The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Enamel matrix derivative inhibits adipocyte differentiation of 3T3-L1 cells via activation of TGF-βRI kinase activity

Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-β can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-β in vitro. Herein, we examined whether TGF-β signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-βRI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-βRI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Effects of Different Fatty Acids and Dietary Lipids on Adiponectin Gene Expression in 3T3-L1 Cells and C57BL/6J Mice Adipose Tissue

Obesity is positively correlated to dietary lipid intake, and the type of lipid may play a causal role in the development of obesity-related pathologies. A major protein secreted by adipose tissue is adiponectin, which has antiatherogenic and antidiabetic properties. The aim of this study was to evaluate the effects of four different high-fat diets (enriched with soybean oil, fish oil, coconut oil, or lard) on adiponectin gene expression and secretion by the white adipose tissue (WAT) of mice fed on a selected diet for either 2 (acute treatment) or 60 days (chronic treatment). Additionally, 3T3-L1 adipocytes were treated for 48 h with six different fatty acids: palmitic, linoleic, eicosapentaenoic (EPA), docosahexaenoic (DHA), lauric, or oleic acid. Serum adiponectin concentration was reduced in the soybean-, coconut-, and lard-enriched diets in both groups. Adiponectin gene expression was lower in retroperitoneal WAT after acute treatment with all diets. The same reduction in levels of adiponectin gene expression was observed in epididymal adipose tissue of animals chronically fed soybean and coconut diets and in 3T3-L1 cells treated with palmitic, linoleic, EPA, and DHA acids. These results indicate that the intake of certain fatty acids may affect serum adiponectin levels in mice and adiponectin gene expression in mouse WAT and 3T3-L1 adipocytes. The effects appear to be time dependent and depot specific. It is postulated that the downregulation of adiponectin expression by dietary enrichment with soybean oil or coconut oil may contribute to the development of insulin resistance and atherosclerosis.

Long Chain Saturated Fatty Acids Increase Haptoglobin Gene Expression in C57BL/6J Mice Adipose Tissue and 3T3-L1 Cells

Background Dietary lipids are directly related to the composition of adipose tissue, aetiology of obesity and arousal of obesity-related pathologies, like chronic inflammation states. Haptoglobin is an acute phase protein secreted by the liver and white adipose tissue, and its blood levels vary according to the volume of fat in the body. Aim of the study To investigate the effect of diets enriched with large amounts of dietary fats, which differ in their fatty acid composition, on the haptoglobin gene expression by visceral and subcutaneous adipose tissue of mice fed for 2 days or 8 weeks. 3T3-L1 cells were treated with fatty acids that are found in those types of dietary fats. Methods Mice were treated acutely (for 2 days) or chronically (for 8 weeks) with diets enriched with soybean oil, fish oil, coconut oil or lard. 3T3-L1 cells were treated with six different fatty acids. Haptoglobin gene expression was quantified by northern blotting. Results Both chronic and acute treatment with lard, which is rich in long chain saturated fatty acids, increased the haptoglobin mRNA expression in the retroperitoneal and epidydimal white adipose tissues. Chronic treatment with coconut oil, which is rich in medium chain saturated fatty acids, increased the haptoglobin expression in the epidydimal and subcutaneous depots. In 3T3-L1, palmitic acid increased the haptoglobin gene expression. Conclusion The type of lipids in the diet can differently modulate the white adipose tissue gene expression of haptoglobin, and saturated fatty acids play a major role in promoting a pro-inflammatory environment. This response is fat pad specific and dependant on the duration of treatment.

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.

Ação de diferentes doses de triiodotironina (T3) sobre a expressão gênica dos receptores de hormônios tireoidianos e leptina em cultura celular de adipócitos, 3T3-L1

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Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

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.

Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.

Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was approximately 1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma.

Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, som: of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected P-32-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 mum nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 mum nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 mum) significantly inhibited basal and insulin-stimulated glucose uptake in adi. pocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 muM nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 muM nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degreesC. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Diferentes doses de Triiodotironina (T3) aumentam a expressão gênica de leptina, adiponectina e TRα com o envolvimento da via fosfatidil inositol 3 quinase (PI3K) em adipócitos, 3T3-L1

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Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast MicroscopyV⃞

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.