Adipocyte Differentiation Process Research Articles

Retinoic acid modulates the retinoblastoma protein during adipocyte terminal differentiation

Terminal differentiation is characterized by a permanent withdrawal of cells from the cell cycle. Retinoblastoma protein (RB) has been involved in cell cycle progression. Accumulating evidence also implicates RB in the promotion of differentiation of many cell types. We present new insights into the role of RB and other cell cycle regulatory proteins in adipocyte differentiation and on the role of retinoic acid (RA) in the regulation of the latter process. It is shown that RA reduces RB expression and enhances RB phosphorylation by a mechanism that involves down-regulation of the cyclin-dependent kinase inhibitor (CKI) p21(Cip1), having this fact as important consequences for both the cell cycle progression and the adipocyte differentiation process. The effects of RA result in the blockage of adipogenesis, but may also favor the retention of a pool of adipose cells able to re-enter the cell cycle, which may be important for the developmental dynamics of adipose tissue in vivo. In addition, these results reinforce the idea that there is a cross-talk between the cell cycle machinery and the adipocyte differentiation machinery that can be modulated by external signals, including nutrients.

Protease Inhibitor Treatments Reveal Specific Involvement of Matrix Metalloproteinase-9 in Human Adipocyte Differentiation

We previously showed that human and murine 3T3-F442A preadipocytes produced and released matrix metalloproteinases (MMPs) 2 and 9 and that a treatment by MMP inhibitors resulted in the blockade of murine fat cell adipose conversion. In parallel, investigators reported that other protease inhibitors, the human immunodeficiency virus (HIV) protease inhibitors (PIs) involved in lipodystrophy in humans, also reduced the adipocyte differentiation process of several murine cell lines. The present work was performed to define the effects of MMP inhibitors and HIV-PIs on the human adipocyte differentiation process, to clarify the involvement of MMPs in the control of human adipogenesis, and to determine whether HIV-PIs interact with MMPs in the control of this process. The effect of two MMP inhibitor and four HIV-PI treatments on the differentiation of primary culture human preadipocytes, as well as the putative relationships between HIV-PIs and MMP-2 and -9 expression, release, or activity were investigated. We showed that MMP inhibitors and HIV-PIs reduced the human adipocyte differentiation process as assessed by the decrease of cell protein and/or triglyceride contents and expression of fatty acid binding protein and hormone-sensitive lipase, two adipocyte markers. Unlike MMP inhibitors, HIV-PIs were devoid of any effect per se on recombinant MMP-2 and 9 activities but reduced the expression and release of MMP-9 by human preadipocytes. Thus, the present study indicates that the modulation of the extracellular matrix components through the production and/or activity of MMPs, and, more precisely, MMP-9 might be a key factor in the regulation of human adipose tissue development.

Microarray evaluation of EP4 receptor-mediated prostaglandin E 2 suppression of 3T3-L1 adipocyte differentiation

Prostaglandin E 2 (PGE 2) has been shown to negatively regulate adipogenesis. To explore to what extent PGE 2 inhibits the differentiation of cells to adipocytes and to examine whether its effect could be due to EP4 receptor signaling, we used microarrays to analyze the gene expression profiles of 3T3-L1 cells exposed to a differentiation cocktail supplemented with PGE 2, AE1-329 (an EP4 agonist), or vehicle. The differentiation-associated responses in genes such as adipocytokines and enzymes related to lipid metabolism were largely weakened upon PGE 2 treatment. In particular, the expression of peroxisome proliferator activated receptor-γ and CCAAT/enhancer binding protein-α, genes playing a central role in adipogenesis, was greatly suppressed. PGE 2 appears to be ineffective to a subclass of insulin target genes such as hexokinase 2 and phosphofructokinase. Similar responses were produced in the differentiation-associated genes upon AE1-329 treatment. These results suggest that PGE 2 inhibits a crucial step of the adipocyte differentiation process by acting on the EP4 receptor in 3T3-L1 cells.

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 PPARγ, 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.

Development and differentiation of adipose tissue

For years adipose tissue has been considered inert, serving only as a depot of energy surplus. However, there have been recent changes, undoubtedly due to advancement of methods for studying the morphology and metabolic activities of adipose tissue (microdialysis and adipose tissue catheterization). In normal-weight subjects, adipose tissue makes 10-12% with males and 15-20% with females. About 80% of adipose tissue is located under the skin, and the rest envelops the internal organs. With humans there are white and brown adipose tissues, which is predominant with infants and small children. From a histological point of view, it is a special form of reticular connective tissue, which contains adipocytes with netlike structure. Human adipose tissue has four types of adrenergic receptors with different topographic dispositions, which manifest different metabolic activity of adipocytes of particular body organs. Changes in adipose tissue are associated with the process of adipocyte differentiation. Critical moments for this process are last months of pregnancy, the first six months of infancy and then puberty. However, the differentiation process may also begin during maturity. Namely, as size of adipocytes can increase to a certain limit, this process can be activated after reaching a "critical" adipocyte volume. The differentiation process is affected by a number of hormones (insulin, glucagon, corticosteroids, somatotropin (STH), thyroid gland hormones, prolactin, testosterone), but also by some other substances (fatty acids, prostaglandins, liposoluble vitamins, butyrate, aspirin, indomethacin, metylxanthine, etc.).

Adipogenesis: a cross-talk between cell proliferation and cell differentiation

The transition between cell proliferation and cell differentiation taking place during adipocyte differentiation is a tightly regulated process where both cell cycle regulators and differentiating factors interact, creating a cascade of events leading to the commitment of the cells into the adipocyte phenotype. Based on in-vitro cell models of adipocyte differentiation, the different stages of adipogenesis have been established, each of them with a particular pattern of gene expression. Re-entry into the cell cycle of growth-arrested preadipocytes is known as the clonal expansion phase. Growth-arrested preadipocytes undergo several rounds of cell cycle before terminally differentiating into adipocytes, suggesting that a cross-talk might exist between the cell cycle or the cell proliferation machinery and the factors controlling cell differentiation. I will focus this review on the influence of the proliferative phase of preadipocytes in the adipocyte differentiation process.

Adipocyte differentiation and transdifferentiation: plasticity of the adipose organ.

In mammals, the adipose organ is a multi-depot organ made of two tissue types, the white and brown adipose tissues, which collaborate in partitioning the energy contained in lipids between thermogenesis and the other metabolic functions. It consists of several sc and visceral depots. Some areas of these depots are brown and correspond to brown adipose tissue, while many are white and correspond to white adipose tissue. White areas contain a variable amount of brown adipocytes and their number varies with age, strain and environmental conditions. Brown and white adipocyte are morphologically different. At light microscopy level, brown adipocytes have cytoplasmic lipids arranged as numerous small droplets (multilocularity), while white adipocytes have cytoplasmic lipids arranged in a unique vacuole (unilocularity). Ultrastructurally, brown adipocytes have numerous big mitochondria packed with cristae and containing the thermogenic uncoupling protein 1 (UCP1). In vivo and in vitro studies have shown that the differentiation process of brown and white adipocytes shows distinctive features. Nevertheless, the origin of the adipocyte precursor is still unknown. Recent data have stressed the plasticity of the adipose organ in adult animals. Indeed, under peculiar conditions fully differentiated, white adipocytes can transdifferentiate into brown adipocytes, and viceversa. The ability of the adipose organ to interconvert its main cytotypes in order to meet changing metabolic needs is highly pertinent to the physiopathology of obesity and related to therapeutic strategies.

Effects of antiretroviral drug combinations on the differentiation of adipocytes.

Preadipocyte cell lines present a cell model with which to understand the physiopathological mechanisms underlying lipodystrophy syndrome, a common complication observed in patients treated with highly active antiretroviral therapy (HAART) that, in general, is associated with the use of protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors (NRTI). The aim of this study was to evaluate the effects of NRTI and of PI and NRTI combinations in this cell model. The differentiation of 3T3-F442A cells was studied by monitoring the expression of specific genes in the presence of therapeutic concentrations of antiretroviral drugs. Messenger RNA (mRNA) was quantified by two reverse transcription-PCR-based methods. In the presence of 2 microM saquinavir, 30 microM ritonavir or 1 microM zidovudine preadipocytes delayed their differentiation, whereas the use of 10 microM nelfinavir led to cell death. Indinavir (10 microM) promoted lipoprotein lipase expression whereas 1 microM lamivudine or 1 microM stavudine enhanced slightly the expression of the malic enzyme gene. However, the combination of indinavir, lamivudine and stavudine led to a large increase in both lipoprotein lipase and malic enzyme mRNA transcription whereas the combination of indinavir, lamivudine and zidovudine led to a 2.5-fold increase in the expression of the lipogenic malic enzyme gene. Similar potentiating effects of NRTI and PI were observed on the expression of the fatty acid synthase gene. Our data suggest that, like PI (although to a lesser extent) NRTI interfere with the differentiation process of adipocytes. In addition, we demonstrate that the effects produced by combinations of NRTI and PI are different from those elicited by each drug separately. This point may be particularly relevant in understanding the physiopathological mechanisms underlying the lipodystrophic syndrome.

Weight loss reduces expression of SREBP1c/ADD1 and PPARgamma2 in adipose tissue of obese women.

Weight loss in obese patients, even if moderate, is clearly beneficial for health and implies a reduction in either adipocyte number or volume. This can be regulated by the key adipose transcription factors, sterol-regulatory-element binding protein-1c/adipocyte differentiation and determination factor-1 (SREBP1c/ADD1), peroxisome proliferator-activated receptor-gamma2 (PPARgamma2) and CCAAT-enhancer binding protein-alpha (C/EBPalpha). which regulate the adipocyte metabolism and differentiation process. The present study was undertaken to obtain insights into the expression of these transcription factors during moderate weight loss in humans. In addition, the adipose depot-related differences and the relation to adipose lipoprotein lipase (LPL) expression and plasma lipids were studied. Using quantitative reverse transcription polymerase chain reaction (RT-PCR), the total amount of each adipose transcription factor messenger ribonucleic acid (mRNA) was determined in the subcutaneous or omental adipose tissue after a controlled, 2-month, bodyweight-reduction trial in 11 obese middle-aged women and 17 comparable obese controls. Weight loss (6% of body weight) was associated with reduced serum insulin and plasma triacylglycerols. Adipose tissue PPARgamma2 and SREBP1c/ADD1 mRNA were lower in the weight-loss group than in controls (by 30% and 28%, respectively), whereas the C/EBPalpha mRNA level did not change. Moreover, PPARgamma2 mRNA was lower only in the subcutaneous adipose depot and was related to both adipose tissue lipoprotein lipase (LPL) mRNA and improvement in plasma triacylglycerols in the weight-loss group. Our results suggest a functional role for SREBP1c/ADD1 and PPARgamma2 in the control of energy metabolism in human adipose tissue.

C-Crk, a Substrate of the Insulin-like Growth Factor-1 Receptor Tyrosine Kinase, Functions as an Early Signal Mediator in the Adipocyte Differentiation Process

Differentiation of 3T3-L1 preadipocytes into adipocytes is induced by a combination of inducers, including a glucocorticoid, an agent that elevates cellular cAMP, and a ligand of the insulin-like growth factor-1 receptor. Previous studies have implicated protein-tyrosine phosphatase (PTPase) HA2, a homologue of PTPase 1B, in the signaling cascade initiated by the differentiation inducers. Vanadate, a potent PTPase inhibitor, blocks adipocyte differentiation at an early stage in the program, but has no effect on the mitotic clonal expansion required for differentiation. Exposure of preadipocytes to vanadate along with the inducing agents led to the accumulation of pp35, a phosphotyrosyl protein that is a substrate for PTPase HA2. pp35 was purified to homogeneity and shown by amino acid sequence and mass analyses of tryptic peptides to be c-Crk, a known cytoplasmic target of the insulin-like growth factor-1 receptor tyrosine kinase. Transfection of 3T3-L1 preadipocytes with a c-Crk antisense RNA expression vector markedly reduced c-Crk levels and prevented differentiation into adipocytes. Studies with C3G, a protein that binds to the SH3 domain in c-Crk, showed that phosphorylation of c-Crk rendered the SH3 domain inaccessible to C3G. Taken together, these findings indicate that locking c-Crk in the phosphorylated state with vanadate prevents its participation in the signaling system that initiates adipocyte differentiation.

Ligand type-specific Interactions of Peroxisome Proliferator-activated Receptor γ with Transcriptional Coactivators

The nuclear peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and acts as a ligand-dependent transcription factor mediating adipocyte differentiation, cell proliferation and inflammatory processes, and modulation of insulin sensitivity. Members of the 160-kDa protein (SRC-1/TIF2/AIB-1) family of coactivators, CBP/p300 and TRAP220/DRIP205, are shown to interact directly with PPARgamma and potentiate nuclear receptor transactivation function in a ligand-dependent fashion. Because PPARgamma ligands exert partially overlapping but distinct subsets of biological action through PPARgamma binding, we wished to examine whether interactions between PPARgamma and known coactivators were induced to the same extent by different classes of PPARgamma ligand. The natural ligand 15-deoxy-Delta12,14-prostaglandin J(2) induced PPARgamma interactions with all coactivators tested (SRC-1, TIF2, AIB-1, p300, TRAP220/DRIP205) in yeast and mammalian two-hybrid assays, as well as in a glutathione S-transferase pull-down assay. However, under the same conditions troglitazone, a synthetic PPARgamma ligand that acts as an antidiabetic agent, did not induce PPARgamma interactions with any of the coactivators. Our findings suggest that ligand binding may alter PPARgamma structure in a ligand type-specific way, resulting in distinct PPARgamma-coactivator interactions.

Endrin inhibits adipocyte differentiation by selectively altering expression pattern of CCAAT/enhancer binding protein-alpha in 3T3-L1 cells.

The effects of selected chlorinated cyclodiene pesticides on the adipocyte differentiation process were examined using the 3T3-L1 adipocyte model in vitro. Endrin was found to cause a dose-dependent inhibition of adipocyte differentiation in 3T3-L1 cells. Aldrin and dieldrin were less potent than endrin in interfering with the adipogenic process. Endrin's inhibitory effect was effective only when the pesticide was present in the medium during the first 48 h after exposure of 3T3-L1 cells to adipogenic inducers. Immunoblots analysis revealed that endrin caused a dose-dependent, selective inhibition of the intracellular levels of CCAAT enhancer binding protein (C/EBP)alpha without altering the expression patterns of C/EBPbeta or C/EBPdelta along the differentiation. Supershift analysis showed that DNA-binding capacity of C/EBPalpha was affected most by endrin treatment. Endrin also caused a decrease in the elevation of the adipogenic factor peroxisome proliferator-activated receptor (PPAR)gamma elicited by the adipogenic inducers. However, the cotreatment with troglitazone, a thiazolidinedione known to activate PPARgamma, did not suppress the antiadipogenic action of endrin, indicating that its direct action site is not PPARgamma receptor. Endrin also altered the pattern of activation of nuclear factor-kappaB, a factor activated by 12-O-tetradecanoylphorbol-13-acetate and tumor necrosis factor-alpha, which are known to interfere with adipocyte differentiation. Thus, endrin inhibited the normal decrease in nuclear factor-kappaB-DNA binding observed as cells are acquiring the adipocyte phenotype at a late stage of differentiation. Our results suggest that endrin inhibits adipocyte differentiation through the specific suppression of C/EBPalpha.

Effects of caffeine on lipoprotein lipase gene expression during the adipocyte differentiation process.

In this study, the effects of caffeine on lipoprotein lipase (LPL) gene expression were investigated in the 3T3-F442A preadipocyte cell line during the adipocyte differentiation process by determining LPL enzymatic activity and its messenger RNA (mRNA) level. The results demonstrate that caffeine acts on the gene expression of LPL, an early marker of adipocyte differentiation. It has a biphasic action: it increases gene expression in terms of mRNA when it is added to preadipocytes during the early stage of differentiation, but this is accompanied by a reduction of enzymatic activity. On the other hand, when caffeine is added for long periods during differentiation and/or when it is added to mature adipocytes, it induces marked inhibition of mRNA levels, correlated with a marked reduction of secreted enzymatic activity. The inhibitory effect of caffeine on LPL mRNA level can be reproduced by theophylline, a phosphodiesterase inhibitor, and by dibutyryl cyclic AMP, a non-metabolizable analog of cyclic AMP. However, the effect of caffeine and theophylline lasts longer than that of cyclic AMP, suggesting that a mechanism other than inhibition of cyclic AMP hydrolysis may be involved in the action of caffeine.

Transcriptional control of adipogenesis

Adipocyte differentiation is coordinatedly regulated by several transription factors. C/EBPβ, C/EBPδ and ADD-1/SREBP-1 are active early during the differentiation process and induce the expression and/or activity of the peroxisome proliferator activated receptor-γ (PPARγ), the pivotal coordinator of the adipocyte differentiation process. Activated PPARγ induces exit from the cell cycle and triggers the expression of adipocyte-specific genes, resulting in increased delivery of energy to the cells. C/EBPα, whose expression coincides with the later stages of differentiation, cooperates with PPARγ in inducing additional target genes and sustains a high level of PPARγ in the mature adipocyte as part of a feedforward loop. Altered activity and/or expression of these transcription factors might underlie the pathogenesis of disorders characterized by increased or decreased adipose tissue depots.

Adipocyte differentiation and leptin expression.

Adipose tissue has long been known to house the largest energy reserves in the animal body. Recent research indicates that in addition to this role, the adipocyte functions as a global regulator of energy metabolism. Adipose tissue is exquisitely sensitive to a variety of endocrine and paracrine signals, e.g. insulin, glucagon, glucocorticoids, and tumor necrosis factor (TNF), that combine to control both the secretion of other regulatory factors and the recruitment and differentiation of new adipocytes. The process of adipocyte differentiation is controlled by a cascade of transcription factors, most notably those of the C/EBP and PPAR families, which combine to regulate each other and to control the expression of adipocyte-specific genes. One such gene, i.e. the obese gene, was recently identified and found to encode a hormone, referred to as leptin, that plays a major role in the regulation of energy intake and expenditure. The hormonal and transcriptional control of adipocyte differentiation is discussed, as is the role of leptin and other factors secreted by the adipocyte that participate in the regulation of adipose homeostasis.

TNF -Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPAR without Effects on Pref-1 Expression

Tumor necrosis factor α (TNFα) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNFα inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor γ (PPARγ) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARγ and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARγ occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNFα-mediated inhibition of adipocyte differentiation, PPARγ messenger RNA (mRNA) expression stays at low levels. In contrast, TNFα treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors[ such as TNFα, basic fibroblast growth factor, transforming growth factor β, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARγ mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNFα to fully differentiated adipocytes results in the rapid disappearance of PPARγ protein expression and the rapid loss of PPARγ DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNFα-generated signals, whereas PPARγ expression remains sensitive to TNFα at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.

TNF alpha-mediated inhibition and reversal of adipocyte differentiation is accompanied by suppressed expression of PPARgamma without effects on Pref-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF alpha inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARgamma and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARgamma occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF alpha-mediated inhibition of adipocyte differentiation, PPARgamma messenger RNA (mRNA) expression stays at low levels. In contrast, TNF alpha treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF alpha, basic fibroblast growth factor, transforming growth factor beta, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARgamma mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF alpha to fully differentiated adipocytes results in the rapid disappearance of PPARgamma protein expression and the rapid loss of PPARgamma DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF alpha-generated signals, whereas PPARgamma expression remains sensitive to TNF alpha at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.

Regulation of Peroxisome Proliferator-activated Receptor γ Activity by Mitogen-activated Protein Kinase

Adipocyte differentiation is regulated both positively and negatively by external growth factors such as insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). A key component of the adipocyte differentiation process is PPARgamma, peroxisomal proliferator-activated receptor gamma. To determine the relationship between PPARgamma activation and growth factor stimulation in adipogenesis, we investigated the effects of PDGF and EGF on PPARgamma1 activity. PDGF treatment decreased ligand-activated PPARgamma1 transcriptional activity in a transient reporter assay. In vivo [32P]orthophosphate labeling experiments demonstrated that PPARgamma1 is a phosphoprotein that undergoes EGF-stimulated MEK/mitogen-activated protein (MAP) kinase-dependent phosphorylation. Purified PPARgamma1 protein was phosphorylated in vitro by recombinant activated MAP kinase. Examination of the PPARgamma1 sequence revealed a single MAP kinase consensus recognition site at Ser82. Mutation of Ser82 to Ala inhibited both in vitro and in vivo phosphorylation and growth factor-mediated transcriptional repression. Therefore, phosphorylation of PPARgamma1 by MAP kinase contributes to the reduction of PPARgamma1 transcriptional activity by growth factor treatment.

Peroxisome Proliferator-activated Receptors α and γ Are Activated by Indomethacin and Other Non-steroidal Anti-inflammatory Drugs

Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) and cyclooxygenase inhibitor that is frequently used as a research tool to study the process of adipocyte differentiation. Treatment of various preadipocyte cell lines with micromolar concentrations of indomethacin in the presence of insulin promotes their terminal differentiation. However, the molecular basis for the adipogenic actions of indomethacin had remained unclear. In this report, we show that indomethacin binds and activates peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor known to play a pivotal role in adipogenesis. The concentration of indomethacin required to activate PPARgamma is in good agreement with that required to induce the differentiation of C3H10T1/2 cells to adipocytes. We demonstrate that several other NSAIDs, including fenoprofen, ibuprofen, and flufenamic acid, are also PPARgamma ligands and induce adipocyte differentiation of C3H10T1/2 cells. Finally, we show that the same NSAIDs that activate PPARgamma are also efficacious activators of PPARalpha, a liver-enriched PPAR subtype that plays a key role in peroxisome proliferation. Interestingly, several NSAIDs have been reported to induce peroxisomal activity in hepatocytes both in vitro and in vivo. Our findings define a novel group of PPARgamma ligands and provide a molecular basis for the biological effects of these drugs on adipogenesis and peroxisome activity.

An ultrastructural study of brown adipose tissue in pre-term human new-borns

The ultrastructure of brown adipose tissue (BAT), the thermogenic type of adipose tissue, was investigated in biopsies from 4 pre-term human new-borns delivered at 25–27 week's gestational age and compared with peri-renal brown fat from 2 adult patients with phaeochromocytoma (a condition of brown fat activation). The cell size of brown adipocytes was smaller in pre-term new-borns than in adult patients; adipocytes were almost exclusively multilocular, suggesting active thermogenesis. In 3 of the pre-term new-borns, brown adipocyte ultrastructure indicated a good to high degree of differentiation (in particular at the level of mitochondria) as compared with activated brown fat cells found in adult patients; in one pre-term infant the tissue morphology was obviously suggestive of an earlier, proliferative phase of development and the differentiation process of brown adipocytes could be traced in some detail. The results suggest that (a) brown adipose tissue may be fairly well-differentiated and thermogenetically active in pre-term human new-borns weighing about 750 g at birth; (b) brown adipocytes apparently develop from vessel-associated cells, the early signs of adipocyte differentiation being glycogen and lipid accumulation; (c) the ultrastructural morphology of mitochondria in well-differentiated BAT from pre-term infants can strictly resemble that found in active brown adipose tissue of adult phaeochromocytoma patients.