Hormone-sensitive adenylyl cyclase in preadipocytes cultured from adipose tissue: comparison with 3T3-L1 cells and adipocytes

The adenylyl cyclase system of preadipocytes derived from the stromal vascular fraction of perirenal rat fat pads was characterized. Unlike mature adipocytes, preadipocyte adenylyl cyclase was only weakly stimulated by catecholamines and adrenocorticotrophic hormone, but was stimulated by guanine nucleotides. Parathyroid hormone and 2-chloroadenosine also stimulated preadipocyte adenylyl cyclase. The adenylyl cyclase system of preadipocytes resembled that of undifferentiated 3T3-L1 cells. However, agents which induced the differentiation of the 3T3-L1 cell adenylyl cyclase system did not have a similar effect on preadipocytes. A medium (CDM6) which induced some differentiation of preadipocyte adenylyl cyclase was developed. The observations that the adenylyl cyclase system of preadipocytes and undifferentiated 3T3-L1 cells are similar, that preadipocyte adenylyl cyclase can be induced to develop along lines similar to early differentiation of 3T3-L1 cells, and that the adenylyl cyclase system of fully-differentiated 3T3-L1 cells has characteristics intermediate between preadipocytes and adipocytes, suggest that the differentiation of preadipocyte and 3T3-L1 adenyly cyclase in vitro mimics adipose adenylyl cyclase development in vivo. The increased catecholamine and ACTH stimulation, and reduced GTP and adenosine sensitivities of adipocytes compared to preadipocytes suggest that a number of genes affecting adenylyl cyclase-associated regulatory and receptor proteins are coordinately repressed and derepressed during development.     

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.     

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpβ and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.     

alpha2- to beta3-Adrenoceptor switch in 3T3-L1 preadipocytes and adipocytes: modulation by testosterone, 17beta-estradiol, and progesterone

Sex steroid hormones are important factors in the determination of fat distribution and accumulation. The aim of this study was to investigate the effect of testosterone (T), 17beta-estradiol (17betaE), and progesterone (P) on adrenergic receptor (AR) gene expression in 3T3-L1 preadipocytes and adipocytes and their relation to the proliferation and differentiation processes. Our data clearly show that alpha(2A)-AR was the highest AR subtype expressed in preadipocytes, whereas in mature adipocytes was by far beta(3)-AR. In the differentiation process to adipocytes, alpha(2A)-AR expression was decreased to 0.3-fold (P < 0.01), whereas beta(3)-AR was upregulated 578-fold (P < 0.001) compared with preadipocytes. In addition, the expression of alpha(2A)-AR in preadipocytes was increased upon incubation with T, 17betaE, and P, and a stimulation of proliferation was also observed in 17betaE- and P-treated cells. In mature adipocytes, 17betaE and P enhanced both alpha(2A)- and beta(3)-AR gene expression (although the effects on beta(3)-AR mRNA levels could be more relevant, since beta(3)-AR was the most highly expressed), whereas T only increased alpha(2A)-AR mRNA levels. Leptin and adipocyte fatty acid-binding protein mRNA levels were higher after 17betaE and P treatment, possibly indicating a proadipogenic effect of these hormones. In conclusion, this study indicates that AR gene expression is affected by these hormones in both preadipocytes and adipocytes, which could have potential importance when considering the role of ARs in the mechanisms underlying the sex-related differences in adipose tissue regional distribution.     

Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose differentiation.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Inhibition of adipocyte differentiation by resistin-like molecule alpha. Biochemical characterization of its oligomeric nature

A novel family of cysteine-rich secreted proteins with unique tissue distribution has recently been identified. One of the members, resistin (for "resistance to insulin"), also called FIZZ3, was identified in a screen for molecules that are down-regulated in mature adipocytes upon administration of thiazolidinediones. The prototypical member of this family was originally identified from bronchoalveolar lavage fluid of inflamed lungs and designated FIZZ1 ("found in inflammatory zone"). This molecule was also found to be highly expressed in adipose tissue and was named resistin-like molecule alpha (RELMalpha). Here we demonstrate that RELMalpha inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. RELMalpha has no effect on proliferation of 3T3-L1 preadipocytes. Pretreatment of 3T3-L1 preadipocytes with RELMalpha does not affect insulin- or platelet-derived growth factor-induced mitogenesis. IRS-1 phosphorylation and glucose transport stimulated by insulin in mature adipocytes were also unaffected by RELMalpha. We show that RELMalpha forms disulfide-linked homooligomers based on results from electrophoresis under reducing and nonreducing conditions, coimmunoprecipitation experiments as well as by mass spectrometry. In addition, RELMalpha is able to form heterooligomers with resistin but not RELMbeta. Since RELMalpha is expressed by adipose tissue and it is a secreted factor, our findings suggest that RELMalpha may be involved in the control of the adipogenesis as well as in the process of muscle differentiation.     

Inhibitory effect of p53 on mitochondrial content and function during adipogenesis

The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.     

Estrogen sulfotransferase inhibits adipocyte differentiation

The estrogen sulfotransferase (EST) is a phase II drug-metabolizing enzyme known to catalyze the sulfoconjugation of estrogens. EST is highly expressed in the white adipose tissue of male mice, but the role of EST in the development and function of adipocytes remains largely unknown. In this report, we showed that EST played an important role in adipocyte differentiation. EST was highly expressed in 3T3-L1 preadipocytes and primary mouse preadipocytes. The expression of EST was dramatically reduced in differentiated 3T3-L1 cells and mature primary adipocytes. Overexpression of EST in 3T3-L1 cells prevented adipocyte differentiation. In contrast, preadipocytes isolated from EST knockout (EST-/-) mice exhibited enhanced differentiation. The inhibitory effect of EST on adipogenesis likely resulted from the sustained activation of ERK1/2 MAPK and inhibition of insulin signaling, leading to a failure of switch from clonal expansion to differentiation. The enzymatic activity of EST was required for the inhibitory effect of EST on adipogenesis, because an enzyme-dead EST mutant failed to inhibit adipocyte differentiation. In vivo, overexpression of EST in the adipose tissue of female transgenic mice resulted in smaller adipocyte size. Taken together, our results suggest that EST functions as a negative regulator of adipogenesis.     

<Emphasis Type="Italic">NYGGF4</Emphasis> homologous gene expression in 3T3-L1 adipocytes: regulation by FFA and adipokines

NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese subjects and is involved in insulin resistance. In the present study, the mRNA expression of NYGGF4 homologous genes was examined in the 3T3-L1 cell line. The NYGGF4 mRNAs were expressed at low levels in the 3T3-L1 preadipocytes. During the conversion of 3T3-L1 preadipocytes to adipocytes, the expression of NYGGF4 mRNA was upregulated. On the 8th day after induction of differentiation, the NYGGF4 mRNA levels peaked and remained high. Free fatty acids (FFA) and tumor necrosis factor-α (TNFα) could upregulate NYGGF4 mRNA expression in 3T3-L1 adipocytes, while interleukin-6 (IL-6), leptin, and resistin exerted an inhibitory effect. The results suggest that the expression of NYGGF4 mRNA is affected by a variety of factors that are related to insulin sensitivity. It is likely that NYGGF4 may be an important mediator in the development of obesity-related insulin resistance.     

Proteasome alterations during adipose differentiation and aging: links to impaired adipocyte differentiation and development of oxidative stress

Intracellular proteins are degraded by a number of proteases, including the ubiquitin-proteasome pathway (UPP). Impairments in the UPP occur during the aging of a variety of tissues, although little is known in regards to age-related alterations to the UPP during the aging of adipose tissue. The UPP is known to be involved in regulating the differentiation of a variety of cell types, although the potential changes in the UPP during adipose differentiation have not been fully elucidated. How the UPP is altered in aging adipose tissue and adipocyte differentiation and the effects of proteasome inhibition on adipocyte homeostasis and differentiation are critical issues to elucidate experimentally. Adipogenesis continues throughout the life of adipose tissue, with continual differentiation of preadipocytes essential to maintaining tissue function during aging, and UPP alterations in mature adipocytes are likely to directly modulate adipose function during aging. In this study we demonstrate that aging induces alterations in the activity and expression of principal components of the UPP. Additionally, we show that multiple changes in the UPP occur during the differentiation of 3T3-L1 cells into adipocytes. In vitro data link observed UPP alterations to increased levels of oxidative stress and altered adipose biology relevant to both aging and differentiation. Taken together, these data demonstrate that changes in the UPP occur in response to adipose aging and adipogenesis and strongly suggest that proteasome inhibition is sufficient to decrease adipose differentiation, as well as increasing oxidative stress in mature adipocytes, both of which probably promote deleterious effects on adipose aging.     

Efficient adenoviral transduction of 3T3-F442A preadipocytes without affecting adipocyte differentiation

The preadipocyte cell lines 3T3-L1 and 3T3-F442A are widely used to study the cellular mechanisms of preadipocyte differentiation and mature adipocyte functions. However, transfection with naked DNA is inefficient in these cell lines. Adenoviral gene transfer is a powerful technique to induce high levels of transgene expression. After failing to obtain 3T3-F442A stable transfectants, we studied different techniques designed to enhance the efficiency of adenoviral transduction in fat cells. First, we compared the effects of two agents known to significantly enhance adenoviral transgene transduction, namely the cationic lipid lipofectamine and the cationic polymer polylysine. We show here that lipofectamine-assisted adenoviral transduction was more efficient in 3T3-F442A than in 3T3-L1 preadipocytes at all tested multiplicity of infection. Lipofectamine, and more efficiently polylysine, yielded high and sustained levels of adenoviral transgene expression in 3T3-F442A preadipocytes. Adenoviral transgene expression was maintained throughout the differentiation process. Furthermore, the two agents also efficiently enhanced adenoviral transduction in mature 3T3-F442A adipocytes. Interestingly, neither protocol affected the differentiation process, morphological features or protein expression of mature adipocytes. These approaches could be of interest to study fat cell differentiation and the functions of mature adipocytes.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

Role of the adipocyte-specific NF-κB activity in the regulation of IP-10 and T cell migration

Infiltration of immune cells into adipose tissue plays a central role in the pathophysiology of obesity-associated low-grade inflammation. The aim of this study was to analyze the role of adipocyte NF-κB signaling in the regulation of the chemokine/adipokine interferon-γ-induced protein 10 kDa (IP-10) and adipocyte-mediated T cell migration. Therefore, the regulation of IP-10 was investigated in adipose tissue of male C57BL/6J mice, primary human and 3T3-L1 preadipocytes/adipocytes. To specifically block the NF-κB pathway, 3T3-L1 cells stably overexpressing a transdominant mutant of IκBα were generated, and the chemical NF-κB inhibitor Bay117082 was used. Adipocyte-mediated T cell migration was assessed by a migration assay. It could be shown that IP-10 expression was higher in mature adipocytes compared with preadipocytes. Induced IP-10 expression and secretion were completely blocked by an NF-κB inhibitor in 3T3-L1 and primary human adipocytes. Stable overexpression of a transdominant mutant of IκBα in 3T3-L1 adipocytes led to an inhibition of basal and stimulated IP-10 expression and secretion. T cell migration was induced by 3T3-L1 adipocyte-conditioned medium, and both basal and induced T cell migration was strongly inhibited by stable overexpression of a transdominant IκBα mutant. In addition, with the use of an anti-IP-10 antibody, a significant decrease of adipocyte-induced T cell migration was shown. In conclusion, in this study, we could demonstrate that the NF-κB pathway is essential for the regulation of IP-10 in 3T3-L1 and primary human adipocytes. Adipocytes rather than preadipocytes contribute to NF-κB-dependent IP-10 expression and secretion. Furthermore, NF-κB-dependent factors and especially IP-10 represent novel signals from adipocytes to induce T cell migration.