Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes

The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.     

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.     

Insulin stimulates the assembly of cytosolic clathrin onto adipocyte plasma membranes

The effects of insulin on the subcellular distribution of the heavy chain of clathrin and on the insulin-like growth factor II (IGF-II) mannose 6-phosphate receptor were investigated in isolated rat adipocytes. Plasma membranes, intracellular membranes, and cytosol were separated by differential centrifugation, and the concentration of clathrin and receptor in each fraction was quantified by sequential immunoblotting with monoclonal and polyclonal antibodies against these proteins. A 3-fold increase in the amount of clathrin heavy chain associated with isolated plasma membranes was found after treatment of cells with low concentrations of insulin. This effect was complete within 2 min of stimulation at 37 degrees C and was abolished at 5-10 degrees C. The insulin-mediated increase in the cell surface concentration of receptors for IGF-II/mannose 6-phosphate displayed a similar time course and temperature dependence. A concomitant decrease in the concentration of IGF-II/mannose 6-phosphate receptors in intracellular membranes was observed. In contrast, no significant changes in the concentration of clathrin in this fraction could be detected. Instead, a marked decrease in the level of unassembled cytosolic clathrin was observed in insulin-treated cells compared with controls. These results suggest that insulin induces an increase in the assembly of cytosolic clathrin onto the plasma membrane in conjunction with its ability to increase the concentration of receptors on the cell surface.     

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.     

Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface-derived vesicle fraction of 3T3-L1 adipocytes. Inhibited lateral mobility of integral plasma membrane proteins in newly inserted membrane areas of differentiated 3T3-L1 cells

The recent demonstration of a large cell surface-derived pool of insulin-sensitive glucose transporters, presumably concentrated in the microvilli of 3T3-L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3-L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle-specific transporter, GluT4, expressed for the first time 3-4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3-L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface-derived vesicle (LDSV) fraction of 3T3-L1 adipocytes most likely consisting of microvilli-derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.     

Regulation of insulin-responsive aminopeptidase expression and targeting in the insulin-responsive vesicle compartment of glucose transporter isoform 4-deficient cardiomyocytes

In adipocytes and cardiac or skeletal muscle, glucose transporter isoform 4 (GLUT4) is targeted to insulin-responsive intracellular membrane vesicles (IRVs) that contain several membrane proteins, including insulin-responsive aminopeptidase (IRAP) that completely colocalizes with GLUT4 in basal and insulin-treated cells. Cardiac GLUT4 content is reduced by 65-85% in IRAP knockout mice, suggesting that IRAP may regulate the targeting or degradation of GLUT4. To determine whether GLUT4 is required for maintenance of IRAP content within IRVs, we studied the expression and cellular localization of IRAP and other GLUT4 vesicle-associated proteins, in hearts of mice with cardiac-specific deletion of GLUT4 (G4H-/-). In G4H-/- hearts, IRAP content was reduced by 60%, but the expression of other vesicle-associated proteins, namely cellugyrin, IGF-II/mannose-6-phosphate, and transferrin receptors, secretory carrier-associated membrane proteins and vesicle-associated membrane protein were unchanged. Using sucrose gradient centrifugation and cell surface biotinylation, we found that IRAP content in 50-80S vesicles where GLUT4 vesicles normally sediment was markedly depleted in G4H-/- hearts, and the remaining IRAP was found in the heavy membrane fraction. Although insulin caused a discernible increase in cell surface IRAP content of G4H-/- cardiomyocytes, cell surface IRAP remained 70% lower than insulin-stimulated controls. Immunoabsorption of intracellular vesicles with anticellugyrin antibodies revealed that IRAP content was reduced by 70% in both cellugyrin-positive and cellugyrin-negative vesicles. Endosomal recycling, as measured by transferrin receptor recycling was normal. Thus, GLUT4 and IRAP content of early endosome-derived sorting vesicles and of IRVs are coordinately regulated, and both proteins are required for maintenance of key constituents of these compartments in cardiac muscle cells in vivo.     

The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin

At maximally active concentrations with 20-min exposure, insulin and phorbol myristate acetate (PMA) stimulated hexose transport in 3T3-L1 adipocytes by 11- and 2-fold, respectively. The potential role of phosphorylation of the glucose transporter (GT) in these stimulations was investigated by the isolation of GT through immunoprecipitation from ortho[32P]phosphate-labeled 3T3-L1 adipocytes. It was found that there was no significant 32P incorporation into GT from basal adipocytes after 2- or 18 h-labeling in the presence of 0.5 mCi of 32Pi/ml. Furthermore, under these labeling conditions, insulin treatment for 1, 4, or 30 min failed to stimulate the phosphorylation of GT. Also, there was no detectable phosphate incorporation into GT upon reversal of insulin-stimulated hexose transport by the removal of insulin (half-time for reversal approximately 8 min). In contrast to these results, exposure of adipocytes to PMA (1 microM) for 20 min elicited a phosphorylation of GT to the extent of about 0.1 phosphate/GT molecule. Exposure of cells to both insulin and PMA resulted in a 3-fold increase in the level of phosphate in GT compared to that seen with PMA alone. Possibly this increase is due to the translocation of GT to the plasma membrane where it is a better substrate for activated protein kinase C. Stimulation of hexose transport was the same with the combined treatment of insulin and PMA compared to that seen with insulin alone. These results indicate that neither a change in the phosphorylation state of the GT nor activation of protein kinase C is involved in the mechanism by which the insulin receptor stimulates glucose transport.     

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.     

The formation of an insulin-responsive vesicular cargo compartment is an early event in 3T3-L1 adipocyte differentiation

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.     

Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes

Insulin regulates glucose transport in muscle and adipose tissue by triggering the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.     

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.     

Evidence for a role of the exocyst in insulin-stimulated Glut4 trafficking in 3T3-L1 adipocytes

Insulin stimulates glucose transport in adipocytes and muscle by inducing the redistribution of Glut4 from intracellular locations to the plasma membrane. The fusion of Glut4-containing vesicles at the plasma membrane is known to involve the target SNAREs syntaxin 4 and SNAP-23 and the vesicle SNARE VAMP2. Little is known about the initial docking of Glut4 vesicles with the plasma membrane. A recent report has implicated Exo70, a component of the mammalian exocyst complex, in the initial interaction of Glut4 vesicles with the adipocyte plasma membrane. Here, we have examined the role of two other exocyst components, rsec6 and rsec8. We show that insulin promotes a redistribution of rsec6 and rsec8 to the plasma membrane and to cytoskeletal fractions within 3T3-L1 adipocytes but does not modulate levels of these proteins co-localized with Glut4. We further show that adenoviral-mediated overexpression of either rsec6 or rsec8 increases the magnitude of insulin-stimulated glucose transport in 3T3-L1 adipocytes. By contrast, overexpression of rsec6 or rsec8 did not increase the extent of the secretion of adipsin or ACRP30 from adipocytes and had no discernible effect on transferrin receptor traffic. Collectively, our data support a role for the exocyst in insulin-stimulated glucose transport and suggest a model by which insulin-dependent relocation of the exocyst to the plasma membrane may contribute to the specificity of Glut4 vesicle docking and fusion with the adipocyte plasma membrane.     

Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.     

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin- sensitive cells

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle- associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP- 2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.     

Selective changes in enzymes of the sn-glycerol 3-phosphate and dihydroxyacetone-phosphate pathways of triacylglycerol biosynthesis during differentiation of 3T3-L1 preadipocytes

Enzyme activities of the sn-glycerol 3-phosphate (glycerol-P) and of the dihydroxyacetone-phosphte (DHAP) pathway of glycerolipid biosynthesis were investigated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Total particulate glycerol-P and DHAP acyltransferase activities increased 70- and 30-fold, respectively, during differentiation induced with methylisobutylxanthine and dexamethasone. The N-ethylmaleimide-sensitive (microsomal) glycerol-P and DHAP acyltransferase activities were virtually undetectable in nondifferentiated cells, and increased in parallel over 70-fold during differentiation. These and several kinetic observations are consistent with the induction of a single microsomal enzyme having dual activity. During differentiaion, the N-ethylmaleimide-resistant DHAP acyltransferase activity increased 10-fold, suggesting the presence of at least two DHAP acyltransferase isoenzymes. Qualitatively similar changes in microsomal glycerol-P and DHAP acyltransferase activities were observed when cell differentiation was induced with insulin or with insulin plus dexamethasone and methylisobutylxanthine. Acyl-DHAP oxidoreductase (EC 1.1.1.101) specific activity increased only 3- to 5-fold during adipocyte differentiation. Alkyl-DHAP synthase activity was not detected. These data demonstrate that selective changes in enzyme activities of the gycerol-P pathways of glycerolipid synthesis occur during the differentiation of 3T3-L1 preadipocytes.     

Dexamethasone regulates the beta-adrenergic receptor subtype expressed by 3T3 L1 preadipocytes and adipocytes

The subtype of the beta-adrenergic receptor expressed in 3T3-L1 preadipocytes and adipocytes differentiated with dexamethasone and methylisobutylxanthine was determined by comparing the affinity of the receptors for epinephrine, norepinephrine, and beta-1 and beta-2 selective antagonist, 8-fold more avidly than adipocyte receptors. In contrast, adipocyte beta-receptors had a 10-fold higher affinity for epinephrine than for norepinephrine and complexed the beta-2 selective agonist zinterol with a 20-fold higher affinity than preadipocyte receptors. Hofstee plots and computer analyses of the binding data revealed that the populations of beta-1 receptors in preadipocytes and beta-2 receptors in adipocytes were nearly homogeneous. Preliminary characterizations of the beta-receptor phenotype in (nondifferentiating) 3T3-C2 cells treated with dexamethasone and methylisobutylxanthine and 3T3-422A adipocytes differentiated with insulin indicated that the expression of beta-2 receptors was not correlated with differentiation, but rather with exposure of the cells to dexamethasone and methylisobutylxanthine. The regulator of beta-receptor subtype was identified as the glucocorticoid analog, dexamethasone, by employing 3T3-L1 adipocytes which were stimulated to differentiate with methylisobutylxanthine and insulin. Detailed binding studies showed that under these conditions the adipocyte receptors retain beta-1 character. Subsequent treatment with 0.5 microM dexamethasone promoted the loss of beta-1 receptors, the appearance of beta-2 receptors, and a net 2- to 3-fold increase in the number of beta-receptors. Dexamethasone effected a complete switch from beta-1 to beta-2 subtype at concentrations as low as 2.5 nM while other steroids were ineffective below a concentration of 10 microM.     

Regulation of triacylglycerol hydrolase expression by dietary fatty acids and peroxisomal proliferator-activated receptors

Triacylglycerol hydrolase (TGH) is an enzyme that catalyzes the lipolysis of intracellular stored triacylglycerol (TG). Peroxisomal proliferator-activated receptors (PPAR) regulate a multitude of genes involved in lipid homeostasis. Polyunsaturated fatty acids (PUFA) are PPAR ligands and fatty acids are produced via TGH activity, so we studied whether dietary fats and PPAR agonists could regulate TGH expression. In 3T3-L1 adipocytes, TGH expression was increased 10-fold upon differentiation, compared to pre-adipocytes. 3T3-L1 cells incubated with a PPARγ agonist during the differentiation process resulted in a 5-fold increase in TGH expression compared to control cells. Evidence for direct regulation of TGH expression by PPARγ could not be demonstrated as TGH expression was not affected by a 24-h incubation of mature 3T3-L1 adipocytes with the PPARγ agonist. Feeding mice diets enriched in fatty acids for 3 weeks did not affect hepatic TGH expression, though a 3-week diet enriched in fatty acids and cholesterol increased hepatic TGH expression 2-fold. Two weeks of clofibrate feeding did not significantly affect hepatic TGH expression or microsomal lipolytic activities in wild-type or PPARα-null mice, indicating that PPARα does not regulate hepatic TGH expression. Therefore, TGH expression does not appear to be directly regulated by PPARs or fatty acids in the liver or adipocytes.     

Insulin-responsive glucose transporters are concentrated in a cell surface-derived membrane fraction of 3T3-L1 adipocytes

The recently proposed mechanistic concept of a receptor-regulated entrance compartment for hexose transport formed by microvilli on 3T3-L1 adipocytes predicted a preferential localization of glucose transporters in these structures. The cytochalasin B-binding technique was used to determine in basal and insulin-stimulated cells the distribution of glucose transporters between plasma membranes, low density microsomes (LDM) and two cell surface-derived membrane fractions prepared by a hydrodynamic shearing technique. The shearing procedure applied prior to homogenization yielded a low density surface-derived vesicle (LDSV) fraction which contained nearly 60% of the cellular glucose transporters and the total insulin-sensitive transporter pool. The rest of the glucose transporter population was localized within the plasma membrane (5%) and the LDM fraction (37%). Pretreatment of the cells with insulin (20 mU/ml for 10 min) reduced the transporter content of the LDSV fraction by 40% and increased that of the plasma membrane fraction 4-fold. The transporter containing LDSV fraction was clearly differentiated from the LDM fraction by its low specific galactosyltransferase activity and its insulin-sensitivity. Scanning electron microscopy revealed that the LDSV fraction contained a rather uniform population of spherical vesicles of 100-200 nm in diameter.     

Regulation of the expression of pp160, a putative insulin receptor signal protein, by insulin, dexamethasone, and 1-methyl-3-isobutylxanthine in 3T3-L1 adipocytes.

pp160, a cytosolic protein with Mr of approximately 160,000, is phosphorylated on tyrosine in response to insulin and is considered to be involved in signaling from the insulin receptor. The expression of pp160 during the differentiation of 3T3-L1 fibroblasts to adipocytes and in adipocytes has been investigated using quantitative immunoblotting with antibodies against a peptide from pp160. Between day 6 and day 8 of differentiation induced by insulin, dexamethasone (Dex), and 1-methyl-3-isobutylxanthine (Mix), pp160 expression increased 10-20-fold over the amount present in confluent fibroblasts. Omission of either insulin or Dex resulted in reduced expression of pp160 and in incomplete adipogenesis. Chronic treatment of fully differentiated adipocytes for 24 h with either insulin, Dex, or Mix alone in the presence of serum resulted in a decrease in the expression of pp160 by 70-85%. Chronic exposure to insulin caused a significant increase in the apparent size of pp160 to 172 kDa. Alkaline phosphatase treatment lowered the Mr of pp160 from both insulin-treated and basal cells to 150,000. These results demonstrate that pp160 is expressed in 3T3-L1 adipocytes during the time when insulin receptors are expressed in large numbers and that the maintenance of pp160 concentrations in adipocytes can be regulated by insulin, Mix, and Dex. The decreased expression of pp160 caused by these factors may be related to postreceptor insulin resistance.