Primary Human Preadipocytes Research Articles

Influence of folate on lipid accumulation in human primary subcutaneous adipocytes (827.8)

Epidemiological studies report inverse associations between serum folate concentrations and body mass index, however the potential influence of changes in folate status on adipose cell development and metabolism has only been examined in animal models or a murine cell line. The objective of this study was to determine the influence of alterations in folate availability on differentiation of human primary preadipocytes. Human primary preadipocytes, derived from subcutaneous adipose tissue of normal weight female donors (ZenBio Inc), were exposed to 0.06, 6 or 60 uM folic acid (FA; Sigma), 5‐methyltetrahydrofolate (MTHF; Merck) or tetrahydrofolate (THF; Merck) during the differentiation period. The impact of treatment on lipid accumulation was determined using AdipoRedTM Adipogenesis assay reagent (Lonza). Results indicate that FA, MTHF and THF decreased lipid accumulation in mature adipocytes, after adjusting for cell number. Interestingly, MTHF and THF, reduced forms of folate, exhibited a greater inhibitory effect on lipid accumulation. These results indicate that biologically active reduced forms of folate may have a greater effect on adipocyte differentiation in human adipocytes. This study also supports preliminary findings in murine cell lines demonstrating an effect of changes in folate availability on adipose cell development.Grant Funding Source: Funded in part by HATCH #GEO00707. MTHF and THF were a kind gift from Merck & Cie, Switzerland.

PKA regulatory subunit R2B is required for murine and human adipocyte differentiation

Adipogenesis is a complex process modulated by several factors, including cAMP signaling. The main cAMP target is protein kinase A (PKA), a tetrameric enzyme with four regulatory subunits showing tissue-specific expression and function: PRKAR2B is the main regulatory subunit in adipose tissue in mice and in adult humans. This study aimed to evaluate the expression of PKA regulatory subunits in human adipose tissue during fetal development and to investigate their role in the differentiation of 3T3-L1 and primary human preadipocytes. The expression of PKA regulatory subunits was evaluated in fetal adipose tissue (immunohistochemistry) and in cultured 3T3-L1 and primary human preadipocytes (western blot analysis). Cultured cells were transiently transfected with siRNA against PRKAR2B and induced to differentiate. Differentiation was evaluated by intracellular triglyceride staining (Oil Red O) and expression of molecular markers of adipocyte differentiation. In this study, we found that PRKAR2B is the main regulatory subunit in human adipose tissue during fetal development, from 12 weeks of gestation to the end of gestation, as well as in 3T3-L1 and primary human preadipocytes. The expression of PRKAR2B increases progressively during in vitro differentiation. The silencing of PRKAR2B abolishes the increase in the expression of peroxisome proliferator-activated receptor gamma (PPARg (PPARG)), fatty acid synthase, aP2 (FABP4), and lipoprotein lipase, as well as intracellular triglyceride accumulation, resulting in impaired adipocyte differentiation in both mouse and human cell systems. In conclusion, PRKAR2B is the key PKA regulatory subunit involved in mouse and human adipose tissue development. The physiological increase in the expression of PRKAR2B is an essential event in adipogenesis in both mice and humans, and it might represent a possible target for future strategies for obesity treatment.

CCL2 mediates anti-fibrotic effects in human fibroblasts independently of CCR2

CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of α-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.

Bisphenol A induces differentiation of human preadipocytes in the absence of glucocorticoid and is inhibited by an estrogen-receptor antagonist

Background:Obesity is a major health concern in the developed world, and increasing evidence suggests that exposures to common environmental substances may enhance the risk for the development of this disease.Objectives:The current study examines the effect of the ubiquitous plastic monomer bisphenol A (BPA) on the differentiation of primary human preadipocytes in vitro and the role of the estrogen and glucocorticoid receptors.Methods:In this study, the mechanism of BPA-induced adipogenesis in preadipocytes from donors with healthy body mass index in the absence of exogenous glucocorticoid was evaluated. The effects of estradiol, the estrogen-receptor (ER) antagonist ICI and the glucocorticoid receptor (GR) antagonist RU486 on BPA-induced adipogenesis were examined. The expression levels of key adipogenic factors were assessed.Results:Treatment of preadipocytes with 1–50 μM BPA induced a dose-dependent increase in differentiation and lipid accumulation as determined by lipid staining and triacylglyceride quantification. BPA also induced expression of the adipogenic markers aP2, adipsin, peroxisome proliferator-activated receptor γ and the CCAAT-enhancer-binding proteins α and β. Co-treatment of cells with ICI inhibited the BPA-induced increase in aP2 levels, while treatment with ICI or estradiol alone had no effect. Treatment of cells with the GR antagonist RU486 had no effect on BPA-induced differentiation as evaluated by aP2 levels.Conclusions:This study is one of the first to show that BPA induces human adipocyte differentiation in the absence of exogenous glucocorticoid through a non-classical ER pathway rather than through GR activation. These studies add to the growing evidence that endocrine-disrupting chemicals such as BPA have the potential to modulate adipogenesis and impact human biology.

Prolonged efficiency of siRNA‐mediated gene silencing in primary cultures of human preadipocytes and adipocytes

ObjectivePrimary human preadipocytes and differentiated adipocytes in culture are valuable cell culture systems to study adipogenesis and adipose function in relation to human adipose biology. To use these systems for mechanistic studies, we studied siRNA-mediated knockdown of genes for its effectiveness.Design and MethodsMethods were developed to effectively deliver siRNA to for gene silencing in primary preadipocytes isolated from human subcutaneous adipose tissue and newly-differentiated adipocytes. Expression level of genes and proteins was measured using quantitative RT-PCR and western blotting. Lipid droplet morphology was observed using microscopy and glycerol release was quantified as a measure of lipolysis.ResultssiRNA-mediated knockdown of genes in primary human preadipocytes resulted in prolonged silencing effects, suppressing genes throughout the process of their differentiation. In newly differentiated adipocytes, siRNA-mediated gene knockdown allowed proteins to stay depleted for at least 5 days. It was possible to re-express a protein after its siRNA-mediated depletion. Importantly, siRNA transfected human adipocytes remained metabolically active, responding to β-adrenergic stimulation to increase lipolysis.ConclusionsOur study describes the methods of gene silencing in primary cultures of human preadipocytes and adipocytes and their prolonged effectiveness.

PKA regulatory subunit R2B is required for murine and human adipocyte differentiation

Adipogenesis is a complex process modulated by several factors, including cAMP signaling. The main cAMP target is protein kinase A (PKA), a tetrameric enzyme with four regulatory subunits showing tissue-specific expression and function: PRKAR2B is the main regulatory subunit in adipose tissue in mice and in adult humans. This study aimed to evaluate the expression of PKA regulatory subunits in human adipose tissue during fetal development and to investigate their role in the differentiation of 3T3-L1 and primary human preadipocytes. The expression of PKA regulatory subunits was evaluated in fetal adipose tissue (immunohistochemistry) and in cultured 3T3-L1 and primary human preadipocytes (western blot analysis). Cultured cells were transiently transfected with siRNA against PRKAR2B and induced to differentiate. Differentiation was evaluated by intracellular triglyceride staining (Oil Red O) and expression of molecular markers of adipocyte differentiation. In this study, we found that PRKAR2B is the main regulatory subunit in human adipose tissue during fetal development, from 12 weeks of gestation to the end of gestation, as well as in 3T3-L1 and primary human preadipocytes. The expression of PRKAR2B increases progressively during in vitro differentiation. The silencing of PRKAR2B abolishes the increase in the expression of peroxisome proliferator-activated receptor gamma (PPARγ (PPARG)), fatty acid synthase, aP2 (FABP4), and lipoprotein lipase, as well as intracellular triglyceride accumulation, resulting in impaired adipocyte differentiation in both mouse and human cell systems. In conclusion, PRKAR2B is the key PKA regulatory subunit involved in mouse and human adipose tissue development. The physiological increase in the expression of PRKAR2B is an essential event in adipogenesis in both mice and humans, and it might represent a possible target for future strategies for obesity treatment.

Staphylococcal Superantigens Stimulate Immortalized Human Adipocytes to Produce Chemokines

BackgroundHuman adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs) on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin.Methodology/Principal FindingsPrimary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT) and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8.Conclusions/SignificanceEndotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to understand further the mechanism by which toxins are involved in wound healing and the development and clinical manifestations of obesity and diabetes.

Adipogenic effect of calcium sensing receptor activation

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24h decreased by 40% the early differentiation marker CCAAT/enhancer-binding protein β. However, upon longer-term (10day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10day effect of cinacalcet exposure for the first 6, 24, 48h, 6, and 10days. Our observations suggest that regardless of the period of exposure, 10day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.

Functional thermogenic beige adipogenesis is inducible in human neck fat

Recent studies suggest human neck brown adipose tissue (BAT) to consist of 'brown adipocyte (BA)-like' or beige adipocytes. However, little is known about their thermogenic function. Within the beige adipocyte transcriptome, fibroblast growth factor-21 (FGF21) is a gene whose protein product acts as an adipokine, regulating cold-induced thermogenesis in animals. Here, we explored (i) the adipogenic potential, thermogenic function and FGF21 secretory capacity of beige adipocytes derived from human neck fat and (ii) the role of FGF21 in modulating adipose bioenergetics. Progenitors isolated from human cervical fat were differentiated into adipocytes with either a BA-like or white adipocyte (WA) phenotype. FGF21 secretion was measured by enzyme-linked immuosorbent assay. Real-time PCR/western blotting was used to determine cellular mRNA/protein levels. Extracellular flux bioanalyzer was used to quantify adipocyte oxygen consumption and fatty acid oxidation. Adipocyte heat production was measured by infrared thermography. Under hormonal manipulation, primary human neck pre-adipocytes differentiated into adipocytes with either BA-like or WA phenotypes, on gene/protein and functional levels. BA-like cells expressed beige but not classic BA markers. During BA differentiation, FGF21 gene expression and secretion were increased, and were augmented following norepinephrine exposure (a cold mimic in vitro). Differentiated WA expressed β-klotho, a critical co-factor mediating FGF21 action. Treatment of WA with FGF21-induced UCP1 expression and increased oxygen consumption, respiratory uncoupling, norepinephrine-mediated thermogenesis, fatty acid oxidation and heat production, thus recapitulating the association between cold-induced FGF21 secretion and cold-induced thermogenesis in vivo. Beige adipocytes are thermogenic in humans. FGF21 is a beige adipokine capable of promoting a brown fat-like thermogenic program in WAs. This study provides first evidence of inducible functional thermogenic beige adipogenesis in human neck fat. FGF21 holds promise as a cold-induced beige adipokine with metabolic benefits of therapeutic relevance through browning of white adipose tissue.

CREB-regulated transcription co-activator family stimulates promoter II-driven aromatase expression in preadipocytes.

The dramatically increased prevalence of breast cancer after menopause is of great concern and is correlated with elevated local levels of estrogens. This is mainly due to an increase in aromatase expression driven by its proximal promoter II (PII). We have previously demonstrated that the CREB co-activator CRTC2 binds directly to PII and stimulates its activity via mechanisms involving LKB1-AMPK in response to prostaglandin E(2) (PGE(2)). There are three members of the CRTC family (CRTC1-3) and this study aimed to characterize the role of other CRTCs in the activation of aromatase PII. The expression and subcellular localization of CRTCs were examined in preadipocytes using qPCR and immunofluorescence. Under basal conditions, CRTC1 expression was the lowest, whereas CRTC3 transcripts were present at higher levels. Basally, CRTC2 and CRTC3 were mainly cytoplasmic and PGE(2) caused their nuclear translocation. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of CRTCs on PII activity and binding. Basal PII activity was significantly increased with all CRTCs. Forskolin (FSK)/phorbol 12-myristate 13-acetate (PMA), to mimic PGE(2), resulted in a further significant increase in PII activity with all CRTCs, with CRTC2 and CRTC3 having greater effects. This was consistent with ChIP data showing an increased binding of CRTCs to PII with FSK/PMA. Moreover, gene silencing of CRTC2 and CRTC3 significantly reduced the FSK/PMA-mediated stimulation of aromatase activity. Interestingly, CRTCs acted cooperatively with CREB1 to increase PII activity, and both CREs were found to be essential for the maximal induction of PII activity by CRTCs. Phosphorylation of CRTC2 at its AMPK target site, Ser 171, dictated its subcellular localization, and the activation of aromatase PII in preadipocytes. In conclusion, this study demonstrates that aromatase regulation in primary human breast preadipocytes involves more than one CRTC.

The role of interleukin 1β in the anti-adipogenic action of macrophages on human preadipocytes

When adipose tissue accumulates in obesity, the ability of preadipocytes to differentiate permits a hyperplastic expansion of functional adipocytes that preserves insulin sensitivity. Adipose infiltration by macrophages is associated with an adipogenic deficit and the appearance of inflamed, insulin-resistant hypertrophied adipocytes. Interleukin 1β (IL1β) has been reported to account for the anti-adipogenic action of macrophages in a mouse model. Using the THP-1 human macrophage cell line and human primary preadipocytes, our objective was to determine whether IL1β was necessary for the ability of conditioned medium from THP-1 macrophages (THP-1-MacCM) to: i) stimulate human preadipocyte inhibitor of κB kinase β (IKKβ) and ii) inhibit human adipocyte differentiation. IL1β is present in THP-1-MacCM, and THP-1-MacCM or IL1β (500 pg/ml; its concentration in THP-1-MacCM) acutely stimulated IKKβ phosphorylation and inhibitor of κB (IκB) degradation in preadipocytes. IL1β was sufficient to inhibit adipogenesis on its own, and this was blocked by SC-514, an IKKβ inhibitor, as has been reported for THP-1-MacCM. IκB degradation by IL1β-immunodepleted THP-1-MacCM was attenuated, whereas IKKβ phosphorylation and the inhibition of adipocyte differentiation were unchanged. Therefore, in contrast to what has been suggested for mouse cell models, IL1β is not required for the ability of MacCM to inhibit adipogenesis in human cell models.

25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 Promote the Differentiation of Human Subcutaneous Preadipocytes

1,25(OH)2D3 inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D3 to 1,25(OH)2D3, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)2D3, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)2D3 enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)2D3 was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0–3), suggesting that 1,25(OH)2D3 promoted maturation. 25(OH)D3 also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)2D3. Consistent with this possibility, incubation of preadipocytes with 25(OH)D3 led to 1,25(OH)2D3 accumulation in the media. 1,25(OH)2D3 also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.

Identification of RIFL, a novel adipocyte-enriched insulin target gene with a role in lipid metabolism

To identify new genes that are important in fat metabolism, we utilized the Lexicon-Genentech knockout database of genes encoding transmembrane and secreted factors and whole murine genome transcriptional profiling data that we generated for 3T3-L1 in vitro adipogenesis. Cross-referencing null models evidencing metabolic phenotypes with genes induced in adipogenesis led to identification of a new gene, which we named RIFL (refeeding induced fat and liver). RIFL-null mice have serum triglyceride levels approximately one-third of wild type. RIFL transcript is induced >100-fold during 3T3-L1 adipogenesis and is also increased markedly during adipogenesis of murine and human primary preadipocytes. siRNA-mediated knockdown of RIFL during 3T3-L1 adipogenesis results in an ~35% decrease in adipocyte triglyceride content. Murine RIFL transcript is highly enriched in white and brown adipose tissue and liver. Fractionation of WAT reveals that RIFL transcript is exclusive to adipocytes with a lack of expression in stromal-vascular cells. Nutritional and hormonal studies are consistent with a prolipogenic function for RIFL. There is evidence of an approximately eightfold increase in RIFL transcript level in WAT in ob/ob mice compared with wild-type mice. RIFL transcript level in WAT and liver is increased ~80- and 12-fold, respectively, following refeeding of fasted mice. Treatment of 3T3-L1 adipocytes with insulin increases RIFL transcript ≤35-fold, whereas agents that stimulate lipolysis downregulate RIFL. Interestingly, the 198-amino acid RIFL protein is predicted to be secreted and shows ~30% overall conservation with the NH(2)-terminal half of angiopoietin-like 3, a liver-secreted protein that impacts lipid metabolism. In summary, our data suggest that RIFL is an important new regulator of lipid metabolism.

The activation state of macrophages alters their ability to suppress preadipocyte apoptosis

Adipose tissue contains macrophages whose state of activation is regulated as obesity develops. Macrophage-secreted factors influence critical processes involved in adipose tissue homeostasis, including preadipocyte proliferation and differentiation into adipocytes. Macrophage-conditioned medium (MacCM) from J774A.1 macrophages protects 3T3-L1 preadipocytes from apoptosis through platelet-derived growth factor (PDGF) signaling. Here, we investigated the effect of macrophage activation on MacCM-dependent preadipocyte survival. MacCM was prepared following activation of either J774A.1 macrophages with lipopolysaccharide (LPS) or human primary monocyte-derived macrophages (MD-macrophages) with LPS or interleukin 4 (IL4). 3T3-L1 and human primary preadipocytes were induced to undergo apoptosis in MacCM, and apoptosis was quantified by cell enumeration or Hoechst nuclear staining. Preadipocyte PDGF signaling was assessed by immunoblot analysis of phosphorylated PDGF receptor, Akt, and ERK1/2. Pro-inflammatory activation of J774A.1 macrophages with LPS inhibited the pro-survival activity of MacCM on 3T3-L1 preadipocytes, despite intact PDGF signaling. Upregulation of macrophage tumor necrosis factor a (TNFα) expression occurred in response to LPS, and TNFα was demonstrated to be responsible for the inability of LPS-J774A.1-MacCM to inhibit preadipocyte apoptosis. Furthermore, MacCM from human MD-macrophages (MD-MacCM) inhibited apoptosis of primary human preadipocytes. MD-MacCM from LPS-treated macrophages, but not IL4-treated anti-inflammatory macrophages, was unable to protect human preadipocytes from cell death. In both murine cell lines and human primary cells, pro-inflammatory activation of macrophages inhibits their pro-survival activity, favoring preadipocyte death. These findings may be relevant to preadipocyte fate and adipose tissue remodeling in obesity.

Identification of BMP and Activin Membrane-Bound Inhibitor (BAMBI) as a Potent Negative Regulator of Adipogenesis and Modulator of Autocrine/Paracrine Adipogenic Factors

Adipose tissue dysfunction underpins the association of obesity with type 2 diabetes. Adipogenesis is required for the maintenance of adipose tissue function. It involves the commitment and subsequent differentiation of preadipocytes and is coordinated by autocrine, paracrine, and endocrine factors. We previously reported that fibroblast growth factor-1 (FGF-1) primes primary human preadipocytes and Simpson Golabi Behmel syndrome (SGBS) preadipocytes and increases adipogenesis through a cascade involving extracellular signal–related kinase 1/2 (ERK1/2). Here, we aimed to use the FGF-1 system to identify novel adipogenic regulators. Expression profiling revealed bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) as a putative FGF-1 effector. BAMBI is a transmembrane protein and modulator of paracrine factors that regulate adipogenesis, including transforming growth factor (TGF) superfamily members (TGF-β and BMP) and Wnt. Functional investigations established BAMBI as a negative regulator of adipogenesis and modulator of the anti- and proadipogenic effects of Wnt3a, TGF-β1, and BMP-4. Further studies showed that BAMBI expression levels are decreased in a mouse model of diet-induced obesity. Collectively, these findings establish BAMBI as a novel, negative regulator of adipogenesis that can act as a nexus to integrate multiple paracrine signals to coordinate adipogenesis. Alterations in BAMBI may play a role in the (patho)physiology of obesity, and manipulation of BAMBI may present a novel therapeutic approach to improve adipose tissue function.

The nuclear receptors NUR77, NURR1 and NOR1 in obesity and during fat loss

Adipose tissue is critical for systemic metabolic health. Identifying key factors regulating adipose tissue function is a research priority. The NR4A subfamily of nuclear receptors (NRs) (NR4A1/NUR77, NR4A2/NURR1 and NR4A3/NOR1) has emerged as important proteins in different disease states and in the regulation of metabolic tissues, particularly in liver and muscle. However, the expression of the NR4A members in human adipose tissue has not previously been described, and their target genes are largely unknown. To determine whether the NR4As are differentially expressed in human adipose tissue in obesity, and identify potential NR4A target genes. Prospective analysis of s.c. adipose tissue before and 1 year after fat loss, and during in vitro differentiation of primary human preadipocytes. Case-control comparison of omental (OM) adipose tissue. A total of 13 extremely obese patients undergoing biliopancreatic diversion with duodenal switch for fat loss, 12 extremely obese patients undergoing laparoscopic sleeve gastrectomy and 37 lean individuals undergoing hernia repair or laparotomy were included in the study. Measurements were done by quantitative PCR gene expression analysis of the NR4A members and in silico promoter analysis based on microarray data. There was a strong upregulation of the NR4As in extreme obesity and normalization after fat loss. The NR4As were expressed at the highest level in stromal-vascular fraction compared with adipocytes, but were downregulated in both fractions after fat loss. Their expression levels were also significantly higher in OM compared with s.c. adipocytes in obesity. The NR4As were downregulated during differentiation of primary human preadipocytes. Moreover, the NR4As were strongly induced within 30 min of tissue incubation. Finally, promoter analysis revealed potential NR4A target genes involved in stress response, immune response, development and other functions. Our data show altered adipose tissue expression of the NR4As in obesity, suggesting that these stress responsive nuclear receptors may modulate pathogenic potential in humans.

Glitazone-like action of glimepiride and glibenclamide in primary human adipocytes

Sulphonylureas (SUs) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. In addition, SUs have pleiotropic effects on other tissues. Conflicting findings have been reported regarding the effects of SUs on adipocytes. We have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. Primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. Lipid accumulation was assessed by Oil Red O staining and determination of triglyceride content; gene expression was measured by RT PCR and Western blotting. Initially, we characterized the genes regulated during human pre-adipocyte differentiation by performing global microarray analysis. Treatment with glimepiride and glibenclamide caused an increased accumulation of lipid droplets and triglycerides. In addition, genes involved in lipid metabolism were induced and chemokine expression was decreased. Interestingly, the effects of SUs were generally qualitatively and quantitatively similar to those of pioglitazone. In direct comparison, glibenclamide was more potent than glimepiride with respect to the induction of fatty acid binding protein 4 (FABP4) (EC(50) 0.32 vs. 2.8 µM), an important adipocyte marker gene. SU-induced differentiation was virtually completely blocked by the peroxisome proliferator-activated receptor γ (PPARγ)-antagonist T0070907 but not affected by diazoxide, indicating PPARγ activation by SUs. Repaglinide had no effect on adipogenesis, although it causes insulin liberation like SUs. In primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating PPARγ. Thus, SUs but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation.

The Antagonists But Not Partial Agonists of Glucocorticoid Receptor Ligands Show Substantial Side Effect Dissociation

A synthetic glucocorticoid receptor (GR) ligand with the efficacy of a glucocorticoid, but without the accompanying side effects, would meet an unmet medical need for the treatment of inflammatory diseases. It was hypothesized that a GR ligand that shifted helix 12 in a manner distinct from an agonist and an antagonist would confer a distinct GR conformation, resulting in differential gene expression and, ultimately, dissociation of antiinflammatory activity from side effects. A structural feature expected to interfere with helix 12 was incorporated into a nonsteroidal, tricyclic scaffold to create novel, high-affinity, and selective GR ligands that manifested a dual function in cellular assays, partial but robust agonist activity for inflammatory cytokine inhibition, and full antagonist activity for reporter gene activation. In contrast, analogs not likely to hinder helix 12 exhibited partial agonist activity for reporter gene activation. The requirement of full antagonist activity for substantial side effect dissociation was demonstrated in primary human preadipocytes, hepatocytes, and osteoblasts in which effects on adipogenesis, key genes involved in gluconeogenesis, and genes important for bone formation were examined, respectively. The dissociated GR ligands, despite lacking significant reporter gene activation, weakly recruit a limited number of coactivators such as peroxisomal proliferator-activated receptor-γ coactivator 1α. Transcriptional activation was sensitive to both peroxisomal proliferator-activated receptor-γ coactivator 1α and GR levels, providing a basis for cell-selective modulation of gene expression. The antiinflammatory activity of the dissociated ligands was further demonstrated in mouse models of inflammation. Together these results suggest that these ligands are promising candidates with robust antiinflammatory activity and likely dissociation against glucocorticoid-induced side effects.

A putative role for endogenous FGF-2 in FGF-1 mediated differentiation of human preadipocytes

The defining characteristic of obesity is increased adipose tissue (AT) mass following chronic positive energy supply. AT mass is determined by adipocyte number and size, which reflect proliferation and differentiation of preadipocytes and hypertrophy of pre-existing adipocytes. The molecular pathways governing AT expansion are incompletely defined. We previously reported that FGF-1 primes proliferating primary human preadipocytes (phPA), thereby increasing adipogenesis. Here we examined whether FGF-1's adipogenic actions were due to modulation of other FGFs. Treatment of phPA with FGF-1 reduced FGF-2 mRNA/protein by 80%. To examine a putative functional role we performed siRNA knockdown studies. Following FGF-2 knockdown preadipocyte proliferation was decreased and expression of adipogenic genes (PPARγ, G3PDH and adiponectin) was increased at day 1 of differentiation. These results suggest that changes in endogenous FGF-2 levels contribute to FGF-1's early adipogenic effects and highlight the complexity of the paracrine interplay between FGFs within human AT.

Curcumin inhibits adipocyte differentiation through modulation of mitotic clonal expansion

Adipocyte differentiation is a key process in determining the number of mature adipocytes in the development of obesity. Here, we examined the function of curcumin, a dietary polyphenol found in turmeric, and its underlying mechanisms in adipocyte differentiation. Our study reveals that curcumin exerts an anti-adipogenic function both in 3T3-L1 murine cells and in human primary preadipocytes as determined by intracellular lipid accumulation assay, quantitative analysis of adipocyte marker gene expression and a noninvasive multimodal Coherent Anti-Stokes Raman Scattering (CARS) microscopic analysis of intracellular curcumin. The inhibitory action of curcumin was largely limited to the early stage of adipocyte differentiation, where curcumin was found to inhibit mitotic clonal expansion (MCE) process as evidenced by impaired proliferation, cell-cycle entry into S phase and the S to G2/M phase transition of confluent cells, and levels of cell cycle-regulating proteins with no significant effect on cell viability and cytotoxicity. This, in turn, resulted in inhibition of mRNA levels of early adipogenic transcription factors, particularly Krüppel-like factor 5 (KLF5), CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptorγ (PPARγ), in the early stage of adipocyte differentiation. Supplementation with rosiglitazone, a PPARγ ligand, during the early stage of adipocyte differentiation partially rescued curcumin-inhibited adipocyte differentiation. Collectively, our results show that curcumin is an anti-adipogenic dietary bioactive component largely involved in the modulation of the MCE process during the early stage of adipocyte differentiation.