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Free access

Paula Fuentes, María José Acuña, Mariana Cifuentes and Cecilia V Rojas

Despite the importance of adipocyte formation for adipose tissue physiology, current knowledge about the mechanisms that regulate the recruitment of progenitor cells to undergo adipogenic differentiation is limited. A role for locally generated angiotensin II emerged from studies with human and murine cells. Preadipose cells from different human fat depots show reduced response to adipogenic stimuli when exposed to angiotensin II. This investigation sought to gain an insight into the intracellular mechanisms involved in the anti-adipogenic response of human preadipose cells from omental fat to angiotensin II. Its effect was evaluated on cells stimulated to adipogenic differentiation in vitro, by assessment of glycerol-3-phosphate dehydrogenase activity and expression of early markers of adipogenesis. Extracellular signal-regulated kinase1,2 (ERK1,2) pathway activation was inferred from the phosphorylated to total ERK1,2 ratio determined by western blot. Exposure to angiotensin II throughout the 10-day differentiation period resulted in a reduced adipogenic response. A similar anti-adipogenic effect was observed when this hormone was present during the first 48 h of induction to differentiation. Angiotensin II treatment had no consequences on CCAAT/enhancer-binding protein β and peroxisome proliferator-activated receptor γ (PPARG) induction, but increased the phosphorylated form of the key adipogenic regulator PPARG. Upon angiotensin II exposure, a raise of phosphorylated ERK1,2 was determined, which was more prominent 8–20 h after induction of adipogenesis (when controls reached negligible values). Chemical inhibition of ERK1,2 phosphorylation prevented angiotensin II-dependent reduction in adipogenesis. These results support the participation of the mitogen-activated protein kinase/ERK1,2 pathway in the anti-adipogenic effect of angiotensin II on preadipose cells from human omental adipose tissue.

Free access

Qinghua Wang, Jing Tang, Shujun Jiang, Zan Huang, Anying Song, Siyuan Hou, Xiang Gao and Hai-Bin Ruan

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipogenesis and a target of the thiazolidinedione (TZD) class of antidiabetic drugs; therefore, identifying novel regulators of PPARγ action in adipocytes is essential for the future development of therapeutics for diabetes. MAGE family member D1 (MAGED1), by acting as an adaptor for ubiquitin-dependent degradation pathways and a co-factor for transcription, plays an important role in neural development, cell differentiation and circadian rhythm. Here, we showed that MAGED1 expression was downregulated during adipogenesis and loss of MAGED1 promoted preadipocyte proliferation and differentiation in vitro. MAGED1 bound to PPARγ and suppressed the stability and transcriptional activity of PPARγ. Compared to WT littermates, MAGED1-deficient mice showed increased levels of PPARγ protein and its target genes, more CD29+CD34+Sca-1+ adipocyte precursors and hyperplasia of white adipose tissues (WATs). Moreover, MAGED1-deficient mice developed late-onset obesity as a result of decreased energy expenditure and physical activity. However, these mice were metabolically healthy as shown by improved glucose clearance and insulin sensitivity, normal levels of serum lipids and enhanced secretion of adipokines such as leptin and adiponectin. Taken together, our data identify MAGED1 as a novel negative regulator of PPARγ activity, adipogenesis and insulin sensitivity in mice. MAGED1 might therefore serve as a novel pharmaceutical target to treat obesity-associated insulin resistance.

Free access

Min Lu and Reigh-Yi Lin

Although TSH is the main regulator of thyroid growth and function, TSH binding activity in fat has long been reported. Since the TSH receptor (TSHR) has been detected in both preadipocytes and adipocytes, we hypothesized that it may play a role in adipose differentiation. Here, we use an in vitro model of adipogenesis from mouse embryonic stem (ES) cells to define TSH function. Directed differentiation of ES cells into the adipose lineage can be achieved over a 3-week period. Although adipocyte differentiation is initiated early in the development of cultured ES cells, TSHR up-regulation is precisely correlated with terminal differentiation of those adipocytes. The adipocytes express TSHR on the cell surface and respond to TSH with increased intracellular cAMP production, suggesting the activation of the protein kinase A signaling pathway. To determine whether TSH impacts adipogenesis, we examined how adipocytes responded to TSH at various points during their differentiation from cultured ES cells. We found that TSH greatly increases adipogenesis when added in the presence of adipogenic factors. More importantly, our data suggest that TSH also stimulates adipogenesis in cultured ES cells even in the absence of adipogenic factors. This finding provides the first evidence of TSH being a pro-adipogenic factor that converts ES cells into adipocytes. It further highlights the potential of ES cells as a model system for use in the study of TSH's role in the regulation of physiologically relevant adipose tissue.

Free access

L Bouraoui, J Gutiérrez and I Navarro

Here, we describe optimal conditions for the culture of rainbow trout (Oncorhynchus mykiss) pre-adipocytes obtained from adipose tissue and their differentiation into mature adipocytes, in order to study the endocrine control of adipogenesis. Pre-adipocytes were isolated by collagenase digestion and cultured on laminin or 1% gelatin substrate. The expression of proliferating cell nuclear antigen was used as a marker of cell proliferation on various days of culture. Insulin growth factor-I stimulated cell proliferation especially on days 5 and 7 of culture. Tumor necrosis factor α (TNFα) slightly enhanced cell proliferation only at a low dose. We verified the differentiation of cells grown in specific medium into mature adipocytes by oil red O (ORO) staining. Quantification of ORO showed an increase in triglycerides throughout culture. Immunofluorescence staining of cells at day 11 revealed the expression of CCAAT/enhancer-binding protein and peroxisome proliferator–activator receptor γ, suggesting that these transcriptional factors are involved in adipocyte differentiation in trout. We also examined the effect of TNFα on the differentiation of these adipocytes in primary culture. TNFα inhibited the differentiation of these cells, as indicated by a decrease in glycerol-3-phosphate dehydrogenase activity, an established marker of adipocyte differentiation. In conclusion, the culture system described here for trout pre-adipocytes is a powerful tool to study the endocrine regulation of adipogenesis in this species.

Free access

Uxía Gurriarán-Rodríguez, Omar Al-Massadi, Ana Belén Crujeiras, Carlos S Mosteiro, María Amil-Diz, Daniel Beiroa, Rubén Nogueiras, Luisa María Seoane, Rosalía Gallego, Yolanda Pazos, Felipe F Casanueva and Jesús P Camiña

This study aimed to investigate the role of preproghrelin-derived peptides in adipogenesis. Immunocytochemical analysis of 3T3-L1 adipocyte cells showed stronger preproghrelin expression compared with that observed in 3T3-L1 preadipocyte cells. Insulin promoted this expression throughout adipogenesis identifying mTORC1 as a critical downstream substrate for this profile. The role of preproghrelin-derived peptides on the differentiation process was supported by preproghrelin knockdown experiments, which revealed its contribution to adipogenesis. Neutralization of endogenous O-acyl ghrelin (acylated ghrelin), unacylated ghrelin, and obestatin by specific antibodies supported their adipogenic potential. Furthermore, a parallel increase in the expression of ghrelin-associated enzymatic machinery, prohormone convertase 1/3 (PC1/3) and membrane-bound O-acyltransferase 4 (MBOAT4), was dependent on the expression of preproghrelin in the course of insulin-induced adipogenesis. The coexpression of preproghrelin system and their receptors, GHSR1a and GPR39, during adipogenesis supports an autocrine/paracrine role for these peptides. Preproghrelin, PC1/3, and MBOAT4 exhibited dissimilar expression depending on the white fat depot, revealing their regulation in a positive energy balance situation in mice. The results underscore a key role for preproghrelin-derived peptides on adipogenesis through an autocrine/paracrine mechanism.

Free access

Nicoleta C Olarescu, Darlene E Berryman, Lara A Householder, Ellen R Lubbers, Edward O List, Fabian Benencia, John J Kopchick and Jens Bollerslev

GH influences adipocyte differentiation, but both stimulatory and inhibitory effects have been described. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are multipotent and are able to differentiate into adipocytes, among other cells. Canonical Wnt/β-catenin signaling activation impairs adipogenesis. The aim of the present study was to elucidate the role of GH on AT-MSC adipogenesis using cells isolated from male GH receptor knockout (GHRKO), bovine GH transgenic (bGH) mice, and wild-type littermate control (WT) mice. AT-MSCs from subcutaneous (sc), epididiymal (epi), and mesenteric (mes) AT depots were identified and isolated by flow cytometry (Pdgfrα + Sca1 + Cd45 Ter119 cells). Their in vitro adipogenic differentiation capacity was determined by cell morphology and real-time RT-PCR. Using identical in vitro conditions, adipogenic differentiation of AT-MSCs was only achieved in the sc depot, and not in epi and mes depots. Notably, we observed an increased differentiation in cells isolated from sc-GHRKO and an impaired differentiation of sc-bGH cells as compared to sc-WT cells. Axin2, a marker of Wnt/β-catenin activation, was increased in mature sc-bGH adipocytes, which suggests that activation of this pathway may be responsible for the decreased adipogenesis. Thus, the present study demonstrates that i) adipose tissue in mice has a well-defined population of Pdgfrα + Sca1 + MSCs; ii) the differentiation capacity of AT-MSCs varies from depot to depot regardless of GH genotype; iii) the lack of GH action increases adipogenesis in the sc depot; and iv) activation of the Wnt/β-catenin pathway might mediate the GH effect on AT-MSCs. Taken together, the present results suggest that GH diminishes fat mass in part by altering adipogenesis of MSCs.

Free access

J Hoppmann, N Perwitz, B Meier, M Fasshauer, D Hadaschik, H Lehnert and J Klein

Obesity is associated with chronic inflammation. Pro-inflammatory adipokines may promote metabolic disorders and cardiovascular morbidity. However, the key mechanisms leading to obesity-related inflammation are poorly understood. The corticosteroid metabolism in adipose tissue plays a crucial role in the pathogenesis of the metabolic syndrome. Both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) mediate corticosteroid action in adipose tissue. The significance of the interplay of these receptors in mediating an inflammatory adipokine response is virtually unexplored. In the present study, we investigated the differential roles of the GR and MR in controlling the key adipose tissue functions including inflammatory adipokine expression and adipogenesis using selective stimulation with receptor agonists, acute receptor knockdown via RNA interference and newly generated knockout adipose cell lines. Selective GR stimulation of white adipocytes with dexamethasone inhibited the expression of interleukin 6 (IL6), monocyte chemoattractant protein-1 (MCP1 or CCL2 as listed in the MGI Database), tumour necrosis factor-α, chemerin and leptin. By contrast, selective MR stimulation with aldosterone promoted the expression of IL6, plasminogen activator inhibitor 1, chemerin and leptin. Furthermore, in the presence of an acute GR knockdown as well as in GR knockout adipocytes, corticosterone increased the gene expression of the pro-inflammatory adipokines IL6 and MCP1. Whereas GR knockout adipocytes displayed a mildly impaired adipogenesis during early differentiation, MR knockout cells completely failed to accumulate lipids. Taken together, our data demonstrate a critical role for the balance between gluco- and mineralocorticoid action in determining adipocyte responses implicated in obesity-associated inflammation and cardiovascular complications.

Free access

Ana María Pino, Juan Manuel Rodríguez, Susana Ríos, Pablo Astudillo, Laura Leiva, Germán Seitz, Mireya Fernández and J Pablo Rodríguez

Human mesenchymal stem cells (hMSCs) are multipotent cells present in bone marrow, which differentiate into osteoblasts and adipocytes, among other lineages. Oestrogens play a critical role in bone metabolism; its action may affect the adipocyte to osteoblast ratio in the bone marrow. In hMSCs, oestrogens are synthesized from C19 steroids by the enzyme aromatase cytochrome P450. In this study, we assessed whether aromatase enzymatic activity varied through early osteogenic (OS) and adipogenic (AD) differentiation. Also, we studied the effect of leptin and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) on aromatase cell activity. Finally, we analysed whether conditions that modify oestrogen generation by cells affected hMSCs differentiation. For these purposes, hMSCs derived from post-menopausal women (65–86 years old) were cultured under basal, OS or AD conditions, in the presence or the absence of leptin and 1,25(OH)2D3. Aromatase activity was measured by the tritiated water release assay and by direct measurement of steroids synthesized from 3H-labelled androstenedione or testosterone. Our results showed that different OS and AD patterns of aromatase activity developed during the first period of differentiation (up to 7 days). A massive and sharp surge of aromatase activity at 24 h characterized early OS differentiation, while increased but constant aromatase activity was increased through adipogenesis. Both leptin and vitamin D increased aromatase activity during osteogenesis, but not during adipogenesis; finally, we showed that favourable aromatase substrates concentration restrained MSCs adipogenesis but improved osteogenesis. Thus, it could be inferred that a high and early increase of local oestrogen concentration in hMSCs affects their commitment either restraining AD or facilitating OS differentiation, or both.

Free access

AnneMarie Gagnon, Charlie Foster, Anne Landry and Alexander Sorisky

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.

Open access

Lei Zhang, Carol Paddon, Mark D Lewis, Fiona Grennan-Jones and Marian Ludgate

Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gβγ signalling may be inhibitory but failed to induce adipogenesis using activated Gsα (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARγ (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARγ1 was reduced and PPARγ2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsα signalling impedes FOXO1 phosphorylation and thus inhibits PPARγ transcription and the alternative promoter usage required to generate PPARγ2, the fat-specific transcription factor necessary for adipogenesis.