Proliferation Of 3T3-L1 Cells Research Articles

Toxicological evaluation of the topoisomerase inhibitor, etoposide, in the model animal Caenorhabditis elegans and 3T3-L1 normal murine cells.

Etoposide, a topoisomerase II inhibitor, has been widely used as a clinical anticancer drug to treat diverse cancer patients. Since not only rapidly dividing cancer cells but also the cells of normal human tissues and every living organism in environmental ecosystems have topoisomerases, it is crucial to study the toxicity of etoposide in other organisms in addition to cancer cells. In this study, we evaluated the toxicity of etoposide in both a soil nematode, Caenorhabditis elegans, and 3T3-L1 normal murine cells. Etoposide significantly retarded the growth, egg laying, and hatching in C. elegans. Etoposide also affected the reproductive gonad tissue, decreased the number of germ cells and induced abnormally enlarged nuclei in C. elegans. In addition, etoposide inhibited 3T3-L1 cell proliferation, with IC50 values of 37.8 ± 7.3 and 9.8 ± 1.8 μM after 24 and 48 hours of treatment, respectively, via the induction of cell cycle arrest at the G2/M phase and apoptotic cell death. Etoposide also induced nuclear enlargement in 3T3-L1 normal murine cells. The reproductive toxicity and abnormal nuclear morphological changes seemed to correlate with the adverse effects of etoposide. We suggest that these experimental platforms, i.e., the toxicological evaluation of both nematodes and 3T3-L1 cells, may be useful to study the mechanisms underlying the side effects of chemicals, including topoisomerase inhibitors.

Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach.

BackgroundFrom ancient times, marine algae have emerged as alternative medicine and foods, contains the rich source of natural products like proteins, vitamins, and secondary metabolites, especially Chlorella vulgaris (C. vulgaris) contains numerous anti-inflammatory, antioxidants and wound healing substances. Type 2 diabetes mellitus is closely associated with adipogenesis and their factors. Hence, we aimed to investigate the chemical constituents and adipogenic modulatory properties of C. vulgaris in 3T3-L1 pre-adipocytes.ResultsWe analysed chemical constituents in ethanolic extract of C. vulgaris (EECV) by LC–MS. Results revealed that the EECV contains few triterpenoids and saponin compounds. Further, the effect of EECV on lipid accumulation along with genes and proteins expressions which are associated with adipogenesis and lipogenesis were evaluated using oil red O staining, qPCR and western blot techniques. The data indicated that that EECV treatment increased differentiation and lipid accumulation in 3T3-L1 cells, which indicates positive regulation of adipogenic and lipogenic activity. These increases were associated with up-regulation of PPAR-γ2, C/EBP-α, adiponectin, FAS, and leptin mRNA and protein expressions. Also, EECV treatments increased the concentration of glycerol releases as compared with control cells. Troglitazone is a PPAR-γ agonist that stimulates the PPAR-γ2, adiponectin, and GLUT-4 expressions. Similarly, EECV treatments significantly upregulated PPAR-γ2, adiponectin, GLUT-4 expressions and glucose utilization. Further, EECV treatment decreased AMPK-α expression as compared with control and metformin treated cells.ConclusionThe present research findings confirmed that the EECV effectively modulates the lipid accumulation and differentiation in 3T3-L1 cells through AMPK-α mediated signalling pathway.

Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes.

The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 μg/mL or 100 μg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts.

Effects of chemerin on the proliferation of 3T3-L1 adipocytes

To explore the effects of chemerin on the proliferation of 333-Li preadipocytes and elucidate its possible mechanism. Recombinant lentivirus-mediated silencing or over-expression of chemerin gene were constructed in 3T3-L1 cells. The proliferation of 3T3-L1 cells was examined by the assays of methyl thiazolyl tetrazolium (MTT) and 5-bromo-2-deoxyuridine (BrdU) incorporation. The expressions of chemerin, ERK1, ERK2 and cyclinD1 in 3T3-L1 cells were detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The protein levels of chemerin, ERK1/2 and p-ERK1/2 were detected by Western blot. MT and BrdU results showed that absorbance (A) value and BrdU incorporation increased in chemerin over-expression group compared with over-expression control group [1. 09 ± 0. 08 vs 0. 90 ± 0. 09, (126. 5 ± 14. 6)% vs (100. 0 ± 7. 0) %, both P <0. 05]. The increase of A value and BrdU incorporation in chemerin over-expression group could be inhibited by PD98059, a blocker of ERK1/2 signal pathway. A value (0. 91 ± 0. 13) and BrdU incorporation [(104. 3 ± 10. 7)%] decreased compared with over-expression group (P <0. 05). They also decreased in chemerin gene silencing group compared to silencing control group [ 0. 72 ± 0. 11 vs 0. 90 ± 0. 09, (77. 6 ± 11. 8) % vs ( 99. 7 ± 6. 3) %, both P < 0. 05]. Quantitative real-time PCR revealed that over-expression group had a higher expression in chemerin, ERK, ERK2 and cyclinDl than over-expression control group (2. 77 ± 0. 31 vs 1. 01 ± 0. 12, 1. 39 ± 0. 19 vs 0. 76 ± 0. 30, 1. 46 ± 0. 14 vs 0. 88 ± 0. 14, 1. 44 ± 0. 17 vs 1. 03 0. 15, all P <0. 05). Chemerin gene silencing group had lower expressions of chemerin, ERKI, ERK2 and cyclinD1 than silencing control group (0. 35 ± 0. 12 vs 1. 25 ± 0. 31, 0. 64 ± 0. 15 vs 1. 03 ± 0. 14, 0. 56 ± 0. 10 vs 1. 06 ± 0. 29, 0. 66 ± 0. 13 vs 1. 09 0. 19, all P <0. 05). Western blot showed that the expressiond of chemerin, ERK1/2 and p-ERK1/2 increased in chemerin over-expression group versus over-expression control group (2. 18 ± 0. 32 vs 1. 18 ± 0. 14, 1. 37 ± 0. 05 vs 0. 97 ± 0. 12, 1. 06 ± 0. 09 vs 0. 56 ± 0. 08, all P < 0. 05). The expressiond of chemerin, ERK1/2 and p-ERK1/2 decreased in chemerin gene silencing group versus silencing control group (1. 00 ± 0. 07 vs 1. 40 ± 0. 17, 0. 87 ± 0. 15 vs 1. 20 ± 0. 12, 0. 49 ± 0. 07 vs 0. 91 ± 0. 11, all P < 0. 05). Chemerin may promote the proliferation of 3T3-L1 cells. And it may be achieved via an up-regulated expression of ERK1/2.

Hormone-sensitive lipase is involved in the action of hydroxysafflor yellow A (HYSA) inhibiting adipogenesis of 3T3-L1cells

BackgroundSafflor yellow A (SY) has been demonstrated to be beneficial to cardiovascular system. Our previous study showed that hydroxysafflor yellow A (HSYA), a main component of SY, could increase peroxisome proliferator-activated receptor γ mRNA expression. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. MethodsThe proliferation and adipogenesis of 3T3-L1 cells treated with HSYA was studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining and intracellular triglyceride assay methods. HSL mRNA expression and promoter activity were studied by real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. ResultsHSYA (0.1mg/L) significantly inhibited the proliferation of 3T3-L1 cells when compared with control cells in 8h. This effect was further enhanced with the extension time (24 to 96h) and an increase of concentration of HSYA (1–10mg/L). The maximal inhibitory action was observed at 0.1mg/L HSYA in 72h (86±11.8% vs. 100±4.1%, p<0.01). HSYA notably reduced the amount of intracellular lipid and triglyceride content in adipocytes to 85% (1mg/L) and 75% (100mg/L) on Day 4 following the differentiation, respectively, while increased HSL mRNA expression and promoter activities to 2.7 fold and 1.55 fold, respectively (p<0.01), in differentiated 3T3-L1 adipocytes. ConclusionsHSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.

Methylglyoxal Mediates Adipocyte Proliferation by Increasing Phosphorylation of Akt1

Methylglyoxal (MG) is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5–20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE) breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity.

Bioactive constituents and anti-proliferative properties of supercritical carbon dioxide Salvia miltiorrhiza extract in 3T3-L1 adipocytes

The present study examined the anti-proliferative properties of aqueous (WESM), ethanolic (EESM) and supercritical carbon dioxide (SCESM-5) extracts of Salvia miltiorrhiza in 3T3-L1 adipocytes, and the contents of bioactive compounds salvianolic acid B (SAB), cryptotanshinone (CTS), tanshinone I (TS-I) and tanshinone IIA (TS-IIA). Results showed that WESM contained mainly SAB, whereas EESM possessed all the four bioactive compounds, but its concentrations of CTS, TS-I and TS-IIA were 2–3-fold lower than SCESM-5. SCESM-5 demonstrated the most potent inhibitory effect on 3T3-L1 cell proliferation, and induction of cell apoptosis and cell cycle arrest. Further studies showed that it enhanced intracellular ROS level, down-regulated CD95 (APO-1/CD95), Bcl-2 and PPAR-γ, up-regulated Bax, AMPK and p-AMPK, and caused activation of caspase-3 and PARP cleavage, as well as JNK and ERK activation and phosphorylation. Pretreatments with antioxidant (Vit E), caspase-3 (z-DEVD-fmk) and ERK (PD98059) inhibitors significantly suppressed the SCESM-5-induced ROS production and cell death. Caspase-3 inhibitor efficiently blocked the Bax expression, caspase-3 activation and PARP cleavage, while ERK inhibitor suppressed the ERK phosphorylation after SCESM-5 treatment. This study concludes that CTS, TS-I and TS-IIA of SCESM-5 may have contributed to the potent anti-proliferative activity through the MAPK and AMPK pathways in 3T3-L1 adipocytes.

Thaumatococcus daiellii 열매 유래 토마틴의 3T3-L1 지방전구세포 분화 억제에 의한 항비만 효과

3T3-L1 지방전구세포 분화 억제에 대한 Thaumatococcus daiellii 열매 유래 토마틴의 항비만 효과를 검토하였다. 3T3-L1 지방전구세포에 토마틴을 0-5 <TEX>${\mu}M$</TEX> 농도로 처리한 결과 세포생존율은 처리 농도 의존적으로 감소하였는데, 토마틴 8일 처리 후 1 및 3 <TEX>${\mu}M$</TEX> 농도에서 각각 97% 및 88.3%의 세포생존율을 보였다. 또한 토마틴 3 <TEX>${\mu}M$</TEX> 처리 농도에서 Oil-Red-O염색 지방구가 현저히 감소된 것으로 나타났다. 3T3-L1 세포 내 중성지방 농도는 양성 대조군에 비해 농도 의존적으로 감소하였다. 따라서 천연 식물성 토마틴은 3T3-L1 지방전구세포의 세포증식 억제 및 중성지방 농도 감소 효과를 보여 항비만 효과가 있는 것으로 밝혀졌다. The effects of thaumatin isolated from katemfe fruit of Thaumatococcus daiellii Benth on 3T3-L1 preadipocyte differentiation was investigated in vitro. 3T3-L1 adipocytes were treated with various concentrations of thaumatin ranging in 0-5 <TEX>${\mu}M$</TEX>. Thaumatin reduced fat accumulation in differentiated 3T3-L1 adipocytes in a dose-dependent manner. 3T3-L1 cell proliferation was 97.0 and 88.3% at 1 and 3 <TEX>${\mu}M$</TEX> after 8 days of thaumatin treatment, respectively. Thaumatin showed a potent inhibitory effect on stained lipid droplets at a concentration of 3 <TEX>${\mu}M$</TEX>. Thaumatin reduced triglyceride accumulation in differentiated 3T3-L1 cells in a dose-dependent manner, compared with positive control cells. This study provides basic information on the anti-obesity activity of thaumatin.

The Effect of Monascus Secondary Polyketide Metabolites, Monascin and Ankaflavin, on Adipogenesis and Lipolysis Activity in 3T3-L1

The aim of the present work is to investigate the effects of Monascus secondary metabolites, monascin (MS) and ankaflavin (AK), on cell proliferation, adipogenesis, lipolysis and heparin-releasable lipoprotein lipase (HR-LPL) in 3T3-L1 preadipocyte. MS and AK inhibit the proliferation of 3T3-L1 cells in a dose-dependent fashion. At 8 μg/mL concentration MS inhibits proliferation for 80.5% after 48 h, whereas the value for AK is 69.2%. Adipogenesis is inhibited by MS and AK without dose-dependency. Triglyceride is decreased 37.1% and 41.1% respectively by treating 0.125 μg/mL MS and AK. Adipocyte-specific transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein β (C/EBPβ), C/EBPδ and C/EBPα mRNA levels are measured by real-time polymerase chain reaction. The expression of the four transcriptional factors analyzed (PPARγ, C/EBPβ, C/EBPδ and C/EBPα) is reduced at the initial and the middle period. At the later period, there is no effect on the expression of PPARγ and C/EBPα by treating MS and AK. Furthermore, both MS and AK increase basal lipolysis of mature adipocytes by 113.2% and 278.3% upregulation, respectively. And both MS and AK reduce the activity of HR-LPL, by 45.3% and 58.1% reduction, respectively. This study reveals for the first time that Monascus secondary metabolites, MS and AK, can prevent the differentiation of preadipocyte and stimulate basal lipolysis of mature adipocytes, avoiding the accumulation of lipid.

Effects of sub-sonic vibration on the proliferation and maturation of 3T3-L1 cells

Aims Although low and high intensity sub-sonic vibrations (SSV) have been shown to facilitate wound healing, very few studies have investigated the effects of SSV on 3T3-L1 preadipocytes. Therefore, the present study was undertaken to investigate the influence of SSV on the proliferation and maturation of 3T3-L1 preadipocytes. Main methods To evaluate the effect of SSV on 3T3-L1 cell proliferation, the cells were maintained in an apparatus that administered SSV (0.5 V) for 3 days at a frequency of 10, 20, 30, or 40 Hz. In addition, to study the effect of SSV on 3T3-L1 cell maturation, the cells were stimulated with SSV for 6 days at a frequency of 10, 20, 30, or 45 Hz. Key findings Sub-sonic vibrations inhibited the proliferation of 3T3-L1 preadipocytes at frequencies of 20 and 30 Hz. Triglyceride levels in cells subjected to SSV at frequencies ranging from 10 to 30 Hz increased compared with those measured in control cells. The expression of adipogenic genes, such as PPAR-γ and C/EBP-α, markedly increased in response to SSV at 20 Hz and 30 Hz during maturation. Significance These results suggest that SSV affected adipogenic gene expression at 20 and 30 Hz.

성별 특이 소 혈청이 세포 배양에 미치는 영향

Dong Mok Lee*,**, Moon Seok Choi*, Gyung Il Woo*, Yu Mi Shin**, Ki Ho Lee***, Yong Pil Cheon****,Taehoon Chun***** and Inho Choi*,** School of Biotechnology, Yeungnam University*, Bovine Genome Resources Bank**, Department of Biochemistry and Molecular Biology, Eulji University***, Department of Biology, Sungshin Women’s University****,School of Life Sciences and Biotechnology, Korea University*****ABSTRACTThe research has been aimed to investigate the effect of different sera including fetal bovine serum(FBS), male bovine serum (MS), female bovine serum(FS), and castrated-male bovine serum(C-MS) on cell proliferation, follicular maturation and ovulation in vitro. Established cell lines and primary cells were cultured in the culture media supplemented with different sera and cells proliferation was observed by cell counting and MTT assay. The results indicated that cell proliferation was significantly different for different serum source. Proliferation of bovine and human myogenic satellite cells was highest in MS. In contrast, proliferation of breast cancer cells and immune cells were the highest in FS and FBS, respectively. There was no difference in the rate of follicular growth, whereas the rate of ovulation was higher in FBS and C-MS. Finally, the wound healing effect and cell proliferation of 3T3-L1 cells showed that wound healing was fastest in FS and cell proliferation was higher in MS. These results suggest the importance of an optimal serum selection in the experiments involving cell culture system, and gender-specific Hanwoo sera may be used as a substitute to FBS.(Key words : Bovine serum, Proliferation, MTT assay)

Effects of capsaicin on the lipid metabolism and gene regulation in differentiated 3T3‐L1 adipocytes

In this study, we investigated the effects of capsaicin on the lipid metabolism and gene expression in differentiated 3T3‐L1 adipocytes. Capsaicin did not show any effect on the 3T3‐L1 cell proliferation and cytotoxicity. Intracellular lipid accumulation was significantly decreased by the addition of capsaicin. Glycerol and free fatty acids release into the medium significantly increased by the addition of capsaicin. The expression of PPAR‐γ, CEBP‐α and aP2 genes, which are involved in adipogenesis, was significantly down‐regulated by capsaicin treatment. However, mRNA levels of HSL, CPTI‐α and UCP2 genes involved in lipid oxidation, were significantly increased by capsaicin treatment. These results suggested that capsaicin may enhance lipolytic activity through modulation of gene expression involved in lipolysis and thermogenesis in 3T3‐L1 cells. "This work supported by the Food Nanotechnology Development Project (2007) and KOSEF (No. M10510130005‐08N1013‐00510)."

Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes

We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237–11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468–475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a “versican-like” molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from non-differentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.

Effects of estrogens and the phytoestrogen genistein on adipogenesis and lipogenesis in males and females

Effects of estrogens and the phytoestrogen genistein on adipogenesis and lipogenesis in males and females

αvβ3 Integrins and Pyk2 Mediate Insulin-Like Growth Factor I Activation of Src and Mitogen-Activated Protein Kinase in 3T3-L1 Cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.

Down-regulation of aortic carboxypeptidase-like protein during the early phase of 3T3-L1 adipogenesis.

Aortic carboxypeptidase-like protein (ACLP) is a 175-kDa protein that is expressed in vascular smooth muscle cells and contains a signal peptide sequence, a lysine- and proline-rich repeating motif, a discoidin-like domain with 35% identity to discoidin I, and a carboxypeptidase-like domain that is 39% identical with carboxypeptidase E. It is secreted into the extracellular matrix and may play a role in abdominal wall development and dermal wound healing. ACLP is also expressed in adipose tissue, but at lower levels. In this study we demonstrate that ACLP protein and mRNA are severely down-regulated in the early phase of 3T3-L1 preadipocyte differentiation induced by insulin, dexamethasone, and isobutylmethylxanthine. Neither dexamethasone, isobutylmethylxanthine, nor insulin treatment alone reduced the level of ACLP protein, suggesting that ACLP down-regulation is a differentiation-associated event. ACLP down-regulation coincided with the onset of the postconfluent mitotic clonal expansion phase of adipogenesis. In contrast, subconfluent 3T3-L1 cell proliferation did not alter ACLP expression, suggesting a specific linkage between ACLP and differentiation-induced clonal expansion. Stable overexpression of ACLP had no effect on preadipocyte differentiation assessed by triacylglycerol accumulation and peroxisome proliferator-activated receptor-gamma levels. The role of ACLP and its marked reduction during adipogenesis merit further study.

Alpha 2-Macroglobulin: a new component in the insulin-like growth factor/insulin-like growth factor binding protein-1 axis.

Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism yet control of their activity by IGF-binding proteins (IGFBPs) remains controversial. The effect of IGFBP-1 depends on its phosphorylation status; phosphorylated IGFBP-1 inhibits IGF actions whereas the nonphosphorylated isoform is stimulatory. In order to understand this phenomenon, we purified phosphorylated IGFBP-1 from normal human plasma by immunoaffinity chromatography. Unexpectedly, the resulting preparation enhanced IGF-stimulated 3T3-L1 fibroblast proliferation, due to the presence of a co-purified protein of approximately 700 kDa. Matrix-assisted laser desorption ionization-mass spectrometry and Western immunoblotting analysis identified this co-purified protein as alpha(2)-macroglobulin (alpha(2)M). Anti-alpha(2)M antibodies co-immunoprecipitated IGFBP-1 from human plasma and from (125)I-IGFBP-1.alpha(2)M complexes formed in vitro. The (125)I-IGFBP-1/alpha(2)M association could be inhibited with excess unlabeled IGFBP-1. Surface plasmon resonance analysis indicated that alpha(2)M preferentially associates with the phosphorylated isoform of IGFBP-1 and that when complexed to alpha(2)M, IGFBP-1 can still bind IGF-I. These findings have functional significance since alpha(2)M protects IGFBP-1 from proteolysis and abrogates the inhibitory effect of phosphorylated IGFBP-1 on IGF-I stimulated 3T3-L1 cell proliferation. We conclude that alpha(2)M is a binding protein of IGFBP-1 which modifies IGF-I/IGFBP-1 actions resulting in enhanced IGF effects. In line with its role in regulating the clearance and activity of other growth factors, we predict that alpha(2)M has a novel and important role in controlling the transport and biological activity of IGFs.