Basal Adipocytes Research Articles

On the Validity of Adipogenic Cell Lines as Model Systems for Browning Processes: In Authentic Brown, Brite/Beige, and White Preadipocytes, There is No Cell-Autonomous Thermogenic Recruitment by Green Tea Compounds.

The potential ability of nutritional compounds to induce or enhance the browning of adipocytes has attracted large interest as a workable means of combatting the obesity epidemic. Green tea compounds are discussed as such inducers of an enhanced thermogenic capacity and activity. However, the cell-autonomous effects of green tea compounds on adipocytes have until now only been demonstrated in adipogenic cell lines (3T3-L1 and 3T3-F442A), i.e., cells of undefined tissue lineage. In this study, we examine the ability of green tea compounds to cell-autonomously induce thermogenic recruitment in authentic brown and brite/beige adipocytes in vitro. In primary brown adipocytes, the green tea compounds suppressed basal UCP1 gene expression, and there was no positive interaction between the compounds and adrenergic stimulation. In white adipocytes, green tea compounds decreased both basal and norepinephrine-induced UCP1 mRNA levels, and this was associated with the suppression of cell differentiation, indicated by reduced lipogenic gene expression and lipid accumulation. A lack of interaction between rosiglitazone and green tea compounds suggests that the green tea compounds do not directly interact with the PPARγ pathway. We conclude that there is a negative effect of the green tea compounds on basal UCP1 gene expression, in both brown and white primary adipocytes, in contrast to the positive effects earlier reported from studies in adipogenic cell lines. We posit that the epigenetic status of the adipogenic cell lines is fundamentally different from that of genuine brown and white adipocytes, reflected, e.g., in several-thousand-fold differences in UCP1 gene expression levels. Thus, results obtained with adipogenic cell lines cannot unreservedly be extrapolated as being relevant for authentic effects in brown and white adipocytes. We suggest that this conclusion can be of general concern for studies attempting to establish physiologically relevant cell-autonomous effects.

Pseudolipomatosis Cutis: A Retrospective Case Series of Skin Biopsies Showing a Distinctive Vacuolar Artifactual Change.

Pseudolipomatosis cutis is a distinctive artifact characterized by the presence of adipocyte-like vacuoles in the biopsy specimens. We describe this change in a series of skin biopsies clustered over 2 periods, November 2017-December 2017 and February 2019. Hematoxylin and eosin-stained sections from 21 skin biopsies were examined by 2 dermatopathologists. Variable-sized vacuoles were noted in the epidermis, dermis, and/or subcutaneous fat in all biopsies. Vacuoles were seen in the stratum corneum in 2 biopsies, epidermis in 15 biopsies, papillary dermis in 19 biopsies, reticular dermis in 20 biopsies, and subcutis in 9 biopsies. The vacuoles resembled a true histological change in many biopsies: vacuolated keratinocytes (n = 6), basal cell vacuolization (n = 6), foamy histiocytes (n = 12), and adipocytes (n = 20). Other changes included dermal collagen sclerosis and empty ectatic vascular channels. The clustering of this peculiar change in biopsies from patients with unrelated skin diseases that stopped on changing the formalin solution in both instances suggests that it is an artifactual change due to improper tissue fixation.

Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.

Insulin-regulated Glut4 Translocation: MEMBRANE PROTEIN TRAFFICKING WITH SIX DISTINCTIVE STEPS

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min(-1) versus 0.6 min(-1)). Differentiation decreases Glut4 ken 40% (ken = 0.12 min(-1)). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025-0.038 min(-1)), not through the fast Tf receptor pathway (kex = 0.2 min(-1)). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min(-1)). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.

Loss of AS160 Akt Substrate Causes Glut4 Protein to Accumulate in Compartments That Are Primed for Fusion in Basal Adipocytes

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulin-mimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k(ex)). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k(ex)), endocytosis (k(en)), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k(ex) or k(en). Insulin increased k(ex) in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k(ex) and release from retention in control cells but only k(ex) in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulin-regulated step.

GLUT4 Is Sorted to Vesicles Whose Accumulation Beneath and Insertion into the Plasma Membrane Are Differentially Regulated by Insulin and Selectively Affected by Insulin Resistance

Insulin stimulates glucose transport by recruiting the GLUT4 glucose transporter to the plasma membrane. Here we use total internal reflection fluorescence microscopy to show that two trafficking motifs of GLUT4, a FQQI motif and a TELE-based motif, target GLUT4 to specialized vesicles that accumulate adjacent to the plasma membrane of unstimulated adipocytes. Mutations of these motifs redistributed GLUT4 to transferrin-containing recycling vesicles adjacent to the plasma membrane, and the degree of redistribution correlated with the increases of the GLUT4 mutants in the plasma membrane of basal adipocytes. These results establish that GLUT4 defaults to recycling endosomes when trafficking to specialized vesicles is disrupted, supporting the hypothesis that the specialized vesicles are derived from an endosomal compartment. Insulin stimulates both the accumulation of GLUT4 in the evanescent field and the fraction of this GLUT4 that is inserted into the plasma membrane. Unexpectedly, these two steps are differentially affected by the development of insulin resistance. We ascribe this selective insulin resistance to inherent differences in the sensitivities of GLUT4 vesicle accumulation and insertion into the plasma membrane to insulin. Differences in insulin sensitivities of various processes may be a general mechanism for the development of the physiologically important phenomenon of selective insulin resistance.

Molecular mechanisms controlling GLUT4 intracellular retention.

In basal adipocytes, glucose transporter 4 (GLUT4) is sequestered intracellularly by an insulin-reversible retention mechanism. Here, we analyze the roles of three GLUT4 trafficking motifs (FQQI, TELEY, and LL), providing molecular links between insulin signaling, cellular trafficking machinery, and the motifs in the specialized trafficking of GLUT4. Our results support a GLUT4 retention model that involves two linked intracellular cycles: one between endosomes and a retention compartment, and the other between endosomes and specialized GLUT4 transport vesicles. Targeting of GLUT4 to the former is dependent on the FQQI motif and its targeting to the latter is dependent on the TELEY motif. These two motifs act independently in retention, with the TELEY-dependent step being under the control of signaling downstream of the AS160 rab GTPase activating protein. Segregation of GLUT4 from endosomes, although positively correlated with the degree of basal retention, does not completely account for GLUT4 retention or insulin-responsiveness. Mutation of the LL motif slows return to basal intracellular retention after insulin withdrawal. Knockdown of clathrin adaptin protein complex-1 (AP-1) causes a delay in the return to intracellular retention after insulin withdrawal. The effects of mutating the LL motif and knockdown of AP-1 were not additive, establishing that AP-1 regulation of GLUT4 trafficking requires the LL motif.

Rab10, a Target of the AS160 Rab GAP, Is Required for Insulin-Stimulated Translocation of GLUT4 to the Adipocyte Plasma Membrane

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a ;reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.

GLUT4 is internalized by a cholesterol-dependent nystatin-sensitive mechanism inhibited by insulin

Insulin slows GLUT4 internalization by an unknown mechanism. Here we show that in unstimulated adipocytes, GLUT4 is internalized by two mechanisms. Approximately 80% of GLUT4 is internalized by a mechanism that is sensitive to the cholesterol-aggregating drug nystatin, and is independent of AP-2 clathrin adaptor and two putative GLUT4 endocytic motifs. The remaining GLUT4 is internalized by an AP-2-dependent, nystatin-resistant pathway that requires the FQQI GLUT4 motif. Insulin inhibits GLUT4 uptake by the nystatin-sensitive pathway and, consequently, GLUT4 is internalized by the AP-2-dependent pathway in stimulated adipocytes. The phenylalanine-based FQQI GLUT4 motif promotes AP-2-dependent internalization less rapidly than a tyrosine-based motif, the classic form of aromatic-based motifs. Thus, both a change in the predominant endocytosis pathway and the specific use of a suboptimal internalization motif contribute to the slowing of GLUT4 internalization in insulin-stimulated adipocytes. Insulin also inhibits the uptake of cholera-toxin B, indicating that insulin broadly regulates cholesterol-dependent uptake mechanisms rather than specially targeting GLUT4. Our work thus identifies cholesterol-dependent uptake as a novel target of insulin action in adipocytes.

Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation.

Somatostatin (SRIF) is a well-known neuroendocrine secretion product. SRIF expression and secretion are induced after inflammation in murine macrophages and in endotoxin-injected sheep and pigs. Because adipocytes have been demonstrated to produce numerous cytokines and peptide hormones, we investigated the expression of SRIF and its receptors (SSTR1-5) in human adipose tissue after inflammatory stimulation in vitro and in tissues from patients with septic disease.Preadipocyte-derived adipocytes, mesenchymal stem cell-derived adipocytes, and mature explanted adipocytes expressed SRIF-mRNA after endotoxin [lipopolysaccharide (LPS)] or IL-1beta treatments. LPS- and IL-1beta-mediated SRIF-mRNA induction was blocked by pretreatment with dexamethasone. Using cocultures and quantitative real-time PCR, we demonstrate adipocyte SRIF induction by secretion factors from activated peripheral blood mononuclear cell-derived macrophages. In contrast to basal adipocytes, SRIF protein was detected in culture supernatants of LPS-treated and of combined TNFalpha/IL-1beta/LPS-treated adipocytes. SRIF protein was visualized by immunohistochemistry in explanted minced adipose tissue after overnight incubation in culture medium supplemented with combined IL-1beta and LPS. In septic patients, expression of SRIF-mRNA and SRIF protein was found in visceral, but not in sc, adipose tissue. Adipocyte mRNA abundance of SSTR 1-5 was differentially regulated by inflammatory treatments.Thus, human visceral adipose tissue secretes SRIF during inflammation and sepsis and expresses several SSTRs. It is tempting to speculate that visceral adipose tissue-derived SRIF plays a modulatory role in the immunological and metabolic response to inflammation.

Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway.

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool.

Effects of crude drugs on glucose uptake in 3T3-L1 adipocytes.

In this study, various water-extracted crude drugs from Radix Asparagi, Radix Ginseng, Radix Scutellariae, Cortex Lycii Radicis, Cortex Phellodendri and Radix Ophiopogonis were investigated in their effects on [3H]-2-deoxyglucose uptake in 3T3-L1 adipocytes. Following treatment of cells with various crude drugs for 60 mim, the basal [3H]-2-deoxyglucose uptake in cultured 3T3-L1 cells was changed by Radix Asparagi from 140 pmole/min/mg protein of control to 513 (0.1 mg/ml), 201 (1 mg/ml) and 97 (10 mg/ml). Glucose uptake was changed to 324 (0.1 mg/ml), 146 (1 mg/ml) and 46 (10 mg/ml) with Radix Ginseng. In the presence of Radix Scutellariae, glucose uptake was changed to 215 (0.1 mg/ml), 213 (1 mg/ml) and 34 (10 mg/ml). In the presence of Cortex Lycii Radicis, glucose uptake was 230 (0.1 mg/ml), 188 (1 mg/ml) and 38 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, uptake was changed to 142 (0.1 mg/ml), 132 (1 mg/ml), 24 (10 mg/ml) and 489 (0.1 mg/ml), 374 (1 mg/ml), 344 (10 mg/ml), respectively. In insulin-stimulated cells, the [3H]-2-deoxyglucose uptake was changed by Radix Asparagi from 570 pmole/min/mg protein of the control to 816 (0.1 mg/ml), 674 (1 mg/ml) and 532 (10 mg/ml). After incubation with Radix Ginseng, the glucose uptake was changed to 254 (0.1 mg/mi), 123 (1 mg/mi) to 76 (10 mg/mi). In the presence of Radix Scutellariae, the glucose uptake was changed to 315 (0.1 mg/ml), 265 (1 mg/ml) and 33 (10 mg/ml). After incubation of Cortex Lycii Radicis, the uptake activity was changed to 281 (0.1 mg/ml), 248 (1 mg/ml) and 37 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, the activity of glucose uptake was measured as 747 (0.1 mg/ml), 523 (1 mg/ml), 33 (10 mg/ml) and 753 (0.1 mg/ml), 740 (1 mg/ml), and 421 (10 mg/ml), respectively. These results indicate that the water-extracted materials of Radix Asparagi and Radix Ophiopogonis increase the glucose uptake in basal and insulin-stimulated 3T3-L1 adipocytes.

Separation and Partial Characterization of Three Distinct Intracellular GLUT4 Compartments in Rat Adipocytes: SUBCELLULAR FRACTIONATION WITHOUT HOMOGENIZATION

Insulin recruits GLUT4 from an intracellular location to the plasma membrane in rat adipocytes. The process involves multiple intracellular compartments and multiple protein functions, details of which are largely unknown partly due to our inability to separate individual GLUT4 compartments. Here, by hypotonic lysis, differential centrifugation, and glycerol density gradient sedimentation, we separated intracellular GLUT4 compartments in rat adipocytes into three fractions: plasma membrane-containing fraction T and plasma membrane-free fractions H and L. The GLUT4 contents in fractions T, H, and L were approximately 25, 56, and 18% of total GLUT4, respectively, in basal adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes. The plasma membrane GLUT4 contents estimated separately further revealed that intracellular GLUT4 in fraction T amounts to approximately 20% in both basal and insulin-stimulated adipocytes. Organelle-specific marker and membrane traffic-related protein distribution data suggested that intracellular GLUT4 in fraction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subcellular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the associated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.

Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using an endosomal ablation technique.

The targeting of the insulin-responsive glucose transporter, GLUT-4, to an intracellular compartment in adipocytes and muscle is one of the key features responsible for the unique insulin sensitivity of this transporter. Through expression of epitope-tagged GLUT-4 mutants in 3T3-L1 adipocytes, two motifs have been identified as playing a central role in GLUT-4 targeting: FQQI in the amino terminus and a di-leucine motif in the carboxy terminus. The goal of this study was to explore the role of these targeting motifs in the intracellular sorting of GLUT-4 using the Tf-HRP ablation technique. This technique provides a quantitative assessment of the amount of GLUT-4 located in recycling endosomes. In basal adipocytes, we find that approximately 40% of GLUT-4 is ablated following Tf-HRP loading. In contrast, here we demonstrate that the intracellular pool of a mutant in which F5 was mutated to A5 is localized to the recycling endosomal pathway, suggesting that the amino terminal FQQI motif functions in trafficking GLUT-4 from early endosomes. In contrast, GLUT-4 in which L489L490 was mutated to A489A490 was localized predominantly to a nonablated compartment. These data imply a role for the di-leucine motif in sorting from a separate intracellular compartment, such as the TGN. Our findings are discussed within the context of a revised multicompartment model for GLUT-4 trafficking in adipocytes, in which mutations in either the FQQI or LL motifs result in the altered subcellular trafficking of GLUT-4 between multiple intracellular compartments.

Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes.

The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived gamma-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.

A Synthetic Peptide Corresponding to the GLUT4 C-terminal Cytoplasmic Domain Causes Insulin-like Glucose Transport Stimulation and GLUT4 Recruitment in Rat Adipocytes

In rat epididymal adipocytes, practically all of the major glucose transporter isoform GLUT4 is constitutively sequestered in intracellular membranes and moves to the plasma membrane in response to insulin, whereas about half of GLUT1, the minor isoform, is constitutively functional at the plasma membrane and thus less affected by insulin. Transfection studies using cells whose glucose transport is normally not regulated by insulin have suggested that the C-terminal cytoplasmic domain of GLUT4 is responsible for its constitutive intracellular sequestration. To test if this was also the case in a classical insulin target cell, we introduced synthetic peptides corresponding to the C-terminal cytoplasmic domain of GLUT4 and GLUT1 (GLUT4C and GLUT1C, respectively) into rat adipocytes and studied their effects on the glucose transport activity and the steady state GLUT4 and GLUT1 distribution between the plasma membrane and intracellular membranes in host cells. GLUT4C introduced into basal adipocytes caused a large (up to 4.5-fold) and dose-dependent increase in the plasma membrane GLUT4, with a proportional reduction in microsomal GLUT4, without affecting GLUT1 distribution. GLUT4C incorporation also caused a large (up to 3-fold) dose-dependent stimulation of 3-O-methyl D-glucose (3OMG) flux in host cells. GLUT4C caused little if any GLUT4 or GLUT1 redistribution and changes in 3OMG flux in insulin-stimulated adipocytes. GLUT1C, on the other hand, did not affect GLUT1 or GLUT4 targeting and 3OMG flux in host cells. These findings not only underscore the importance of the C-terminal cytoplasmic domain of GLUT4 in its constitutive intracellular sequestration in a classical insulin target cell but also suggest the existence of a regulatory protein in adipocytes that interacts with GLUT4 at its cytoplasmic domain, thus participating in the constitutive intracellular sequestration of GLUT4.

Ligand-induced conformational changes modify proteolytic cleavage of the adipocyte insulin-sensitive glucose transporter

The transport conformation of the human erythrocyte glucose transporter (GLUT1) modifies rates of proteolytic cleavage of this protein by a variety of enzymes. We investigated the effects of ligand-induced conformational change on the susceptibility to enzymic cleavage of the insulin-sensitive rat adipocyte glucose transporter (GLUT4). A GLUT4-enriched slow sedimenting microsomal fraction was prepared from basal adipocytes and subjected to PAGE and immunoblotting. The GLUT4 protein was detected in these immunoblots with a C-terminal-specific antiserum as an M(r)-46,000-50,000 doublet. GLUT1 protein was not detected by a GLUT1-specific antiserum in these membranes. Tryptic digestion caused loss of the GLUT4 signal in immunoblots in a time- and concentration-dependent fashion. Low-M(r) membrane-bound fragments were not observed in electrophoretic gels, whether detection was attempted by immunoblotting or by counting radioactivity in gel slices following photolabelling with [3H]cytochalasin B. Transport-specific ligands known to induce an outward-facing conformation in the human erythrocyte GLUT1 protein retarded cleavage of the GLUT4 protein by submaximal concentrations of trypsin, whereas ligands known to induce an inward-facing conformation increased the extent of cleavage. The transported substrate D-glucose retarded tryptic cleavage of GLUT4. This result contrasts with the known behaviour of GLUT1, in which D-glucose accelerates cleavage. Cleavage of GLUT4 by thermolysin was also retarded by the outward-binding analogue 4,6-O-ethylidene glucose. These results show that the conformational sensitivity to proteolysis of GLUT4 mirrors that of GLUT1, except that the glucose-loaded GLUT4 has a different steady-state configuration, which may reflect underlying kinetic differences between the two proteins.

Role of Two Types of Glucose Transporters in Enlarged Adipocytes From Aged Obese Rats

The mechanism of insulin-resistant glucose-transport activity in enlarged aged adipocytes was examined. Glucose-transport activity was assessed by measuring 3-O-methylglucose transport and the concentration of HepG2 erythrocyte/glucose transporter (GLUT1), and the muscle/adipose tissue transporter (GLUT4) was estimated by immunoblotting. Basal glucose-transport activity increased 6.3-fold/cell but remained constant per unit cellular surface area due to cell enlargement. Maximal insulin-stimulated transport activity remained constant per cell but decreased per unit cellular surface area. On a per protein basis, GLUT1 and GLUT4 from aged rats decreased to approximately 60 and 10% of those from young rats, respectively. However, when the protein content of each fraction and the recoveries of marker enzymes were used for estimating the amount of transporters in intact adipocytes, the amount of GLUT1 per cell remained relatively constant, whereas that of GLUT4 decreased. In basal cells from young rats, 31% of the total GLUT1 per cell was located in the plasma membrane, whereas in those from aged rats, 63% was located in the plasma membrane. Thus, in comparing basal adipocytes from aged rats with those from young rats, GLUT1 per cell in the plasma membrane increased 2.8-fold, but this increase was less than that of transport activity (6.3-fold). In basal cells from young rats, 8% of the total GLUT4 was located in the plasma membrane, and a 4.5-fold increase was observed with insulin treatment, but the amount of GLUT4 in each fraction from aged rats markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

The Role of Insulin Receptor Sulphydryl Groups in Insulin Binding and Cellular Response in Rat Adipocytes

Phenylarsine oxide (PAO), an agent which reacts with vicinal sulphydryl groups and dithiothreitol (DTT), a disulphide reducing agent, inhibited insulin binding to intact adipocytes with half maximal inhibition occurring at 28 microM and 340 microM, respectively. Pretreatment of adipocytes with DTT (2mM) prevented insulin stimulation of glucose uptake by approximately 50%. The marked inhibition of insulin binding to adipocytes by PAO and DTT is consistent with the involvement of the receptor cysteine-rich region of hormone binding. Furthermore, DTT inhibition of insulin binding suggests that the integrity of disulphide bridges is critical for insulin binding. The inhibitory effect of DTT and PAO on insulin binding were not additive, instead addition of DTT to PAO-treated adipocytes effected 15% reversal of binding inhibition. The marked inhibition of insulin binding by addition of low concentrations of DTT (0.2-2.0mM) to intact adipocytes is in contrast to the previously reported biphasic response for the effect of DTT on insulin binding to isolated plasma membranes from rat adipocytes (Schweitzer et al. Proc. Natl. Acad. Sci. U.S.A. 77, 4692-4696, 1980). Scatchard plots for 125I-iodoinsulin binding to adipocytes in the basal state were linear. In contrast, Scatchard analysis of insulin binding to plasma membranes prepared from both basal and insulin-stimulated adipocytes yielded severely curvilinear plots. The data suggests that (i) fundamental differences exist between the receptor state in intact cells and isolated plasma membranes and (ii) that a disulphide-rich region within the insulin receptor, other than the previously reported class I and class II disulphide bridges, is critical for insulin binding and cellular response.