L6 Muscle Cells Research Articles

Validation of Fatty Acids Regulated Genes in Adipocytes and Muscle Cells

The prevalence of obesity/type 2 diabetes has dramatically increased during last two decades in the United States. Environmental factors including diets may play an important role in developing of these conditions. Adipose and muscle tissues are both significantly affected with these diseases. In order to study epigenetic modifications induced by environmental factors, we have been validating genes screened out from adipocytes and muscle cells by microarrays. In this study, 3T3‐L1 adipocytes and L6 muscle cells were cultured in media containing saturated fatty acids to mimic hyperlipidemia conditions, which occur during the development of obesity and type 2 diabetes. DNA microarrays were used to identify differentially expressed genes in adipocytes and muscle cells by saturated and poly unsaturated fatty acids. We have validated some of the genes that were screened out by DNA microarrays including RASA2, MEF2C, GRP78, etc by real time PCR. RASA2 functions as a suppressor of Ras. It may be involved in growth suppression of muscle cells by saturated fatty acids. In addition, it has been confirmed by using Western Blots that saturated fatty acid palmitic acid induces stress in muscle cells and unsaturated fatty acid linoleic acid does not induce cell death through apoptosis pathway. We will further look into if epigenetic mechanisms are involved in regulation of the validated genes.Support or Funding InformationStartup funds from Texas A&M University‐Kingsville

Applications of calcium electroporation to effective apoptosis induction in fibrosarcoma cells and stimulation of normal muscle cells

The electroporation (EP) supports various types of anticancer therapies by the selective transport of cytostatics. Increase in intracellular calcium level by EP may be a new approach to fibrosarcoma treatment. Calcium is one of the most important factors of cell proliferation, differentiation and cell death (apoptosis or necrosis). Calcium level balanced by electroporation can cause different effects on normal and pathological cells. The efficiency and safety of electroporation combined with Ca2+ ions were examined in our study. The two muscle cell lines were used: normal rat skeletal muscle cells — L6 and cancer muscle cells — Wehi-164 (fibrosarcoma). Two CaCl2 concentrations were tested: 0.5mM and 5mM combined with EP parameters: 1000V/cm, 1200V/cm, and 1500V/cm. The results show that EP supported by Ca2+ is cytotoxic for Wehi-164 cells and simultaneously safe for normal muscle cells. The main type of cell death – apoptosis – was confirmed by Tunnel and Annexin V/PI assay. Additionally, sPLA2 pro-tumorigenic influence was proved by immunocytochemistry. Moreover, EP with 0.5mM of Ca2+ slightly stimulates the normal muscle cells — L6 to increase proliferation.

Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM) was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM), GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor) and U73122 (PLC inhibitor). However, 2-APB (IP3R blocker) only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation-contraction coupling.

The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation-contraction coupling. However, the mechanism for subsequent Ca(2+) release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca(2+) under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation-contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca(2+) from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca(2+) was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca(2+) concentration in the media. Ca(2+) entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca(2+) entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca(2+) release from the ryanodine receptor to the cytosol.

Polymeric carrier-mediated intracellular delivery of phosphatidylinositol-3,4,5-trisphosphate to overcome insulin resistance

Background: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is a major lipid second messenger in insulin-mediated signalling towards the metabolic actions of this hormone in muscle and fat.Purpose: Assessing the intracellular transport of exogenous PIP3 attached to a polymeric carrier in an attempt to overcome cellular insulin resistance.Methods: Artificial chromatic bio-mimetic membrane vesicles composed of dimyristoylphosphatidylcholine and polydiacetylene were applied to screen the polymeric carriers. PIP3 cellular localization and bio-activity was assessed by fluorescent and live-cell microscopy in L6 muscle cells and in 3T3-L1 adipocytes.Results and discussion: We demonstrate that a specific-branched polyethylenimine (PEI-25, 25 kDa) carrier forms complexes with PIP3 that interact with the bio-mimetic membrane vesicles in a manner predictive of their interaction with cells: In L6 muscle cells, PEI-25/fluorescent-PIP3 complexes are retarded at the cell perimeter. PEI-25/PIP3 complexes retain their bio-activity, engaging signalling steps downstream of PIP3, even in muscle cells rendered insulin resistant by exposure to high glucose/high insulin.Conclusions: Inducing insulin actions by intracellular PIP3 delivery (PEI-25/PIP3 complexes) in some forms of insulin-resistant cells provides the first proof-of-principle for the potential therapeutic use of PIP3 in a “second-messenger agonist” approach. In addition, utilization of an artificial bio-mimetic membrane platform to screen for highly efficient PIP3 delivery predicts biological function in cells.

Preconditioning L6 Muscle Cells with Naringin Ameliorates Oxidative Stress and Increases Glucose Uptake.

Enhanced oxidative stress contributes to pathological changes in diabetes and its complications. Thus, strategies to reduce oxidative stress may alleviate these pathogenic processes. Herein, we have investigated Naringin mediated regulation of glutathione (GSH) & intracellular free radical levels and modulation of glucose uptake under oxidative stress in L6 cell lines. The results from the study demonstrated a marked decrease in glutathione with a subsequent increase in free radical levels, which was reversed by the pretreatment of Naringin. We also observed that the increased malondialdehyde level, the marker of lipid peroxidation on induction of oxidative stress was retrieved on Naringin pretreatment. Addition of Naringin (100 μM) showed approximately 40% reduction in protein glycation in vitro. Furthermore, we observed a twofold increase in uptake of fluorescent labeled glucose namely 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2 - NBDG) on Naringin treatment in differentiated L6 myoblast. The increased uptake of 2-NBDG by L6 myotubes may be attributed due to the enhanced translocation of GLUT4. Our results demonstrate that Naringin activate GSH synthesis through a novel antioxidant defense mechanism against excessive Reactive Oxygen Species (ROS) production, contributing to the prevention of oxidative damage in addition to its effect on glycemic control.

Sustainable Resource based Hyperbranched Epoxy Nanocomposite as an Infection Resistant, Biodegradable, Implantable Muscle Scaffold

Use of sustainable resources in the realm of materials science has opened newer possibilities for fabricating biodegradable biomaterials. Cellulose is an abundantly available bioresource that proved excellent potential in composite science and the biomaterials domain. Hence, in this report the potentiality of a “green” CuO–nanofibrillar cellulose/glycerol based hyperbranched epoxy nanocomposite was presented as an implantable muscle tissue scaffold. Muscles reconstruction has been a longstanding problem in medical science. However, with the evolution of polymeric scaffolds, repairing of damaged or lost tissues becomes possible with the aid of tissue engineering. The prepared material possessed high mechanical properties (tensile strength 55 MPa) to support cell growth. In vitro studies revealed that the material could support the growth and proliferation of L6 muscle cells, without causing any detriment to their cellular morphology. Further, both in vitro and in vivo toxicity assessments established the profound biocompatibility of the material. Moreover, the nanocomposite exhibited inhibitory effect against Staphylococcus aureus, Escherichia coli and Candida albicans, the microorganisms responsible for various surgical site infections. In vivo investigation revealed the biodegradability of the prepared material which overrules the need of repeated surgery for any implantable biomaterial. Thus, the overall work endorses the sustainable resource based nanocomposite as a high performance, biodegradable, antimicrobial scaffold material for reconstruction of muscles tissues.

ShRNA-mediated gene silencing of lipoprotein lipase improves insulin sensitivity in L6 skeletal muscle cells

In previous studies, we demonstrated that down-regulation of lipoprotein lipase in L6 muscle cells increased insulin-stimulated glucose uptake. In the current study, we used RNA interference technology to silence the LPL gene in L6 cells and generate a LPL-knock-down (LPL-KD) cell line. ShRNA transfected cells showed a 88% reduction in the level of LPL expression. The metabolic response to insulin was compared in wild-type (WT) and LPL-KD cells. Insulin-stimulated glycogen synthesis and glucose oxidation were respectively, 2.4-fold and 2.6-fold greater in LPL-KD cells compared to WT cells. Oxidation of oleic acid was reduced by 50% in LPL-KD cells compared to WT cells even in the absence of insulin. The contribution of LPL in regulating fuel metabolism was confirmed by adding back purified LPL to the culture media of LPL-KD cells. The presence of 10 μg/mL LPL resulted in LPL-KD cells reverting back to lower glycogen synthesis and glucose oxidation and increased fatty acid oxidation. Thus, LPL depletion appeared to mimic the action of insulin. These finding suggests an inverse correlation between muscle LPL levels and insulin-stimulated fuel homeostasis.

Amelioration of palmitate-induced metabolic dysfunction in L6 muscle cells expressing low levels of receptor-interacting protein 140.

We have shown that reduced expression of receptor-interacting protein 140 (RIP140) alters the regulation of fatty-acid (FA) oxidation in muscle. To determine whether a high level of FA availability alters the effects of RIP140 on metabolic regulation, L6 myotubes were transfected with or without RNA interference oligonucleotide sequences to reduce RIP140 expression, and then incubated with high levels of palmitic acid, with or without insulin. High levels of palmitate reduced basal (53%-58%) and insulin-treated (24%-44%) FA uptake and oxidation, and increased basal glucose uptake (88%). In cells incubated with high levels of palmitate, low RIP140 increased basal FA uptake and insulin-treated FA oxidation and glucose uptake, and decreased basal glucose uptake and insulin-treated FA uptake. Under basal conditions, low RIP140 increased the mRNA content of FAT/CD36 (159%) and COX4 (61%), as well as the protein content of Nur77 (68%), whereas the mRNA expression of FGF21 (50%) was decreased, as was the protein content of CPT1b (35%) and FGF21 (44%). Under insulin-treated conditions, low RIP140 expression increased the mRNA content of MCAD (84%) and Nur77 (84%), as well as the protein content of Nur77 (23%). Thus, a low level of RIP140 restores the rates of FA uptake in the basal state, in part via a reduction in upstream insulin signaling. Our data also indicate that the protein expression of Nur77 may be modulated by RIP140 when muscle cells are metabolically challenged by high levels of palmitate.

Metformin attenuates palmitic acid-induced insulin resistance in L6 cells through the AMP-activated protein kinase/sterol regulatory element-binding protein-1c pathway

AMP-activated protein kinase (AMPK) is an important effector of metformin action on glucose uptake in skeletal muscle cells. We recently reported that metformin improved insulin receptor substrate-1 (IRS-1)-associated insulin signaling by downregulating sterol regulatory element-binding protein-1c (SREBP-1c) expression. In this study, we investigated whether AMPK activation and SREBP-1c inhibition contribute to the beneficial effects of metformin on IRS-1-associated insulin signaling in L6 myotubes. L6 muscle cells were incubated with palmitic acid (PA) to induce insulin resistance and then treated with metformin and/or the AMPK inhibitor, compound C. AMPK, SREBP-1c, IRS-1 and Akt protein expression levels were determined by western blot analysis. The effects of metformin on SREBP-1c gene transcription were determined by a luciferase reporter assay. Glucose uptake was evaluated using the 2-NBDG method. In the PA-treated L6 cells, metformin treatment enhanced AMPK phosphorylation, reduced SREBP-1c expression and increased IRS-1 and Akt protein expression, whereas treatment with compound C blocked the effects of metformin on SREBP-1c expression and the IRS-1 and Akt levels. Moreover, metformin suppressed SREBP-1c promoter activity and promoted glucose uptake through AMPK. The results from this study demonstrate that metformin ameliorates PA-induced insulin resistance through the activation of AMPK and the suppression of SREBP-1c in skeletal muscle cells.

Oleic and linoleic fatty acids downregulate Slc2a4/GLUT4 expression via NFKB and SREBP1 in skeletal muscle cells

Oleic (OA) and linoleic (LA) fatty acids may be important regulators of Slc2a4 gene (GLUT4 protein) in skeletal muscle, thus participating in insulin resistance. We investigated the effect of OA and LA on the Slc2a4/GLUT4 expression in L6 muscle cells; as well as potential transcriptional regulators. OA and LA (50–400 µM) decreased the Slc2a4/GLUT4 expression in a dose-dependent way (maximum of ~50%, P < 0.001). OA and LA did not alter the Slc2a4-binding activity of oxysterols-receptor-LXR-alpha and peroxisome-proliferator-activated-receptor-gamma; but decreased the Slc2a4-binding activity of the sterol-regulatory-element-binding-protein-1 (SREBP1) enhancer (50%, P < 0.001), and increased (~30%, P < 0.001) the nuclear proteins binding into the Slc2a4-nuclear-factor-NF-kappa-B-binding site (repressor), and the phosphorylation of the inhibitors of nuclear-factor-kappa-B-kinase alpha/beta (150–300%, P < 0.001). In sum, OA and LA are potent inhibitors of the Slc2a4/GLUT4 expression in muscle cells; an effect involving reduced SREBP1 and increased NFKB transcriptional activity. These regulations may participate in the fatty acid-related pathophysiology of insulin resistance.

Hypoglycemic activity of Piper sarmentosum Roxp Extract in L6 muscle cells and 3T3-L1 adipose cells

Piper sarmentosum (Cha Phluu) is very popular and more widely used. It is a common plant used in Thai traditional medicine and cooking. Piper sarmentosumis was long been used to relieve the symptoms of malaria, toothache, diuretic, coughs, flu, rheumatism and diabetic treatment. The objective of this study was to investigate the in vitro hypoglycemic activity of its ethanolic extract. The glucose uptake of radioactive 2-deoxyglucose in muscle cells (L6) and adipose cells (3T3-L1) were used to measure the hypoglycemic activity. The hypoglycemic activity of P. sarmentosum in both muscle cells (L6) and adipose cells (3T3-L1) showed the glucose uptake increasing level by fold of basal. The ethanolic extract at concentration 50 µg/ml exhibited the highest glucose uptake level by 1.33 fold in L6-muscle and 1.73 fold in 3T3-L1 adipose cells respectively. This result can support using the ethanolic extract of P. sarmentosum as hypoglycemic drug to increase glucose uptake in both L6 muscle and 3T3-L1 adipose cells. The further isolation of active hypoglycemic compounds from this plant should be continued and developed to antidiabetic drug, health products to prevent diabetes or promote in daily food consumption.

Effects of crude water-soluble extract of Momordica charantia on viability, caspase activity, cytochrome c release and on cytosolic calcium levels in different cancer cell lines

Prior to the availability of chemotherapeutic agents, dietary measures, including traditional medicines derived from plants, were the major forms of cancer treatment. One such plant is Momrdica charantia (Family: Cucurbitaceae), whose fruit is known as corilla or bitter gourd/melon. M. charantia possesses anti-carcinogenic properties and it can modulate its effect via xenobiotic metabolism and oxidative stress. This study investigated the anti-cancer effect of an active water soluble extract (s) of M. charantia on cell viability and its cellular mechanism of action in inducing cell death. The fruit was washed and cut in to small pieces, liquidised in deionised water using a blender and subsequently, dried using a rota evaporator and oven. Both time course (800 µg/ml) and dose- dependent (200 µg/ml-800 µg/ml) experiments were performed treating six different cancer cell lines, namely 1321N1, Gos-3, U87-MG, Sk Mel, Corl -23, Weri Rb-1 and normal L6 muscle cell line with the crude fruit extract for 24 hours at 37 °C. Cell viability was measured using the MTT assay. The results show that the crude water soluble extract of M. charantia can evoke both time-course at (800 µg/ml) and dose-dependent (20 µg/ml - 800 µg/ml) decreases in cell viability (cell death) with maximal increases in cell death with 800 µg/ml over a period of 24 hours following incubation. The results of this study have also shown that the crude water-soluble extract of M. charantia (800 µg/ml) can elicit marked and significant (p < 0.050) increases in the activities of caspase - 3 and caspase - 9 in all the cell lines. The crude water soluble extract of M. charantia can stimulate the release of cytochrome-c and elevated intracellular free calcium concentrations [Ca 2+ ] i in the different cancer cell lines compared to untreated cell lines. These results clearly show that M. charantia is exerting its anti- cancer effect via an insult to the mitochondria resulting in apoptosis, calcium overloading and subsequently, cell death.

Anti-diabetic potential of a Sasa quelpaertensis Nakai extract in L6 skeletal muscle cells

The anti-diabetic potential of a Sasa quelpaertensis Nakai extract (SQE) in L6 muscle cells was evaluated. The SQE stimulated glucose uptake via the AMP-activated protein kinase-activating pathway, but not via the insulinstimulating pathway. The SQE increased the expression of fatty acid translocase and peroxisome proliferator-activated receptor γ, and induced phosphorylation of acetyl-CoA carboxylase. The SQE attenuated oleic acid-induced intracellular triglyceride contents by decreasing the expression of sterol regulatory element-binding protein-1c and fatty acid synthase. These results indicate that the SQE probably has an anti-diabetic potential by enhancing glucose uptake and reducing triglyceride content in L6 skeletal muscle cells.

Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models: a mechanistic insight.

Ceramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies.

Ca²⁺ signals promote GLUT4 exocytosis and reduce its endocytosis in muscle cells.

Elevating cytosolic Ca(2+) stimulates glucose uptake in skeletal muscle, but how Ca(2+) affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca(2+) leading to contraction preclude analysis of the impact of individual, Ca(2+)-derived signals. In L6 muscle cells stably expressing GLUT4myc, the Ca(2+) ionophore ionomycin raised cytosolic Ca(2+) and caused a gain in cell surface GLUT4myc. Extra- and intracellular Ca(2+) chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4myc. Ionomycin stimulated GLUT4myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4myc endocytosis triggered by ionomycin. In summary, rapid Ca(2+) influx into muscle cells accelerates GLUT4myc exocytosis while slowing GLUT4myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca(2+)-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

Insulin sensitivity is inversely related to cellular energy status, as revealed by biotin deprivation.

We have reported an early decrease in glycemia in rats fed a biotin-deficient diet with reduced cellular ATP levels, suggesting increased insulin sensitivity. Here, we show that biotin-deprived rats are more tolerant of glucose, as shown by both oral and intraperitoneal glucose tolerance tests, during which insulin plasma levels were significantly diminished in deficient rats compared with controls. Biotin-deficient rats had lower blood glucose concentrations during intraperitoneal insulin sensitivity tests than controls. Furthermore, more glucose was infused to maintain euglycemia in the biotin-deficient rats during hyperinsulinemic euglycemic clamp studies. These results demonstrate augmented sensitivity to insulin in biotin-deprived rats. They are most likely the consequence of an insulin-independent effect of AMPK activation on GLUT4 membrane translocation with increased glucose uptake. In biotin-deficient cultured L6 muscle cells, there was increased phosphorylation of the energy sensor AMPK. We have now confirmed the augmented AMPK activation in both biotin-deprived in vivo muscle and cultured muscle cells. In these cells, glucose uptake is increased by AMPK activation by AICAR and diminished by its knockdown by the specific siRNAs directed against its α1- and α2-catalytic subunits, with all of these effects being largely independent of the activity of the insulin-signaling pathway that was inhibited with wortmannin. The enhanced insulin sensitivity in biotin deficiency likely has adaptive value for organisms due to the hormone promotion of uptake and utilization of not only glucose but other nutrients such as branched-chain amino acids, whose deficiency has been reported to increase insulin tolerance.

Roles of GILZ in protein metabolism of L6 muscle cells exposed to serum from septic rats.

Sepsis is a complex inflammatory response to infection, associating with dramatic metabolic disorders. Although the mechanisms of immune response during sepsis have been largely clarified, current studies rarely pay attention to the disordered protein metabolism in sepsis. In this study, L6 rat skeletal muscle cells treated with serum from septic rats were used as an in vitro model for sepsis-like condition in skeletal muscle. We found that the expression of glucocorticoid-induced leucine zipper (GILZ) positively correlates with glucocorticoid receptor and negatively correlates with myosin heavy chain expression in L6 muscle cells upon septic serum induction. Moreover, we propose that GILZ may associate with cytokines such as TNF-α, IL-1β as well as IL-10 to cooperatively modulate the glucocorticoid/glucocorticoid receptor-mediated regulation of protein metabolism during sepsis. So the present study provides a new approach and theoretical basis for further studies on the regulation of protein metabolism of skeletal muscle during sepsis.

Involvement of Amp Kinase in Glucose Uptake and Palmitate Oxidation in L6 Muscle Cell Cultures

AICAR (5-aminoimidazole-4-carboxamide 1-s-D-ribonucleoside) is known to activate the AMP-activated protein kinase (AMPK) in many cell types, including skeletal muscle. We showed that AICAR activated glucose uptake, fatty acid oxidation, and lactate formation by L6 skeletal muscle cell cultures. All of the actions were duplicated by adiponectin, another known activator of AMPK, demonstrating selectivity of AICAR. Moreover, AICAR and adiponectin similarly caused phosphorylation of AMPK, and its target proteins acetyl CoA carboxylase and S6Kinase. While creatine treatment of cells is known to greatly enhance creatine and creatine phosphate levels, it was proposed to paradoxically activate AMPK, and yet not affect glucose transport. However, we found creatine treatment to be without effect on glucose transport, fatty acid oxidation, or phosphorylation of AMPK or its target proteins. Unlike AICAR and adiponectin, creatine treatment stimulated glycogen formation from glucose and inhibited lactate formation. Consistent with this, we observed a diminution in phosphofructokinase expression. Thus, we conclude that creatine and creatine phosphate are not significant allosteric modulators of AMPK in intact cells. We investigated the role of AMPK in glycogen metabolism, using an inhibitor of glycogen phosphorylase. While the inhibitor enhanced glucose incorporation into glycogen, AICAR and adiponectin still diminished glycogen formation, supporting a direct action of AMPK on glycogen synthase. To probe the mechanism for adiponectin, we used siRNA constructs against adenyate kinase and LKB1 (the AMPK kinase). We concluded that adiponectin likely acts through an activation of LKB1, rather than indirectly increasing the turnover of ATP, involving adenylate kinase.

Metformin and Resveratrol Induce Beneficial Metabolic Adaptations in L6 Muscle Cells Treated with HIV Protease Inhibitors: Evidence for Inhibition of JNK1/2 Signaling

C-Jun-N-terminal kinase (JNK1/2) pro-inflammatory signaling has been shown to play a role in highly active antiretroviral therapy (HAART)-induced insulin resistance in skeletal muscle cells. To determine the effectiveness of metformin and resveratrol in blunting HAART-induced insulin resistance, L6 skeletal muscle cells were treated ± (with or without) HIV protease inhibitors (ritonavir+atazanavir sulfate; RA) and either metformin, resveratrol, or the JNK1/2 inhibitor SP600125 and incubated ± insulin (100 nM). SP600125 as well as metformin and resveratrol reduced (P<0.05) JNK1/2 phosphorylation in RA-treated cells but not in healthy cells. In basal and insulin-stimulated cells, RA treatment significantly increased JNK1/2 phosophorylation (P<0.05). Additionally, when RA-stimulated cells were treated with metformin, resveratrol or SP6000125, JNK1/2 phosphorylation was reduced (P<0.05) indicating reduced inflammation. Basal and insulin-stimulated glucose uptake was increased in RA-treated cells (P<0.05). The presence of insulin resistance in RA-treated cells was partially abrogated (P>0.05) by metformin and SP600125 treatment. RA-treatment increased FA (fatty acid) uptake and oxidation in both the basal and insulin-stimulated state. Increased percent change of AKTSer473 phosphorylation indicated increased insulin sensitivity in RA+metformin (P=0.08) and RA+resveratrol (P<0.05) treated cells. Treatment with metformin, resveratrol or SP600125 had very little impact on p38 phosphorylation in basal and insulin-stimulated cells made insulin-resistant by incubation with RA. Our data indicate that metformin and resveratrol induce beneficial metabolic changes in cells made insulin resistant by treatment with protease inhibitors in part via inhibition of JNK1/2 pro-inflammatory signaling and stimulation of insulin-mediated AKTSer473 phosphorylation.