scholarly journals The Effects of Insulin and Retinoic Acid on the Content of Glucose Transporter 4 in Different Preparation Lysates of L6 Muscle Cells (P15-013-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Tiannan Wang ◽  
Guoxun Chen
Keyword(s):  
Retinoic Acid ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Total Cell ◽  
Control Group ◽  
Protein Levels ◽  
L6 Cells ◽  
Glut4 Expression ◽  
Lysis Buffer ◽  
L6 Muscle Cells

Abstract Objectives Glucose enters the skeletal muscle cells through glucose transporters (GLUTs), a process that is stimulated by insulin through the movement of GLUT4 to the cell membrane. Here, we studied the effects of retinoic acid (RA) and insulin on GLUT4 expression in L6 muscle cells. Methods Rat L6 muscle cells were induced to differentiation after confluency. Cells were incubated in medium containing 2% HS containing in the absence or presence of 1 μM RA without or with 10 nM insulin for 4 or 6 days with replacement of fresh media every two days. Cells were lyzed and prepared in the following ways. 1) Cells were lyzed in lysis buffer (50 mM HEPES pH 7.5, 10 mM EDTA, 10% Glycerol, 2% NP-40 and 2% Triton X-100) on ice for 20 minutes, vortex cells every 10 minutes to help lyse completely. After that, the lysate was centrifuged at 12,000 x g for 15 minutes at 4°C for the collection of both the supernatant and pellet. 2) Cells were directly lyzed in 1 x SDS loading buffer (designated as total cell lysate here). The GLUT4 protein levels in those samples were determined via Western Blot using anti-GLUT4 antibody (#07-1404, C-terminus) from EMD Millipore Corp. Results On day 4 or 6, the supernatants of cells treated with RA + insulin had a significantly less GLUT4 expression that that of the control group. Additional, on day 4, the supernatant of cells treated with insulin alone also had less GLUT4 protein than that of the control group. The pellets of cells on day 4 treated with RA had a significantly less GLUT4 protein level than that of the control group. On day 4 or 6, the GLUT4 protein levels in total cell lysates were not significantly different among the four treatment groups. Conclusions The current lysis buffer allowed us to observe that the treatments of RA and/or insulin can significantly affect GLUT4 protein levels in the supernatants. Given the fact that a significant amount of GLUT4 protein remained in the pellets, the lysis buffer used here could not completely solubilize differentiated L6 cells, suggesting that cautions lysis buffers and methods were chosen carefully to prepare L6 cells for the analysis of GLUT4 protein. Additionally, it also indicates that the differentially solubilized fractions probably provide us tools to study the effects of RA and insulin on the subcellular movement of GLUT4 in L6 muscle cells. Funding Sources The University of Tennessee, Knoxville.

Retinoic acid stimulates glucose transporter expression in L6 muscle cells

1995 ◽  
Vol 108 (1-2) ◽  
pp. 161-167 ◽  
Author(s):  
Mark W. Sleeman ◽  
Hong Zhou ◽  
Suzanne Rogers ◽  
Kong Wah Ng ◽  
James D. Best
Keyword(s):  
Retinoic Acid ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
L6 Muscle Cells ◽  
Transporter Expression

Long-term treatment with insulin and retinoic acid increased glucose utilization in L6 muscle cells via glycogenesis

2020 ◽  
Vol 98 (6) ◽  
pp. 683-697
Author(s):  
Matthew Goff ◽  
Guoxun Chen
Keyword(s):  
Retinoic Acid ◽  
Glucose Utilization ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Muscle Cells ◽  
Mrna Levels ◽  
Glucose Levels ◽  
L6 Cells ◽  
Long Term Treatment ◽  
L6 Muscle Cells

The skeletal muscle regulates glucose homeostasis. Here, the effects of vitamin A metabolites including retinoic acid (RA) alone, and in combination with insulin, on glucose utilization were investigated in rat L6 muscle cells during the differentiation process. L6 cells were treated with differentiation medium containing retinol, retinal, RA, and (or) insulin. The glucose levels and pH values in the medium were measured every 2 days. The expression levels of insulin signaling and glycogen synthesis proteins, as well as glycogen content were determined. Retinal and RA reduced the glucose content and pH levels in the medium of the L6 cells. RA acted synergistically with insulin to reduce glucose and pH levels in the medium. The RA- and insulin-mediated reduction of glucose in the medium only occurred when glucose levels were at or above 15 mmol/L. Insulin-induced phosphorylation of Akt Thr308 was further enhanced by RA treatment through the activation of retinoic acid receptor. RA acted synergistically with insulin to phosphorylate glycogen synthase kinase 3β, and dephosphorylate glycogen synthase (GS), which was associated with increases in the protein and mRNA levels of GS. Increases in glycogen content were induced by insulin, and was further enhanced in the presence of RA. We conclude that activation of the RA signaling pathway enhanced insulin-induced glucose utilization in differentiating L6 cells through increases in glycogenesis.

scholarly journals Gundelia tournefortii: Fractionation, Chemical Composition and GLUT4 Translocation Enhancement in Muscle Cell Line

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3785
Author(s):  
Sleman Kadan ◽  
Sarit Melamed ◽  
Shoshana Benvalid ◽  
Zipora Tietel ◽  
Yoel Sasson ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Rapid Development ◽  
Muscle Cells ◽  
Flash Chromatography ◽  
Glucose Disposal ◽  
Gas Chromatography Mass Spectrometry ◽  
Glut4 Translocation ◽  
Toxic Concentrations ◽  
L6 Muscle Cells

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 μg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography–mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.

scholarly journals Artemisia princeps Extract Promoted Glucose Uptake in Cultured L6 Muscle Cells via Glucose Transporter 4 Translocation

2010 ◽  
Vol 74 (10) ◽  
pp. 2036-2042 ◽  
Author(s):  
Norio YAMAMOTO ◽  
Manabu UEDA ◽  
Kyuichi KAWABATA ◽  
Takuya SATO ◽  
Kengo KAWASAKI ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Glucose Transporter 4 ◽  
Artemisia Princeps ◽  
L6 Muscle Cells

scholarly journals Mammalian Tribbles homolog 3 impairs insulin action in skeletal muscle: role in glucose-induced insulin resistance

2010 ◽  
Vol 298 (3) ◽  
pp. E565-E576 ◽  
Author(s):  
Jiarong Liu ◽  
Xuxia Wu ◽  
John L. Franklin ◽  
Joseph L. Messina ◽  
Helliner S. Hill ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Transporter ◽  
Vastus Lateralis ◽  
Glucose Deprivation ◽  
Muscle Cells ◽  
Diabetic Rats ◽  
Glucose Disposal ◽  
Insulin Resistant ◽  
Protein Levels

Tribbles homolog 3 (TRIB3) was found to inhibit insulin-stimulated Akt phosphorylation and modulate gluconeogenesis in rodent liver. Currently, we examined a role for TRIB3 in skeletal muscle insulin resistance. Ten insulin-sensitive, ten insulin-resistant, and ten untreated type 2 diabetic (T2DM) patients were metabolically characterized by hyperinsulinemic euglycemic glucose clamps, and biopsies of vastus lateralis were obtained. Skeletal muscle samples were also collected from rodent models including streptozotocin (STZ)-induced diabetic rats, db/db mice, and Zucker fatty rats. Finally, L6 muscle cells were used to examine regulation of TRIB3 by glucose, and stable cell lines hyperexpressing TRIB3 were generated to identify mechanisms underlying TRIB3-induced insulin resistance. We found that 1) skeletal muscle TRIB3 protein levels are significantly elevated in T2DM patients; 2) muscle TRIB3 protein content is inversely correlated with glucose disposal rates and positively correlated with fasting glucose; 3) skeletal muscle TRIB3 protein levels are increased in STZ-diabetic rats, db/db mice, and Zucker fatty rats; 4) stable TRIB3 hyperexpression in muscle cells blocks insulin-stimulated glucose transport and glucose transporter 4 (GLUT4) translocation and impairs phosphorylation of Akt, ERK, and insulin receptor substrate-1 in insulin signal transduction; and 5) TRIB3 mRNA and protein levels are increased by high glucose concentrations, as well as by glucose deprivation in muscle cells. These data identify TRIB3 induction as a novel molecular mechanism in human insulin resistance and diabetes. TRIB3 acts as a nutrient sensor and could mediate the component of insulin resistance attributable to hyperglycemia (i.e., glucose toxicity) in diabetes.

scholarly journals Graphene oxide nanofilm and the addition of L-glutamine can promote development of embryonic muscle cells

2020 ◽  
Author(s):  
Marlena Zielińska-Górska ◽  
Anna Hotowy ◽  
Mateusz Wierzbicki ◽  
Jaśmina Bałaban ◽  
Malwina Sosnowska ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Surface Properties ◽  
Satellite Cells ◽  
Chicken Embryo ◽  
Culture Medium ◽  
Muscle Cells ◽  
Control Group ◽  
Protein Levels ◽  
Myogenic Potential

Abstract Background: Formation of muscular pseudo-tissue depends on muscle precursor cells, the extracellular matrix (ECM)-mimicking structure and factors stimulating cell differentiation. These three things cooperate and can create a tissue-like structure, however, their interrelationships are relatively unknown. The objective was to study the interaction between surface properties, culture medium composition and heterogeneous cell culture. We would like to demonstrate that changing the surface properties by coating with graphene oxide nanofilm (nGO) can affect cell behaviour and especially their need for the key amino acid L-glutamine (L-Glu).Results: Chicken embryo muscle cells and their precursors, cultured in vitro, were used as the experimental model. The mesenchymal stem cell, collected from the hind limb of the chicken embryo at day 8 were divided into 4 groups; the control group and groups treated with nGO, L-Glu and nGO supplied with L-Glu (nGOxL-Glu). The roughness of the surface of the plastic plate covered with nGO was much lower than a standard plate. The test of nGO biocompatibility demonstrated that the cells were willing to settle on the nGO without any toxic effects. Moreover, nGO by increasing hydrophilicity and reducing roughness and presumably through chemical bonds available on the GO surface stimulated the colonisation of primary stromal cells that promote embryonic satellite cells. The viability significantly increased in cells cultured on nGOxL-Glu. Observations of cell morphology showed that the most mature state of myogenesis was characteristic for the group nGOxL-Glu. This result was confirmed by increasing the expression of MYF5 genes at mRNA and protein levels. nGO also increased the expression of MYF5 and also very strongly the expression of PAX7 at mRNA and protein levels. However, when analysing the expression of PAX7, a positive link was observed between the nGO surface and the addition of L-Glu.Conclusions: The use of nGO and L-Glu supplement may improve myogenesis and also the myogenic potential of myocytes and their precursors by promoting the formation of satellite cells. Studies have, for the first time, demonstrated positive cooperation between surface properties nGO and L-Glu supplementation to the culture medium regarding the myogenic potential of cells involved in muscle formation.

Expression of lipoprotein lipase during differentiation of cultured L6 muscle cells

1994 ◽  
Vol 72 (3) ◽  
pp. 243-247 ◽  
Author(s):  
Mary Jo LaDu ◽  
Warren K. Palmer
Keyword(s):  
Lipoprotein Lipase ◽  
Lipase Activity ◽  
Time Course ◽  
Muscle Cells ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
L6 Cells ◽  
Highly Correlated ◽  
L6 Muscle Cells

The presence of lipoprotein lipase (LPL) in L6 muscle cells is equivocal. Analysis of a 21-day time course indicates that these cells express both LPL activity and mRNA. Lipase activity peaked at 4 days after plating and decreased to a nadir at day 21 after plating. Characterization of lipase activity at 4 and 19 days after plating, corresponding to myoblasts and myotubes, respectively, indicated that most of the enzyme activity had the properties of LPL, including an alkaline pH optimum, a serum requirement, and inhibition by NaCl. LPL mRNA expression peaked at 7 days after plating and fell slightly (24%) at day 21. The primary LPL mRNA species in these cells is 3.7 kb in length. Lipase activity and LPL mRNA were highly correlated during the time course (r = +0.82), suggesting transcriptional regulation of the enzyme. These data clearly demonstrate that L6 cells express LPL during differentiation.Key words: myoblasts, myotubes, mRNA, total protein, total RNA.

scholarly journals Unique mechanism of GLUT3 glucose transporter regulation by prolonged energy demand: increased protein half-life

1998 ◽  
Vol 333 (3) ◽  
pp. 713-718 ◽  
Author(s):  
Zayna A. KHAYAT ◽  
Anthony L. McCALL ◽  
Amira KLIP
Keyword(s):  
Glucose Uptake ◽  
Protein Content ◽  
Glucose Transport ◽  
Half Life ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Mrna Levels ◽  
Glut1 Protein ◽  
L6 Muscle Cells

L6 muscle cells survive long-term (18 h) disruption of oxidative phosphorylation by the mitochondrial uncoupler 2,4-dinitrophenol (DNP) because, in response to this metabolic stress, they increase their rate of glucose transport. This response is associated with an elevation of the protein content of glucose transporter isoforms GLUT3 and GLUT1, but not GLUT4. Previously we have reported that the rise in GLUT1 expression is likely to be a result of de novo biosynthesis of the transporter, since the uncoupler increases GLUT1 mRNA levels. Unlike GLUT1, very little is known about how interfering with mitochondrial ATP production regulates GLUT3 protein expression. Here we examine the mechanisms employed by DNP to increase GLUT3 protein content and glucose uptake in L6 muscle cells. We report that, in contrast with GLUT1, continuous exposure to DNP had no effect on GLUT3 mRNA levels. DNP-stimulated glucose transport was unaffected by the protein-synthesis inhibitor cycloheximide. The increase in GLUT3 protein mediated by DNP was also insensitive to cycloheximide, paralleling the response of glucose uptake, whereas the rise in GLUT1 protein levels was blocked by the inhibitor. The GLUT3 glucose transporter may therefore provide the majority of the glucose transport stimulation by DNP, despite elevated levels of GLUT1 protein. The half-lives of GLUT3 and GLUT1 proteins in L6 myotubes were determined to be about 15 h and 6 h respectively. DNP prolonged the half-life of both proteins. After 24 h of DNP treatment, 88% of GLUT3 protein and 57% of GLUT1 protein had not turned over, compared with 25% in untreated cells. We conclude that the long-term stimulation of glucose transport by DNP arises from an elevation of GLUT3 protein content associated with an increase in GLUT3 protein half-life. These findings suggest that disruption of the oxidative chain of L6 muscle cells leads to an adaptive response of glucose transport that is distinct from the insulin response, involving specific glucose transporter isoforms that are regulated by different mechanisms.

scholarly journals Graphene oxide nanofilm and the addition of L-glutamine can promote development of embryonic muscle cells

2020 ◽  
Author(s):  
Marlena Zielińska-Górska ◽  
Anna Hotowy ◽  
Mateusz Wierzbicki ◽  
Jaśmina Bałaban ◽  
Malwina Sosnowska ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Surface Properties ◽  
Satellite Cells ◽  
Chicken Embryo ◽  
Culture Medium ◽  
Muscle Cells ◽  
Control Group ◽  
Protein Levels ◽  
Myogenic Potential

Abstract Background: Formation of muscular pseudo-tissue depends on muscle precursor cells, the extracellular matrix (ECM)-mimicking structure and factors stimulating cell differentiation. These three things cooperate and can create a tissue-like structure, however, their interrelationships are relatively unknown. The objective was to study the interaction between surface properties, culture medium composition and heterogeneous cell culture. We would like to demonstrate that changing the surface properties by coating with graphene oxide nanofilm (nGO) can affect cell behaviour and especially their need for the key amino acid L-glutamine (L-Glu). Results: Chicken embryo muscle cells and their precursors, cultured in vitro, were used as the experimental model. The mesenchymal stem cell, collected from the hind limb of the chicken embryo at day 8 were divided into 4 groups; the control group and groups treated with nGO, L-Glu and nGO supplied with L-Glu (nGOxL-Glu). The roughness of the surface of the plastic plate covered with nGO was much lower than a standard plate. The test of nGO biocompatibility demonstrated that the cells were willing to settle on the nGO without any toxic effects. Moreover, nGO by increasing hydrophilicity and reducing roughness stimulated the colonisation of primary stromal cells that promote embryonic satellite cells. The viability significantly increased in cells cultured on nGOxL-Glu. Observations of cell morphology showed that the most mature state of myogenesis was characteristic for the group nGOxL-Glu. This result was confirmed by increasing the expression of MYF5 genes at mRNA and protein levels. nGO also increased the expression of MYF5 and also very strongly the expression of PAX7 at mRNA and protein levels. However, when analysing the expression of PAX7 , a positive link was observed between the nGO surface and the addition of L-Glu. Conclusions: The use of nGO and L-Glu supplement may improve myogenesis and also the myogenic potential of myocytes and their precursors by promoting the formation of satellite cells. Studies have, for the first time, demonstrated positive cooperation between surface properties nGO and L-Glu supplementation to the culture medium regarding the myogenic potential of cells involved in muscle formation.

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