Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue

Aim: Previous studies have shown that exercise training reduced white adipose tissue (WAT) mass compared to that in sedentary controls, and that the smaller mass contained fewer adipocytes. However, the effect of exercise training on adipogenesis is not completely clear. Therefore, we re-examined the effect of exercise training on adipocyte numbers in WAT and, if such an effect was found tested the adipogenic responses of stromal-vascular fraction (SVF) cells containing adipose tissue-derived stem cells (ADSC) in epididymal WAT from exercise-trained (TR) rats. Methods: Wistar male rats were divided into two groups: control (C) and TR. The TR rats were subjected to exercise on a treadmill for 9 weeks. SVF cells containing ADSC were separated from epididymal WAT by centrifugation. Expression of adipocyte differentiation-related genes and adipogenesis of SVF cells were examined. Results: In SVF cells of TR rats, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and that of PPARγ target lipogenic genes was dramatically downregulated, whereas that of preadipocyte factor-1 gene was significantly upregulated. Lipid accumulation in SVF cells of TR rats after the induction of adipocyte differentiation was significantly suppressed in comparison with that of C rats. Moreover, increased expression of hypoxia-inducible factor-1α (HIF-1α) protein was observed in SVF cells of TR rats. Pre-treatment of YC-1, a potent HIF-1α inhibitor, in SVF cells of TR rats restored adipogenesis. Conclusion: These results suggest that exercise training suppresses the ability of SVF cells to differentiate into adipocytes, and that underlying mechanisms involve the upregulation of HIF-1α expression.     

Src family kinases suppress differentiation of brown adipocytes and browning of white adipocytes

Brown adipocytes and beige adipocytes can expend energy, generate heat, and increase whole‐body energy expenditure. The detailed mechanisms of adipogenesis and thermogenesis of these cells are still obscure. Here, we show that Src family kinases (SFKs) regulate both brown adipogenesis and browning of white adipocytes. To identify factors involved in brown adipogenesis, we first examined the effect of several chemical inhibitors on the differentiation of brown preadipocytes isolated from mouse brown adipose tissue (BAT) and found that treatment with PP2, the specific inhibitor of SFKs, promoted the differentiation. Another inhibitor of SFKs, PP1, also promoted the brown adipogenesis, whereas an inactive analogue of PP2, PP3, did not. Moreover, over‐expression of C‐terminal Src kinase (CSK), the negative regulator of SFKs, also promoted brown adipogenesis. Next, we examined the effect of inhibition of SFKs on the differentiation of white preadipocytes isolated from white adipose tissue (WAT). Our results showed that either PP2 treatment or CSK‐over‐expression generated Ucp1‐positive beige adipocytes, thus inducing browning of white adipocytes. Finally, our analysis showed that the expression levels and activity of SFKs in WAT were much higher than in BAT. These results taken together suggest that SFKs regulate differentiation and browning of fat cells in vivo.     

Mitochondrial (Dys)function in Adipocyte (De)differentiation and Systemic Metabolic Alterations

In mammals, adipose tissue, composed of BAT and WAT, collaborates in energy partitioning and performs metabolic regulatory functions. It is the most flexible tissue in the body, because it is remodeled in size and shape by modifications in adipocyte cell size and/or number, depending on developmental status and energy fluxes. Although numerous reviews have focused on the differentiation program of both brown and white adipocytes as well as on the pathophysiological role of white adipose tissues, the importance of mitochondrial activity in the differentiation or the dedifferentiation programs of adipose cells and in systemic metabolic alterations has not been extensively reviewed previously. Here, we address the crucial role of mitochondrial functions during adipogenesis and in mature adipocytes and discuss the cellular responses of white adipocytes to mitochondrial activity impairment. In addition, we discuss the increase in scientific knowledge regarding mitochondrial functions in the last 10 years and the recent suspicion of mitochondrial dysfunction in several 21st century epidemics (ie, obesity and diabetes), as well as in lipodystrophy found in HIV-treated patients, which can contribute to the development of new therapeutic strategies targeting adipocyte mitochondria.Adipocytes and, more generally, the adipose tissues are major actors in both obesity and the emergence of a cluster of associated diseases such as insulin resistance and type 2 diabetes mellitus (T2DM), cardiovascular diseases, hypertension, dyslipidemia, and even some cancers. Obesity and diabetes are now recognized as worldwide epidemics,¹ with 1.6 billion people being overweight, of which 400 million are obese (body mass index ≥30) (World Health Organization, Geneva 2006).Attention for adipocytes has increased ever since it has been found that these differentiated cells are not only able to store and release triglycerides (TGs) but also have an important endocrine activity. Indeed, adipocytes secrete “adipokines” (specific hormones and proinflammatory cytokines) to communicate systemically with other cell types and thus, importantly, contribute to the regulation of energy homeostasis.² Adipose tissue is present in different interacting depots in the body. In addition to white adipose tissue (WAT), brown adipose tissue (BAT) can also be distinguished.³ Although BAT originates from the myogenic lineage,⁴ it shares many features of WAT that are discussed in this review. Because WAT is by far the largest depot in humans and, as a metabolically active, lipid storage and endocrine organ, its proper functioning is essential for health maintenance and is of primary importance for pharmaceutical and food industries.A better understanding of the mechanisms involved in adipocyte differentiation, dedifferentiation (defined as the acquisition of a more primitive phenotype and gain of cell proliferative ability)⁵ and even trans-differentiation (a process related to reversion of one cell phenotype into another, ie, from white to brown adipocytes,^6,7 which is still poorly experimentally documented), is required to unravel mechanisms underlying obesity and its symptomatic cohort of associated pathologies. This understanding may be used to develop new, original, and more effective therapeutic approaches that directly target intracellular pathways in adipocytes. Although the adipocyte differentiation program,⁸ as well as the activity/function and dys/malfunction of the endoplasmic reticulum (which play an important role in the adipocyte physiology) have been recently reviewed,⁹ the role of mitochondrial activity or dysfunction during preadipocyte differentiation and its consequences in mature adipocytes has hardly been addressed. Subcutaneous and visceral WATs have a different metabolic activity, depending on their anatomical position and mitochondrial content: epididymal (in the visceral depot) adipocytes are richer in mitochondria than inguinal (s.c.) adipocytes.¹⁰ In addition, mitochondria play a key role in physiological processes and are involved in the pathology of many diseases.¹¹ In compiling knowledge on mitochondria in the context of adipose tissue, we hope to stimulate thoughts in regards to the impact of mitochondrial activity in adipocyte biology, the effects of mitochondrial dysfunction/stress on adipocytes, and the subsequent alterations of systemic metabolic functions.     

Integrin αV Mediates the Effects of Irisin on Human Mature Adipocytes

Introdution and AimsThe myokine irisin is critical to modulating adipocytes thermogenesis and influence whole-body metabolism. However, whether there is difference in the effects of irisin on adipocytes derived from different depots remains unknown, and the receptor of irisin on adipocytes is still unclear. In this study, we determine the browning effect of irisin on adipocytes of subcutaneous and visceral human adipose tissue and explore the possibility that integrin αV was the receptor of irisin on human adipocytes.MethodsHuman adipose-derived stem cells were isolated from human subcutaneous and visceral white adipose tissues and induced to differentiate into mature adipocytes, and the expression of UCP1 and thermogenic genes in mature adipocytes were examined with or without irisin treatment and compared between groups of different adiposity and different spots. Immunoprecipitation analysis was used to detect the interaction between irisin and integrin αV on adipocytes, and the protein expression of integrin αV in adipocytes was also compared between groups of different adiposity and anatomic position.ResultsIrisin treatment could increase the expression level of beige adipocyte marker protein UCP1 and specific thermogenic genes in mature adipocytes derived from subcutaneous white adipose tissue but not in visceral adipose tissue. The results of immunoprecipitation showed that irisin could be attached to integrin αV on mature adipocytes, and there was no significant difference in the gene and protein expression of integrin αV in adipocytes, either derived from subcutaneous and visceral adipose tissue, or derived from obese and normal-weight individuals.ConclusionThe results of the present study indicated that irisin contributed to the transformation of mature white adipocytes to beige adipocytes in human subcutaneous adipose tissue but not in visceral adipose tissue. Integrin αV may mediate the browning effects of irisin on human mature adipocytes, which could provide the potential therapeutic targets for obesity and metabolic syndrome by promoting human brown adipose tissue activity.     

Altered expression of master regulatory genes of adipogenesis in lipomas from patients bearing tRNA(Lys) point mutations in mitochondrial DNA

The mechanisms underlying the appearance of lipomas in patients bearing mutations in the tRNA(Lys) gene of mitochondrial DNA are unknown. We investigated changes in gene expression patterns in lipomas from three patients bearing A8344G or G8363A tRNA(Lys) gene mutations. Uncoupling protein-1 mRNA was detected in the lipomas, in contrast with undetectable expression in normal adipose tissue. However, expression of other markers of brown fat, such as PGC-1alpha, was unaltered. PPARgamma and retinoblastoma gene expression was down regulated in the lipomas, but C/EBPalpha mRNA was not affected. The expression of Pref-1 was dramatically down regulated. Thus, lipomatosis due to tRNA(Lys) mutations is associated with a pattern of altered expression of master regulators of adipogenesis consistent with enhanced proliferation but maintenance of adipocyte features, and with a distorted pattern of brown versus white adipocyte differentiation.     

Multiple symmetric lipomatosis. Ultrastructural investigation of the tissue and preadipocytes in primary culture

Multiple symmetric lipomatosis (MSL) is characterized by enlarging, painless fat deposits in the neck and upper trunk. The pathogenesis of MSL is unknown. Owing to localization of MSL fat deposits in the neck and interscapular region, it has been suggested that they could originate from brown fat. However, the histological appearance of MSL adipose tissue is that of white fat, with prevailing monovacuolar adipocytes. Nevertheless, MSL adipocytes are smaller than adipocytes of the common white adipose tissue and show peculiar metabolic features. The ultrastructure of MSL lesions has been not described. The present work investigated the ultrastructural morphology of MSL adipose tissue and lipomatous adipocyte precursors maintained in long-term culture. Samples of lipomatous tissue were obtained from patients affected with MSL undergoing surgical lipectomy. Portion of the tissue was processed for electron microscopy; the rest was digested with collagenase, and isolated preadipocytes from the stromal-vascular fraction were cultured up to 15 days. Cultured cells were prepared for electron microscopy in situ and their morphology compared with human white adipose tissue preadipocytes and rat brown preadipocytes cultured in parallel. Results show the following. 1) Adipocytes of MSL are not monovacuolar and resemble the largest adipocytes that can be found in rat and human brown fat. 2) Some morphological features of MSL adipocyte precursors resemble brown adipocyte more than white: cultured MSL preadipocytes transiently develop large mitochondria with parallel cristae resembling those of the brown fat cell and maintain a multivacuolar lipid deposit in culture, i.e. a typical feature of brown preadipocytes. 3) Some morphological features suggest a neoplastic nature of MSL adipocytes. Taken together, these findings suggest that MSL is a neoplastic disease which could originate in brown fat.     

Molecular determinants of brown adipocyte formation and function

Humans contain essentially two types of adipose tissue: brown adipose tissue (BAT) and white adipose tissue (WAT). The function of WAT is to store fat while that of BAT is to burn fat for heat production. A potential strategy to combat obesity and its related disorders is to induce the conversion of WAT into BAT. In this issue of Genes & Development, Kajimura and colleagues (pp. 1397-1409) have identified a mechanism by which PRDM16, the principal regulator of brown adipocyte formation and function, can simultaneously induce BAT gene expression, while suppressing WAT gene expression. The studies suggest that PRDM16 and its associated coregulators PPARgamma coactivator-1alpha (PGC-1alpha) and C-terminal-binding protein 1/2 (CtBP1/2), which control the switch from WAT to BAT, are potential targets for development of obesity-related therapeutics.     

In vivo differentiation of brown adipocytes in adult mice: An electron microscopic study

The differentiation of brown adipocyte precursor cells was studied in interscapular brown adipose tissue of adult mice by electron microscopy. Different stages of cell differentiation were characterized in situ. Previous autoradiographic studies suggested that interstitial cells represent the precursor cells of fully differentiated brown adipocytes. The present observations provide morphological evidence for a progressive differentiation of interstitial stem cells into mature brown adipocytes. Four typical stages of development were identified: (1) interstitial cells, (2) protoadipocytes, (3) preadipocytes, and (4) mature brown adipocytes. Interstitial stem cells were small spindle shaped cells, situated between brown adipocytes and characterized by a high nuclear-cytoplasmic ratio, the scarcity of organelles, and the absence of lipid inclusions. Protoadipocytes resembled interstitial cells except that they contained a few tiny lipid droplets in their cytoplasm. Preadipocytes had a larger cytoplasm enclosing many mitochondria and lipid droplets; the smooth endoplasmic reticulum was well developed surrounding the lipid droplets, and was closely associated with the mitochondria. Preadipocytes had the typical structure of growing cells, developing long cytoplasmic processes between and around blood capillaries. Mature brown adipocytes represented the final stage of differentiation. Almost all their cellular volume was occupied by lipid droplets and numerous mitochondria with very dense cristae. Brown adipocytes were also characterized by a tight association with blood capillaries, as expected from metabolically active cells requiring oxygen and substrates. These observations provide direct ultrastructural evidence for a progressive differentiation of interstitial cells into brown adipocytes with a continuum of intermediate cellular types.     

NYGGF4基因在人前体脂肪细胞分化过程中的表达及肿瘤坏死因子α对其调控的研究

目的 观察人前体脂肪细胞诱导分化过程中NYGGF4基因mRNA表达水平的变化,探讨重组肿瘤坏死因子α(tumor necrosis factor α,TNFα)对成熟脂肪细胞中NYGGF4基因表达水平的调节作用.方法 体外培养人内脏来源的原代前体脂肪细胞(human preadipocytes-visceral,HPA-v),在诱导HPA-v分化成熟的基础上,应用不同浓度重组TNFα干预成熟脂肪细胞16 h,或以10 ng/mL TNFα分别干预不同时间,采用实时荧光定量逆转录PCR技术检测的NYGGF4 mRNA表达水平.结果 (1)HPA-v诱导分化至第17 d,具备成熟脂肪细胞特征;(2)NYGGF4基因低表达于人前体脂肪细胞中,随脂肪细胞分化成熟其表达水平逐渐升高;(3)随TNFα干预浓度增高及干预时间延长,人成熟脂肪细胞中NYGGF4mRNA水平呈现逐渐升高的趋势.结论 NYGGF4基因具随脂肪细胞分化成熟表达逐渐上调的特征,TNFα能显著上调成熟脂肪细胞中NYGGF4基因的表达,其效应具有剂量依赖和时间反应性.     

The pre- and postnatal development and ageing of interscapular brown adipose tissue in the rat

Summary Developmental and ageing changes in interscapular brown adipose tissue have been studied in white Wistar rats by light, fluorescence and electron microscopy.Cellular aggregation was noted in the fetal interscapular area on the 15th day of gestation and vascularised primitive lobules of brown adipose tissue became unequivocally identifiable on the 17th day in utero. Brown adipocyte precursors appeared to be derived directly from mesenchymal cells and were uniquely characterised by larger (0.8–1.7 m diameter) mitochondria. Numbers of these precursor cells (pre-adipocytes) were seen in mitosis during intrauterine life. Pericapillary cells similar in appearance to embryonic pre-adipocytes were regularly observed within brown fat lobules throughout later life.Cardinal features noted in mature brown adipose tissue were parenchymal cells with a multilocular lipid distribution and numerous large mitochondria with distinctive inparallel cristae, as well as an extensive vascular network and a dense catecholaminergic vasomotor and parenchymal innervation.Brown adipocytes generally retained a multilocular lipid distribution into old age, and although the catecholaminergic fluorescence of the nerves supplying the tissue was reduced, a widespread distribution of noradrenergic vasomotor and parenchymal nerves and of nexuses between brown adipocytes continued to be demonstrable by electron microscopy in the brown adipose tissue of senile rats.     

Coenzyme Q as an antiadipogenic factor

Coenzyme Q (CoQ) is not only the single antioxidant synthesized in humans but also an obligatory element of mitochondrial functions. We have previously reported CoQ deficiency in white adipose tissue of ob/ob mice. We sought to determine (i) whether this deficit exists in all species and its relevance in human obesity and (ii) to what extent CoQ could be involved in adipocyte differentiation. Here we identified in rodents as well as in humans a specific very strong nonlinear negative correlation between CoQ content in subcutaneous adipose tissue and obesity indexes. This striking correlation reveals a threshold value similar in both species. This relative deficit in CoQ content in adipose tissue rapidly took place during the time course of high-fat-diet-induced obesity in mice. Adipocyte differentiation was assessed in vitro using the preadipocyte 3T3-F442A cell line. When CoQ synthesis was inhibited by a pharmacological approach using chlorobenzoic acid, this strongly triggered adipose differentiation. In contrast, adipogenesis was strongly inhibited when a long-term increase in CoQ content was obtained by overexpressing human 4-hydroxy benzoate acid polyprenyltransferase gene. Altogether, these data suggest that a strict level of CoQ remains essential for adipocyte differentiation, and its impairment is associated with obesity.     

Brown adipocyte precursor cells: a morphological study

The origin of brown adipocyte precursor cells is to date unknown. Some authors believe they arise from vascular cells, others from interstitial cells. The purpose of the present ultrastructural study was to find markers in rat fetal and perinatal adipose tissue that can be used to identify brown adipose precursor cells. The study was carried out on the interscapular brown adipose tissue of fetal (fetuses of 19 and 21 days) and perinatal rats (pups of 4 and 12 hours and of 1, 3, 5, 7, 9, 11, 13, and 15 days). The analysis focused on stem cells and showed the characteristic presence of typical mitochondria which make their identification as brown adipocyte precursor cells inequivocal. These cells were frequently observed in a pericytic position. Also some endothelial cells were characterised by typical mitochondria and abundant glycogen. These data seem to support the hypothesis that brown adipocytes originate from vascular cells.     

Enhanced proliferation of human umbilical vein endothelial cells and differentiation of 3T3-L1 adipocytes in coculture

The interactions between adipocytes and endothelial cells in adipose tissue development are poorly understood. In this study, we characterized the growth and differentiation of 3T3-L1 preadipocytes and human umbilical vein endothelial cells (HUVECs) in planar and collagen gel cocultures. In planar coculture, preadipocyte proliferation was up to three times as great as in the control culture with only preadipocytes, where the increase was proportional to the HUVEC fraction in the seeding mixture. In the collagen gel coculture, triglyceride (TG) content (per adipocyte) was up to 3.4 times as much as in the control with only adipocytes. This effect depended on the total density and composition of the seeding mixture, with the largest increase observed at the highest density (2 x 10(6) cells/mL collagen) and preadipocyte:HUVEC ratio (90:10) tested in this study. Immunostaining showed that the collagen gel coculture also supported the elongation of endothelial cells. Blockade of vascular endothelial growth factor receptor 2 (VEGFR2) abolished the adipogenesis- and neovascularization-related effects of the coculture. Taken together, our results indicate that endothelial cell-mediated enhancement of adipocyte differentiation requires the activation of VEGFR2.     

Gene expression in human brown adipose tissue

Brown adipose tissue (BAT) has profound effects on body weight and metabolism in rodents. Recent reports show that human adults have significant amounts of BAT. Our aim was to study the gene expression profile of human BAT. Biopsies of adipose tissue with brown-red color and subcutaneous white adipose tissue (WAT) were obtained from 24 patients undergoing surgery in the thyroid region. Intrascapular BAT and epididymal WAT biopsies were obtained from 10 mice. Expression was analyzed by DNA microarray, real-time PCR and immunohistochemistry. Using the expression of the brown adipocyte-specific gene uncoupling protein 1 (UCP1) as a marker, approximately half of the human brown-red adipose tissue biopsies taken in the thyroid region contained BAT, and the presence of cells with brown adipocyte morphology was also verified by histology. Microarray analysis of 9 paired human BAT and WAT samples showed that 17 genes had at least a 4-fold higher expression in BAT compared to WAT and five of them (CKMT1, KCNK3, COBL, HMGCS2, TGM2) were verified using real-time PCR (P<0.05 for all). In addition, immunohistochemistry showed that the UCP1, KCNK3 and CKMT1 proteins are expressed in brown adipocytes. Except for UCP1 and KCNK3, the genes overexpressed in human BAT were not overexpressed in mouse BAT compared to mouse WAT. Our analysis identified genes that are differentially expressed in human BAT compared to WAT. The results also show that there are species-specific differences in BAT gene expression and this emphasizes the need for further molecular characterization of human BAT to clarify the mechanisms involved in regulated heat production in humans.     

Basic fibroblast growth factor (bFGF) contributes to the enlargement of brown adipose tissue during cold acclimation

The contribution of basic fibroblast growth factor to brown adipose tissue (BAT) enlargement during cold acclimation was investigated using rat brown adipocytes in primary culture. After cold exposure (at 5° C) for 28 days, the level of bFGF messenger ribonucleic acid (mRNA) in BAT of cold-acclimated rats was markedly increased with the increase in the BAT weight. In addition, the blood plasma from cold-acclimated rats considerably enhanced the expression of basic fibroblast growth factor mRNA in rat brown adipocytes. Likewise, the blood plasma from cold-acclimated rats significantly stimulated the growth of rat brown adipocyte precursor cells compared with that from warm-acclimated rats, whereas there was no difference of effect between the two blood plasmas on the growth of bovine capillary endothelial cells. Basic fibroblast growth factor, but not platelet-derived growth factor stimulated the growth of brown adipocyte precursor cells. The conditioned medium from brown adipocyte primary culture markedly stimulated the growth of bovine capillary endothelial cells and the effect was inhibited considerably by antibasic fibroblast growth factor antibody. These results suggest that some factors concerned with the growth of brown adipocyte precursor cells are present in the blood plasma from cold-acclimated rats, and that basic fibroblast growth factor produced by brown adipocytes may significantly contribute to BAT enlargement by autocrine mechanisms during cold exposure.     

Elucidating the Preadipocyte and Its Role in Adipocyte Formation: a Comprehensive Review

Adipogenesis is a complex process whereby the multipotent adipose-derived stem cell is converted to a preadipocyte before terminal differentiation into the mature adipocyte. Preadipocytes are present throughout adult life, exhibit adipose fat depot specificity, and differentiate and proliferate from distinct progenitor cells. The mechanisms that promote preadipocyte commitment and maturation involve numerous protein factor regulators, epigenetic factors, and miRNAs. Detailed characterization of this process is currently an area of intense research and understanding the roles of preadipocytes in tissue plasticity may provide insight into novel approaches for tissue engineering, regenerative medicine and treating a host of obesity-related conditions. In the current study, we analyzed the current literature and present a review of the characteristics of transitioning adipocytes and detail how local microenvironments influence their progression towards terminal differentiation and maturation. Specifically, we detail the characterization of preadipocyte via surface markers, examine the signaling cascades and regulation behind adipogenesis and cell maturation, and survey their role in tissue plasticity and health and disease.