scholarly journals Dandelion Chloroform Extract Promotes Glucose Uptake via the AMPK/GLUT4 Pathway in L6 Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Ping Zhao ◽  
Qian Ming ◽  
Mingrui Xiong ◽  
Guanjun Song ◽  
Li Tan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Laser Scanning ◽  
Chloroform Extract ◽  
Cell Culture Supernatant ◽  
Glut4 Translocation ◽  
L6 Cells ◽  
Glut4 Expression ◽  
Compound C ◽  
Number Of Patients

The number of patients with type 2 diabetes mellitus (T2DM) is increasing rapidly worldwide. Glucose transporter 4 (GLUT4) is one of the main proteins that transport blood glucose into the cells and is a target in the treatment of T2DM. In this study, we investigated the mechanism of action of dandelion chloroform extract (DCE) on glucose uptake in L6 cells. The glucose consumption of L6 cell culture supernatant was measured by a glucose uptake assay kit, and the dynamic changes of intracellular GLUT4 and calcium (Ca2+) levels were monitored by laser scanning confocal microscopy in L6 cell lines stably expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM) was traced via myc-GLUT4-mOrange. GLUT4 expression and AMP-activated protein kinase (AMPK), protein kinase B (PKB/Akt), protein kinase C (PKC), and phosphorylation levels were determined by performing western blotting. GLUT4 mRNA expression was detected by real-time PCR. DCE up-regulated GLUT4 expression, promoted GLUT4 translocation and fusion to the membrane eventually leading to glucose uptake, and induced AMPK phosphorylation in L6 cells. The AMPK inhibitory compound C significantly inhibited DCE-induced GLUT4 expression and translocation while no inhibitory effect was observed by the phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin and PKC inhibitor Gö6983. These data suggested that DCE promoted GLUT4 expression and transport to the membrane through the AMPK signaling pathway, thereby stimulating GLUT4 fusion with PM to enhance glucose uptake in L6 cells. DCE-induced GLUT4 translocation was also found to be Ca2+-independent. Together, these findings indicate that DCE could be a new hypoglycemic agent for the treatment of T2DM.

scholarly journals Ethanolic Extract of Folium Sennae Mediates the Glucose Uptake of L6 Cells by GLUT4 and Ca2+

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2934 ◽  
Author(s):  
Ping Zhao ◽  
Qian Ming ◽  
Junying Qiu ◽  
Di Tian ◽  
Jia Liu ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
G Protein ◽  
Glucose Transporter ◽  
Ethanolic Extract ◽  
High Morbidity ◽  
L6 Cells ◽  
Glut4 Expression ◽  
Compound C

In today’s world, diabetes mellitus (DM) is on the rise, especially type 2 diabetes mellitus (T2DM), which is characterized by insulin resistance. T2DM has high morbidity, and therapies with natural products have attracted much attention in the recent past. In this paper, we aimed to study the hypoglycemic effect and the mechanism of an ethanolic extract of Folium Sennae (FSE) on L6 cells. The glucose uptake of L6 cells was investigated using a glucose assay kit. We studied glucose transporter 4 (GLUT4) expression and AMP-activated protein kinase (AMPK), protein kinase B (PKB/Akt), and protein kinase C (PKC) phosphorylation levels using western blot analysis. GLUT4 trafficking and intracellular Ca2+ levels were monitored by laser confocal microscopy in L6 cells stably expressing IRAP-mOrange. GLUT4 fusion with plasma membrane (PM) was observed by myc-GLUT4-mOrange. FSE stimulated glucose uptake; GLUT4 expression and translocation; PM fusion; intracellular Ca2+ elevation; and the phosphorylation of AMPK, Akt, and PKC in L6 cells. GLUT4 translocation was weakened by the AMPK inhibitor compound C, PI3K inhibitor Wortmannin, PKC inhibitor Gö6983, G protein inhibitor PTX/Gallein, and PLC inhibitor U73122. Similarly, in addition to PTX/Gallein and U73122, the IP3R inhibitor 2-APB and a 0 mM Ca2+-EGTA solution partially inhibited the elevation of intracellular Ca2+ levels. BAPTA-AM had a significant inhibitory effect on FSE-mediated GLUT4 activities. In summary, FSE regulates GLUT4 expression and translocation by activating the AMPK, PI3K/Akt, and G protein–PLC–PKC pathways. FSE causes increasing Ca2+ concentration to complete the fusion of GLUT4 vesicles with PM, allowing glucose uptake. Therefore, FSE may be a potential drug for improving T2DM.

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

2005 ◽  
Vol 289 (4) ◽  
pp. E643-E649 ◽  
Author(s):  
Shinya Yamaguchi ◽  
Hiroshi Katahira ◽  
Sachihiko Ozawa ◽  
Yoko Nakamichi ◽  
Toshiaki Tanaka ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Mitogen Activated Protein Kinase ◽  
Intrinsic Activity ◽  
Glut4 Translocation ◽  
Amp Activated Protein Kinase ◽  
Sb 203580

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin ( P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP ( P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin ( P < 0.05–0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin ( P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.

scholarly journals AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells

2015 ◽  
Vol 228 (2) ◽  
pp. 105-114 ◽  
Author(s):  
Nami Kim ◽  
Jung Ok Lee ◽  
Hye Jeong Lee ◽  
Yong Woo Lee ◽  
Hyung Ip Kim ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Gene Encoding ◽  
Beneficial Effects ◽  
Calcium Calmodulin Dependent ◽  
L6 Myotubes ◽  
Glut4 Expression ◽  
Compound C ◽  
Dependent Protein

Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phosphorylation of protein kinase C-α (PKCα). Chelation of calcium with BAPTA-AM blocked isoeugenol-induced AMPK phosphorylation and glucose uptake. Isoeugenol stimulated p38MAPK phosphorylation that was inhibited after pretreatment with compound C, an AMPK inhibitor. Isoeugenol also increased glucose transporter type 4 (GLUT4) expression and its translocation to the plasma membrane. GLUT4 translocation was not observed after the inhibition of AMPK and CaMKK. In addition, isoeugenol activated the Akt substrate 160 (AS160) pathway, which is downstream of the p38MAPK pathway. Knockdown of the gene encoding AS160 inhibited isoeugenol-induced glucose uptake. Together, these results indicate that isoeugenol exerts beneficial health effects by activating the AMPK/p38MAPK/AS160 pathways in skeletal muscle.

scholarly journals Inhibition of the Protein Tyrosine Phosphatase SHP-1 Increases Glucose Uptake in Skeletal Muscle Cells by Augmenting Insulin Receptor Signaling and GLUT4 Expression

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 4581-4588 ◽  
Author(s):  
Sébastien Bergeron ◽  
Marie-Julie Dubois ◽  
Kerstin Bellmann ◽  
Michael Schwab ◽  
Nancy Larochelle ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Glucose Uptake ◽  
Protein Tyrosine Phosphatase ◽  
Insulin Action ◽  
Tyrosine Phosphatase ◽  
Glycogen Synthesis ◽  
Glut4 Translocation ◽  
Protein Tyrosine ◽  
Glut4 Expression

The protein tyrosine phosphatase (PTPase) Src-homology 2-domain-containing phosphatase (SHP)-1 was recently reported to be a novel regulator of insulin's metabolic action. In order to examine the role of this PTPase in skeletal muscle, we used adenovirus (AdV)-mediated gene transfer to express an interfering mutant of SHP-1 [dominant negative (DN)SHP-1; mutation C453S] in L6 myocytes. Expression of DNSHP-1 increased insulin-induced Akt serine-threonine kinase phosphorylation and augmented glucose uptake and glycogen synthesis. Pharmacological inhibition of glucose transporter type 4 (GLUT4) activity using indinavir and GLUT4 translocation assays revealed an important role for this transporter in the increased insulin-induced glucose uptake in DNSHP-1-expressing myocytes. Both GLUT4 mRNA and protein expression were also found to be increased by DNSHP-1 expression. Furthermore, AdV-mediated delivery of DNSHP-1 in skeletal muscle of transgenic mice overexpressing Coxsackie and AdV receptor also enhanced GLUT4 protein expression. Together, these findings confirm that SHP-1 regulates muscle insulin action in a cell-autonomous manner and further suggest that the PTPase negatively modulates insulin action through down-regulation of both insulin signaling to Akt and GLUT4 translocation, as well as GLUT4 expression.

Loss of HIF-1α impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells

2014 ◽  
Vol 306 (9) ◽  
pp. E1065-E1076 ◽  
Author(s):  
Hidemitsu Sakagami ◽  
Yuichi Makino ◽  
Katsutoshi Mizumoto ◽  
Tsubasa Isoe ◽  
Yasutaka Takeda ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Intracellular Signaling ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Glut4 Translocation

Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12myocytes in which HIF-1α is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase

2001 ◽  
Vol 359 (3) ◽  
pp. 639-649 ◽  
Author(s):  
Romel SOMWAR ◽  
David Y. KIM ◽  
Gary SWEENEY ◽  
Carol HUANG ◽  
Wenyan NIU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Glut4 Translocation ◽  
L6 Myotubes ◽  
Mitogen Activated Protein ◽  
Stimulation Of

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t1/2 = 2.5min) preceded the stimulation of 2-deoxyglucose uptake (t1/2 = 6min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22°C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40–60%. Pretreatment with SB203580 lowered the apparent transport Vmax of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent Km for either hexose. The IC50 values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2μM respectively, and correlated with the IC50 for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4μM, respectively). Insulin caused a dose- (EC50 = 15nM) and time- (t1/2 = 3min) dependent increase in p38 MAPK phosphorylation, which peaked at 10min (2.3±0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38α (2.6±0.3-fold) and p38β (2.3±0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

scholarly journals A Crucial Role for the Small GTPase Rac1 Downstream of the Protein Kinase Akt2 in Insulin Signaling that Regulates Glucose Uptake in Mouse Adipocytes

2019 ◽  
Vol 20 (21) ◽  
pp. 5443 ◽  
Author(s):  
Takenaka ◽  
Nakao ◽  
Matsui ◽  
Satoh
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Glut4 Translocation ◽  
Insulin Dependent ◽  
Mouse Adipocytes ◽  
Phosphoinositide 3 Kinase

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.

Abstract 13103: A Novel Anchor Protein TUG Mediates GLUT4 Translocation by AMP-activated Protein Kinase in the Ischemic Heart

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yina Ma ◽  
Wanqing Sun ◽  
Nanhu Quan ◽  
Lin Wang ◽  
Xingchi Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Ex Vivo ◽  
Ischemic Heart ◽  
Ischemia And Reperfusion ◽  
Ampk Activation ◽  
Glut4 Translocation ◽  
Heart Perfusion ◽  
Amp Activated Protein Kinase

Introduction: Ischemic heart disease is a leading cause of death, and it is caused by reduced blood flow to the ischemic area. Thus, an increasing nutrient uptake is a key approach to increase cardiomyocyte survival rate during the ischemia and reperfusion (I/R) period. TUG (tether containing a UBX domain, for GLUT4, 60 KDa) is a regulator of GLUT4 trafficking, it can be cleaved to mobilize GLUT4 from intracellular membranes to the cell surface after insulin stimulation in skeletal muscle. The energy sensor AMP-activated protein kinase (AMPK) is known to play an important cardioprotective role during myocardial I/R by regulating GLUT4 translocation and glucose uptake. Hypothesis: TUG is one of the downstream targets of AMPK, which can be phosphorylated by hypoxia/ischemia induced AMPK activation. Phosphorylation of TUG accelerates its cleavage and increases GLUT4 translocation during ischemia/reperfusion in the heart. Methods: In vitro hypoxia chamber and ex vivo isolated mouse heart perfusion Langendorff system were used to test the hypothesis. Antithrombin (AT) is an endogenous AMPK agonist in the heart, which was used to define the role of TUG in regulating GLUT4 trafficking during ischemia and reperfusion in the heart. Results: The ex vivo heart perfusion data demonstrated that AT triggered AMPK activation and significantly increase glucose uptake and GLUT4 translocation during ischemia and reperfusion (p<0.05 vs. vehicle). Intriguingly, GLUT4 immunoprecipitation data showed that AT treatment caused a dissociation of TUG from GLUT4. Moreover, AT treatment increased abundance of a TUG cleavage product (42 KDa) in response to I/R. All of these glucose transporter trafficking events are blunted in the AMPK kinase dead (KD) transgenic hearts. In HL-1 cardiomyocytes, TUG proteins were phosphorylated by activated AMPK during hypoxia. Moreover, TUG siRNA knockdown the TUG of HL-1 cells caused significantly increased cell surface GLUT4 and glucose uptake. Conclusions: Cardiac AMPK activation stimulates TUG cleavage and causes the dissociation between TUG and GLUT4 in the intracellular vesicles. TUG is a critical mediator that modulates cardiac GLUT4 translocation to cell surface and enhances glucose uptake by AMPK signaling pathway.

scholarly journals MicroRNAs-Mediated Regulation of Skeletal Muscle GLUT4 Expression and Translocation in Insulin Resistance

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
João Victor Esteves ◽  
Francisco Javier Enguita ◽  
Ubiratan Fabres Machado
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glycemic Control ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Regulation Of Gene Expression ◽  
Glut4 Translocation ◽  
Therapeutic Approaches ◽  
Glut4 Expression ◽  
Solute Carrier

The solute carrier family 2 facilitated glucose transporter member 4 (GLUT4) plays a key role in the insulin-induced glucose uptake by muscle and adipose tissues. In prediabetes and diabetes, GLUT4 expression/translocation has been detected as reduced, participating in mechanisms that impair glycemic control. Recently, a class of short endogenous noncoding RNAs named microRNAs (miRNAs) has been increasingly described as involved in the posttranscriptional epigenetic regulation of gene expression. The present review focuses on miRNAs potentially involved in the expression of GLUT4 expression, and proteins related to GLUT4 and translocation in skeletal muscle, seeking to correlate them with insulin resistance and diabetes. So far, miR-21a-5p, miR-29a-3p, miR-29c-3p, miR-93-5p, miR-106b-5p, miR-133a-3p, miR-133b-3p, miR-222-3p, and miR-223-3p have been reported to directly and/or indirectly regulate the GLUT4 expression; and their expression is altered under diabetes-related conditions. Besides, some miRNAs that have been linked to the expression of proteins involved in GLUT4 translocation machinery in muscle could also impact glucose uptake. That makes these miRNAs promising targets for preventive and/or therapeutic approaches, which could improve glycemic control, thus deserving future new investigations.

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