Phorbol ester only partially mimics the effects of insulin on glucose transport and glucose-transporter distribution in 3T3-L1 adipocytes.
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ABSTRACT:
SUBMITTER: Gibbs EM
PROVIDER: S-EPMC1150025 | biostudies-other | 1991 Apr
REPOSITORIES: biostudies-other
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Project description:The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.
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Project description:The cDNAs for two putative glucose transporters from mouse 3T3-L1 adipocytes were isolated and sequenced. One of these cDNAs encodes the murine homolog of the human hepG2/erythrocyte glucose transporter, termed GT1. GT1 mRNA is most abundant in mouse brain and is expressed in both 3T3-L1 preadipocytes and adipocytes. The other cDNA encodes a glucose transporter-like protein, termed GT2, that has a unique amino acid sequence and tissue distribution. GT2 cDNA encodes a protein with 63% amino acid sequence identity and a similar structural organization to GT1. GT2 mRNA is found at high levels in mouse skeletal muscle, heart, and adipose tissue, all of which exhibit insulin-stimulated glucose uptake. GT2 mRNA is absent from 3T3-L1 preadipocytes but is induced dramatically during differentiation into adipocytes. This increase in mRNA content correlates closely with the acquisition of insulin-stimulated glucose uptake. We propose that GT2 is an insulin-regulated glucose transporter.
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Project description:We have expressed the liver-type facilitative glucose transporter, GLUT2, in the insulin-sensitive 3T3-L1 adipocyte clonal cell line in an effort to address the importance of transporter isoform and cellular environment on the ability of insulin to mediate glucose-transporter translocation. Analysis of non-differentiated fibroblastic cell clones transfected with the GLUT2 cDNA identified the presence of this isoform in several independent clones. These clones exhibited increased deoxyglucose and fructose transport rates compared with control cells. Upon differentiation, the fibroblastic clones selected for study achieved > 95% phenotypic conversion into adipocytes. Expression of the GLUT2 protein was maintained throughout the differentiation protocol. Subcellular fractionation revealed that in response to insulin, unlike the native GLUT4, GLUT2 protein did not undergo significant translocation to the plasma membrane; furthermore, the subcellular distribution of the expressed GLUT2 was quite distinct from that of the endogenous GLUT4. 3T3-L1 adipocytes expressing GLUT2 only exhibited a 2-fold increase in insulin-stimulated fructose uptake, further suggesting that GLUT2 does not undergo insulin-stimulated translocation.
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Project description:The effects of insulin and phorbol 12-myristate 13-acetate (PMA) on the levels of cellular phosphoinositides were investigated in 3T3-L1 adipocytes. Stimulation for 4 min with PMA (1 microM) or insulin (10 nM) increased levels of PtdIns(3,4,5)P3 approx. 2-fold and 6-fold respectively. PMA also had a small effect on the cellular levels of PtdIns4P, whereas insulin had no effect on PtdIns4P levels; levels of PtdIns(4,5)P2 and PtdIns3P were not significantly affected by either agent. Insulin increased the levels of the p85 alpha subunit of phosphoinositide (PI) 3-kinase associated with membranes, whereas PMA decreased levels of membrane-associated p85 alpha. PMA did not increase PI 3-kinase activity in anti-phosphotyrosine or anti-p85 immunoprecipitates. The stimulation of glucose transport by insulin or PMA was blocked by 100 nM wortmannin or 10 ng/ml LY294002, indicating that PI 3-kinase is essential for stimulation by both agents. In summary, these results demonstrate: (1) that PMA and insulin stimulate PtdIns(3,4,5)P3 production by distinct mechanisms in 3T3-L1 adipocytes, and (2) that stimulation of PtdIns(3,4,5)P3 production by PMA is likely to be important in signalling pathways leading from PMA stimulation to end-point responses such as glucose transport.
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