bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

The Farnesoid X Receptor Regulates Adipocyte Differentiation and Function by Promoting Peroxisome Proliferator-activated Receptor-γ and Interfering with the Wnt/β-Catenin Pathways

The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipocyte differentiation and function. The aim of this study was to analyze the role of FXR in adipocyte function and to assess whether it modulates PPARγ action. Therefore, we tested the responsiveness of FXR-deficient mice (FXR^−/−) and cells to the PPARγ activator rosiglitazone. Our results show that genetically obese FXR^−/−/ob/ob mice displayed a resistance to rosiglitazone treatment. In vitro, rosiglitazone treatment did not induce normal adipocyte differentiation and lipid droplet formation in FXR^−/− mouse embryonic fibroblasts (MEFs) and preadipocytes. Moreover, FXR^−/− MEFs displayed both an increased lipolysis and a decreased de novo lipogenesis, resulting in reduced intracellular triglyceride content, even upon PPARγ activation. Retroviral-mediated FXR re-expression in FXR^−/− MEFs restored the induction of adipogenic marker genes during rosiglitazone-forced adipocyte differentiation. The expression of Wnt/β-catenin pathway and target genes was increased in FXR^−/− adipose tissue and MEFs. Moreover, the expression of several endogenous inhibitors of this pathway was decreased early during the adipocyte differentiation of FXR^−/− MEFs. These findings demonstrate that FXR regulates adipocyte differentiation and function by regulating two counteracting pathways of adipocyte differentiation, the PPARγ and Wnt/β-catenin pathways.     

Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE₂. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE₂ levels. In turn, PGE₂ suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE₂ on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B₄). Taken together, these findings identify PGE₂ as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.     

Direct Angiotensin II Type 2 Receptor Stimulation Ameliorates Insulin Resistance in Type 2 Diabetes Mice with PPARγ Activation

Objectives

The role of angiotensin II type 2 (AT₂) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT₂ receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue.

Methods

T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[³H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined.

Results

Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored β cell damage in diabetic pancreatic tissue.

Conclusion

The present study demonstrated that direct AT₂ receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic β cells.     

Involvement of Inducible 6-Phosphofructo-2-kinase in the Anti-diabetic Effect of Peroxisome Proliferator-activated Receptor γ Activation in Mice

PFKFB3 is the gene that codes for the inducible isoform of 6-phosphofructo-2-kinase (iPFK2), a key regulatory enzyme of glycolysis. As one of the targets of peroxisome proliferator-activated receptor γ (PPARγ), PFKFB3/iPFK2 is up-regulated by thiazolidinediones. In the present study, using PFKFB3/iPFK2-disrupted mice, the role of PFKFB3/iPFK2 in the anti-diabetic effect of PPARγ activation was determined. In wild-type littermate mice, PPARγ activation (i.e. treatment with rosiglitazone) restored euglycemia and reversed high fat diet-induced insulin resistance and glucose intolerance. In contrast, PPARγ activation did not reduce high fat diet-induced hyperglycemia and failed to reverse insulin resistance and glucose intolerance in PFKFB3^+/− mice. The lack of anti-diabetic effect in PFKFB3^+/− mice was associated with the inability of PPARγ activation to suppress adipose tissue lipolysis and proinflammatory cytokine production, stimulate visceral fat accumulation, enhance adipose tissue insulin signaling, and appropriately regulate adipokine expression. Similarly, in cultured 3T3-L1 adipocytes, knockdown of PFKFB3/iPFK2 lessened the effect of PPARγ activation on stimulating lipid accumulation. Furthermore, PPARγ activation did not suppress inflammatory signaling in PFKFB3/iPFK2-knockdown adipocytes as it did in control adipocytes. Upon inhibition of excessive fatty acid oxidation in PFKFB3/iPFK2-knockdown adipocytes, PPARγ activation was able to significantly reverse inflammatory signaling and proinflammatory cytokine expression and restore insulin signaling. Together, these data demonstrate that PFKFB3/iPFK2 is critically involved in the anti-diabetic effect of PPARγ activation.     

Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.