Preadipose Cells Research Articles

Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development.

Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells

Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development.

Transfection of adipocytes and preparation of nuclear extracts.

AbstractThe uniqueness of the adipose cell type must be considered before choosing a procedure for gene transfer into mature adipocytes. Because adipocytes isolated from adipose tissue (AT) are filled with so many lipids that cells cannot be plated on a culture support, because of their low density, and because such terminally differentiated cells do not divide, the procedure of choice for introducing DNA into fat cells is electroporation. The detailed protocol described below has been published in references (1,2). One must keep in mind however, that primary adipocytes isolated from the animal have a limited life-span in culture (not exceeding 1 wk), so that only transient transfection can be envisaged in such a system. When stable transfectants are needed, other cellular models must be used, such as established preadipose cell lines. Among the most popular, 3T3-L1, 3T3-F442A, and Ob17 can also be transfected by electroporation (3). A protocol adapted to these situations is also given. These techniques are simple to perform, but require special equipment for electric shock delivery. Finally, it is important to realize that only a small proportion of the cell population will incorporate foreign DNA, using these transfection procedures. High-efficiency gene transfer in mature adipocytes, using recombinant adenoviral systems is also possible (3,4), but will not be described here. KeywordsNuclear ExtractMature AdipocyteHypotonic BufferElectroporation CuvetsPrimary AdipocyteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Alterations of Peroxisome Proliferator-activated Receptor δ Activity Affect Fatty Acid-controlled Adipose Differentiation

Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPARgamma or PPARdelta promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPARdelta in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPARdelta mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPARdelta enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPARgamma and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPARdelta mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPARdelta plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPARdelta expression or activity could affect adaptive responses of white adipose tissue to nutritional changes.

Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow

In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E2(PGE2). The difference of these two lines for osteoclast formation was not related with their abilities of PGE2production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE2. In TMS-12 cells, ODF expression was detected in the PGE2-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor γ (PPARγ), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)2D3. The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.

LPA as a paracrine mediator of adipocyte growth and function.

Adipogenesis corresponds to the recruitment of new adipocytes in adipose tissue, and results from the proliferation/differentiation of preadipocytes. Production of paracrine and autocrine factors by adipocytes plays an important role in adipogenesis. We recently demonstrated the existence of adipocyte production of lysophosphatidic acid (LPA) both in vitro and in situ. This production is modulated by catecholamines via alpha 2-adrenergic receptors. Adipocyte-LPA present in conditioned media increases the growth of a preadipose cell line in culture. This growth is associated with an activation of mitogen-activated protein kinases, and of the focal adhesion kinase. Because of the close proximity of preadipocytes and adipocytes within adipose tissue, adipocyte-LPA could play an important role in autocrine/paracrine control of adipogenesis.

Prostacyclin IP receptor up-regulates the early expression of C/EBPβ and C/EBPδ in preadipose cells

Prostacyclin (PGI 2) and its stable analogue carbacyclin (cPGI 2) are known to trigger the protein kinase A pathway after binding to the cell surface IP receptor and to promote or enhance terminal differentiation of adipose precursor cells to adipose cells. The early expression of C/EBPβ and C/EBPδ is known to be critical for adipocyte differentiation in vitro as well as in vivo. We report herein that in Ob1771 and 3T3-F442A preadipose cells, activation of the IP receptor by specific agonists (PGI 2, cPGI 2 and BMY 45778) is sufficient to up-regulate rapidly the expression of C/EBPβ and C/EBPδ. Cyclic AMP-elevating agents are able to substitute for IP receptor agonists, in agreement with the coupling of IP receptor to adenylate cyclase. Consistent with the fact that PGI 2 is released from preadipose cells and behaves as a paracrine/autocrine effector of adipose cell differentiation, the present results favor a key role of prostacyclin by means of the IP receptor and its intracellular signaling pathway in eliciting the critical early expression of both transcription factors.

The role of the epidermal growth factor-like protein dlk in cell differentiation.

This review focuses on the current knowledge about the function of the EGF-like homeotic protein dlk. dlk is a transmembrane protein that possesses six Epidermal Growth Factor-like sequences at the extracellular domain, a single transmembrane domain and a short intracellular tail. Because of its overall structure and amino acid homology, dlk belongs to the EGF-like homeotic protein family. This family includes proteins such as the Notch receptor and its homologues, as well as Notch ligands, such as Delta, Serrate, and their mammalian homologues Dll1, Dll2 and Dll3 and Jagged 1 and Jagged 2. (For a recent review see Fleming, 1998). dlk is highly expressed by preadipose cell lines, and neuroendocrine tumors, such as pheochromocytomas and neuroblastomas. dlk has been involved in several differentiation processes, such as adipogenesis, hematopoiesis and B cell lymphopoiesis, and neuroendocrine differentiation, including the differentiation of pancreas and the adrenal gland. The extracellular region of dlk can be released by action of an unknown protease and this soluble dlk variant accumulates in the amniotic fluid and is able to inhibit adipocyte differentiation in vitro. Recent evidence indicates, however, that membrane-associated dlk variants play a positive role in the differentiation process. These findings suggest that dlk plays an important role in differentiation and tumorigenesis of several cellular types.

Leptin Expression during the Differentiation of Subcutaneous Adipose Cells of Human Embryos in situ

In this study, the immunocytochemical expression of leptin in the developing subcutaneous tissue of human embryos at 6–10 weeks of gestation was investigated using the avidin-biotin peroxidase method. Immunocytochemical staining for leptin was observed in the cytoplasm of the differentiating preadipose cells. The other cells present in the embryonal subcutis (mesenchymal cells differentiating into fibroblasts, fibrocytes, endothelial cells) were leptin-negative. The developing skin epithelium lying above it and the blood cells in the capillaries also did not express leptin. The results suggest that the leptin is produced by the developing fat cells from the beginning of lipidogenesis and differentiation. It possibly acts as a hormonal factor regulating the embryonal growth, development and body fat storage.

The adipose conversion process: regulation by extracellular and intracellular factors.

White adipose tissue regulates lipid metabolism and acts as a secretory organ. Because of its importance for human health and animal production, many studies have attempted to better understand its development at the cellular and molecular levels by culturing preadipose cells in vitro. This synthesis article describes our current knowledge, acquired by this approach, concerning the regulation of the different steps of the adipocyte differentiation program by extracellular (hormones, cytokines, growth factors, retinoids and fatty acids) and intracellular agents (second messengers and transcription factors). The discrepant effects that have been observed for some of these factors are also discussed. This information is very important in the perspective of a better control of fat deposits in human and breeding species.

Regulation of porcine adipogenesis in vitro, as compared with other species

In vitro studies, mostly performed on murine cell lines, allowed us to identify the role played by hormonal agents, second-messenger pathways, extracellular matrix proteins, and transcription factors in adipose conversion. Some information has also been reported when studies were conducted on primary cultures that originated from various species. However, because of conflicting results, probably caused, at least in part, by species specificity, developing cultures of preadipose cells from economically important species appeared necessary to better understand and control the animals’ fat development. We reviewed our current knowledge concerning the regulation of cultured porcine preadipose cells by hormones, second-messenger pathways, and extracellular matrix proteins. The results clearly demonstrate that such primary cultures are essential to avoid the establishment of hazardous concepts originated from rodent and aneuploid cell lines in particular.

Cell surface receptors, nuclear receptors and ligands that regulate adipose tissue development

Our knowledge of adipose tissue development has increased dramatically over the last two decades, through a combination of in vitro studies using cellular models and in vivo studies using mouse models with invalidated target genes. Critical early events of the differentiation programme appear to involve in preadipose cells (i) the entry of fatty acids and the production of fatty acid metabolites as activators/ligands of nuclear peroxisome proliferator-activated receptors (PPARs) and (ii) the very early expression of PPARδ and CAAT/enhancer binding proteins (C/EBPs) β and δ. Among fatty acids, prostacyclin produced from arachidonic acid enhances the expression of both C/EBPs through cell surface IP receptor and presumably activates PPARδ. Together, these transcription factors up-regulate the expression of PPARγ and C/EBPα which lead in turn to the acquisition of the adipocyte phenotype. Altogether, these studies have provided a molecular link between high-fat diets and excess of adipose tissue development through hyperplasia and hypertrophy.

Leukemia Inhibitory Factor and Its Receptor Promote Adipocyte Differentiation via the Mitogen-activated Protein Kinase Cascade

Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr(-/-) ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor gamma2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPbeta and C/EBPdelta is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPbeta and C/EBPdelta genes via the mitogen-activated protein kinase cascade.

Selectivity of fatty acids on lipid metabolism and gene expression.

Triacylglycerols represent the main form of storage for a wide spectrum of fatty acids. Their utilization first involves mobilization from adipose tissue through lipolysis. The release of individual fatty acids from adipose tissue is selective in vitro and in vivo in animal studies and also in human subjects. Generally, fatty acids are more readily mobilized from fat cells when they are short-chain and unsaturated. This selectivity could affect the storage of individual fatty acids in adipose tissue, and their subsequent supply to tissues. The nature of the dietary fats could affect lipid homeostasis and body fat deposition. Dietary fish oil influences adipose tissue development in a site-specific manner as a function of diet and feeding period. A diet high in n-3 polyunsaturated fatty acids (PUFA) results in a preferential partitioning of ingested energy towards oxidation at the expense of storage. Fatty acids are important mediators of gene expression in the liver. Indeed, genes encoding both glycolytic and lipogenic enzymes and key metabolic enzymes involved in fatty acid oxidation are regulated by dietary PUFA. White adipose tissue could also be a target for PUFA control of gene expression. The treatment of pre-adipose cells by fatty acids induces the expression of numerous genes that encode proteins involved in fatty acid metabolism. The mechanisms of PUFA-mediated repression of gene expression in adipocytes seem to be different, at least partly, from those described in liver. Tissue-specific and site-specific factors are possibly involved in the specific effect of PUFA on gene expression, although other mechanisms cannot be excluded.

Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation

To clarify the molecular basis for the prostaglandin (PG) mediated effects in adipose cells at various stages of their development, expression of mRNAs encoding receptors specific for prostaglandin E 2, F 2α and I 2 (i.e. EP, FP, and IP receptors) was investigated in differentiating clonal Ob1771 pre-adipocytes, as well as in mouse primary adipose precursor cells and mature adipocytes. We have further characterized the differential expression of mRNAs encoding three subtypes of the EP receptor, i.e. EP 1, EP 3, and EP 4, and examined the expression of mRNAs encoding the three isoforms (α, β, and γ) of the EP 3 receptor. Altogether the results show that the expression of IP, FP, EP 1, and EP 4 receptor mRNAs was considerably more pronounced in pre-adipose cells than in adipose cells, mRNAs encoding the α, β, and γ isoforms of the EP 3 receptor were all exclusively expressed in freshly isolated mature adipocytes. These data may indicate that PGI 2, PGF 2α, and PGE 2 may interact directly with specific receptors in pre-adipose cells, whose transduction mechanisms are known to affect maturation related changes. In mature adipocytes, however, the equipment of mRNAs encoding the EP 3 receptor isoforms is in agreement with the well known effect of PGE 2 on adenylate cyclase and lipolysis in mature adipocytes.

Various stimulators of the cyclic AMP pathway fail to promote adipose conversion of porcine preadipocytes in primary culture

The cyclic adenosine-monophosphate (cAMP) pathway is generally recognized as one of the essential pathways for the adipose conversion of rodent preadipocytes in vitro. However, divergent effects of cAMP on adipocyte differentiation have also been reported. Since there is very little data on non-rodent preadipose cells, the aim of the present work was to analyze the effects of classic activators of the cAMP pathway on the proliferation and differentiation of porcine preadipocytes grown either in serum-free or in serum-containing medium. In both media, the addition of 10 μM forskolin from day 1 after cell plating to day 3 or 7 did not affect cell proliferation. Such stimulations also failed to enhance preadipocyte differentiation, as assessed by the measurement of lipoprotein lipase (LPL) and glycerol 3-phosphate dehydrogenase (GPDH) activities, two markers of adipose conversion. Similar results were obtained when various concentrations of forskolin (0.1 n M–100 μM) were added for 2 days either during the growth phase (days 1–3) or after confluence (days 5–7). Addition of methylisobutylxanthine (MIX) or 8-bromo-cAMP was also found inefficient to stimulate porcine preadipocytes differentiation clearly. By contrast, post-confluence treatment of the murine 3T3-L1 cell line with either forskolin or MIX markedly enhanced lipid accumulation and led to a dramatic increase in GPDH activity (up to 120 times). This indicates that similar culture conditions are adipogenic for the murine 3T3-L1 preadipocytes but not for porcine preadipose cells. In summary, this work clearly highlights the finding that porcine preadipocytes do not respond to classic activators of the cAMP pathway like rodent cells do. This calls in question again the general model proposed for the action of this pathway in adipose conversion and suggests that the mechanisms regulating adipocyte differentiation may differ among species.

Prostacyclin as a critical prostanoid in adipogenesis

Adipose cell differentiation from adipoblasts to preadipose and to adipose cells is a multistep process. Terminal differentiation of preadipose cells expressing early markers to adipose cells expressing late and very late markers and accumulating triacylglycerol requires a combination of circulating and locally-produced hormones. Prostacyclin (PGI 2), one of the major metabolites of arachidonic acid in adipose tissue, has been shown to exert autocrine and paracrine adipogenic effects in vitro. As discussed herein, multiple arguments support the proposition that PGI 2 is a key prostanoid involved in adipogenesis.

Long chain fatty acids as modulators of gene transcription in preadipose cells.

During the last years, it has been clearly established that long-chain fatty acids act as modulators of gene expression in various tissues, such as adipose tissue, intestine and liver. This transcriptional action of fatty acids explains in part adaptation mechanisms of tissues to nutritional changes and especially to high-fat diets by increasing expression of proteins involved in lipid catabolism in liver and fatty acid uptake and utilization in other tissues. It is now clearly demonstrated that some of these transcriptional effects of fatty acids are mediated by activation of specific nuclear hormone receptors, called peroxisome proliferator-activated receptors (PPARs). These findings will be discussed with a special reference to control of gene expression in preadipocytes and adipose tissue development.

Extracellular matrix induced by TGFβ impairs insulin signal transduction in 3T3‐L1 preadipose cells

When 3T3-L1 preadipose cells are exposed to transforming growth factor β (TGFβ), they synthesize more extracellular matrix (ECM) and resist differentiation-inducing stimuli. The mechanism by which ECM suppresses adipose cell differentiation (adipogenesis) remains unknown. Since adipogenesis is an insulin/insulin-like growth factor-1 (IGF-1)-dependent process, we investigated whether TGFβ-induced ECM inhibits insulin signaling. When preadipose cells were pretreated overnight with TGFβ, we observed a 75% decrease in insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) compared to that in control cells. Culturing 3T3-L1 preadipose cells on fibronectin, a component of the ECM induced by TGFβ, also inhibited insulin-dependent IRS-1 tyrosine phosphorylation and adipogenesis, supporting a role for ECM in mediating TGFβ's inhibitory effect on insulin signaling. Since the insulin-stimulated association of phosphoinositide (PI) 3-kinase with IRS-1 depends on IRS-1 tyrosine phosphorylation, we measured the presence of the PI 3-kinase 85 kDa regulatory subunit in anti-IRS-1 immunoprecipitates. Following insulin stimulation, PI 3-kinase-IRS-1 association was reduced by 70% in TGFβ pretreated vs. control preadipose cells. However, insulin-stimulated cellular production of PI(3,4,5)P3 was unaltered by TGFβ pretreatment. This suggests that IRS-1-associated p85-type PI 3-kinase may represent a particular subset of total cellular PI 3-kinase that is specifically inhibited by TGFβ. Reduction of insulin-stimulated association of IRS-1 with p85-type PI 3-kinase by TGFβ may be one potential mechanism through which TGFβ blocks 3T3-L1 adipose cell differentiation. J. Cell. Physiol. 175:370–378, 1998. © 1998 Wiley-Liss, Inc.

Extracellular matrix induced by TGFβ impairs insulin signal transduction in 3T3-L1 preadipose cells

When 3T3-L1 preadipose cells are exposed to transforming growth factor beta (TGFbeta), they synthesize more extracellular matrix (ECM) and resist differentiation-inducing stimuli. The mechanism by which ECM suppresses adipose cell differentiation (adipogenesis) remains unknown. Since adipogenesis is an insulin/insulin-like growth factor-1 (IGF-1)-dependent process, we investigated whether TGFbeta-induced ECM inhibits insulin signaling. When preadipose cells were pretreated overnight with TGFbeta, we observed a 75% decrease in insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) compared to that in control cells. Culturing 3T3-L1 preadipose cells on fibronectin, a component of the ECM induced by TGFbeta, also inhibited insulin-dependent IRS-1 tyrosine phosphorylation and adipogenesis, supporting a role for ECM in mediating TGFbeta's inhibitory effect on insulin signaling. Since the insulin-stimulated association of phosphoinositide (PI) 3-kinase with IRS-1 depends on IRS-1 tyrosine phosphorylation, we measured the presence of the PI 3-kinase 85 kDa regulatory subunit in anti-IRS-1 immunoprecipitates. Following insulin stimulation, PI 3-kinase-IRS-1 association was reduced by 70% in TGFbeta pretreated vs. control preadipose cells. However, insulin-stimulated cellular production of PI(3,4,5)P3 was unaltered by TGFbeta pretreatment. This suggests that IRS-1-associated p85-type PI 3-kinase may represent a particular subset of total cellular PI 3-kinase that is specifically inhibited by TGFbeta. Reduction of insulin-stimulated association of IRS-1 with p85-type PI 3-kinase by TGFbeta may be one potential mechanism through which TGFbeta blocks 3T3-L1 adipose cell differentiation.