Interleukin-4 inhibits platelet-derived growth factor-induced preadipocyte proliferation

Interleukin-4 (IL-4) activates STAT6 in 3T3-L1 preadipocytes but its functional role is not known. In this report, we first assessed interleukin-4 receptor alpha (IL-4Ralpha) expression during adipogenesis. IL-4Ralpha was highly expressed in proliferating 3T3-L1 preadipocytes. Receptor expression was down-regulated in post-confluent growth arrested preadipocytes. Induction of differentiation led to a transient 36-h increase in expression, but then levels decreased to undetectable amounts 3-8 days after induction of differentiation. Depending on the cell type, IL-4 either increases or decreases cell proliferation. In growth arrested preconfluent 3T3-L1 preadipocytes, IL-4 alone had no effect on preadipocyte proliferation. In contrast, IL-4 inhibited platelet-derived growth factor (PDGF-BB) induced preadipocyte proliferation. PDGF-BB, but not IL-4, induced STAT3 tyrosine and AKT serine phosphorylation. Both PDGF-BB and IL-4 induced STAT6 tyrosine phosphorylation, but the bands showed distinct electrophoretic migration patterns. IL-4 alone and IL-4 added to the differentiation cocktail had no effect on adipocyte formation or PPARgamma expression. Collectively, these studies demonstrate that IL-4 inhibits PDGF-BB-induced preadipocyte proliferation, possibly through STAT6 activation. The pattern of IL-4 receptor expression suggests that the effects of IL-4 are targeted primarily towards preadipocytes.     

LRP1 Receptor Controls Adipogenesis and Is Up-Regulated In Human and Mouse Obese Adipose Tissue

The cell surface low-density lipoprotein receptor-related protein 1, LRP1, plays a major role in lipid metabolism. The question that remains open concerns the function of LRP1 in adipogenesis. Here, we show that LRP1 is highly expressed in murine preadipocytes as well as in primary culture of human adipocytes. Moreover, LRP1 remains abundantly synthesised during mouse and human adipocyte differentiation. We demonstrate that LRP1 silencing in 3T3F442A murine preadipocytes significantly inhibits the expression of PPARγ, HSL and aP2 adipocyte differentiation markers after adipogenesis induction, and leads to lipid-depleted cells. We further show that the absence of lipids in LRP1-silenced preadipocytes is not caused by lipolysis induction. In addition, we provide the first evidences that LRP1 is significantly up-regulated in obese C57BI6/J mouse adipocytes and obese human adipose tissues. Interestingly, silencing of LRP1 in fully-differentiated adipocytes also reduces cellular lipid level and is associated with an increase of basal lipolysis. However, the ability of mature adipocytes to induce lipolysis is independent of LRP1 expression. Altogether, our findings highlight the dual role of LRP1 in the control of adipogenesis and lipid homeostasis, and suggest that LRP1 may be an important therapeutic target in obesity.     

Regulation of Wnt signaling during adipogenesis

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.     

Culture and differentiation of preadipocytes in two-dimensional and three-dimensional in vitro systems

Adipogenesis is a complex process that involves the differentiation of preadipocytes into mature adipocytes. We have developed two-dimensional (2D) and three-dimensional (3D) cell culture systems for the purpose of culturing and differentiating primary preadipocytes in vitro. Differentiating preadipocytes show multiple lipid droplet accumulation and comparable protein expression patterns to mature adipocytes in vivo. We report that in both in vitro systems terminally differentiated adipocytes show characteristics similar to those of mature adipocytes in vivo, assessed by the expression of the S100alpha/beta protein, insulin receptor and caveolin-1, and receptors for inflammatory mediators, namely tumor necrosis factor-alpha receptors I and II (TNFRI and TNFRII) and chemokine receptor 5 (CCR5). Our results demonstrate that the S100 protein, caveolin-1, and insulin receptor are expressed and up-regulated in differentiating and terminally differentiated cells. In addition, the receptors for TNFalpha are not present in preadipocytes but are expressed in differentiating preadipocytes and in differentiated adipocytes. Similarly, CCR5 was exclusively expressed in differentiating preadipocytes and terminally differentiated adipocytes, but not in preadipocytes. Both 2D and 3D culture models are highly robust and reproducible and offer the potential to study adipogenesis and cellular interactions closely resembling and comparable to those in vivo. Our 3D collagen system offers a distinct advantage over the 2D system in that the adipocytes remain confined within the matrix and remain intact during biochemical analysis. Moreover, the collagen matrix allows adipocytes to closely simulate morphological characteristics and behavior as in vivo whilst permitting manipulation of the microenvironment in vitro to study adipogenesis.     

Cathepsin-D, a key protease in breast cancer, is up-regulated in obese mouse and human adipose tissue, and controls adipogenesis

The aspartic protease cathepsin-D (cath-D) is overexpressed by human epithelial breast cancer cells and is closely correlated with poor prognosis in breast cancer. The adipocyte is one of the most prominent cell types in the tumor-microenvironment of breast cancer, and clinical studies have shown that obesity increases the incidence of breast cancer. Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse. Cath-D expression is also increased during human and mouse adipocyte differentiation. We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers. Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity.     

SiRNA against Fabp5 induces 3T3-L1 cells apoptosis during adipocytic induction

Fatty acid-binding protein 5 (Fabp5), exhibits an important role in binding free fatty acids, as well as regulating lipid metabolism and transport. The purpose of this study was to evaluate the role of Fabp5 during adipogenesis. 3T3-L1 preadipocytes were selected as cell differentiation model and short interfering RNAs (siRNA) against Fabp5 (siFabp5) were prepared. Our results showed that two potent siFabp5 specifically inhibited endogenous expression of Fabp5 at both mRNA and protein level. SiFabp5 had little effect on undifferentiated 3T3-L1 fibroblasts. However, during adipocytic induction, 3T3-L1 preadipocytes transfected with siFabp5 significantly reduced cell viability, as well as increased both caspase-3 activity and procaspase-3 cleavage. Furthermore, we illustrated that knockdown Fabp5 inhibited the expression of PPARγ and C/EBPα during adipocytic induction. In conclusion, our data suggests that Fabp5 is crucial in maintaining the viability of preadipocytes during adipogenesis via the activation of Akt cascade, and decreased Fabp5 expression induce differentiating preadipocytes apoptosis via caspase-3 activation.     

Aortic carboxypeptidase-like protein is regulated by transforming growth factor beta in 3T3-L1 preadipocytes

Adipogenesis is characterized by early remodeling of the extracellular matrix, allowing preadipocytes to adopt a more spherical shape and optimize lipid accumulation as they mature. Aortic carboxypeptidase-like protein (ACLP), found in collagen-rich tissues including adipose tissue, is expressed in 3T3-L1 and 3T3-F442A preadipocytes, and is downregulated during adipogenesis. We now report that ACLP is found in medium conditioned by 3T3-L1 preadipocytes. Transforming growth factor (TGF) beta, a known modulator of fibrillar matrix protein production, increased ACLP expression by 2.4+/-0.4-fold (mean+/-SE; n=3) in 3T3-L1 preadipocytes, through a mechanism that requires p42/44 MAPK activity. Addition of TGFbeta to differentiation medium, which inhibits adipogenesis, raised ACLP levels in 3T3-L1 cells. However, sustained expression of ACLP in stable clones of 3T3-L1 or 3T3-F442A preadipocytes did not interfere with adipogenesis.     

Expression of p107 and p130 during human adipose-derived stem cell adipogenesis

Within the first 24 h of hormonally stimulated adipocyte differentiation, murine 3T3-L1 preadipocytes undergo a mitotic expansion phase prior to terminal differentiation. During this time, the cell cycle regulatory proteins, p130 and p107 undergo dramatic differential expression and the transient increase in expression of p107 appears to be required for terminal differentiation. Recently, human adipose-derived human stem cells (hASC) of mesenchymal origin have been used as a model of human adipocyte differentiation and we sought to determine if differentiating hASC undergo clonal expansion and if the regulated expression of p130/p107 was similar to that observed during 3T3-L1 adipogenesis. Results indicate that differentiating hASC, unlike 3T3-L1 cells do not undergo clonal expansion and p130 expression gradually diminishes across differentiation. However, p107 expression is transiently increased during hASC differentiation in a manner analogous to 3T3-L1 cells suggesting a similar role for p107 in terminal differentiation in human adipocytes.     

p38MAP Kinase activity is required for human primary adipocyte differentiation

Little is known about the role of p38MAPK in human adipocyte differentiation. Here we showed that p38MAPK activity increases during human preadipocytes differentiation. Pharmacological inhibition of p38MAPK during adipocyte differentiation of primary human preadipocytes markedly reduced triglycerides accumulation and adipocyte markers expression. Cell cycle arrest or proliferation was not affected by p38MAPK inhibition. Although induction of C/EBPbeta was not altered by the p38MAPK inhibitor, its phosphorylation on Threonine(188) was decreased as well as PPARgamma expression. These results indicate that p38MAPK plays a positive role in human adipogenesis through regulation of C/EBPbeta and PPARgamma factors.