Browse
Submit Data
Databases
API
Help

Dataset Information

33 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Knockout of syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion.

ABSTRACT: Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper.

SUBMITTER: Black HL

PROVIDER: S-EPMC8767277 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking.

Project description:The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

| S-EPMC4310743 | biostudies-literature

Cyclodextrin-Mediated Cholesterol Depletion Induces Adiponectin Secretion in 3T3-L1 Adipocytes.

Project description:Adipocytes store a significant amount of cholesterol and triglycerides. However, whether cholesterol modulates adipocyte function remains largely unknown. We modulated the cholesterol level in adipocytes to examine its effect on the secretion of adiponectin, an important hormone specifically secreted by adipocytes. Treating differentiated 3T3-L1 adipocytes with 4 mM methyl-β-cyclodextrin (MβCD), a molecule with a high affinity for cholesterol, rapidly depleted cholesterol in adipocytes. Interestingly, MβCD treatment increased adiponectin in the medium without affecting its intracellular level, suggesting a modulation of secretion. By contrast, cholesterol addition did not affect adiponectin secretion, suggesting that cholesterol-depletion-induced intracellular cholesterol trafficking, but not reduced cholesterol level, accounted for MβCD-induced adiponectin secretion. MβCD-induced adiponectin secretion was reduced after 10 μg/mL U18666A treatment that suppressed cholesterol transport out of late endosomes/lysosomes. Depleting Niemann-Pick type C1 (NPC1) or NPC2 proteins, which mediate endosomal/lysosomal cholesterol export, consistently reduced MβCD-induced adiponectin secretion. Furthermore, treatment with 1 μM bafilomycin A1, which neutralized acidic endosomes/lysosomes, also attenuated MβCD-induced adiponectin secretion. Finally, MβCD treatment redistributed cellular adiponectin to lower-density fractions in sucrose gradient fractionation. Our results show that MβCD-mediated cholesterol depletion elevates the secretion of adiponectin, highlighting the involvement of endosomes and lysosomes in adiponectin secretion in adipocytes.

| S-EPMC10572842 | biostudies-literature

A role for Rab14 in the endocytic trafficking of GLUT4 in 3T3-L1 adipocytes.

Project description:Insulin enhances the uptake of glucose into adipocytes and muscle cells by promoting the redistribution of the glucose transporter isoform 4 (GLUT4) from intracellular compartments to the cell surface. Rab GTPases regulate the trafficking itinerary of GLUT4 and several have been found on immunopurified GLUT4 vesicles. Specifically, Rab14 has previously been implicated in GLUT4 trafficking in muscle although its role, if any, in adipocytes is poorly understood. Analysis of 3T3-L1 adipocytes using confocal microscopy demonstrated that endogenous GLUT4 and endogenous Rab14 exhibited a partial colocalisation. However, when wild-type Rab14 or a constitutively-active Rab14Q70L mutant were overexpressed in these cells, the colocalisation with both GLUT4 and IRAP became extensive. Interestingly, this colocalisation was restricted to enlarged 'ring-like' vesicular structures (mean diameter 1.3 µm), which were observed in the presence of overexpressed wild-type Rab14 and Rab14Q70L, but not an inactive Rab14S25N mutant. These enlarged vesicles contained markers of early endosomes and were rapidly filled by GLUT4 and transferrin undergoing endocytosis from the plasma membrane. The Rab14Q70L mutant reduced basal and insulin-stimulated cell surface GLUT4 levels, probably by retaining GLUT4 in an insulin-insensitive early endosomal compartment. Furthermore, shRNA-mediated depletion of Rab14 inhibited the transit of GLUT4 through early endosomal compartments towards vesicles and tubules in the perinuclear region. Given the previously reported role of Rab14 in trafficking between endosomes and the Golgi complex, we propose that the primary role of Rab14 in GLUT4 trafficking is to control the transit of internalised GLUT4 from early endosomes into the Golgi complex, rather than direct GLUT4 translocation to the plasma membrane.

| S-EPMC3666250 | biostudies-literature

Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes.

Project description:Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA-GLUT4-GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA-GLUT4-GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.

| S-EPMC7060922 | biostudies-literature

Pig testis extract augments the expression and secretion of adiponectin in 3T3-L1 adipocytes

Project description:Adiponectin is known as a key molecule to ameliorate symptoms of the type 2 diabetes mellitus and disorder of lipid metabolism. In this study, we investigated whether hot water extracts of some livestock by-products induced expression of adiponectin using 3T3-L1 adipocytes. Out of 11 extracts tested, pig testis extracts (PT) enhanced adiponectin mRNA expression and secretion of adiponectin protein from 3T3-L1 cells. Furthermore, simultaneous treatment with PT and daidzein, soy phytoestrogen, enhanced synergistically adiponectin secretion. Moreover, pretreatment with an estrogen receptor β (ERβ) antagonist (PHTPP) diminished adiponectin secretion in daidzein treated cell, but not in PT treated cells. Transcriptome analyses revealed that daidzein and PT commonly regulated PPAR signaling pathway, although gene expression was differently regulated in PT-treated cells and daidzein-treated cells. The expression of 476 and 380 genes significantly up-regulated in daidzein and PT treatment, although commonly regulated genes were only 86 genes. These results suggested that PT may ameliorate lipid metabolic dysfunction via promote adipocytes differentiation and enhance adiponectin secretion through different mechanism from daidzein.

2017-06-30 | GSE92630 | GEO

A novel IRS-1-associated protein, DGK? regulates GLUT4 translocation in 3T3-L1 adipocytes.

Project description:Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGK?) as an IRS-1-associated protein, and examined roles of DGK? in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGK? was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1? (PIP5K1?), which had been reported to interact with DGK?, also inhibited insulin-induced GLUT4 translocation. Moreover, DGK? interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGK? (short-form DGK?), which competed out DGK? from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGK? prevents GLUT4 translocation in the absence of insulin and that the DGK? dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1? activity.

| S-EPMC5064357 | biostudies-literature

Repression of GLUT4 expression by the endoplasmic reticulum stress response in 3T3-L1 adipocytes.

Project description:Expression of GLUT4 is decreased in adipocytes in obesity and type 2 diabetes, contributing to the insulin resistance of these states. Recent investigations suggest a role for activation of the ER stress response in the pathophysiology of type 2 diabetes. We investigated activation of the ER stress response in 3T3-L1 adipocytes. We show that activation of the ER stress response decreased GLUT4 expression at the level of gene transcription. Activation of the ER stress response also increased the expression of CHOP10, an inhibitor of the activity and expression of C/EBPalpha. As expected, activation of the ER stress response decreased expression of C/EBPalpha, an activator of GLUT4 expression, providing a mechanism to account for the repression of GLUT4 by ER stress activation. Our studies identify repression of GLUT4 expression as another potential mechanism for obesity-induced activation of the ER stress response to contribute to the insulin resistance of obesity.

| S-EPMC2018654 | biostudies-literature

Trafficking of Glut4-green fluorescent protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface glut4 levels.

Project description:Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement ('translocation') of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red(R). K(+)-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3'-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.

| S-EPMC1220673 | biostudies-other

Pig testis extract augments adiponectin expression and secretion through the peroxisome proliferator-activated receptor signaling pathway in 3T3-L1 adipocytes.

Project description:Adiponectin is a key molecule whose upregulation may ameliorate symptoms of type 2 diabetes mellitus and disorders of lipid metabolism. Several plant-derived components have been shown to enhance adiponectin secretion; however, there have been no reports on the effects of animal-derived products. Therefore, in the current study, we investigated whether hot-water extracts of specific livestock by-products induce the expression of adiponectin in mouse 3T3-L1 adipocytes. Out of the 11 extracts tested, pig testis extract (PTE) was found to enhance adiponectin expression and secretion by 3T3-L1 cells. Furthermore, simultaneous treatment with PTE and daidzein, a soy phytoestrogen, synergistically enhanced adiponectin secretion. Moreover, pretreatment with an estrogen receptor β antagonist (PHTPP) diminished adiponectin secretion from daidzein-treated cells but not from PTE-treated cells. Transcriptome analyses revealed that both daidzein and PTE regulate the peroxisome proliferator-activated receptor signaling pathway, although differences in the regulation of gene expression were observed between PTE- and daidzein-treated cells. These results suggest that PTE ameliorates lipid metabolic dysfunction by promoting adipocyte differentiation and enhancing adiponectin secretion via a mechanism different from that of daidzein.

| S-EPMC6021293 | biostudies-literature

Insulin action on GLUT4 traffic visualized in single 3T3-l1 adipocytes by using ultra-fast microscopy.

Project description:A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20-30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5-7 min at 37 degrees C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.

| S-EPMC30573 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data