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

Abstract

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...

Regulation of lipid accumulation in 3T3-L1 cells: insulin-independent and combined effects of fatty acids and insulin

“…This observation is consistent with other studies in 3T3-L1 cells (El-Jack et al, 1999;Gross et al, 2004) in transition from preadipocyte to adipocyte morphology. Interestingly, in the present study no insulin effect on total cellular GLUT-4 expression was observed as might have been expected; although it may be assumed that GLUT-4 would be recruited to the plasma membrane in response to insulin.…”

p115 Interacts with the GLUT4 Vesicle Protein, IRAP, and Plays a Critical Role in Insulin-stimulated GLUT4 Translocation

“…Importantly and as cited in the introduction, IRAP was also identified in this way as marker for GSV trafficking that is apparently more abundant than GLUT4 itself. Indeed, we have shown that IRAP is directed to GSVs during the course of 3T3-L1 adipocyte differentiation before significant GLUT4 expression occurs (El-Jack et al, 1999). Moreover, in engineered adipocytes, IRAP can become essentially the only major cargo constituent of GSVs and it responds to insulin in a manner similar or identical to GSVs from 3T3-L1 cells (Gross et al, 2004).…”

“…Efforts to further understand this phenomenon are underway. The development of GSVs occurs during the differentiation of adipocytes, presumably as a result of the expression of an as yet unknown protein or proteins that initially results in the sequestration of IRAP, then GLUT4 (El-Jack et al, 1999). It is unlikely that p115 is one of these putative proteins as its expression is unchanged over the 8 -10 d required for adipocytes to fully differentiate (unpublished data)…”

“…IRAP undergoes increased intracellular sequestration upon differentiation of 3T3-L1 adipocytes in correlation with the development of an insulin responsive compartment whose formation precedes GLUT4 expression during the differentiation process El-Jack et al, 1999). Recently, we reported that GLUT4 is not absolutely necessary for the formation of GSVs in engineered adipocytes and that it is targeted to a preexisting vesicle, which can form independently of GLUT4 as adipocytes differentiate (Gross et al, 2004).…”

“…IRAP displays similar trafficking kinetics to GLUT4 in 3T3-L1 adipocytes (Garza and Birnbaum, 2000), although there may be some differences in the internalization rate of these two proteins in rat adipocytes (Kandror, 1999). In any case, these citations document considerable evidence that IRAP is a marker for insulindependent GLUT4/GSV trafficking.IRAP undergoes increased intracellular sequestration upon differentiation of 3T3-L1 adipocytes in correlation with the development of an insulin responsive compartment whose formation precedes GLUT4 expression during the differentiation process El-Jack et al, 1999). Recently, we reported that GLUT4 is not absolutely necessary for the formation of GSVs in engineered adipocytes and that it is targeted to a preexisting vesicle, which can form independently of GLUT4 as adipocytes differentiate (Gross et al, 2004).…”