Insulin and insulin-like growth factor 1 stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein in 3T3-L1 adipocytes.
ABSTRACT: Insulin and IGF-1 (insulin-like growth factor 1) rapidly stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein (pp160) in intact 3T3-L1 adipocytes. Half-maximal phosphorylation of pp160 is attained with either 4 nM-insulin or 20 nM-IGF-1. A semi-quantitative immunoblotting procedure using anti-phosphotyrosine antibody revealed that the insulin-stimulated 3T3-L1 adipocyte possesses approx. 3 x 10(5) and 0.6 x 10(5) phosphotyrosyl sites, respectively, in pp160 and insulin receptor beta-subunit. Removal of insulin from stimulated cells results in the rapid (within 15 min) loss of phosphate groups from tyrosyl residues in both pp160 and receptor beta-subunit. Whereas pp160 remains maximally phosphorylated on tyrosine for up to 60 min in the presence of 100 nM-insulin, IGF-1 at the same concentration induces only a transient response that is maximally 50% of that observed with insulin. pp160 is not phosphorylated on tyrosine in response to platelet-derived growth factor or epidermal growth factor. Although pp160 appears to be a soluble cytoplasmic protein, in the presence of 1 mM-ZnCl2 it becomes membrane-associated. In view of its apparent cytoplasmic localization and its inability to bind to either wheat-germ agglutinin or concanavalin A, pp160 does not appear to be a typical glycoprotein growth-factor receptor. Our results suggest that pp160 may be a physiologically important cellular substrate of the insulin-receptor tyrosine kinase in the intact 3T3-L1 adipocyte.
Project description:We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1-2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3'-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes.
Project description:During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50-70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (alpha2,beta2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor beta subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.
Project description:Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -19 of the FAS gene were compared. Deletions of the promoter sequences from -136 to -19 resulted in a step-wise decrease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the -136 to -19 region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 fibroblasts transiently transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.
Project description:The development of a hormone-responsive glucose transport activity during differentiation of 3T3-L1 murine fibroblasts to an insulin-sensitive adipocyte-like phenotype was studied. Glucose transport activity was insensitive to insulin or insulin-like growth factor I (IGF-I) before differentiation, and was increased by 8-10-fold after differentiation by both insulin and IGF-I via their own respective receptors. In contrast, in undifferentiated cells insulin and IGF-I stimulated a large increase of [3H]thymidine incorporation into DNA via IGF-I receptors, indicating that undifferentiated 3T3-L1 cells are equipped with fully functioning hormone (IGF-I) receptors. Thus the previously described increase in expression of insulin receptors during differentiation cannot solely account for the development of hormone-sensitive glucose transport in the 3T3-L1 cell. The total glucose transport activity reconstituted from membrane fractions was increased by about 3-fold during differentiation. In differentiated cells, more than 80% of the total reconstitutable glucose transport activity was detected in an intracellular compartment (200,000 g microsomes) as compared with about 20% in undifferentiated cells. Immunoblots with specific antiserum confirmed previous reports indicating that the adipose tissue/muscle glucose transporter (GT3) was exclusively present in the differentiated cells, whereas the erythrocyte/brain glucose transporter (GT1) was detected in both differentiated and undifferentiated cells. Upon differentiation, GT1 was redistributed from plasma membranes to the intracellular compartment. In addition, the newly formed GT3 was predominantly found (greater than 80% of total) in the microsomal fraction of differentiated cells. Both GT1 and GT3 appeared to be hormone-sensitive, since in differentiated cells insulin as well as IGF-I gave rise to their translocation from the intracellular compartment to the plasma membrane. These data suggest that, in addition to the specific expression of the GT3 transporter, the formation of a large pool of intracellular glucose transporters comprising both GT1 and GT3 contributes to the development of insulin sensitivity in the 3T3-L1 cell.
Project description:Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that exerts insulinotropic and growth and survival effects on pancreatic ?-cells. Additionally, there is increasing evidence supporting an important role for GIP in the regulation of adipocyte metabolism. In the current study we examined the molecular mechanisms involved in the regulation of GIP receptor (GIPR) expression in 3T3-L1 cells. GIP acted synergistically with insulin to increase neutral lipid accumulation during progression of 3T3-L1 preadipocytes to the adipocyte phenotype. Both GIPR protein and mRNA expression increased during 3T3-L1 cell differentiation, and this increase was associated with upregulation of nuclear levels of sterol response element binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor ? (PPAR?), as well as acetylation of histones H3/H4. The PPAR? receptor agonists LY171883 and rosiglitazone increased GIPR expression in differentiated 3T3-L1 adipocytes, whereas the antagonist GW9662 ablated expression. Additionally, both PPAR? and acetylated histones H3/H4 were shown to bind to a region of the GIPR promoter containing the peroxisome proliferator response element (PPRE). Knockdown of PPAR? in differentiated 3T3-L1 adipocytes, using RNA interference, reduced GIPR expression, supporting a functional regulatory role. Taken together, these studies show that GIP and insulin act in a synergistic manner on 3T3-L1 cell development and that adipocyte GIPR expression is upregulated through a mechanism involving interactions between PPAR? and a GIPR promoter region containing an acetylated histone region.
Project description:BACKGROUND:Obesity and metabolic syndrome are important public concerns, and there is increasing demand for effective therapeutic strategies. Flavonoids are expected to improve the risk factors associated with metabolic syndrome. Anthocyanidins are a kind of flavonoids; well known for their anti-oxidative, anti-inflammatory and anti-tumor properties. However, their effects on adipocytes and molecular systems are not well defined. In this study, we examined the effects of anthocyanidins-enriched bilberry extracts on adipocyte differentiation. METHODS:Utilizing 3T3-L1 cell line, we investigated that bilberry extracts and anthocyanidins induced inhibition of lipid accumulation during adipogenesis. To identify what is the most important bilberry mediated-effect, we analyzed the expressions of key transcriptional factors associated with adipocyte differentiation by Real Time (RT)-PCR. From the results of RT-PCR, we hypothesized that bilberry extracts and anthocyanidins blocks insulin signal, we determined the phosphorylation of tyrosine residues of insulin receptor substrate 1 (IRS1) protein by western blotting analysis. In addition, we compared the whole-genome expression profiles of early stage of adipocyte differentiation under four different growth conditions (DMSO, bilberry, two anthocyanidins) by microarray analyses and Gene Set Enrichment Analysis (GSEA). RESULTS:Exposure to bilberry extracts and anthocyanidins during adipocyte differentiation inhibited 3T3-L1 differentiation. During this period, bilberry extracts and anthocyanidin significantly decreased a key adipocyte differentiation-associated marker, peroxisome proliferator-activated receptor- ? (Ppar ? ) and sterol regulatory element-binding protein 1c (Srebp1c). Western blotting analysis showed that bilberry extracts and anthocyanidin decreased the phosphorylation of tyrosine residues of IRS1. In addition, microarray experiments and GSEA data revealed significantly altered expression of the known genes of the insulin pathway in cells treated with bilberry extracts or anthocyanidins in the early differentiation stages. CONCLUSIONS:Our data demonstrate that anthocyanidin enriched bilberry extracts strongly inhibit the adipocyte differentiation via the insulin pathway. Furthermore, bilberry extracts might be used as a potential complementary treatment for the obese patients with metabolic syndrome.
Project description:Metabolic syndrome is an important public concern and demand for effective therapeutic strategies. Abdominal obesity, especially an increase in the visceral adipose tissue, is the main cause of this syndrome. Flavonoids are expected to improve risk factors for metabolic syndrome. Bilberry, original species of blueberry containing anthocyanidin flavonoids have been used for centuries in Europe to ameliorate the symptoms of diabetes, but their effects and the mechanisms on lipid accumulation of adipocyte cells are not well defined. In the present study, we investigated effects of the Bilberry extract on differentiation of adipocytes using 3T3-L1 adipocyte cell line. Exposure to Bilberry extract during the early period of adipogenesis (6 days) was significantly inhibited adipocyte differentiation of 3T3-L1. During this period, Bilberry extract greatly down-regulated the mRNA levels of the key adipogenesis-associated markers peroxisome proliferator-activated receptor-γ (PPARγ). Furthermore, Bilberry extract significantly decreased expression of the transcription factor Sterol Regulatory Element Binding Protein 1c (SREBP1c), which plays a central role in adipocyte differentiation including the induction of PPARγ. The expression of SREBP1c is remarkably enhanced in response to insulin, thus raises the possibility that Bilberry extract might inhibit the Insulin pathway. So, We investigated whether Billberry extract and anthocyanidines turned the insulin signaling pathway using microarray. As a result, Gene Set Enrichment Analysis (GSEA) shows that these additives turned insulin signaling pathway certainly. We quantified expression profiles of whole mRNAs by microarray in the adipocyte from 3T3-L1 with or without additives. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) were grown to confluence in DMEM media (Sigma Aldrich, Tokyo, Japan) with 10% calf serum and penicillin (100 U/ml)/streptomycin (100 ug/ml). Adipogenesis was induced using an adipogenesis assay kit (Chemicon International, Temecula, CA). On day 0, cells were induced with initiation media (10 ug/ml insulin, 1 uM dexamethasone and 0.5 uM IBMX in DMEM media added with 1: 0.1% DMSO, 2: 100 ug/ml Bilberry extract, 3: 100 nM Delphinidin or 4: 100 nM Cyanidin (total 4 samples). On day 2, harvested the cells, extracted total RNA and did microarray experiments.
Project description:We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5'-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5' flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 +/- 0.2-, 2.6 +/- 0.1-, 2.0 +/- 0.2- and 1.7 +/- 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 +/- 0.3- and 3.5 +/- 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 +/- 0.8-, 3.0 +/- 0.7- and 2.3 +/- 0.5-fold respectively in serum-free media, and by 2.6 +/- 0.4-, 3.3 +/- 0.9-, 3.1 +/- 0.4- and 2.9 +/- 0.6-fold respectively in the presence of 0.5% serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.
Project description:The peroxisome proliferator?activated receptor ? (PPAR?) plays an important role in insulin sensitivity and adipocyte differentiation. It is known as ligand?receptor that improves insulin sensitivity in type 2 diabetes mellitus. Several kinds of indigo plant have been already used to treat diabetes in oriental traditional medicine, but its mechanism has not been clarified yet. To investigate the effect of indirubin, which is a component of Polygonum tinctorium on the cell differentiation and adipprocess in 3T3?L1 cells, 3T3?L1 cells were cultured to determine the effect of cell differentiation and glucose uptake with indirubin. As a result, Indirubin compound enhanced adipocyte differentiation in 3T3?L1 cells similar to rosiglitazone. This effect was terminated by cotreatment with GW9662, a PPAR? antagonist. In mature 3T3?L1 adipocytes, the lipid droplet size and accumulation were reduced by this compound. The basal and insulin?stimulated glucose uptakes were also significantly increased. In addition, indirubin treatment significantly enhanced estrogen level by 1.64?fold with mature adipocytes which can be attributed to its aromatase activity. Conclutionaly, this finding suggested that indirubin is a potential anti?diabetic compound for type 2 diabetes mellitus by promoting adipocyte differentiation and glucose uptake via PPAR?.
Project description:Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.