Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes

Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation of Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism, and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.     

Organelle relationships in cultured 3T3-L1 preadipocytes

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha- naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.     

Endothelin-1 inhibits adipogenic differentiation of 3T3-L1 preadipocytes

The effect of endothelin (ET)-1 on the adipogenic differentiation of 3T3-L1 preadipocytes was examined. Cellular morphology and lipoprotein lipase activity were used as differentiation markers. ET-1 inhibited the hormone-induced adipogenic differentiation of 3T3-L1 preadipocytes morphologically and biochemically in a dose-dependent manner. These findings promote ET-1 as a potent inhibitor of adipogenic differentiation, playing an important role in cellular differentiation of preadipocytes and making it a significant regulator of lipid metabolism.     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

Proteomic analysis of 3T3-L1 preadipocytes having a higher cell proliferation rate after treatment with low-molecular-weight silk fibroin peptides

Objectives: Previous studies have reported that fibroin peptides can be used in a new strategy for development of anti‐diabetic peptide drugs. In this study, we separated silk fibroin hydrolysates (SFH) containing silk fibroin peptides into four components according to their molecular weight and tested the effects of these together with three synthetic silk fibroin hexapeptides GAGAGS, GAGAGY, GAGAGA on cell proliferation of 3T3‐L1 preadipocytes. The aim of this study was to investigate protein expression profiles of 3T3‐L1 preadipocytes and those treated with SFH component Fraction I and the synthetic silk fibroin hexapeptide GAGAGS to be able to elucidate difference in protein expression between the 3T3‐L1 preadipocytes and those treated with fibroin peptides Fraction I and GAGAGS. Materials and methods: SFH was separated by dialysis. MTT assays were performed to test effects of SFH components and synthetic silk fibroin hexapeptides on 3T3‐L1 preadipocyte proliferation. We generated proteome maps using two‐dimensional gel electrophoresis and analysed them by peptide mass fingerprinting. Results: GAGAGS and peptide mixtures, Fraction I and Fraction II, had significant effect in promoting 3T3‐L1 preadipocyte proliferation. In the proteomic analysis, 73 protein spots were successfully identified, including 15 which were differentially expressed. Conclusions: Our results show that some silk fibroin peptides of low molecular weight SFH and hexapeptide GAGAGS affected 3T3‐L1 preadipocyte proliferation.     

Proteomic analysis of 3T3-L1 adipocyte mitochondria during differentiation and enlargement

The increase in adipose tissue mass arises in part from progressive lipid loading and triglyceride accumulation in adipocytes. Enlarged adipocytes produce the highest levels of pro-inflammatory molecules and reactive oxygen species (ROS). Since mitochondria are the site for major metabolic processes (e.g., TCA cycle) that govern the extent of triglyceride accumulation as well as the primary site of ROS generation, we quantitatively investigated changes in the adipocyte mitochondrial proteome during different stages of differentiation and enlargement. Mitochondrial proteins from 3T3-L1 adipocytes at different stages of lipid accumulation (days 0-18) were digested and labeled using the iTRAQ 8-plex kit. The labeled peptides were fractionated using a liquid phase isoelectric fractionation system (MSWIFT) to increase the depth of proteome coverage and analyzed using LC-MS/MS. A total of 631 proteins in the mitochondrial fraction, including endoplasmic reticulum-associated and golgi-related mitochondrial proteins, were identified and classified into 12 functional categories. A total of 123 proteins demonstrated a statistically significant change in expression in at least one of the time points over the course of the experiment. The identified proteins included enzymes and transporters involved in the TCA cycle, fatty acid oxidation, and ATP synthesis. Our results indicate that cultured adipocytes enter a state of metabolic-overdrive where increased flux through the TCA cycle and increased fatty acid oxidation occur simultaneously. The proteomic data also suggest that accumulation of reduced electron carriers and the resultant oxidative stress may be attractive targets for modulating adipocyte function in metabolic disorders.     

Catecholamine-stimulated protein phosphorylation in 3T3-L1 preadipocytes and adipocytes

The endogenous protein phosphorylation stimulated by catecholamines was compared in 3T3-L1 preadipocytes and adipocytes. Phosphorylation of a protein with an approximate molecular weight of 57,000 was stimulated both in preadipocytes and adipocytes of 3T3-L1. Stimulated phosphorylation of four other proteins with approximate molecular weights of 90,000, 62,000, 48,000, and 32,000 was observed only in 3T3-L1 adipocytes. All of these proteins appeared to be localized in the microsomal fraction. Phosphorylation of these proteins was stimulated by norepinephrine, epinephrine, isoproterenol, dibutyryl cyclic AMP, theophylline, or 1-methyl-3-isobutylxanthine, but not by A23187. Among the phosphorylated proteins in 3T3-L1 adipocytes, the 62,000 dalton protein was most evident. Using this protein as a marker, it appeared that epinephrine and norepinephrine were effective in stimulating the phosphorylation at the same concentration range. This result was in clear contrast to the different affinities of these catecholamines for beta-receptors of 3T3-L1 adipocytes reported by Lai, Rosen, and Rubin (J. Biol. Chem. (1982) 257, 6691-6696). The phosphorylation of the 62,000 dalton protein in 3T3-L1 adipocytes was observed 1 min after the addition of norepinephrine, and dephosphorylation was observed within 10 min after the addition of propranolol.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.     

Adipocyte conversion of cultured 3T3-L1 preadipocytes by bezafibrate

Transient exposure of cultured 3T3-L1 preadipocytes to hypolipidemic fibrate drugs results in extensive adipocyte conversion. Adipocyte conversion in culture was characterized by an increase in neutral lipids content and in adipocyte marker enzymes like hormone-sensitive lipase and glycerol-3-phosphate dehydrogenase. Adipocyte conversion in culture was also accompanied by induction of cyanide-insensitive peroxisomal palmitoyl-CoA oxidation. The conversion pattern exerted by fibrate drugs in 3T3-L1 cells was similar to that reported previously for primary cultured epididymal preadipocytes (R. Brandes, R. Arad and J. Bar-Tana, Biochim. Biophys. Acta, 877, 314-321 (1986)), and seems to refute clonal selection in the conversion sequel initiated by fibrate drugs in primary cultured preadipocytes.     

Regulation of tristetraprolin during differentiation of 3T3-L1 preadipocytes

Tristetraprolin is a zinc-finger-containing RNA-binding protein. Tristetraprolin binds to AU-rich elements of target mRNAs such as proto-oncogenes, cytokines and growth factors, and then induces mRNA rapid degradation. It was observed as an immediate-early gene that was induced in response to several kinds of stimulus, such as insulin and other growth factors and stimulators of innate immunity such as lipopolysaccharides. We observed that tristetraprolin was briefly expressed during a 1-8 h period after induction of differentiation in 3T3-L1 preadipocytes. Detailed analysis showed that tristetraprolin mRNA expression was stimulated by fetal bovine serum and differentiation inducers, and was followed by rapid degradation. The 3'UTR of tristetraprolin mRNAs contain adenine- and uridine-rich elements. Biochemical analyses using RNA pull-down, RNA immunoprecipitation and gel shift experiments demonstrated that adenine- and uridine-rich element-binding proteins, HuR and tristetraprolin itself, were associated with tristetraprolin adenine- and uridine-rich elements. Functional characterization confirmed that tristetraprolin negatively regulated its own expression. Thus, our results indicated that the tight autoregulation of tristetraprolin expression correlated with its critical functional role in 3T3-L1 differentiation.     

LMO4 modulates proliferation and differentiation of 3T3-L1 preadipocytes

Previous microarray analyses revealed that LMO4 is expressed in 3T3-L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT-PCR sequencing and quantitative real-time RT-PCR, we confirmed that LMO4 gene is expressed in 3T3-L1 preadipocytes and its expression peaks at the early stage of 3T3-L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3-L1 preadipocytes, and attenuated the differentiation of 3T3-L1 preadipocytes, as evidenced by reduced lipid accumulation and down-regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.     

High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes

We studied the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_e) on adipocyte differentiation. Preadipocytes exposed to continuous [Ca²⁺]_e higher than 2.5 mmol/l accumulated little or no cytoplasmic lipid compared to controls in 1.8 mmol/l [Ca²⁺]_e. Differentiation was monitored by Oil Red O staining of cytoplasmic lipid and triglyceride assay of accumulated lipid, by RT-PCR analysis of adipogenic markers, and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). Elevated [Ca²⁺]_e inhibited expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and steroid regulatory binding element protein. High [Ca²⁺]_e significantly inhibited differentiation marker expression including adipocyte fatty acid binding protein, and GPDH. The decrease in Pref-1 expression that accompanied differentiation also was prevented by high [Ca²⁺]_e. Treatment of 3T3-L1 cells with high [Ca²⁺]_e did not significantly affect cell number or viability and did not trigger apoptosis. Levels of intracellular Ca⁺² remained unchanged in various [Ca²⁺]_e. Treatment of 3T3-L1 with pertussis toxin (PTX) partially restored lipid accumulation and increased differentiation markers in cells treated with 5 mmol/l [Ca²⁺]_e. ‘Classical’ parathyroid cell Ca²⁺ sensing receptors (CaSR) were not detected either by RT-PCR or by Western blotting. These results suggest that continuos exposure to high [Ca²⁺]_e inhibits preadipocyte differentiation and that this may involve a G-protein-coupled mechanism mediated by a novel Ca²⁺ sensor or receptor.     

Enhanced pro-apoptotic and anti-adipogenic effects of genistein plus guggulsterone in 3T3-L1 adipocytes

Genistein (G), an isoflavone, and guggulsterone (GS), the active substance in guggulipid, have been reported to possess therapeutic effects for obesity. In the present study, we investigated the effects of combinations of G plus GS on apoptosis and adipogenesis in 3T3-L1 cells. In mature adipocytes, G and GS individually caused apoptosis, but combination of G plus GS significantly increased apoptosis, more than either compound alone. Furthermore, G plus GS caused a greater increase in procaspase-3 cleavage, Bax expression, cytochrome c release, and proteolytic cleavage of PARP than either compound alone. In maturing preadipocytes G and GS each suppressed lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. These results suggest that combination of genistein and guggulsterone may exert anti-obesity effects by inhibiting adipogenesis and inducing apoptosis in adipocytes.     

Flow cytometric analysis of DNA replication during the differentiation of 3T3-L1 preadipocytes

We have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line 3T3-L1. When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI-treated cells are incubated in iododeoxyuridine (IdUrd)- or bromodeoxyuridine (BrdUrd)-containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitored. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3-L1 cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.     

Functional ion channels and cell proliferation in 3T3-L1 preadipocytes

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation.     

Characterization of heterokaryons between skeletal myoblasts and preadipocytes: myogenic potential of 3T3-L1 preadipocytes

It has been shown previously that heterokaryons between myoblasts and non-myogenic cells disturb myogenic differentiation (Hirayama et al. (2001); Cell Struct. Funct. 26, 37-47), suggesting that some myogenesis inhibitory factors exist in non-myogenic cells. Skeletal myoblasts and adipose cells are derived from a common mesodermal stem cell, indicating that both cells have a closer relationship in the developmental lineage than the other somatic cells. To investigate the functional relationship between myoblasts and adipose cells, heterokaryons between quail myoblasts and 3T3-L1 cells, a mouse preadipocyte cell line, were prepared and examined for characteristics of myogenic differentiation. Myogenic differentiation was inhibited in the heterokaryons between quail myoblasts and well-differentiated (adipocytes) 3T3-L1 cells. On the contrary, normal myogenic differentiation proceeded in the heterokaryons between quail myoblasts and undifferentiated (preadipocytes) 3T3-L1 cells. Further investigation showed that the mouse myogenin gene from 3T3-L1 cells was transactivated in the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells. The results demonstrated that undifferentiated 3T3-L1 cells have no myogenesis inhibitory factors but acquire these during terminal differentiation into adipocytes.     

Protein kinase A suppresses the differentiation of 3T3-L1 preadipocytes

cAMP and protein kinase A （PKA） are widely known as signaling molecules that are important for the induction of adipogenesis. Here we show that a strong increase in the amount of cAMP inhibits the adipogenesis of 3T3-L1 fibroblast cells. Stimulation of PKA activity suppresses adipogenesis and, in contrast, inhibition of PKA activity markedly accelerates the adipogenic process. As adipogenesis progresses, there is a significant increase in the expression level of PKA regulatory and a corresponding decrease in PKA activity. Moreover, treatment of 3T3-L1 cells with epidermal growth factor （EGF） stimulates PKA activity and blocks adipogenesis. Inhibition of PKA activity abolishes this suppressive effect of EGF on adipogenesis. Moreover, asubunits ctivation of PKA induces serine/threonine phosphorylation, reduces tyrosine phosphorylation of insulin receptor substrate 1 （IRS-1） and the association between PKA and IRS-1. Taken together, our study demonstrates that PKA has a pivotal role in the suppression of adipogenesis, cAMP at high concentrations can suppress adipogenesis through PKA activation. These findings could be important and useful for understanding the mechanisms of adipogenesis and the relevant physiological events.     

Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27^kip1, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBPβ were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.