Glut4 Is Targeted to Specific Vesicles in Adipocytes of Transgenic Mice Overexpressing Glut4 Selectively in Adipose Tissue

Tozzo, Effie; Kahn, Barbara B.; Pilch, Paul F.; Kandror, Konstantin V.

doi:10.1074/jbc.271.18.10490

Cited by 18 publications

(14 citation statements)

References 37 publications

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“…With this moderate level of overexpression, it is unlikely that the normal sorting mechanisms were saturated. Indeed, in adipocytes of transgenic mice that markedly overexpressed Glut 4 (15-20 fold) in fat tissue, the transporters appeared to be targeted to the same unique structurally defined vesicles as in native cells (25).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of a Constitutively Active Form of Phosphatidylinositol 3-Kinase Is Sufficient to Promote Glut 4 Translocation in Adipocytes

Tanti

Grémeaux

Grillo

et al. 1996

Journal of Biological Chemistry

151

109

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Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitopetagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.Insulin promotes glucose uptake in muscle and adipose tissue by increasing the translocation of the glucose transporter Glut 4 from an intracellular compartment to the plasma membrane, but the exact molecular mechanism is still undetermined (1, 2). Following insulin binding, the insulin receptor phosphorylates on tyrosine residues different substrates, including insulin receptor substrate 1 and 2 (IRS1 1 and IRS2)and Src homology collagen (SHC). The phosphorylated IRS1 docks proteins containing Src homology 2 (SH2) domains, such as phosphatidylinositol 3-kinase (PI 3-kinase), Nck, Syp, and Grb2 (3). Thus, the p85 subunit of PI 3-kinase binds through its SH2 domains to the tyrosine phosphorylated YMXM motifs of IRS1 (3); the consequences are an increase in the catalytic activity of the PI 3-kinase p110 subunit (4) and a rise in the intracellular level of PI 3,4-biphosphate and PI 3,4,5-triphosphate. A series of recent data indicates that PI 3-kinase is involved in insulin-induced Glut 4 translocation. For example, the blockade of Glut 4 translocation by pharmacological inhibitors of PI 3-kinase, such as wortmannin or LY294002 (5-8), or by a dominant negative mutant of PI 3-kinase (9, 10) indicates that PI 3-kinase activation is a necessary step for the insulin stimulation of transporter trans...

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Section: Discussionmentioning

confidence: 99%

Overexpression of a Constitutively Active Form of Phosphatidylinositol 3-Kinase Is Sufficient to Promote Glut 4 Translocation in Adipocytes

Tanti

Grémeaux

Grillo

et al. 1996

Journal of Biological Chemistry

151

109

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“…Taken together, these results demonstrate that intracellular sequestration of GLUT4 in adipocytes is saturable. We showed previously GLUT4 is targeted to specific vesicles in adipocytes of aP2-GLUT4-Tg mice, which have a similar composition to control adipocytes (27). However, we examined only intracellular vesicles, and we could not rule out that GLUT4 might also be present in other structures that are more likely to target to the plasma membrane.…”

Section: Intracellular Sequestration Of Glut4 Is Saturable In Adipo-mentioning

confidence: 99%

“…We have shown previously (27) that GLUT4-containing vesicles from both WT and aP2-GLUT4-Tg adipocytes exclude GLUT1, a glucose transporter that resides in a different vesicle population than GLUT4 and traffics to the PM more constitutively. These data are consistent with the lack of effect of GLUT4 overexpression on GLUT1 subcellular distribution (Fig.…”

Section: Intracellular Sequestration Of Glut4 Is Saturable In Adipo-mentioning

confidence: 99%

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GLUT4 Overexpression or Deficiency in Adipocytes of Transgenic Mice Alters the Composition of GLUT4 Vesicles and the Subcellular Localization of GLUT4 and Insulin-responsive Aminopeptidase

Carvalho

Schellhorn

Zabolotny

et al. 2004

Journal of Biological Chemistry

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The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4؊/؊) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80 -95% in aP2-GLUT4؊/؊ adipocytes and increased ϳ10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4؊/؊ adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4؊/؊ adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4؊/؊ or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4-and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

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“…The fact that there is no change in the sedimentation coefficient of the vesicles in comparing day 3 and day 9 (Figures 4 -6) supports this notion, as does additional data we have obtained. Overexpression of GLUT4 in fat cells from transgenic mice results in GLUT4-containing vesicles indistinguishable from their normal counterparts in sedimentation behavior, insulin responsiveness, and protein content other than GLUT4 (Tozzo et al, 1996). More recently, we have obtained vesicles from denervated rat skeletal muscle, in which GLUT4 expression is dramatically reduced (Coderre et al, 1992), the opposite expression level of the transgenic study, and again, the resultant vesicles show reduced GLUT4 content, but they sediment at the same rate as those from normal muscle and show a normal profile of other vesicle proteins such as IRAP and the receptors for transferrin and mannose-6-phosphate (Zhou et al, 1998) Thus, we think that the GLUT4-containing vesicular compartment represents an insulin-regulatable, postendosomal cargo vesicle whose behavior is not dependent on the presence of the major cargo proteins.…”

Section: Formation Of Insulin-responsive Vesiclesmentioning

confidence: 99%

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Jack

Kandror

Pilch

1999

MBoC

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Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles. INTRODUCTIONThe insulin-stimulated glucose transport that regulates postprandial blood glucose levels occurs principally as a result of the insulin-dependent translocation of glucose transporters from an intracellular storage pool to the cell surface (for review, see Kandror and Pilch, 1996a;Rea and James, 1997). The tissue-specific glucose transporter isoform glucose transporter 4 (GLUT4) 1 (Kandror and Pilch, 1996a;Rea and James, 1997) is responsible for most of the transport function in fat and muscle, but the ubiquitous GLUT1 glucose transporter isoform is expressed to an appreciable extent in adipocytes, where it also shows insulin-dependent translocation to the cell surface (Zorzano et al., 1989;Holman et al., 1990). Despite extensive study (for review, see Kandror and Pilch, 1996a;Rea and James, 1997), much remains unknown about the cellular trafficking pathway of GLUT4, including its biochemical basis and relationship to other established cellular pathways of protein traffic and secretion. In particular, it is not yet clear whether the GLUT4-containing compartment represents a specialized secretory organelle, analogous to synaptic vesicles in the brain and unique to fat and muscle, or whether, during the process of differentiation, insulin sensitivity has been applied to a preexisting recycling pathway(s), such as endosome to cell surface recycling, which is present in nondifferentiated precursors.Morphological studies of GLUT4 distribution in a variety of insulin-sensitive tissues have used immunogold electron microscopy to determine that intracellular GLUT4 is localized principally in small uniform vesicles near the cell surface, as well as in small tubulovesicular elements. These tissue...

show abstract

Glut4 Is Targeted to Specific Vesicles in Adipocytes of Transgenic Mice Overexpressing Glut4 Selectively in Adipose Tissue

Cited by 18 publications

References 37 publications

Overexpression of a Constitutively Active Form of Phosphatidylinositol 3-Kinase Is Sufficient to Promote Glut 4 Translocation in Adipocytes

Overexpression of a Constitutively Active Form of Phosphatidylinositol 3-Kinase Is Sufficient to Promote Glut 4 Translocation in Adipocytes

GLUT4 Overexpression or Deficiency in Adipocytes of Transgenic Mice Alters the Composition of GLUT4 Vesicles and the Subcellular Localization of GLUT4 and Insulin-responsive Aminopeptidase

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

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