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

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.

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

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.100305 ◽

2010 ◽

Vol 74 (10) ◽

pp. 2036-2042 ◽

Cited By ~ 10

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

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Regulation of lactate production and glucose transport as well as of glucose transporter 1 and lactate dehydrogenase A mRNA levels by basic fibroblast growth factor in rat Sertoli cells

Journal of Endocrinology ◽

10.1677/joe.0.1730335 ◽

2002 ◽

Vol 173 (2) ◽

pp. 335-343 ◽

Cited By ~ 31

Author(s):

MF Riera ◽

SB Meroni ◽

HF Schteingart ◽

EH Pellizzari ◽

SB Cigorraga

Keyword(s):

Growth Factor ◽

Glucose Transport ◽

Glucose Transporter ◽

Lactate Production ◽

Mrna Levels ◽

Dependent Manner ◽

Short Term ◽

Glucose Transporter 1 ◽

Ldh Activity

By using cultured rat Sertoli cells as a model, both the action of basic fibroblast growth factor (bFGF) on lactate production and the site of this action were studied. bFGF stimulated Sertoli cell lactate production in a dose-dependent manner (basal: 7.3+/-0.5; 0.1 ng/ml bFGF: 7.5+/-0.5; 1 ng/ml bFGF: 7.5+/-0.6; 10 ng/ml bFGF: 10.3+/-1.0; 30 ng/ml bFGF: 15.2+/-1.5; 50 ng/ml bFGF: 15.4+/-1.6 microg/microg DNA). Two major sites for the action of this growth factor were identified. First, bFGF was shown to exert short- and long-term stimulatory effects on glucose transport (basal: 1170+/-102; 30 ng/ml bFGF for 120 min: 1718+/-152 and basal: 718+/-64; 30 ng/ml bFGF for 48 h: 1069+/-69 d.p.m./microg DNA respectively). Short-term bFGF stimulation of glucose transport was not inhibited by the protein synthesis inhibitor cycloheximide. These results indicate that short-term bFGF stimulation of glucose uptake does not involve an increase in the number of glucose transporters. On the other hand, stimulation with bFGF for periods of time longer than 12 h increased glucose transporter 1 (GLUT1) mRNA levels. These increased mRNA levels were probably ultimately responsible for the increments in glucose uptake that are observed in long-term treated cultures. Secondly, bFGF increased lactate dehydrogenase (LDH) activity (basal: 31.0+/-1.4; 30 ng/ml bFGF: 45.7+/- 2.4 mIU/microg DNA). The principal subunit component of those LDH isozymes that favors the transformation of pyruvate to lactate is subunit A. bFGF increased LDH A mRNA levels in a dose- and time-dependent manner. In summary, the results presented herein show that glucose transport, LDH activity and GLUT1 and LDH A mRNA levels are regulated by bFGF to achieve an increase in lactate production. These observed regulatory actions provide unequivocal evidence of the participation of bFGF in Sertoli cell lactate production which may be related to normal germ cell development.

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Stimulation of glucose transport in L6 muscle cells by long-term intermittent stretch-relaxation

FEBS Letters ◽

10.1016/0014-5793(92)80217-5 ◽

1992 ◽

Vol 301 (1) ◽

pp. 94-98 ◽

Cited By ~ 11

Author(s):

Yasuhide Mitsumoto ◽

Gregory P. Downey ◽

Amira Klip

Keyword(s):

Glucose Transport ◽

Muscle Cells ◽

L6 Muscle Cells ◽

Stimulation Of

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5-Aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent jejunal glucose transport: a possible role for AMPK

Biochemical Journal ◽

10.1042/bj20040694 ◽

2005 ◽

Vol 385 (2) ◽

pp. 485-491 ◽

Cited By ~ 97

Author(s):

John WALKER ◽

Humberto B. JIJON ◽

Hugo DIAZ ◽

Payam SALEHI ◽

Thomas CHURCHILL ◽

...

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transport ◽

Western Blotting ◽

Glucose Transporter ◽

Mitogen Activated Protein Kinase ◽

Mrna Levels ◽

Energy Status ◽

P38 Inhibitor ◽

Mitogen Activated Protein

AMPK (AMP-activated protein kinase) is a key sensor of energy status within the cell. Activated by an increase in the AMP/ATP ratio, AMPK acts to limit cellular energy depletion by down-regulating selective ATP-dependent processes. The purpose of the present study was to determine the role of AMPK in regulating intestinal glucose transport. [3H]3-O-methyl glucose fluxes were measured in murine jejunum in the presence and absence of the AMPK activators AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin and the p38 inhibitor, SB203580. To differentiate between a sodium-coupled (SGLT1) and diffusive (GLUT2) route of entry, fluxes were measured in the presence of the SGLT1 and GLUT2 inhibitors phloridzin and phloretin. Glucose transporter mRNA levels were measured by reverse transcriptase–PCR, and localization by Western blotting. Surface-expressed GLUT2 was assessed by luminal biotinylation. Activation of p38 mitogen-activated protein kinase was analysed by Western blotting. We found that treatment of jejunal tissue with AICAR resulted in enhanced net glucose uptake and was associated with phosphorylation of p38 mitogen-activated protein kinase. Inhibition of p38 abrogated the stimulation of AICAR-stimulated glucose uptake. Phloretin abolished the AICAR-mediated increase in glucose flux, whereas phloridzin had no effect, suggesting the involvement of GLUT2. In addition, AICAR decreased total protein levels of SGLT1, concurrently increasing levels of GLUT2 in the brush-border membrane. The anti-diabetic drug metformin, a known activator of AMPK, also induced the localization of GLUT2 to the luminal surface. We conclude that the activation of AMPK results in an up-regulation of non-energy requiring glucose uptake by GLUT2 and a concurrent down-regulation of sodium-dependent glucose transport.

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4F2hc stabilizes GLUT1 protein and increases glucose transport activity

AJP Cell Physiology ◽

10.1152/ajpcell.00416.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. C1047-C1054 ◽

Cited By ~ 20

Author(s):

Haruya Ohno ◽

Yusuke Nakatsu ◽

Hideyuki Sakoda ◽

Akifumi Kushiyama ◽

Hiraku Ono ◽

...

Keyword(s):

Amino Acid ◽

Glucose Uptake ◽

Glucose Transporter ◽

Fluorescent Protein ◽

Amino Acid Uptake ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Mrna Levels ◽

Glucose Transporter 1 ◽

Glut1 Protein

Glucose transporter 1 (GLUT1) is widely distributed throughout various tissues and contributes to insulin-independent basal glucose uptake. Using a split-ubiquitin membrane yeast two-hybrid system, we newly identified 4F2 heavy chain (4F2hc) as a membrane protein interacting with GLUT1. Though 4F2hc reportedly forms heterodimeric complexes between amino acid transporters, such as LAT1 and LAT2, and regulates amino acid uptake, we investigated the effects of 4F2hc on GLUT1 expression and the associated glucose uptake. First, FLAG-tagged 4F2hc and hemagglutinin-tagged GLUT1 were overexpressed in human embryonic kidney 293 cells and their association was confirmed by coimmunoprecipitation. The green fluorescent protein-tagged 4F2hc and DsRed-tagged GLUT1 showed significant, but incomplete, colocalization at the plasma membrane. In addition, an endogenous association between GLUT1 and 4F2hc was demonstrated using mouse brain tissue and HeLa cells. Interestingly, overexpression of 4F2hc increased the amount of GLUT1 protein in HeLa and HepG2 cells with increased glucose uptake. In contrast, small interfering RNA (siRNA)-mediated 4F2hc gene suppression markedly reduced GLUT1 protein in both cell types, with reduced glucose uptake. While GLUT1 mRNA levels were not affected by overexpression or gene silencing of 4F2hc, GLUT1 degradation after the addition of cycloheximide was significantly suppressed by 4F2hc overexpression and increased by 4F2hc siRNA treatment. Taken together, these observations indicate that 4F2hc is likely to be involved in GLUT1 stabilization and to contribute to the regulation of not only amino acid but also glucose metabolism.

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Protein Kinase Cζ Mediates Insulin-induced Glucose Transport through Actin Remodeling in L6 Muscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0969 ◽

2006 ◽

Vol 17 (5) ◽

pp. 2322-2330 ◽

Cited By ~ 38

Author(s):

Li-Zhong Liu ◽

Hai-Lu Zhao ◽

Jin Zuo ◽

Stanley K.S. Ho ◽

Juliana C.N. Chan ◽

...

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transport ◽

Fold Increase ◽

Muscle Cells ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Insulin Effect ◽

Actin Remodeling ◽

L6 Muscle Cells

Protein kinase C (PKC) ζ has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKCζ on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCζ translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKCζ with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCζ distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCζ pseudosubstrate peptide. In stable transfected cells, overexpression of PKCζ caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKCζ overexpression were abolished by cell-permeable PKCζ pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCζ to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCζ translocation. Together, these results suggest that PKCζ mediates insulin effect on glucose transport through actin remodeling in muscle cells.

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Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)40047-1 ◽

1990 ◽

Vol 265 (3) ◽

pp. 1516-1523

Author(s):

P S Walker ◽

T Ramlal ◽

V Sarabia ◽

U M Koivisto ◽

P J Bilan ◽

...

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Muscle Cells ◽

Transport Activity ◽

Mrna Transcription ◽

Coordinate Control ◽

L6 Muscle Cells

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Testosterone activates glucose metabolism through AMPK and androgen signaling in cardiomyocyte hypertrophy

Biological Research ◽

10.1186/s40659-021-00328-4 ◽

2021 ◽

Vol 54 (1) ◽

Author(s):

Mayarling Francisca Troncoso ◽

Mario Pavez ◽

Carlos Wilson ◽

Daniel Lagos ◽

Javier Duran ◽

...

Keyword(s):

Glucose Metabolism ◽

Cardiac Hypertrophy ◽

Glucose Uptake ◽

Male Rats ◽

Cardiomyocyte Hypertrophy ◽

Mrna Levels ◽

Elevated Glucose ◽

Amp Activated Protein Kinase ◽

Cultured Cardiomyocytes

Abstract Background Testosterone regulates nutrient and energy balance to maintain protein synthesis and metabolism in cardiomyocytes, but supraphysiological concentrations induce cardiac hypertrophy. Previously, we determined that testosterone increased glucose uptake—via AMP-activated protein kinase (AMPK)—after acute treatment in cardiomyocytes. However, whether elevated glucose uptake is involved in long-term changes of glucose metabolism or is required during cardiomyocyte growth remained unknown. In this study, we hypothesized that glucose uptake and glycolysis increase in testosterone-treated cardiomyocytes through AMPK and androgen receptor (AR). Methods Cultured cardiomyocytes were stimulated with 100 nM testosterone for 24 h, and hypertrophy was verified by increased cell size and mRNA levels of β-myosin heavy chain (β-mhc). Glucose uptake was assessed by 2-NBDG. Glycolysis and glycolytic capacity were determined by measuring extracellular acidification rate (ECAR). Results Testosterone induced cardiomyocyte hypertrophy that was accompanied by increased glucose uptake, glycolysis enhancement and upregulated mRNA expression of hexokinase 2. In addition, testosterone increased AMPK phosphorylation (Thr172), while inhibition of both AMPK and AR blocked glycolysis and cardiomyocyte hypertrophy induced by testosterone. Moreover, testosterone supplementation in adult male rats by 5 weeks induced cardiac hypertrophy and upregulated β-mhc, Hk2 and Pfk2 mRNA levels. Conclusion These results indicate that testosterone stimulates glucose metabolism by activation of AMPK and AR signaling which are critical to induce cardiomyocyte hypertrophy.

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

Molecules ◽

10.3390/molecules26133785 ◽

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.

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