Complete Membrane Fractionation of 3T3-L1 Adipocytes

Sadler, Jessica B. A.; Lamb, Christopher A.; Gould, Gwyn W.; Bryant, Nia J.

doi:10.1101/pdb.prot083691

Cited by 8 publications

(7 citation statements)

References 3 publications

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“…Plates were washed using sterile HES buffer, scraped and homogenised using a Dounce homogeniser and subcellular fraction performed as described [ 34 ]. This procedure generates a fraction enriched in the PM (PM), high-density membranes (HDM), low-density membranes (LDM) and soluble protein fraction [ 45–47 ]. Insulin results in a redistribution of GLUT4 from the LDM to the PM fraction.…”

Section: Methodsmentioning

confidence: 99%

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

et al. 2022

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Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIa to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIa impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

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Section: Methodsmentioning

confidence: 99%

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

et al. 2022

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“…This was followed by centrifugation at 16,000 g ; the Supernatant from this fraction (16k S) has been shown to be enriched in GLUT4-containing vesicles, i.e. GSCs ( Sadler et al, 2016a , b ), whereas its pellet (16k P) contains dense membranes, including plasma membranes. Samples were resuspended in buffer for protein determination, and analysed by SDS-PAGE and immunoblotting exactly as described previously ( Sadler et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

“…Adipocytes were homogenised and subjected to a subcellular fractionation protocol by successive centrifugation at 1000 x g to remove large debris and intact cells; some of the plasma membrane is also present within this fraction. This was followed by centrifugation at 16,000 x g. The Supernatant from this fraction has been shown to be enriched in GLUT4-containing vesicles (the GSC) (Sadler et al, 2016a(Sadler et al, , 2016b. The pellet contains dense membranes, including plasma membranes.…”

Section: Subcellular Fractionation and Immunoblottingmentioning

confidence: 99%

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

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Livingstone

Mastick

et al. 2022

Journal of Cell Science

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Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with 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 HA-GLUT4-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.

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“…The pellet was placed onto the high‐sucrose HES cushions and centrifuged at 41 000 g for 60 minutes at 4°C. The plasma membrane fraction was found between the 2 resulting layers of HES …”

Section: Methodsmentioning

confidence: 99%

GLUT12 and adipose tissue: Expression, regulation and its relation with obesity in mice

et al. 2019

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Aim:The facilitative glucose transporter GLUT12 was isolated from the breast cancer cell line MCF-7 by its homology with GLUT4. GLUT12 is expressed in insulin-sensitive tissues such as adipose tissue. The aim of this work was to investigate GLUT12 expression and hormonal regulation in 3T3-L1 adipocytes and in adipose tissue of lean and diet-induced obese mice. Methods: Uptake studies were performed using radio-labelled sugars; α-methyld-glucose (αMG) was used as specific substrate of GLUT12. Expression and localization of GLUT12 in adipocytes were investigated by western blot and immunohistochemical methods. Results: GLUT12 is expressed in the peri-nuclear region of mouse adipocytes.Insulin, by AKT activation, and TNF-α, by AMPK activation, increase αMG uptake by inducing GLUT12 translocation to the membrane. In contrast, leptin and adiponectin decrease GLUT12 activity through its internalization. Under hypoxia conditions GLUT12 expression is upregulated. The response of GLUT12 to TNF-α, leptin, adiponectin and hypoxia is the opposite to that of GLUT4. In diet-induced obese mice and obese subjects, GLUT12 protein is decreased. Intraperitoneal injection of insulin increases AKT phosphorylation and GLUT12 expression, but this effect is lost in obese animals. Conclusion: We hypothesize that GLUT12 would contribute to modulate sugar absorption in physiological and pathophysiological situations such as obesity.

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Complete Membrane Fractionation of 3T3-L1 Adipocytes

Cited by 8 publications

References 3 publications

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

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

GLUT12 and adipose tissue: Expression, regulation and its relation with obesity in mice

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