Insulin Resistance In Muscle Cells Research Articles

Exploration of the main active metabolites from Tinospora crispa (L.) Hook. f. & Thomson stem as insulin sensitizer in L6.C11 skeletal muscle cell by integrating in vitro, metabolomics, and molecular docking

Ethnopharmacological relevanceTinospora crispa (L.) Hook. f. & Thomson stem (TCS) has long been used as folk medicine for the treatment of diabetes mellitus. Previous study revealed that TCS possesses multi-ingredients and multi-targets characteristic potential as insulin sensitizer activity. However, its mechanisms of action and molecular targets are still obscure. Aim of the studyIn the present study, we investigated the effects of TCS against insulin resistance in muscle cells through integrating in vitro experiment and identifying its active biomarker using metabolomics and in molecular docking validation. Materials and methodsWe used centrifugal partition chromatography (CPC) to isolate 33 fractions from methanolic extract of TCS, and then used UHPLC-Orbitrap-HRMS to identify the detectable metabolites in each fraction. We assessed the insulin sensitization activity of each fraction using enzyme-linked immunosorbent assay (ELISA), and then used confocal immunocytochemistry microscopy to measure the translocation of glucose transporter 4 (GLUT4) to the cell membrane. The identified active metabolites were further simulated for its molecular docking interaction using Autodock Tools. ResultsThe polar fractions of TCS significantly increased insulin sensitivity, as measured by the inhibition of phosphorylated insulin receptor substrate-1 (pIRS1) at serine-312 residue (ser312) also the increasing number of translocated GLUT4 and glycogen content. We identified 58 metabolites of TCS, including glycosides, flavonoids, alkaloids, coumarins, and nucleotides groups. The metabolomics and molecular docking simulations showed the presence of minor metabolites consisting of tinoscorside D, higenamine, and tinoscorside A as the active compounds. ConclusionsOur findings suggest that TCS is a promising new treatment for insulin resistance and the identification of the active metabolites in TCS could lead to the development of new drugs therapies for diabetes that target these pathways.

1638-P: Oxidized Lipids Cause Ceramide Accumulation and Insulin Resistance in Skeletal Muscle

Accumulation of muscle lipids promotes insulin resistance and type 2 diabetes (T2D). Ceramides are important muscle lipids that decrease insulin sensitivity and it was previously shown that accumulation of ceramides in the sarcolemma negatively impacts insulin sensitivity in humans. The mechanisms explaining accumulation of lipids in specific compartments in skeletal muscle are unknown, but one possibility is through circulating oxidized lipids. POVPC, a major circulating oxidized phospholipid, was exposed to rat myotubes (1-50 μM) for 4h and the total cellular ceramide levels in myotubes measured by LC-MS/MS. Total ceramide content increased dose dependently at POVPC doses in the physiological range (5 μM, p <0.05). To determine the mechanism of POVPC induced ceramide accumulation, ceramide was measured in myotubes after treatment with POVPC (25 μM) in the presence of inhibitors of neutral and acid sphingomyelinase (GW4869 and ARC39), ceramide synthase (fumonisin B1) and serine palmitoyltransferase (myriocin). The individually administered inhibitors in the presence of POVPC each showed a decrease (41-55%) in ceramide accumulation. When all of inhibitors were administered together along with POVPC, an almost complete inhibition of ceramide accumulation (95%) was observed indicating that the sphingomyelinase, de novo, and salvage pathways of ceramide synthesis are activated by POVPC. The role of oxidized lipids on insulin sensitivity was examined after treatment of rat myotubes with 1-25 μM POVPC. Increasing doses of POVPC at doses as low as 1 μM significantly decreased insulin sensitivity in vitro (p <0.05). These results reveal circulating oxidized lipids promote ceramide accumulation and insulin resistance in muscle cells and may help explain variability in total and localized muscle ceramide levels between individuals. These data also suggest that therapies designed to lower circulating oxidized lipids could be beneficial for the treatment and prevention of T2D. Disclosure K.Zemski berry: None. B.C.Bergman: Research Support; Eli Lilly and Company. Funding National Institutes of Health (P30DK048520); University of Colorado (25N0350)

1554-P: Carnosic Acid Protects against Short-Term Lipid-Infusion–Induced Glucose Intolerance in Mice

Background: Obesity and type 2 diabetes (T2D) are associated with elevated plasma free fatty acids (FFA). In vivo exposure to FFAs, such as palmitate, by lipid infusion results in insulin resistance as observed in obesity and T2D. In recent years, compounds found in herbs and plants have attracted attention for prevention and treatment of insulin resistance and T2D. In recent studies by our group, carnosic acid (CA), found in rosemary, increased glucose uptake and attenuated the FFA (palmitate)-induced insulin resistance in muscle cells and adipocytes. In addition, CA caused a robust activation of AMP-activated protein kinase (AMPK). Our present study aimed to examine the effects of CA in short-term FFA-induced glucose intolerance in mice. Material and Methods: C57Bl/6J mice were cannulated in their jugular veins and infused for 48h with ethylpalmitate (EtP, nontoxic method of elevating circulating palmitate because of hydrolysis to palmitate and ethanol by plasma esterases), mimicking elevated FFA levels seen in obesity. In addition, a group of mice were infused with EtP plus 50 mg/kg CA for 48h. Ethanol vehicle with or without CA were two additional control groups. At the end of the infusions, the mice were subjected to a 2 hr hyperglycemic clamp (20 mM glucose) and the glucose infusion rate was measured to assess glucose tolerance. Results: Preliminary data revealed that mice infused with EtP and CA had a higher glucose infusion rate (GIR; mg/kg*min) (66.5±10, mean±SEM) than mice infused with EtP alone (22.7±10). The EtP and CA was comparable to the GIR of ethanol vehicle infused mice (73±9). Future Directions: Further analysis of collected plasma and tissue will be used to assess insulin resistance and beta cell function in vivo and to elucidate the mechanisms of CA alleviation of FFA-induced insulin resistance. Conclusions: Our data indicate that CA has the potential to counteract FFA-induced glucose intolerance in mice. Disclosure N.Tsakiridis: None. F.Vlavcheski: None. E.Tsiani: None. A.Giacca: None.

Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

Muscle Cell Insulin Resistance Is Attenuated by Rosmarinic Acid: Elucidating the Mechanisms Involved

Obesity and elevated blood free fatty acid (FFA) levels lead to impaired insulin action causing insulin resistance in skeletal muscle, and contributing to the development of type 2 diabetes mellitus (T2DM). Mechanistically, insulin resistance is associated with increased serine phosphorylation of the insulin receptor substrate (IRS) mediated by serine/threonine kinases including mTOR and p70S6K. Evidence demonstrated that activation of the energy sensor AMP-activated protein kinase (AMPK) may be an attractive target to counteract insulin resistance. We reported previously that rosemary extract (RE) and the RE polyphenol carnosic acid (CA) activated AMPK and counteracted the FFA-induced insulin resistance in muscle cells. The effect of rosmarinic acid (RA), another polyphenolic constituent of RE, on FFA-induced muscle insulin resistance has never been examined and is the focus of the current study. Muscle cell (L6) exposure to FFA palmitate resulted in increased serine phosphorylation of IRS-1 and reduced insulin-mediated (i) Akt activation, (ii) GLUT4 glucose transporter translocation, and (iii) glucose uptake. Notably, RA treatment abolished these effects, and restored the insulin-stimulated glucose uptake. Palmitate treatment increased the phosphorylation/activation of mTOR and p70S6K, kinases known to be involved in insulin resistance and RA significantly reduced these effects. RA increased the phosphorylation of AMPK, even in the presence of palmitate. Our data indicate that RA has the potential to counteract the palmitate-induced insulin resistance in muscle cells, and further studies are required to explore its antidiabetic properties.

A bioactive component of Portulaca Oleracea L., HM-chromanone, improves palmitate-induced insulin resistance by inhibiting mTOR/S6K1 through activation of the AMPK pathway in L6 skeletal muscle cells.

Increased free fatty acid levels in the blood are common in obesity and cause insulin resistance associated with type 2 diabetes in the muscles. Previous studies have confirmed the antidiabetic and anti-obesity potential of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone). However, it is unknown how HM-chromanone alleviates obesity-related insulin resistance in L6 skeletal muscle cells. Palmitate induced insulin resistance and reduced glucose uptake, whereas HM-chromanone significantly increased glucose uptake. In palmitate-treated L6 skeletal muscle cells, HM-chromanone stimulated liver kinase B1 (LKB1) and 5'-adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. The AMPK inhibitor compound C, and the LKB1 inhibitor radicicol blocked the effects of HM-chromanone. Furthermore, HM-chromanone significantly inhibited mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase 1 (S6K1) activation, but there was no change in protein kinase C θ (PKC θ) expression. When pAMPK was inhibited with compound C, the effect of HM-chromanone on the inhibition of mTOR and S6K1 was significantly diminished. This indicates that HM-chromanone inhibits mTOR and S6K1 activation through pAMPK activation. Inhibition of mTOR and S6K1 by HM-chromanone significantly reduced IRS-1Ser307 and IRS-1Ser632 phosphorylation, leading to insulin resistance. This resulted in an increase in PM-GLUT4 (glucose transporter 4) expression, thereby stimulating glucose uptake in insulin-resistant muscle cells. HM-chromanone can improve palmitate-induced insulin resistance by inhibiting mTOR and S6K1 through activation of the AMPK pathway in L6 skeletal muscle cells. These results show the therapeutic potential of HM-chromanone for improving insulin resistance in type 2 diabetes.

Carnosic Acid Attenuates the Free Fatty Acid-Induced Insulin Resistance in Muscle Cells and Adipocytes

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine kinases including JNK, mTOR, and p70 S6K cause serine phosphorylation of the insulin receptor substrate (IRS) and have been implicated in insulin resistance. Activation of AMP-activated protein kinase (AMPK) increases glucose uptake, and in recent years, AMPK has been viewed as an important target to counteract insulin resistance. We reported previously that carnosic acid (CA) found in rosemary extract (RE) and RE increased glucose uptake and activated AMPK in muscle cells. In the present study, we examined the effects of CA on palmitate-induced insulin-resistant L6 myotubes and 3T3L1 adipocytes. Exposure of cells to palmitate reduced the insulin-stimulated glucose uptake, GLUT4 transporter levels on the plasma membrane, and Akt activation. Importantly, CA attenuated the deleterious effect of palmitate and restored the insulin-stimulated glucose uptake, the activation of Akt, and GLUT4 levels. Additionally, CA markedly attenuated the palmitate-induced phosphorylation/activation of JNK, mTOR, and p70S6K and activated AMPK. Our data indicate that CA has the potential to counteract the palmitate-induced muscle and fat cell insulin resistance.

A novel phenolic formulation for treating hepatic and peripheral insulin resistance by regulating GLUT4-mediated glucose uptake

Chronic insulin resistance suppresses muscle and liver response to insulin, which is partially due to impaired vesicle trafficking. We report here that a formula consisting of resveratrol, ferulic acid and epigallocatechin-3-O-gallate is more effective in ameliorating muscle and hepatic insulin resistance than the anti-diabetic drugs, metformin and AICAR. The formula enhanced glucose transporter-4 (GLUT4) translocation to the plasma membrane in the insulin-resistant muscle cells by regulating both insulin-independent (calcium and AMPK) and insulin-dependent (PI3K) signaling molecules. Particularly, it regulated the subcellular location of GLUT4 through endosomes to increase glucose uptake under insulin-resistant condition. Meanwhile, this phytochemicals combination increased glycogen synthesis and decreased glucose production in the insulin-resistant liver cells. On the other hand, this formula also showed anti-diabetic potential by the reduction of lipid content in the myotubes, hepatocytes, and adipocytes. This study demonstrated that the three phenolic compounds in the formula could work in distinct mechanisms and enhance both insulin-dependent and independent vesicles trafficking and glucose transport mechanisms to improve carbohydrate and lipid metabolism.

Ursolic Acid Lactone Obtained from Eucalyptus tereticornis Increases Glucose Uptake and Reduces Inflammatory Activity and Intracellular Neutral Fat: An In Vitro Study.

Obesity has a strong relationship to insulin resistance and diabetes mellitus, a chronic metabolic disease that alters many physiological functions. Naturally derived drugs have aroused great interest in treating obesity, and triterpenoids are natural compounds with multiple biological activities and antidiabetic mechanisms. Here, we evaluated the bioactivity of ursolic acid lactone (UAL), a lesser-known triterpenoid, obtained from Eucalyptus tereticornis. We used different cell lines to show for the first time that this molecule exhibits anti-inflammatory properties in a macrophage model, increases glucose uptake in insulin-resistant muscle cells, and reduces triglyceride content in hepatocytes and adipocytes. In 3T3-L1 adipocytes, UAL inhibited the expression of genes involved in adipogenesis and lipogenesis, enhanced the expression of genes involved in fat oxidation, and increased AMP-activated protein kinase phosphorylation. The range of biological activities demonstrated in vitro indicates that UAL is a promising molecule for fighting diabetes.

Rosemary extract activates AMPK, inhibits mTOR and attenuates the high glucose and high insulin-induced muscle cell insulin resistance.

Impaired action of insulin in skeletal muscle, termed insulin resistance, leads to increased blood glucose levels resulting in compensatory increase in insulin levels. The elevated blood glucose and insulin levels exacerbate insulin resistance and contribute to the pathogenesis of type 2 diabetes mellitus. In previous studies we found attenuation of free fatty acid-induced muscle cell insulin resistance by rosemary extract (RE). In the present study we investigated the effects of RE on high glucose (HG) and high insulin (HI)-induced muscle cell insulin resistance. Exposure of L6 myotubes to 25mmol/L glucose and 100nmol/L insulin for 24h, to mimic hyperglycemia and hyperinsulinemia, abolished the acute insulin-stimulated glucose uptake, increased the serine phosphorylation of IRS-1 and the phosphorylation/activation of mTOR and p70S6K. Treatment with RE significantly improved the insulin-stimulated glucose uptake and increased the acute insulin-stimulated tyrosine phosphorylation while reducing the HG+HI-induced serine phosphorylation of IRS-1 and phosphorylation of mTOR and p70S6K. Additionally, treatment with RE significantly increased the phosphorylation of AMPK, its downstream effector ACC and the plasma membrane GLUT4 levels. Our data indicate a potential of RE to counteract muscle cell insulin resistance and more studies are required to investigate its effectiveness in vivo. Novelty: RE phosphorylated muscle cell AMPK and ACC under both normal and HG+HI conditions. The HG+HI-induced serine phosphorylation of IRS-1 and activation of mTOR and p70S6K were attenuated by RE. RE restored the insulin-stimulated glucose uptake by enhancing GLUT4 glucose transporter translocation to plasma membrane.

Pyrroloquinoline Quinone Modifies Lipid Profile, but Not Insulin Sensitivity, of Palmitic Acid-Treated L6 Myotubes.

Pyrroloquinoline quinone (PQQ) is a novel stimulator of mitochondrial biogenesis and cellular energy metabolism. This is the first study investigating regulatory mechanisms and metabolic responses underlying PQQ’s action in palmitate-exposed L6 myotubes. Particularly, we assessed alterations in lipid content and composition, expression of metabolic enzymes, and changes in glucose transport. The experiments were conducted using muscle cells subjected to short (2 h) and prolonged (24 h) incubation with PQQ in a sequence of pre- and post-palmitic acid (PA) exposure. We demonstrated the opposite effects of 2 and 24 h treatments with PQQ on lipid content, i.e., a decline in the level of free fatty acids and triacylglycerols in response to short-time PQQ incubation as compared to increases in diacylglycerol and triacylglycerol levels observed after 24 h. We did not demonstrate a significant impact of PQQ on fatty acid transport. The analysis of metabolic enzyme expression showed that the vast majority of PQQ-dependent alterations cumulated in the PA/PQQ 24 h group, including elevated protein amount of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α), sirtuin-1 (SIRT1), phosphorylated 5′AMP-activated protein kinase (pAMPK), carnitine palmitoyltransferase I (CPT1), citrate synthase (CS), fatty acid synthase (FAS), and serine palmitoyltransferase, long chain base subunit 1 (SPT1). In conclusion, the results mentioned above indicate PQQ-dependent activation of both fatty acid oxidation and lipid synthesis in order to adapt cells to palmitic acid-rich medium, although PQQ did not attenuate insulin resistance in muscle cells.

Attenuation of Free Fatty Acid (FFA)-Induced Skeletal Muscle Cell Insulin Resistance by Resveratrol is Linked to Activation of AMPK and Inhibition of mTOR and p70 S6K.

Insulin resistance, a main characteristic of type 2 diabetes mellitus (T2DM), is linked to obesity and excessive levels of plasma free fatty acids (FFA). Studies indicated that significantly elevated levels of FFAs lead to skeletal muscle insulin resistance, by dysregulating the steps in the insulin signaling cascade. The polyphenol resveratrol (RSV) was shown to have antidiabetic properties but the exact mechanism(s) involved are not clearly understood. In the present study, we examined the effect of RSV on FFA-induced insulin resistance in skeletal muscle cells in vitro and investigated the mechanisms involved. Parental and GLUT4myc-overexpressing L6 rat skeletal myotubes were used. [3H]2-deoxyglucose (2DG) uptake was measured, and total and phosphorylated levels of specific proteins were examined by immunoblotting. Exposure of L6 cells to FFA palmitate decreased the insulin-stimulated glucose uptake, indicating insulin resistance. Palmitate increased ser307 (131% ± 1.84% of control, p < 0.001) and ser636/639 (148% ± 10.1% of control, p < 0.01) phosphorylation of IRS-1, and increased the phosphorylation levels of mTOR (174% ± 15.4% of control, p < 0.01) and p70 S6K (162% ± 20.2% of control, p < 0.05). Treatment with RSV completely abolished these palmitate-induced responses. In addition, RSV increased the activation of AMPK and restored the insulin-mediated increase in (a) plasma membrane GLUT4 glucose transporter levels and (b) glucose uptake. These data suggest that RSV has the potential to counteract the FFA-induced muscle insulin resistance.

2300-PUB: Activation of GLP-1 Signaling Attenuates FFAs-Induced Muscle Dysfunction and Senescence

Obesity and insulin resistance are frequently associated with type 2 diabetes (T2D). Evidence is now revealed that plasma free fatty acid (FFA) levels are often elevated in most T2D patients, and this can cause peripheral insulin resistance directly. As a result, these FFAs inhibit insulin-stimulated glucose uptake, thus contributing to the onset of insufficient energy within cells. Among various T2D-related complications, the continuously loss of muscle is one of the most important adverse effects. Blocked insulin signaling in skeletal muscle is manifested by decreased insulin sensitivity and impaired muscle cell function including glucose transport, glucose phosphorylation, reduced glucose oxidation and glycogen synthesis that might ultimately lead to muscle loss. Interestingly, recent studies have found that GLP-1 agonists appear to slow muscle wasting during T2D. Since GLP-1 receptors are abundantly expressed on the membrane of muscle cells, indicating that they may play some important roles in muscle physiology. However, the detailed mechanism is still not well understood. In this study, we investigated the effect of GLP-1 agonist against FFA-induced muscle wasting. We used mouse C2C12 myotubes to evaluate effects of GLP-1 agonists FFA-induced muscle loss and dysfunction. Our data indicated that FFA induces insulin resistance, oxidative stress muscle loss and cell senescence in muscle cells. Moreover, FFA also decreased glucose uptake and reduced myotube mass and function. Conversely, GLP-1 agonists could protect against FFAs-induced myotube dysfunctions, including insulin resistance, oxidative stress, cell loss and senescence, that eventually reversed myotube mass and function through AMPK/SIRT1 pathway. We expect our results can extend the contributing role of GLP-1 signaling in FFA-induced insulin resistance of muscle cells, and the modulation of GLP-1 signaling may be a workable strategy for muscle wasting prevention. Disclosure H. Li: None. C. Lin: None. C. Huang: None. E. Kornelius: None. Y. Yang: None. C. Peng: None. C. Li: None. S. Liu: None. Funding Ministry of Science and Technology of Taiwan (108-2320-B-040-029-MY2)

Amelioration of High-Insulin-Induced Skeletal Muscle Cell Insulin Resistance by Resveratrol Is Linked to Activation of AMPK and Restoration of GLUT4 Translocation.

Insulin resistance, the hallmark of type 2 diabetes mellitus (T2DM), is linked to hyperinsulinemia, which develops to counterbalance initial peripheral hormone resistance. Studies indicate that chronically elevated levels of insulin lead to skeletal muscle insulin resistance by deregulating steps within the insulin signaling cascade. The polyphenol resveratrol (RSV) has been shown to have antidiabetic properties in vitro and in vivo. In the present study, we examined the effect of RSV on high insulin (HI)-induced insulin resistance in skeletal muscle cells in vitro and investigated the mechanisms involved. Parental and GLUT4myc-overexpressing L6 rat skeletal muscle cells were used. [3H]2-deoxyglucose (2DG) uptake was measured, and total and phosphorylated levels of specific proteins were examined by immunoblotting. Exposure of L6 cells to HI levels (100 nM) for 24 h decreased the acute-insulin-stimulated 2DG uptake, indicating insulin resistance. HI increased ser307 and ser636/639 phosphorylation of IRS-1 (to 184% ± 12% and 225% ± 28.9% of control, with p < 0.001 and p < 0.01, respectively) and increased the phosphorylation levels of mTOR (174% ± 6.7% of control, p < 0.01) and p70 S6K (228% ± 33.5% of control, p < 0.01). Treatment with RSV abolished these HI-induced responses. Furthermore, RSV increased the activation of AMPK and restored the insulin-mediated increase in plasma membrane GLUT4 glucose transporter levels. These data suggest that RSV has a potential to counteract the HI-induced muscle insulin resistance.

Whey Peptides Stimulate Differentiation and Lipid Metabolism in Adipocytes and Ameliorate Lipotoxicity-Induced Insulin Resistance in Muscle Cells.

Deregulation of lipid metabolism and insulin function in muscle and adipose tissue are hallmarks of systemic insulin resistance, which can progress to type 2 diabetes. While previous studies suggested that milk proteins influence systemic glucose homeostasis and insulin function, it remains unclear whether bioactive peptides generated from whey alter lipid metabolism and its accumulation in muscle and adipose tissue. Therefore, we incubated murine 3T3-L1 preadipocytes and C2C12 myotubes with a whey peptide mixture produced through pepsin-pancreatin digestion, mimicking peptides generated in the gut from whey protein hydrolysis, and examined its effect on indicators of lipid metabolism and insulin sensitivity. Whey peptides, particularly those derived from bovine serum albumin (BSA), promoted 3T3-L1 adipocyte differentiation and triacylglycerol (TG) accumulation in accordance with peroxisome proliferator-activated receptor γ (PPARγ) upregulation. Whey/BSA peptides also increased lipolysis and mitochondrial fat oxidation in adipocytes, which was associated with the upregulation of peroxisome proliferator-activated receptor δ (PPARδ). In C2C12 myotubes, whey but not BSA peptides ameliorated palmitate-induced insulin resistance, which was associated with reduced inflammation and diacylglycerol accumulation, and increased sequestration of fatty acids in the TG pool. Taken together, our study suggests that whey peptides generated via pepsin-pancreatin digestion profoundly alter lipid metabolism and accumulation in adipocytes and skeletal myotubes.

Amelioration of insulin resistance using the additive effect of ferulic acid and resveratrol on vesicle trafficking for skeletal muscle glucose metabolism.

Dysregulation of vesicle trafficking in muscle is one of the factors responsible for the pathogenesis of insulin resistance (IR). Ferulic acid (FER) and resveratrol (RSV) are known to have hypoglycemic property. In this study, differentiated L6 myotubes were induced with palmitate as a model of IR. Chemical ablation of muscle vesicles was used to investigate how FER and RSV influence glucose utilization. Results showed that both FER and RSV elicit glucose uptake and promote glycogen synthesis in insulin-resistant muscle cells. Mechanistic studies further showed that FER markedly enhances the transferrin receptor-containing endosomal compartment activities via phosphoinositide 3-kinase (PI3K)/atypical protein kinase C-dependent pathway, while RSV facilitates glucose transporter storage vesicles (GSV) trafficking via an exercise-like effect of conventional protein kinase C/5'-adenosine monophosphate-activated protein kinase (AMPK) modulation. Therefore, these two phenolic compounds promoted glucose transport through two separate routes, and they had an additive effect on the increase of glucose uptake in insulin-resistant muscle cells. These findings provide a basis for the understanding of the antidiabetic potential of RSV and FER combination.

Effect of Subtoxic DDT Exposure on Glucose Uptake and Insulin Signaling in Rat L6 Myoblast-Derived Myotubes.

Exposure to persistent organic pollutants including dichlorodiphenyltrichloroethane (DDT) induces insulin resistance. But the mechanism is not clearly known. The present study was designed to explore the effect of subtoxic DDT exposure on (1) insulin-stimulated glucose uptake, (2) malondialdehyde (MDA) level and total antioxidant content, (3) activation of redox sensitive kinases (RSKs), and (4) insulin signaling in rat L6 myoblast-derived myotubes. Exposure to 30 mg/L and 60 mg/L of DDT for 18 hours dose dependently decreased glucose uptake and antioxidant content in myotubes and increased MDA levels. The exposures did not alter tumor necrosis factor α (TNF-α) level as determined by enzyme-linked immunosorbent assay, despite decreased messenger RNA expression following DDT exposures. Phosphorylation of c-Jun N-terminal kinases and IκBα, an inhibitory component of nuclear factor κB (NFκB), was increased, suggesting activation of RSKs. The level of tyrosine phosphorylation of insulin receptor substrate 1 and serine phosphorylation of protein kinase B (Akt) on insulin stimulation decreased in myotubes with exposure to subtoxic concentrations of DDT, but there was no change in tyrosine phosphorylation level of insulin receptors. We conclude that subtoxic DDT exposure impairs insulin signaling and thereby induces insulin resistance in muscle cells. Data show that oxidative stress-induced activation of RSKs is responsible for impairment of insulin signaling on DDT exposure.

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.

Modulation of Insulin Resistance and the Adipocyte-Skeletal Muscle Cell Cross-Talk by LCn-3PUFA.

The cross-talk between skeletal muscle and adipose tissue is involved in the development of insulin resistance (IR) in skeletal muscle, leading to the decrease in the anabolic effect of insulin. We investigated if the long chain polyunsaturated n-3 fatty acids (LCn-3PUFA), eicosapentaenoic and docosapentaenoic acids (EPA and DPA, respectively) could (1) regulate the development of IR in 3T3-L1 adipocytes and C2C12 muscle cells and (2) inhibit IR in muscle cells exposed to conditioned media (CM) from insulin-resistant adipocytes. Chronic insulin (CI) treatment of adipocytes and palmitic acid (PAL) exposure of myotubes were used to induce IR in the presence, or not, of LCn-3PUFA. EPA (50 µM) and DPA (10 µM) improved PAL-induced IR in myotubes, but had only a partial effect in adipocytes. CM from adipocytes exposed to CI induced IR in C2C12 myotubes. Although DPA increased the mRNA levels of genes involved in fatty acid (FA) beta-oxidation and insulin signaling in adipocytes, it was not sufficient to reduce the secretion of inflammatory cytokines and prevent the induction of IR in myotubes exposed to adipocyte’s CM. Treatment with DPA was able to increase the release of adiponectin by adipocytes into CM. In conclusion, DPA is able to protect myotubes from PAL-induced IR, but not from IR induced by CM from adipocytes.

Improvement of palmitate-induced insulin resistance in C2C12 skeletal muscle cells using Platycodon grandiflorum seed extracts

Platycodon grandiflorum seeds (PGSE) and P. grandiflorum root extracts (PGRE) were prepared and their effect on glucose uptake were evaluated in C2C12 myotubes. PGSE had significantly higher total phenolic and flavonoid contents than PGRE (P < 0.05), whereas crude saponin contents did not differ significantly between extracts. IC50 values of PGSE and PGRE for α-glucosidase were 0.269 and 1.47 mg/mL, respectively. PGSE treatments led to greater glucose uptake-promoting effects than PGRE treatments in normal and palmitate-treated C2C12 myotubes and luteolin was identified as an active PGSE compound. The insulin signaling molecules and insulin-independent AMP kinase were significantly activated by PGSE treatments and GLUT4 translocation on the plasma membrane was increased by 1.7 fold in palmitate-treated C2C12 myotubes. Based on the α-glucosidase inhibitory activity and glucose uptake promoting effects of PGSE in insulin-resistant muscle cells, these extract have a great potential to be developed as an anti-diabetic agent.