Rat L6 Myotubes Research Articles

Oxidised phosphatidylcholine induces sarcolemmal ceramide accumulation and insulin resistance in skeletal muscle.

Intracellular ceramide accumulation in specific cellular compartments is a potential mechanism explaining muscle insulin resistance in the pathogenesis of type 2 diabetes. Muscle sarcolemmal ceramide accumulation negatively impacts insulin sensitivity in humans, but the mechanism explaining this localised accumulation is unknown. Previous reports revealed that circulating oxidised LDL is elevated in serum of individuals with obesity and type 2 diabetes. Oxidised phosphatidylcholine, which is present in oxidised LDL, has previously been linked to ceramide pathway activation, and could contribute to localised ceramide accumulation in skeletal muscle. We hypothesised that oxidised phosphatidylcholine inversely correlates with insulin sensitivity in serum, and induces sarcolemmal ceramide accumulation and decreases insulin sensitivity in muscle. We used LC-MS/MS to quantify specific oxidised phosphatidylcholine species in serum from a cross-sectional study of 58 well-characterised individuals spanning the physiological range of insulin sensitivity. We also performed in vitro experiments in rat L6 myotubes interrogating the role of specific oxidised phosphatidylcholine species in promoting sarcolemmal ceramide accumulation, inflammation and insulin resistance in skeletal muscle cells. Human serum oxidised phosphatidylcholine levels are elevated in individuals with obesity and type 2 diabetes, inversely correlated with insulin sensitivity, and positively correlated with sarcolemmal C18:0 ceramide levels in skeletal muscle. Specific oxidised phosphatidylcholine species, particularly 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), increase total ceramide and dihydroceramide and decrease total sphingomyelin in the sarcolemma of L6 myotubes by de novo ceramide synthesis and sphingomyelinase activation. POVPC also increases inflammatory signalling and causes insulin resistance in L6 myotubes. These data suggest that circulating oxidised phosphatidylcholine species promote ceramide accumulation and decrease insulin sensitivity in muscle, help explain localised sphingolipid accumulation and muscle inflammatory response, and highlight oxidised phosphatidylcholine species as potential targets to combat insulin resistance.

Inhibition of the ubiquitin-proteasome system reduces the abundance of pyruvate dehydrogenase kinase 1 in cultured myotubes

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.

AMPK and glucose deprivation exert an isoform-specific effect on the expression of Na+,K+-ATPase subunits in cultured myotubes

In skeletal muscle, Na+,K+-ATPase (NKA), a heterodimeric (α/β) P-type ATPase, has an essential role in maintenance of Na+ and K+ homeostasis, excitability, and contractility. AMP-activated protein kinase (AMPK), an energy sensor, increases the membrane abundance and activity of NKA in L6 myotubes, but its potential role in regulation of NKA content in skeletal muscle, which determines maximum capacity for Na+ and K+ transport, has not been clearly delineated. We examined whether energy stress and/or AMPK affect expression of NKA subunits in rat L6 and primary human myotubes. Energy stress, induced by glucose deprivation, increased protein content of NKAα1 and NKAα2 in L6 myotubes, while decreasing the content of NKAα1 in human myotubes. Pharmacological AMPK activators (AICAR, A-769662, and diflunisal) modulated expression of NKA subunits, but their effects only partially mimicked those that occurred in response to glucose deprivation, indicating that AMPK does not mediate all effects of energy stress on NKA expression. Gene silencing of AMPKα1/α2 increased protein levels of NKAα1 in L6 myotubes and NKAα1 mRNA levels in human myotubes, while decreasing NKAα2 protein levels in L6 myotubes. Collectively, our results suggest a role for energy stress and AMPK in modulation of NKA expression in skeletal muscle. However, their modulatory effects were not conserved between L6 myotubes and primary human myotubes, which suggests that coupling between energy stress, AMPK, and regulation of NKA expression in vitro depends on skeletal muscle cell model.

LCMS Determination and Cytotoxicity of Abrus precatorius on L6 and SK-N-MC Cell Lines.

The present study aimed to investigate the cytotoxic effect of various extracts derived from Abrus precatorius Linn. leaves on rat L6 and human SK-N-MC neuroblastoma cell lines and determine the secondary metabolites responsible for the cytotoxicity of Abrus precatorius. Successive solvent extraction of A.precatorius leaves was carried out using the Soxhlet apparatus with solvents such as petroleum ether, chloroform, ethyl acetate, and ethanol. HPTLC fingerprinting and LC-MS studies were performed to assess the presence of secondary metabolites, such as flavonoids and phenols, in the ethyl acetate extract. Furthermore, the cytotoxic effect of extracts was tested on rat skeletal muscle cell line L6 and human neuroblastoma cell line SK-N-MC using MTT assay. The total phenolic content of ethyl acetate and ethanol extracts of A.precatorius were 72.67 and 60.73 mg, respectively, of GAE/g dry weight of the extract. The total flavonoid content of ethyl acetate and ethanol extract of A.precatorius were 107.33 and 40.66 mg of Quercetin equivalents/g dry weight of the extract. LCMS analysis demonstrated that the flavonoids in specific Naringenin, Diosmetin, Glycitin, and Genistein might play a prominent role in the cytotoxicity of A.precatorius. The cytotoxicity study revealed that the extracts of A.precatorius were non-toxic to rat L6 myotubes, and the IC50 values of the various extracts, such as APPE, APCH, APEA, and APET, were >100 µg/ml. The extracts exhibited cytotoxic activity against human neuroblastoma SK-N-MC cells, and the IC50 values of APPE, APCH, APEA, APET, and the standard drug "Cisplatin" were >100, >100, 64.88, >100, and 3.72 µg/ml, respectively. It was concluded from the study that the extracts of Abrus precatorius were cytotoxic to neuroblastoma cell lines but non-toxic to normal cell lines. HPTLC and LC-MS studies confirmed that flavonoids in the ethyl acetate extract could be responsible for the biological activity.

Moderately lipophilic 2-(Het)aryl-6-dithioacetals, 2-phenyl-1,4-benzodioxane-6-dithioacetals and 2-phenylbenzofuran-5-dithioacetals: Synthesis and primary evaluation as potential antidiabetic AMPK-activators

Since the 1950′s, AMP-kinase (AMPK) has been used as a promising target for the development of antidiabetic drugs against Type 2 diabetes mellitus (T2D). Indeed, the canonical antidiabetic drug metformin recruits, at least partially, AMPK activation for its therapeutic effect. Herein we present design and synthesis of 20 novel relatively polar cyclic and acyclic dithioacetals of 2-(Het)arylchroman-6-carbaldehydes, 2-phenyl-1,4-benzodioxane-6-carbaldehyde, and 2-phenylbenzofuran-5-carbaldehyde, which were developed as potential AMPK activators. Three of the synthesized dithioacetals demonstrated significant enhancement (≥70%) of glucose uptake in rat L6 myotubes. Noteworthy, one of the dithioacetals, namely 4-(6-(1,3-dithian-2-yl)chroman-2-yl)pyridine, exhibited high potency comparing to other molecules. It increased the rate of glucose uptake in rat L6 myotubes and augmented insulin secretion from rat INS-1E cells in pharmacological relevant concentrations (up to 2 μM). Both effects were mediated by activation of AMPK. In addition, the compound showed excellent pharmacokinetic profile in healthy mice, including maximal oral bioavailability. Such bifunctionality (increased glucose uptake and insulin secretion) can be used as a starting point for the development of a novel class of antidiabetic drugs with dual activity that is relevant for T2D treatment.

Thiourea derivatives inhibit key diabetes-associated enzymes and advanced glycation end-product formation as a treatment for diabetes mellitus.

This study was designed to screen novel thiourea derivatives against different enzymes, such as α-amylase, α-glucosidase, protein tyrosine phosphatase 1 B, and advanced glycated end product (AGEs). A cytotoxicity analysis was performed using rat L6 myotubes and molecular docking analysis was performed to map the binding interactions between the active compounds and α-amylase and α-glucosidase. The data revealed the potency of five compounds, including E (1-(2,4-difluorophenyl)-3-(3,4-dimethyl phenyl) thiourea), AG (1-(2-methoxy-5-(trifluoromethyl) phenyl)-3-(3-methoxy phenyl) thiourea), AF (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), AD (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), and AH (1-(2,4-difluorophenyl)-3-(2-iodophenyl) thiourea), showed activity against α-amylase. The corresponding percentage inhibitions were found to be 85 ± 1.9, 82 ± 0.7, 75 ± 1.2, 72 ± 0.4, and 65 ± 1.1%, respectively. These compounds were then screened using in vitro assays. Among them, AH showed the highest activity against α-glucosidase, AGEs, and PTP1B, with percentage inhibitions of 86 ± 0.4% (IC50 =47.9μM), 85 ± 0.7% (IC50 =49.51 μM), and 85 ± 0.5% (IC50 =79.74 μM), respectively. Compound AH showed an increased glucose uptake at a concentration of 100 μM. Finally, an in vivo study was conducted using a streptozotocin-induced diabetic mouse model and PTP1B expression was assessed using real-time PCR. Additionally, we examined the hypoglycemic effect of compound AH in diabetic rats compared to the standard drug glibenclamide.

Additional effects of simultaneous treatment with C14-Cblin and celastrol on the clinorotation-induced rat L6 myotube atrophy.

Two novel reagents, N-myristoylated Cbl-b inhibitory peptide (C14-Cblin) and celastrol, a quinone methide triterpene, are reported to be effective in preventing myotube atrophy. The combined effects of C14-Cblin and celastrol on rat L6 myotubes atrophy induced by 3D-clinorotation, a simulated microgravity model, was investigated in the present study. We first examined their effects on expression in atrogenes. Increase in MAFbx1/atrogin-1 and MuRF-1 by 3D-clinorotation was significantly suppressed by treatment with C14-Cblin or celastrol, but there was no additive effect of simultaneous treatment. However, celastrol significantly suppressed the upregulation of Cbl-b and HSP70 by 3D-clinorotation. Whereas 3D-clinorotation decreased the protein level of IRS-1 in L6 myotubes, C14-Cblin and celastrol inhibited the degradation of IRS-1. C14-Cblin and celastrol promoted the phosphorylation of FOXO3a even in microgravity condition. Simultaneous administration of C14-Cblin and celastrol had shown little additive effect in reversing the impairment of IGF-1 signaling by 3D-clinorotation. While 3D-clinorotation-induced marked oxidative stress in L6 myotubes, celastrol suppressed 3D-clinorotation-induced ROS production. Finally, the C14-Cblin and celastrol-treated groups were inhibited decrease in L6 myotube diameter and increased the protein content of slow-twitch MyHC cultured under 3D-clinorotation. The simultaneous treatment of C14-Cblin and celastrol additively prevented 3D-clinorotation-induced myotube atrophy than single treatment. J. Med. Invest. 69 : 127-134, February, 2022.

Laminarin From Salicornia herbacea Stimulates Glucose Uptake Through AMPK-p38 MAPK Pathways in L6 Muscle Cells

Laminarin is a component of brown seaweed, especially isolated from Salicornia herbacea. Laminarin was known to have various physiological functions, however, the molecular mechanism is still unclear. In this study, we report that laminarin stimulates an activation of AMP-activated protein kinase (AMPK) and increases glucose uptake in rat L6 myotubes. Laminarin also increases an intracellular calcium release. Inhibition of Ca2+ release, using with CaMKK inhibitor, STO-609, blocked laminarin-induced AMPK activity, indicating that laminarin stimulated AMPK activity via calcium. In addition, laminarin activates p38 mitogen-activated protein kinase (MAPK) signaling pathways depending on AMPK activity. Moreover, the inhibition of either AMPK or p38 MAPK blocked laminarin-induced glucose uptake in rat L6 myotubes. Taken together, these results demonstrate that the hypoglycemic effect of laminarin is caused by its ability to activate AMPK-p38 MAPK pathways in skeletal muscles.

Isolation and Total Synthesis of Mabuniamide, a Lipopeptide from an Okeania sp. Marine Cyanobacterium.

The bioassay-guided fractionation of an Okeania sp. marine cyanobacterium collected in Okinawa led to the isolation of the lipopeptide mabuniamide (1). The gross structure of 1 was determined by spectroscopic analyses, and its absolute configuration was determined using Marfey's analysis of the acid hydrolysate of 1. The absolute configuration of 1 was confirmed by total synthesis. Mabuniamide (1) stimulated glucose uptake in cultured rat L6 myotubes. In addition, mabuniamide (1) and its stereoisomer (2) exhibited moderate antimalarial activity.

1779-P: Characterization of Insulin-Regulated CLASP2 Phosphorylation

During an unbiased proteomics screen of rat L6 myotubes, we previously discovered the first member of the microtubule plus-end interacting protein (+TIP) family that undergoes insulin-stimulated phosphorylation, CLIP-associating protein 2 (CLASP2). Here we report that CLASP2 protein is expressed in several muscle groups as well as epididymal fat in mouse and we have successfully confirmed the expression of CLASP2 protein in human vastus lateralis muscle by mass spectrometry. In addition, insulin resistant, 60% fat diet-fed mice exhibit a loss of CLASP2 expression in epididymal adipose tissue versus control animals. We performed an insulin time course and discovered that insulin stimulates the phosphorylation of both endogenous and overexpressed CLASP2 in 3T3-L1 adipocytes in a manner to that similarly observed in L6 myotubes. Inhibition of either PI 3-kinase with wortmannin or mTOR with rapamycin had no effect on insulin-stimulated, proline-directed serine phosphorylation of CLASP2, whereas inhibition of the MAPK pathway reduced proline-directed serine phosphorylation of endogenous CLASP2 to basal levels. Quantitative phosphoproteomics identified Ser884/Ser1004/Ser1005/Ser1021 as significant insulin-stimulated CLASP2 phosphorylation sites that can be decreased after inhibition of the MAPK pathway. These findings indicate that CLASP2 undergoes insulin-stimulated proline-directed serine phosphorylation that is under control of the MAPK pathway in 3T3-L1 adipocytes. In addition, we also present new evidence of CLASP2 protein expression in mammalian tissues that are specifically targeted for insulin-stimulated glucose uptake and suggest that CLASP2 is altered within in vivo models of insulin resistance and type 2 diabetes. Disclosure N.K. Barker: None. J.L. Krantz: None. P.R. Langlais: None. Funding National Institutes of Health

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

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

Gingerenone A Sensitizes the Insulin Receptor and Increases Glucose Uptake by Inhibiting the Activity of p70 S6 Kinase.

The bioactive constituents in ginger extract are responsible for anti-hyperglycemic effects and the underlying mechanisms are incompletely understood. Gingerenone A (Gin A) has been identified as an inhibitor of p70 S6 (S6K1), a kinase that plays a critical role in the pathogenesis of insulin resistance. This study aims to evaluate if Gin A can sensitize the insulin receptor by inhibiting S6K1 activity. Western blot analysis reveals that Gin A induces phosphatidylinositide-3 kinase (PI3K) feedback activation in murine 3T3-L1 adipocytes and rat L6 myotubes, as evidenced by increased AKTS473 and S6K1T389 but decreases S6S235/236 and insulin receptor substrate 1 (IRS-1)S1101 phosphorylation. Western blot and immunoprecipitation analysis reveal that Gin A increases insulin receptor tyrosine phosphorylation in L6 myotubes and IRS-1 binding to the PI3K in 3T3-L1 adipocytes. Confocal microscopy reveals that Gin A enhances insulin-induced translocation of glucose transporter 4 (GLUT4) into the cell membrane in L6 cells. 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) Fluorescent assay reveals that Gin A enhances insulin-stimulated glucose uptake in 3T3-L1 adipocytes and L6 myotubes. Gin A overcomes insulin resistance and increases glucose uptake by inhibiting S6K1 activity. Gin A or other plant-derived S6K1 inhibitors could be developed as novel antidiabetic agents.

Alpha-linolenic acid and linoleic acid differentially regulate the skeletal muscle secretome of obese Zucker rats

Evidence shows that proteins secreted from skeletal muscle influence a broad range of metabolic signaling pathways. We previously reported that essential polyunsaturated fatty acids (PUFA) improved whole-body glucose homeostasis in obese Zucker rats; however, the mechanisms underlying these benefits remain enigmatic. While PUFA and obesity influence skeletal muscle function, their effects on the secretome are unknown. The aim of this work was to determine if improvements in whole-body glucose homeostasis in obese Zucker rats fed diets supplemented with either linoleic acid (LA) or alpha-linolenic acid (ALA) for 12 wk are related to changes in the skeletal muscle secretome. Secreted proteins were identified with a predictive bioinformatic analysis of microarray gene expression from red tibialis anterior skeletal muscle. Approximately 130 genes were differentially expressed (false discovery rate = 0.05) in obese rats compared with lean controls. The expression of 15 genes encoding secreted proteins was differentially regulated in obese controls, obese LA-supplemented, and obese ALA-supplemented rats compared with lean controls. Five secreted proteins ( Col3a1, Col15a1, Pdgfd, Lyz2, and Angptl4) were differentially regulated by LA and ALA. Most notably, ALA supplementation reduced Angptl4 gene expression compared with obese control and obese-LA supplemented rats and reduced circulating ANGPTL4 serum concentrations. ALA also influenced Angptl4 gene expression and ANGPTL4 secretion from differentiated rat L6 myotubes. Altogether, the present data indicate that obesity has a greater global impact on skeletal muscle gene expression than either essential PUFA; however, LA and ALA may exert their metabolic benefits in part by regulating the skeletal muscle secretome.

Ypaoamides B and C, Linear Lipopeptides from an Okeania sp. Marine Cyanobacterium

Two new pyrrolinone-containing lipopeptides, ypaoamides B (1) and C (2), were isolated from an Okeania sp. marine cyanobacterium collected in Okinawa. Their structures were determined by spectroscopic analysis and Marfey's analysis of acid hydrolysates. Ypaoamides B (1) and C (2) stimulated glucose uptake in cultured rat L6 myotubes. In particular, ypaoamide B (1) showed potent activity and activated AMP-activated protein kinase.

Long non‐coding RNAs are Transcriptional Regulators of Contractile Protein‐coding Genes in Skeletal Muscle

Contractile properties of skeletal muscle determine strength, power and endurance. Muscle disuse and disease causes muscle atrophy and a shift in fiber‐type that diminish muscle strength and endurance. Better understanding of the transcriptional regulation of contractile protein genes by long non‐coding (lnc) RNAs may lead to development of promising new therapeutic treatment options. We previously identified and characterized four novel lncRNAs associated with key contractile protein‐coding genes by examining the transcriptome response to seven days of hindlimb unloading suspension (HS) in rat soleus (SOL) muscle using RNA‐Seq followed by validation with RT‐PCR. We showed that HS induced alterations in sense (S) and antisense (AS) lncRNAs associated with Myh7, Myl2, Tnni1, and Tnnc1. We hypothesized that these muscle disuse‐responsive lncRNAs regulate transcription of contractile genes by cis‐acting mechanisms. We sought to test this hypothesis using rat L6 myotubes to determine the cellular localization and function of the lncRNAs. We used strand‐specific Antisense Oligonucleotides (ASOs) in the form of Locked Nucleic Acid (LNA) GapmeRs to induce RNase‐H mediated degradation of lncRNAs. Real‐time PCR was used to assess RNA levels.Examination of nuclear and cytoplasmic RNA fractions revealed that each of the lncRNAs was <5‐fold higher in the nuclear fraction, indicating transcription‐mediated roles. We previously showed that an AS lncRNA at an intergenic location between Myh7 and Ngdn was upregulated during HS while Myh7 expression was decreased. Knockdown of this lncRNA resulted in a significant increase in Ngdn expression, suggesting that this lncRNA normally inhibits Ngdn. Thus, this lncRNA may serve to coordinate expression of Myh7 and Ngdn, which itself is implicated in translational regulation. Knockdown of a S lncRNA near TnnC1 resulted in elevated transcription of overlapping AS RNA and TnnC1 pre‐mRNA (NS). Further study is needed to determine how the lncRNA may regulate TnnC1. Gapmer‐mediated knockdown of AS lncRNA in the upstream‐promoter region of Tnni1 resulted in a 7‐fold increase in Tnni1 pre‐mRNA, suggesting that this AS lncRNA mediates transcriptional repression of Tnni1. Moreover, this AS lncRNA may hold potential as a potent and accessible target of therapeutic intervention in cases of disease or disuse where manipulation of slow‐myofiber specific Troponin I expression may be utilized to enhance contractile properties. In summary, we characterized novel lncRNAs thought to transcriptionally regulate expression of contractile genes in response to disuse. We conclude that transcriptional regulation of the examined contractile protein coding genes is influenced by associated lncRNAs.Support or Funding InformationMercer University School of MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphate stimulates myotube atrophy through autophagy activation: evidence of hyperphosphatemia contributing to skeletal muscle wasting in chronic kidney disease

BackgroundAccelerated muscle atrophy is associated with a three-fold increase in mortality in chronic kidney disease (CKD) patients. It is suggested that hyperphosphatemia might contribute to muscle wasting, but the underlying mechanisms remain unclear. Although evidence indicates that autophagy is involved in the maintenance of muscle homeostasis, it is not known if high phosphate levels can result in activation of autophagy, leading to muscle protein loss.MethodsImmortalized rat L6 myotubes were exposed to a high concentration of phosphate, with or without autophagy inhibition. Myotube atrophy was examined by phase contrast microscopy. Autophagic activity was assessed by measuring the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 using quantitative real-time polymerase chain reaction and western blot.ResultsPhosphate induced cell atrophy in L6 myotubes in a dose- and time-dependent manner, and these responses were not associated with calcification or osteogenesis. Phosphate also dose- and time-dependently increased the LC3-II/LC3-I ratio. Inhibition of autophagy with wortmannin or knockdown of Atg5 significantly suppressed myotube atrophy caused by high phosphate concentration.ConclusionsHigh phosphate concentration induces muscle cell atrophy through the activation of autophagy. Targeting autophagy could be a therapeutic strategy for preventing muscle wasting caused by hyperphosphatemia.

Antidiabetic and nephroprotective potential of polyherbal self-fermented Ariṣṭa formulation: Evidence-based research

Background: Ayurvedic Ariṣṭa preparations hold an age-old heritage in the prevention and cure of metabolic disorders and associated complications. Aim: The aim of the study was to study the antidiabetic activity of a self-fermented Ariṣṭa preparation (DB-07) and its potential in preventing or curing diabetes-induced nephropathy in experimental models. Materials and Methods: Rats with Streptozotocin-induced diabetes were used for studying the various dose effects of DB-07 (2.5, 3, and 3.5 ml/kg/day) in preventive and therapeutic treatment schedules. Metformin (500 mg/kg) was used as a standard drug. Fasting blood glucose and biochemical parameters such as blood protein, urea, uric acid, and creatinine were accessed by various biochemical tests. In vitro α-amylase and α-glucosidase inhibition assay and 2-deoxy glucose uptake assay using rat L6 myotubes were studied for determining the antidiabetic mechanism of action. Results: A dose-dependent and statistically significant effect on lowering the fasting blood glucose level was observed in both preventive and therapeutic scheduled treatments. Treatment with DB-07 significantly reversed the altered biochemical parameters and the effects were comparable to metformin-treated rats. DB-07 exhibited concentration-dependent inhibition of both α-amylase and α-glucosidase enzymes with an IC50 values of 10.93 μg/ml and 8.73 μg/ml, respectively. DB-07 also showed glucose uptake potential (ca. 70% activity at 125 μg/ml) in differentiated L6 myotubes. Conclusions: These observations have revealed the antidiabetic and nephroprotective potential of DB-07. DB-07 can be a potential herbal alternative to maintain a healthy blood glucose level and will prove useful in diabetes-associated renal complications.

Differential expression of microRNA let-7b-5p regulates burn-induced hyperglycemia.

One of the classical features observed in patients with burn injury is hyperglycemia. There have been previous reports that a cohort of microRNAs (miRNAs) is differentially expressed in the dermis of patients with burn injury. More specifically, it has been shown that the miR-194 can target the insulin-like growth factor receptor 1 (IGF1R) and silence its protein expression resulting in hyperglycemia. The objective of the current study was to discover if additional miRNA-mediated post-transcriptional mechanism exists that lead to suppression of IGF1R protein expression post-burn injury. Using the 30% total body surface area (TBSA) model of burn injury in rats we found that the miRNA let-7b can target IGF1R and downregulate its protein expression, in turn attenuating PI3K/Akt and Gsk3β activation leading to hyperglycemia. Increased let-7b expression was significantly more than the previously reported miR-194 both in the burn rats compared to sham and in patients with burn injury compared to healthy subjects. Serum from burn rats also resulted in decreased IGF1R protein expression in rat L6 myotubes. In vivo targeting of let-7b by antagomir mitigated the effect of increased let-7b expression on IGF1R protein expression and hyperglycemia. Thus targeting let-7b might be a promising approach to treat hyperglycemia in patients with burn injury.

Effect of Sucralose on Glucose Uptake in Rat L6 Myotubes

ABSTRACT Introduction With growing awareness of the link between diet and health and the problem of obesity, public concern over sugar levels in the diet is forcing a worldwide trend toward cutting down on sugar by using artificial sweeteners (AS). Aim To study the effect of increasing concentrations of sucralose (an AS) on glucose uptake in rat L6 myotubes. Materials and methods The L6 cell line from American type cell culture (ATCC) was grown in Dulbecco's Modified Eagle's Medium (DMEM) and differentiated into myotubes. The wells were exposed to either 0, 1 nM, 1 μM, or 1 mM of sucralose alone or with 10 nM insulin for 24 hours. Glucose uptake was studied after this period. Results Significant decrease was seen between the insulin-stimulated basal glucose uptake and insulin-stimulated glucose uptake across all the concentrations of sucralose treatment. Conclusion Increased concentration of sucralose appears to decrease glucose uptake even on insulin stimulation. Clinical significance It may not be beneficial to use sucralose in certain groups of people who have insulin resistance or are prone to it. How to cite this article Prakash SN, Shanthakumari J, Devanath A. Effect of Sucralose on Glucose Uptake in Rat L6 Myotubes. Indian J Med Biochem 2017;21(2):162-165.

Triterpenoids Isolated from Ziziphus jujuba Enhance Glucose Uptake Activity in Skeletal Muscle Cells.

Jujube (Ziziphus jujuba Mill.), a traditional folk medicine and functional food in China and South Korea, is known for its beneficial properties, which include anti-cancer, anti-oxidative, and anti-obesity effects. To assess the anti-hyperglycemic effect of jujube in this study, we investigated the glucose uptake-promoting activity of jujube in rat L6 myotubes. After determining that the jujube extract induces muscle glucose uptake, we identified the following active compounds by bioassay-guided fractionation: betulonic acid, betulinic acid, and oleanonic acid. Ursonic acid, known to be present in jujube, was semi-synthesized from ursolic acid and also observed to enhance glucose uptake. These four triterpenic acids induced glucose uptake in a glucose transporter 4-dependent manner. Comparison experiments of jujube fruits from three countries, namely, China, South Korea, and Japan, revealed that Japanese jujube has a higher content of active triterpenoids and is the most potent enhancer of glucose uptake.