Pref-1 (preadipocyte factor 1) activates the MEK/extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation

Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore, treatment of Pref-1 null MEF with Pref-1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation primarily by preventing peroxisome proliferator-activated receptor gamma2 induction. However, in the presence of PD98059 or depletion of ERK1/2, exogenous Pref-1 cannot inhibit adipocyte differentiation in Pref-1 null MEF. We conclude that Pref-1 activates MEK/ERK signaling, which is required for Pref-1 inhibition of adipogenesis.     

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.     

Cancer cells, adipocytes and matrix metalloproteinase 11: a vicious tumor progression cycle

This brief review focuses on the emerging role of matrix metalloproteinase 11 (MMP-11) in cancer progression. It has recently been shown that MMP-11 is induced in adipose tissue by cancer cells as they invade their surrounding environment. MMP-11 negatively regulates adipogenesis by reducing pre-adipocyte differentiation and reversing mature adipocyte differentiation. Adipocyte dedifferentiation in turn leads to the accumulation of nonmalignant peritumoral fibroblast-like cells, which favor cancer cell survival and tumor progression. This MMP-11-mediated bi-directional cross-talk between invading cancer cells and adjacent adipocytes/pre-adipocytes highlights the central role that MMP-11 plays during tumor desmoplasia and represents a molecular link between obesity and cancer.     

Obestatin as a regulator of adipocyte metabolism and adipogenesis

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/β, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPβ, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.     

Ablation of NG2 proteoglycan leads to deficits in brown fat function and to adult onset obesity

Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O(2) consumption and CO(2) production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes.     

Regulation of pre-adipocyte proliferation and apoptosis by the small leucine-rich proteoglycans, biglycan and decorin

Evidence for a functional role for extracellular matrix (ECM) proteins in adipose tissue is demonstrated in dynamic changes in expression of ECM genes during adipocyte differentiation and in obesity. Components of the ECM may regulate adipose cell number expansion by restricting pre-adipocyte proliferation, regulating apoptosis and inhibiting adipogenesis. Although pre-adipocytes express multiple proteoglycans, their role in pre-adipocyte proliferation up to now has remained unknown. The study described here was conducted to characterize roles of small leucine-rich proteoglycans (SLRPs) in adipocyte proliferation. Pre-adipocytes were seeded on plates coated with biglycan and decorin and were allowed to differentiate. In addition, pre-adipocytes were incubated on plates coated with biglycan, decorin, or fibronectin and measurements were made of cell proliferation and apoptosis. We are able to report that SLRPs decorin and biglycan did not affect differentiation of our 3T3-L1 cells; however, biglycan and decorin did reduce proliferation of pre-adipocytes, partly by induction of apoptosis. Furthermore, anti-proliferative capabilities of decorin and biglycan were nullified with removal of GAG side-chains suggesting that the chains played key roles in anti-proliferative effects of the SLRPs. We also found that co-treatment of decorin or biglycan with the proteoglycan fibronectin restored normal proliferation, an indication that multiple ECM proteins may act in concert to regulate overall proliferation rates of pre-adipocytes. These studies indicate that SLRPs may compose a regulatory factor in adipose tissue expansion, through hyperplasia.     

Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.     

Adipocyte differentiation is modulated by secreted delta-like (dlk) variants and requires the expression of membrane-associated dlk

Previous studies demonstrate that the delta-like (dlk) and preadipocyte factor 1 (Pref-1) genes encode similar proteins. Pref-1 is downregulated during adipocyte differentiation, and expression of ectopic Pref-1 inhibits adipogenesis. We explored whether dlk functions similarly to Pref-1 and studied the role of alternately spliced dlk variants encoding membrane-associated or -secreted forms. We also studied whether enforced downregulation of dlk/Pref-1 may enhance the differentiation response of non-committed cells. Ectopic expression of a potentially secreted dlk variant, conditioned media from dlk expressing cells or several individual epidermal-growth-factor-dlk peptides inhibited 3T3-L1 differentiation. This demonstrates that dlk and Pref-1 are functionally equivalent. dlk gene mRNA encoding for secreted variants decreased much faster than total dlk gene mRNA during differentiation of 3T3-L1 cells. In fact, total dlk or membrane-associated dlk protein expression increased during the first hours of differentiation. Cells sorted for lowest levels of dlk protein diminished or lost their ability to differentiate. These data suggest that membrane and secreted dlk protein variants play opposite roles in the control of adipogenesis. In addition, enforced downregulation of dlk protein expression in the weakly adipogenic Balb/c 3T3 cell line dramatically enhanced adipogenesis in response to insulin. These results indicate that dlk protein not only participates in processes leading to inhibition of adipogenesis but that the control of its expression and different spliced variants is essential for the adipogenic response to extracellular signals.     

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells简

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue (WAT) or brown adipose tissue (BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brown-like adipocytes were discovered in WAT. These brown-like adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expression pattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation (adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.     

Zic1 mRNA is transiently upregulated in subcutaneous fat of acutely cold-exposed mice

In the mammalian adipose organ cold exposure not only activates typical brown adipose tissue, but also induces browning, that is the formation of thermogenic multilocular adipocytes in white, or predominantly white, adipose depots such as subcutaneous fat. Unlike typical brown adipocytes, newly formed thermogenic adipocytes have been reported not to express the gene zinc finger of the cerebellum 1 (Zic1). Here, a time course approach enabled us to document a significant increase in Zic1 messenger RNA in inguinal subcutaneous fat from acutely (24 hr) cold-exposed mice, which was paralleled by an increase in multilocular and paucilocular uncoupling protein 1-positive adipocytes and in parenchymal noradrenergic innervation. This transient, depot-specific molecular signature was associated not to Zic1 promoter demethylation, but to chromatin remodeling through an H3K9me3 histone modification. These findings challenge the notion that Zic1 is exclusively expressed by typical brown adipocytes and suggest its involvement in brown adipocyte precursor differentiation and/or white-to-brown adipocyte transdifferentiation.     

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue(WAT) or brown adipose tissue(BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brownlike adipocytes were discovered in WAT. These brownlike adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expressionpattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation(adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.     

Myostatin inhibits brown adipocyte differentiation via regulation of Smad3-mediated β-catenin stabilization

Brown adipocytes play an important role in regulating energy balance, and there is a good correlation between obesity and the amount of brown adipose tissue. Although the molecular mechanism of white adipocyte differentiation has been well characterized, brown adipogenesis has not been studied extensively. Moreover, extracellular factors that regulate brown adipogenic differentiation are not fully understood. Here, we assessed the mechanism of the regulatory action of myostatin in brown adipogenic differentiation using primary brown preadipocytes. Our results clearly showed that differentiation of brown adipocytes was significantly inhibited by myostatin treatment. In addition, myostatin-induced suppression of brown adipogenesis was observed during the early phase of differentiation. Myostatin induced the phosphorylation of Smad3, which led to increased β-catenin stabilization. These effects were blocked by treatment with a Smad3 inhibitor. Expression of brown adipocyte-related genes, such as PPAR-γ, UCP-1, PGC-1α, and PRDM16, were dramatically down-regulated by treatment with myostatin, and further down-regulated by co-treatment with a β-catenin activator. Taken together, the present study demonstrated that myostatin is a potent negative regulator of brown adipogenic differentiation by modulation of Smad3-induced β-catenin stabilization. Our findings suggest that myostatin could be used as an extracellular factor in the control of brown adipocyte differentiation.     

Pref-1 and ADSF/resistin: two secreted factors inhibiting adipose tissue development.

Adipose tissue is the source of a wide array of factors of great biological significance that are involved in many aspects of organism physiology, including appetite control and peripheral metabolism. Here, we describe two secreted factors from adipose tissue that inhibit adipogenesis. Pref-1 is a preadipocyte secreted factor synthesized as a transmembrane protein that undergoes proteolitic cleavage to generate two distinct soluble forms. In vitro assays have demonstrated that only the large soluble form of Pref-1 is biologically active and inhibits adipocyte differentiation. In vivo, mice lacking Pref-1 expression show accelerated fat deposition, perinatal mortality and growth retardation as well as distinct skeletal malformations, highlighting the importance of Pref-1 during mouse development in addition to its role in adipose tissue development. ADSF/resistin is secreted by adipocytes and inhibits adipose cells differentiation in vitro. Its function is still unclear, but its expression and high circulating levels have been associated with an impairment of insulin action. The findings show that Pref-1 and possibly ADSF/resistin secretion control fat cell differentiation and adipose tissue development.     

Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs)

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARγ (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARγ expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.