Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.     

A Modified Protocol to Maximize Differentiation of Human Preadipocytes and Improve Metabolic Phenotypes

Adipose stromal cells proliferate and differentiate into adipocytes, providing a valuable model system for studies of adipocyte biology. We compared differentiation protocols for human preadipocytes and report on their metabolic phenotypes. By simply prolonging the adipogenic induction period from the first 3 to 7 days, the proportion of cells acquiring adipocyte morphology increased from 30–70% to over 80% in human subcutaneous preadipocytes (passages 5–6). These morphological changes were accompanied by increases in the adipogenic marker expression and improved adipocyte metabolic phenotypes: enhanced responses to β‐adrenergically stimulated lipolysis and to insulin‐stimulated glucose metabolism into triglyceride (TG). Confirming previous studies, fetal bovine serum (FBS) dose‐dependently inhibited adipogenesis. However, in subcutaneous preadipocytes that differentiate well (donor‐dependant high capacity and subcultured fewer than two times), the use of 7d‐induction protocols in both 3% FBS and serum‐free conditions allowed >80% differentiation. Responsiveness to β‐adrenergically stimulated lipolysis was lower in 3% FBS. Rates of insulin‐stimulated glucose uptake were higher in adipocytes differentiated with 3% FBS, whereas the sensitivity to insulin was almost identical between the two groups. In summary, extending the length of the induction period in adipogenic cocktail improves the degree of differentiation and responses to key metabolic hormones. This protocol permits functional analysis of metabolic phenotypes in valuable primary human adipocyte cultures through multiple passages.     

Adiponectin expression in humans is dependent on differentiation of adipocytes and down-regulated by humoral serum components of high molecular weight

Adiponectin is an adipocytokine with profound anti-diabetic and anti-atherogenic effects. Even though adiponectin expression is restricted to adipocytes, serum levels are paradoxically decreased in obesity. We characterized how adiponectin expression and regulation relates to adipocyte differentiation in a human adipocyte cell culture model. Adiponectin was not expressed by human preadipocytes. Differentiation into adipocytes was necessary to induce an increasing expression of adiponectin (359 +/- 64-fold, P < 0.001) in parallel to an increasing expression of adipocyte differentiation markers. Adiponectin protein synthesis and secretion occurred specifically in mature adipocytes and may thus serve as a distinctive marker of adipocyte differentiation. Addition of serum during the course of differentiation as well as acutely to mature adipocytes significantly and concentration-dependently suppressed adiponectin to almost non-detectable levels (to 9.8 +/- 0.03%, P = 0.0043), suggesting a strong humoral serum component of adiponectin down-regulation. This serum component is present in both obese and lean individuals with a tendency to a stronger effect in obese men and women. Separation by molecular size suggests that higher molecular weight (>30 kDa) fractions exert inhibition of adiponectin. Withdrawal of adipogenic ingredients from the culture medium also resulted in a decrease of adiponectin expression and secretion to 62.01 +/- 0.09% and 70.86 +/- 0.05%, respectively. We identified insulin as a critical component to maintain adiponectin expression with a down-regulation to 61.6 +/- 0.1% (P = 0.0011) in the absence of insulin. These dynamic changes of adiponectin expression and regulation with adipocyte differentiation are of physiological interest in the light of the paradoxical decrease of adiponectin levels and the continuous recruitment of preadipocytes for differentiation in obesity.     

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 adipocyte differentiation and insulin action with rapamycin

Here, we demonstrated that inhibition of mTOR with rapamycin has negative effects on adipocyte differentiation and insulin signaling. Rapamycin significantly reduced expression of most adipocyte marker genes including PPARgamma, adipsin, aP2, ADD1/SREBP1c, and FAS, and decreased intracellular lipid accumulation in 3T3-L1 and 3T3-F442A cells, suggesting that rapamycin would affect both lipogenesis and adipogenesis. Contrary to the previous report that suppressive effect of rapamycin on adipogenesis is limited to the clonal expansion, we revealed that its inhibitory effect persisted throughout the process of adipocyte differentiation. Thus, it is likely that constitutive activation of mTOR might be required for the execution of adipogenic programming. In differentiated 3T3-L1 adipocytes, chronic treatment of rapamycin blunted the phosphorylation of AKT and GSK, which is stimulated by insulin, and reduced insulin-dependent glucose uptake activity. Taken together, these results suggest that rapamycin not only prevents adipocyte differentiation by decrease of adipogenesis and lipogenesis but also downregulates insulin action in adipocytes, implying that mTOR would play important roles in adipogenesis and insulin action.     

Measuring committed preadipocytes in human adipose tissue from severely obese patients by using adipocyte fatty acid binding protein

To understand the significance of the reported depot differences in preadipocyte dynamics, we developed a procedure to identify committed preadipocytes in the stromovascular fraction of fresh human adipose tissue. We documented that adipocyte fatty acid binding protein (aP2) is expressed in human preadipocyte clones capable of replication, indicating that can be used as a marker of committed preadipocytes. Because aP2 expression can be induced in macrophages, stromovascular cells were also stained for the macrophage marker CD68. We found aP2+CD68- cells (designated as committed preadipocytes) that did not have lipid droplets (true preadipocytes) and that did have lipid droplets < 6.5 microm in diameter (very immature adipocytes). Adipose tissue from subcutaneous, omental, and mesenteric depots was obtained from nine patients undergoing bariatric surgery for measurement of stromovascular cell number, the number of committed preadipocytes (aP2+CD68-), aP2+ macrophages (aP2+CD68+), and aP2- macrophages (aP2-CD68+). The number of committed preadipocytes did not differ significantly between depots but varied >20-fold among individuals. Total cell number, stromovascular cell number, and the number of aP2- macrophages was less (P < 0.05) in subcutaneous than in omental fat (means +/- SE, in millions: subcutaneous, 2.3 +/- 0.3, 1.4 +/- 0.3, and 0.17 +/- 0.08; and omental, 4.8 +/- 0.7, 3.8 +/- 0.5, and 0.34 +/- 0.06); mesenteric depot was intermediate. These data indicate that the cellular composition of adipose tissue varies between depots and between individuals. The ability to quantify committed preadipocytes in fresh adipose tissue should facilitate study of adipose tissue biology.     

Rehmannia inhibits adipocyte differentiation and adipogenesis

Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPβ, as well as C/EBPα and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPβ to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.     

An Evi1-C/EBPβ complex controls peroxisome proliferator-activated receptor γ2 gene expression to initiate white fat cell differentiation

Fibroblastic preadipocyte cells are recruited to differentiate into new adipocytes during the formation and hyperplastic growth of white adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, is expressed at low levels in preadipocytes, and its levels increase dramatically and rapidly during the differentiation process. However, the mechanisms controlling the dynamic and selective expression of PPARγ in the adipocyte lineage remain largely unknown. We show here that the zinc finger protein Evi1 increases in preadipocytes at the onset of differentiation prior to increases in PPARγ levels. Evi1 expression converts nonadipogenic cells into adipocytes via an increase in the predifferentiation levels of PPARγ2, the adipose-selective isoform of PPARγ. Conversely, loss of Evi1 in preadipocytes blocks the induction of PPARγ2 and suppresses adipocyte differentiation. Evi1 binds with C/EBPβ to regulatory sites in the Pparγ locus at early stages of adipocyte differentiation, coincident with the induction of Pparγ2 expression. These results indicate that Evi1 is a key regulator of adipogenic competency.     

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.     

过表达RBP4抑制体外培养的猪前体脂肪细胞分化

为研究视黄醇结合蛋白4(retinol binding protein 4,RBP4)对猪前体脂肪细胞分化的影响,实验构建了RBP4重组腺病毒表达载体,包装并感染猪前体细胞,采用油红O染色和Real-time PCR等方法,检测了过表达RBP4对成脂分化的作用. 研究结果显示,重组腺病毒RBP4载体构建成功,转染猪前体脂肪细胞后,使RBP4的mRNA水平和蛋白水平分别增加了约400倍和20倍. 过表达RBP4能减少脂肪细胞的脂质积累,降低成脂关键基因过氧化物酶体增生物激活受体γ (peroxisome proliferator-activated receptor gamma, PPARγ)和脂肪酸结合蛋白2 (adipocyte protein 2, aP2)的表达. 结果表明,RBP4对猪前体脂肪细胞分化有抑制作用,为进一步研究RBP4对猪前体脂肪细胞分化的作用机制奠定基础.     

Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice-including relationship of sex differences

We have previously demonstrated that adipose-derived stromal cells (ASCs) as well as bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. Both types are considered to include mesenchymal stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have also previously reported the plasticity of BSCs and ASCs. In this study, we focused on adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice. Moreover, preadipocytes and mature adipocytes were harvested at the same time, and the cells were cultured to compare them with ASCs. Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs, preadipocytes, and mature adipocytes. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks. Adipogenic differentiation of ASCs was assessed by Oil Red O staining and the expression of the adipocyte specific peroxisome proliferative activated receptor gamma2 (PPAR-gamma2) gene. These ASCs stained positively, and expression of PPAR-gamma2 was detected. Moreover, we also tried to characterize the influence of sex differences on the adipogenic differentiation of ASCs harvested from both male and female mice. This was assessed by the expression levels of the PPAR-gamma2 gene using real-time PCR. The results showed that the expression levels of ASCs harvested from female mice were a maximum of 2.89 times greater than those harvested from male mice. This suggests that the adipogenic differentiation of ASCs is closely related to sex differences.     

OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype

Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.