Insulin-like growth factor-1/insulin bypasses Pref-1/FA1-mediated inhibition of adipocyte differentiation

Pref-1 is a highly glycosylated Delta-like transmembrane protein containing six epidermal growth factor-like repeats in the extracellular domain. Pref-1 is abundantly expressed in preadipocytes, but expression is down-regulated during adipocyte differentiation. Forced expression of Pref-1 in 3T3-L1 cells was reported to inhibit adipocyte differentiation. Here we show that efficient and regulated processing of Pref-1 occurs in 3T3-L1 preadipocytes releasing most of the extracellular domain as a 50-kDa heterogeneous protein, previously isolated and characterized as FA1. Unexpectedly, we found that forced expression of the soluble form, FA1, or full-length Pref-1 did not inhibit adipocyte differentiation of 3T3-L1 cells when differentiation was induced by standard treatment with methylisobutylxanthine, dexamethasone, and high concentrations of insulin. However, forced expression of either form of Pref-1/FA1 in 3T3-L1 or 3T3-F442A cells inhibited adipocyte differentiation when insulin or insulin-like growth factor-1 (IGF-1) was omitted from the differentiation mixture. We demonstrate that the level of the mature form of the IGF-1 receptor is reduced and that IGF-1-dependent activation of p42/p44 mitogen-activated protein kinases (MAPKs) is compromised in preadipocytes with forced expression of Pref-1. This is accompanied by suppression of clonal expansion and terminal differentiation. Accordingly, supplementation with insulin or IGF-1 rescued p42/p44 MAPK activation, clonal expansion, and adipocyte differentiation in a dose-dependent manner.     

The Effect of Glucose Concentration on Insulin-Induced 3T3-L1 Adipose Cell Differentiation

We examined the effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation. Oil Red O staining of neutral lipid, cellular triglyceride mass, and glycerol phosphate dehydrogenase (GPDH) activity, were greater in 3T3-L1 cells cultured at 5 mM vs. 25 mM glucose. GPDH activity was 2- to 4-fold higher at 5 mM vs. 25 mM glucose over a range of insulin concentrations (0. 1 to 100 nM). Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was 1. 7-fold greater, and insulinstimulated phosphoinositide 3-kinase association with IRS-1 was 2. 3-fold higher, at 5 mM vs. 25 mM glucose. These effects of glucose were not caused by alterations in IRS-1 mass or cell-surface insulin binding. In preadipose cells at 5 mM glucose, expression of the leukocyte antigen-related (LAR) protein tyrosine phosphatase (negative regulator of insulin signaling) was 63% of the level at 25 mM glucose. Our data demonstrate that glucose concentration affects insulin-induced 3T3-L1 adipose cell differentiation as well as differentiation-directed insulin signaling pathways. Alterations in LAR expression potentially may be involved in modulating these responses.     

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.     

Autophagy participants in the dedifferentiation of mouse 3T3-L1 adipocytes triggered by hypofunction of insulin signaling

Our previous data indicate that both insulin and IGF-1 signallings dysfunction promotes the dedifferentiation of primary human and mouse white adipocytes. Based on the fact that insulin activates mTOR and inhibits autophagy, and autophagy deficiency can inhibit the differentiation of white adipocytes, we speculate that autophagy may be related to the dedifferentiation of white adipocytes. We investigated the underlying mechanism of autophagy during dedifferentiation of mouse 3T3-L1 adipocytes. After incomplete inhibition of insulin and IGF-1 signallings, 3T3-L1 adipocytes manifest dedifferentiation accompanied with an increase of autophagy level. If induction only of autophagy in the adipocytes, then the cells also occur somewhat dedifferentiation, and with a slight decrease of insulin signal, while its degree was weaker than insulin signal inhibited cells. Notably, after inhibition of the insulin and IGF-1 signallings and simultaneously inducing autophagy, the dedifferentiation of 3T3-L1 adipocytes was the most obvious compared with other groups, and the insulin and IGF-1 signallings decreases was greater than the cells with inhibition only of insulin signalling. If inhibition of both insulin signal and autophagy simultaneously, the dedifferentiation of the adipocytes reveals similar tendencies to the cells that insulin signal was inhibited. No significant dedifferentiation occurs of 3T3-L1 cells if only inhibition of autophagy. Taken all together, in this study, we proved that autophagy is positively related to the dedifferentiation of 3T3-L1 adipocytes and is regulated through the insulin-PI3K-AKT-mTOCR1-autophagy pathway. Autophagy may also has a certain degree of negative feedback affect on the insulin signalling of 3T3-L1 cells. Our work may help to better understand the biological properties of mature adipocytes and may help formulate anti-obesity strategies by regulating insulin and insulin signaling level.     

3T3-L1 preadipocyte differentiation and poly(ADP-ribose) synthetase

Differentiation of 3T3-L1 preadipocytes, induced by methyl-isobutylxanthine (MIX), dexamethasone (DEX), and insulin, results in cells with the morphological and biochemical characteristics of adipocytes. Following incubation of 3T3-L1 cells with MIX, DEX, and insulin, poly(ADP-ribose) synthetase activity decreased abruptly, remained low for several hours and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents. It was not observed when preparations were assayed in the presence of DNase I, indicating that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin. The change in synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. It appears to be differentiation specific since nondifferentiating 3T3-C2 control cells did not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.     

黄芩素对3T3-L1小鼠前脂肪细胞分化及对脂肪酸合成酶活性的影响

目的 研究黄芩素对3T3-L1小鼠前脂肪细胞分化及对脂肪酸合成酶活性的影响。方法 油红O染色法测定细胞分化速度;分光光度法测定脂肪酸合成酶活性。结果 黄芩素对3T3-L1小鼠前脂肪细胞向脂肪细胞分化以及对脂肪酸合成酶活性有抑制作用。结论 黄芩素通过阻断脂肪合成而抑制小鼠前脂肪细胞分化;黄芩素具有开发成减肥药物的潜力。     

Insulin and TNF alpha induce expression of the forkhead transcription factor gene Foxc2 in 3T3-L1 adipocytes via PI3K and ERK 1/2-dependent pathways

We have recently identified the winged helix/forkhead gene Foxc2 as a key regulator of adipocyte metabolism that counteracts obesity and diet-induced insulin resistance. This study was performed to elucidate the hormonal regulation of Foxc2 in adipocytes. We find that TNF alpha and insulin induce Foxc2 mRNA in differentiated 3T3-L1 cells with the kinetics of an immediate early response (1-2 h with 100 ng/ml insulin or 5 ng/ml TNF alpha). This induction is, in both cases, attenuated by the PI3K inhibitor wortmannin as well as the MAPK kinase inhibitor PD98059. Furthermore, we show that stimulation of 3T3-L1 adipocytes with phorbol-12-myristate-13-acetate or 8-(4-chlorophenyl)thio-cAMP induces the expression of Foxc2. Interestingly, we find that the basal level of Foxc2 mRNA is down-regulated whereas hormonal responsiveness increases during differentiation of 3T3-L1 from preadipocytes to adipocytes. At the protein level, immunoblots with Foxc2 antibody demonstrated an induction of Foxc2 by insulin and TNF alpha in nuclear extracts of 3T3-L1 adipocytes. EMSA of nuclear proteins from phorbol-12-myristate-13-acetate- and TNF alpha-treated 3T3-L1 adipocytes using a forkhead consensus oligonucleotide revealed specific binding of a Foxc2/DNA complex. In conclusion, our data suggest that insulin and TNF alpha regulate the expression of Foxc2 via a PI3K- and ERK 1/2-dependent pathway in 3T3-L1 adipocytes. Also, signaling pathways downstream of PKA and PKC induce the expression of Foxc2 mRNA.     

Endothelin-1 inhibits resistin secretion in 3T3-L1 adipocytes

Resistin is an adipocyte-derived hormone whose role in the development of insulin resistance is controversial. Endothelin-1 (ET-1) is a 21 amino acid peptide demonstrated to possess vasoconstrictor, positive inotropic, mitogenic, and metabolic properties. In numerous disease states, including congestive heart failure, obesity, and diabetes, elevated levels of ET-1 have been reported and are thought to contribute to the pathology of the disease. A recent study demonstrated that ET-1 induces the expression and stimulates the secretion of the adipose tissue-derived hormone leptin. However, the effect of ET-1 on resistin secretion has not been determined. To characterize the effect of ET-1 on resistin secretion, 3T3-L1 fibroblasts were differentiated into adipocytes and allowed to mature for 14 days. Cells were incubated for 24h with ET-1 (1-100 nM), insulin (1-100 nM), insulin+ET-1 (100 nM I+E) or the appropriate vehicle or antagonist. At the end of the incubation period, resistin secretion was determined in the media by immunoblotting and densitometric analysis. ET-1 (1-100 nM) significantly decreased basal resistin secretion by 49% (1 nM), 43% (10nM), and 59% (100 nM). Insulin (1-100 nM) produced a concentration-dependent increase in resistin secretion from 3T3-L1 adipocytes (1 nM-42%, 10nM-55%, and 100 nM-86% vs. control). Insulin-stimulated resistin secretion (100 nM) was almost completely inhibited (94%) by ET-1 (100 nM). The effects of ET-1 on resistin protein secretion were inhibited by co-incubation with the ET(A) receptor antagonist BQ-610. In conclusion, our studies demonstrate that basal and hormonal stimulation of resistin secretion by insulin are inhibited by ET-1. Such findings demonstrate that resistin secretion is regulated in a similar manner to other adipose tissue factors, including leptin, in 3T3-L1 adipocytes. In addition, our findings suggest that vascular factors such as ET-1 may regulate whole body energy metabolism through adipocyte-derived hormones, including leptin and resistin.     

The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis.

The receptors for insulin and insulin-like growth factor I (IGF-I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF-I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA-synthesis, and expression of proto-oncogene c-fos in 3T3-L1 adipocytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, KI of IGF-I greater than 500 nM; IGF-I receptor, KD = 1.1 nM, KI of insulin = 191 nM). Induction of proto-oncogene c-fos mRNA by insulin and IGF-I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF-I, 3.9 nM). Similarly, both insulin and IGF-I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF-I, 4.0 nM), glucose transport activity (EC50 of insulin, 1.7 nM; IGF-I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3-L1 cells the alpha-subunits of insulin and IGF-I receptors have an equal potential to stimulate a metabolic and a mitogenic response.     

The 3-Phosphorylated Phosphoinositide Response of 3T3-L1 Preadipose Cells Exposed to Insulin,Insulin-Like Growth Factor-1, or Platelet-Derived Growth Factor

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P₂ either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P₃ peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P₃ response. Insulin failed to increase PI(3,4)P₂ at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P₂ and PI(3,4,5)P₃ rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P₃ with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P₂.     

dlk modulates mitogen-activated protein kinase signaling to allow or prevent differentiation

The EGF-like membrane protein dlk plays a crucial role in the control of cell differentiation. Overexpression of the protein prevents, whereas inhibition of its expression increases, adipocyte differentiation of 3T3-L1 cells in response to Insulin-like Growth Factor I (IGF-1) or insulin. We have investigated whether dlk modulates the signaling pathways known to control this process. We found that the levels of dlk expression modulated signaling through the IGF-1 receptor, causing changes in the activation levels and kinetics of Extracellular-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) that correlated with differentiation outcome. These changes occurred in response to IGF-1 or insulin but not in response to Epidermal Growth Factor. However, the levels of expression of IGF-1 receptor, or the activation of Insulin Receptor Substrate-1 in response to IGF-1, were not affected by the levels of dlk expression. Therefore, dlk appears to modulate ERK/MAPK signaling in response to specific differentiation signals.     

Possible involvement of pertussis toxin-sensitive GTP-binding protein(s) in c-fos expression during differentiation of 3T3-L1 fibroblasts to adipocytes.

Following the differentiation of 3T3-L1 fibroblasts by insulin/dexamethasone/methylisobutylxanthine, marked increases in cAMP levels by isoproterenol but not forskolin and in 2-deoxyglucose uptake by insulin occurred. Pertussis toxin-pretreatment prior to addition of insulin/dexamethasone/methylisobutylxanthine and exposure of cells to pertussis toxin during differentiation attenuated glycerophosphate dehydrogenase activity as a differentiation marker enzyme and the responses to isoproterenol and insulin by approximately 50% of those in pertussis toxin-untreated cells. On the other hand, insulin/dexamethasone/methylisobutylxanthine caused induction of c-fos proto-oncogene in confluent 3T3-L1 fibroblasts. This induction was also reduced in pertussis toxin-pretreated cells. These results suggested that pertussis toxin-sensitive GTP-binding protein(s) is involved in expression of c-fos mRNA accompanied by differentiation. In addition, accumulation of c-fos mRNA by insulin/dexamethasone/methylisobutylxanthine was enhanced in protein kinase C-depleted cells pretreated with phorbol 12-myristate 13-acetate, indicating that protein kinase C may negatively regulate c-fos expression induced by insulin/dexamethasone/methylisobutylxanthine.     

dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells

dlk1 is an epidermal growth factor (EGF)-like homeotic protein containing an intracellular region, a single transmembrane domain, and an extracellular region possessing six EGF-like repeats and a protease-target sequence. dlk1 functions as a modulator of adipogenesis, and other differentiation processes. The molecular mechanisms by which dlk1 regulates these processes are unclear. It has been reported that different Dlk1 mRNA spliced variants, encoding for isoforms possessing the protease-target sequence or not, determine the production of membrane-associated or soluble, secreted extracellular dlk1 proteins that appear to affect adipogenesis of 3T3-L1 cells differently. In particular, only soluble variants inhibit this process. Some recent evidence suggest that dlk1 may modulate extracellular stimuli inducing differentiation. Thus, an enforced decrease of Dlk1 expression in BALB/c 3T3 cells, which results in an increase of their adipogenic potential in response to insulin-like growth factor 1 (IGF-1), modifies the kinetics and levels of activation of ERK1/2 triggered by it. In this work, we identified a strong and specific interaction between the protease-target dlk1 region and the non-IGF binding region of IGF binding protein 1 (IGFBP1), a protein that binds to IGFs and modulates their action. We also observed that the increased adipogenic potential of 3T3-L1 cells caused by diminishing Dlk1 expression through transfection with an antisense Dlk1 expression construct was inhibited by the presence of IGFBP1 in the differentiation medium. On the other hand, the presence of IGFBP1 in the culture medium slightly increased the adipogenic potential of control 3T3-L1 cells, expressing regular levels of Dlk1. These data suggest that membrane dlk1 variants bind to extracellular IGFBP1/IGF-1 complexes, which may favor the release of IGF-1 and increase the local concentration of free IGF-1 that can enhance IGF receptor signaling, leading to adipogenesis.     

Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome.     

The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue.

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.     

胰岛素对3T3-L1脂肪细胞中极低密度脂蛋白受体基因表达的影响

体外培养3T3-L1细胞分化模型,研究不同浓度胰岛素及慢性胰岛素刺激对3T3-L1脂肪细胞中极低密度脂蛋白受体（VLDLR）基因表达的影响.在不同浓度胰岛素及胰岛素慢性刺激的干预下,用半定量RT-PCR检测细胞VLDLR mRNA水平的变化.微量化GOD-PAP法检测培养基中残存的葡萄糖.在细胞诱导分化过程中,胰岛素浓度的增高促进VLDLR的表达;胰岛素慢性刺激下,VLDLR表达因浓度差异呈现不同变化.研究结果表明,胰岛素的浓度及慢性刺激对3T3-L1脂肪细胞的成熟和VLDLR基因的表达有显著作用,而胰岛素抵抗明显减低成熟脂肪细胞VLDLR的表达.