using LAR short hairpin RNA. The levels of LAR protein and phosphorylation of insulin receptor substrate-1 (IRS1) and Akt were detected by western blot analysis. 2-Deoxyglucose uptake was measured in LAR knockdown and control cells using d-[2-(3)H]glucose. LAR protein level was decreased by 65% in the stable cell line compared with the control cells. Palmitate (0.5 mM) significantly induced LAR mRNA (65%) and protein levels (40%) in myotubes compared with untreated cells. Palmitate significantly reduced insulin-stimulated glucose uptake in both the control and LAR knockdown cells by 33 and 51% respectively. However, LAR depletion improved insulin-stimulated glucose uptake in myotubes treated with palmitate. Furthermore, the inhibition of LAR prevented palmitate-induced decreases in phosphorylation of IRS1(Tyr632) and Akt(Ser473) in C2C12 cells. In conclusion, these results reveal that palmitate induces LAR expression in C2C12 cells. We also provided evidence that the inhibition of LAR attenuates palmitate-induced insulin resistance in myotubes.