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.

Dibutyryl cAMP-induced Increases in triacylglycerol lipase activity in developing L8 myotube cultures

1990 ◽  
Vol 68 (6) ◽  
pp. 689-693 ◽  
Author(s):  
Warren K. Palmer ◽  
Lawrence B. Oscai ◽  
Peter J. Bechtel ◽  
Glenn A. Fisher
Keyword(s):  
Enzyme Activity ◽  
Lipoprotein Lipase ◽  
Lipase Activity ◽  
Culture Medium ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Dibutyryl Camp ◽  
Ph Optimum ◽  
Triacylglycerol Lipase

Triacylglycerol (TG) lipase activity, with an alkaline pH optimum, has been identified in the cellular fraction of L8 myotube cultures. This TG lipase activity was stimulated by serum and inhibited by NaCl and protamine sulfate. These characteristics have been classically described for lipoprotein lipase. It was possible to increase the activity of this TG lipase three- to fivefold by incubating the cells with dibutyryl cAMP Maximal enzyme activity was observed 16 h following the addition of 10–100 μM dibutyryl cAMP to the cultured cells. Enzyme activity returned to control levels 24 h after removal of the nucleotide from the culture medium. Serum-sensitive alkaline TG lipase activity was also identified in five other myotube preparations of cultured muscle cells. The highest levels of activity were found in rat skeletal muscle primary, H9, and L6 cell types. The finding that dibutyryl cAMP is an effective inducer of alkaline TG lipase activity provides us with a valuable model to investigate mechanisms regulating synthesis, compartmentalization, and transport of lipoprotein lipase in muscle.Key words: lipoprotein lipase, cultured muscle cells, enzyme induction.

Characterization of Lipoprotein Lipase Activity in the Newborn Rat Liver

Neonatology ◽  
1987 ◽  
Vol 51 (1) ◽  
pp. 45-52 ◽  
Author(s):  
Manuel Reina ◽  
Senén Vilaró ◽  
Ignasi Ramirez ◽  
Miquel Llobera
Keyword(s):  
Lipoprotein Lipase ◽  
Rat Liver ◽  
Lipase Activity ◽  
Lipoprotein Lipase Activity ◽  
Newborn Rat

Characterization of a novel insect digestive DNase with a highly alkaline pH optimum

2002 ◽  
Vol 32 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Johann P Schernthaner ◽  
Ross E Milne ◽  
Harvey Kaplan
Keyword(s):  
Alkaline Ph ◽  
Ph Optimum

Characterization of the triacylglycerol lipase activity in three types of rat skeletal muscle

1987 ◽  
Vol 65 (3) ◽  
pp. 317-322 ◽  
Author(s):  
Wayne C. Miller ◽  
Warren K. Palmer ◽  
David A. Arnall ◽  
Lawrence B. Oscai
Keyword(s):  
Skeletal Muscle ◽  
Lipoprotein Lipase ◽  
Lipase Activity ◽  
Lipolytic Activity ◽  
Vastus Lateralis ◽  
High Energy ◽  
Glycolytic Flux ◽  
Ph Optimum ◽  
Triacylglycerol Lipase ◽  
Triglyceride Lipase

The purpose of this study was to characterize the lipolytic activity of the alkaline triglyceride lipase in homogenates of three types of skeletal muscle obtained from heparin-perfused rat hindlimb. Specifically, the red portion of the vastus lateralis, the white portion of the vastus lateralis, and the soleus muscles were examined. To remove capillary-bound lipoprotein lipase from the capillary beds, muscle was perfused with an erythrocyte-free buffer containing 4% albumin, 5 units of heparin/mL, and 7.5 μM adenosine. Adenosine reduced perfusion pressure from 117 ± 5 to 86 ± 6 mmHg (1 mmHg = 133.32 Pa), providing evidence for an effective vasodilation. This vasodilation increased the amount of lipoprotein lipase removed from the capillary beds. By the end of the experiment, perfusates were lipoprotein lipase-free. Oxygen supply to the perfused hindlimb appeared adequate as evidenced by similar high energy phosphate values for perfused and contralateral control tissues. For example, in soleus muscle, ATP content was 4.5 ± 0.6 vs. 4.2 ± 0.3 μmol/g, ADP concentration was 1.0 ± 0.2 vs. 1.4 ± 0.2 μmol/g, and creatine phosphate level was 12.9 ± 0.7 vs. 11.0 ± 0.6 μmol/g for perfused and contralateral control soleus, respectively. In addition, K+ output by the hindlimb was negligible, while glycolytic flux of perfused muscle was similar to that measured in control tissue. The findings that triglyceride levels of soleus and red vastus lateralis were decreased suggest that endogenous triglyceride was providing energy for the hindlimb during perfusion. Skeletal muscle triglyceride lipase activity was stimulated by serum and heparin, inhibited by NaCl and protamine, and had a pH optimum of 8.1. These results are consistent with the hypothesis that the major lipolytic activity present in the intracellular compartment of skeletal muscle is the alkaline triglyceride lipase with characteristics similar to those of lipoprotein lipase.

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 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.

scholarly journals Triacylglycerol Lipase Activities of Cultured Rat L6 Myoblasts

1985 ◽  
Vol 38 (1) ◽  
pp. 41
Author(s):  
RK Tume ◽  
F D Shaw
Keyword(s):  
Lipoprotein Lipase ◽  
Cell Lines ◽  
Lipase Activity ◽  
Maximum Velocity ◽  
Acid Lipase ◽  
Ph Optimum ◽  
Triacylglycerol Lipase ◽  
Inhibitory Component ◽  
Cell Homogenate ◽  
Hydrolysis Of

The utilization of exogenous triacylglycerol by fusing and non-fusing rat L6 myoblasts grown in culture was investigated. Although small quantities of triacylglycerol were accumulated by both cell lines during an incubation of 2 h, no evidence could be found for the presence of lipoprotein lipase, either in the cells or released into the medium. Cell homogenate studies confirmed the absence of lipoprotein lipase but revealed the presence of an acid lipase having a pH optimum at 4�6. Acid lipase activity was mainly associated with a 15 000 g pellet and was capable of hydrolysing triolein at maximum velocity in the millimolar range. Unlike lipoprotein lipase, acid lipase was strongly inhibited by serum and preliminary investigations suggest that the inhibitory component of serum is located amongst the higher density lipoproteins. It is likely that the acid lipase is of lysosomal origin and is responsible for the hydrolysis of internalized triacylglycerol for subsequent utilization by the cell.

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