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.     

Leukemia inhibitory factor influences the fate choice of mesenchymal progenitor cells

Osteoblasts and adipocytes derive from a common mesenchymal precursor, and in at least some circumstances, differentiation along these two lineages is inversely related. For example, we have recently observed that concomitant with inhibition of osteoblast differentiation and bone nodule formation, leukemia inhibitory factor (LIF) induces genes regulating lipid metabolism in fetal rat calvaria (RC) cell cultures. In this study, we further investigated the adipogenic capacity of LIF-treated RC cells. Quantitative analyses revealed that LIF increased the adipocyte differentiation induced by the peroxisome proliferator-activated receptor gamma agonist BRL49653 (BRL) in RC cell populations. Gene expression profiling of individual RC cell colonies in untreated cells or cells treated with LIF, BRL, or combined LIF-BRL suggested that some adipocytes arose from bipotential or other primitive precursors, including osteoprogenitors, since many colonies co-expressed osteoblast and adipocyte differentiation markers, whereas some arose from other cell pools, most likely committed preadipocytes present in the population. These analyses further suggested that LIF and BRL do not act at the same stages of the mesenchymal hierarchy, but rather that LIF modifies differentiation of precursor cells, whereas BRL acts later to favor adipocyte differentiation. Taken together, our data suggest that LIF increased adipocyte differentiation at least in part by altering the fate of osteoblastic cells and their precursors.     

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSC) can differentiate into osteoblasts, adipocytes, chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro, MSC differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering, but may also be relevant in the search for new treatments of osteoporosis.     

Embryonic stem cell-derived adipogenesis

Key events leading to terminal differentiation of preadipocytes into adipocytes have been characterized in the recent years. However, master genes that commit progression from multipotent mesenchymal stem cell to the adipoblast stage of development have not yet been identified. The use of embryonic stem (ES) cells as a route to study early events in adipogenesis and to characterize factors involved in the decision of stem cells to follow the adipogenic pathway is described in this paper. The capacity of lif-/- and lifr-/- ES cells to undergo adipocyte differentiation is reported as an application of mutant ES cells to study gene function during the development of adipose cells.     

Fine structural studies on white adipocyte differentiation

The differentiation of mammalian white adipocytes from prenatal through early postnatal periods was studied by light and electron microscopy in C57BL mice. Anatomical regions chosen for this study were the epididymal, mesometrial, mesenteric and inguinal fat pads. In each of these regions, adipocytes differentiated from fibroblast-like cells (preadipocytes) characterized by an ovoid nucleus, profiles of rough endoplasmic reticulum, microtubules, microfilaments, spherical mitochondria, and small multiple lipid inclusions. Preadipocytes of the inguinal fat pad were first observed prior to birth (17–19 days), whereas, in the other anatomical sites, these cells were not observed until one to three days postnatally. As differentiation proceeded, and as the adipocytes assumed a spherical shape, there was a progressive decrease in the amount of rough endoplasmic reticulum and microfilaments concomitant with transient glycogen storage and an increase in the size of lipid droplets. Mature unilocular adipocytes were observed in the inguinal fat pads at three days of age. On the other hand, these cells did not appear until seven days after birth in the epididymal fat pad, mesometrium and mesentery. Regardless of the anatomical region studied, the differentiation of preadipocytes to adipocytes proceeded similarly. Preadipocytes could not be distinguished from fibroblasts morphologically within the fat depots studied. Adipocytes at the mid-stages of differentiation and in all regions studied occasionally exhibited close intercellular contacts of varying morphology.     

Thoughts on the source of tissue on subsequent cell culture success

This paper describes attempts to initiate equine adipocyte cultures from necropsy cases with varying intervals from time of death to isolation and culture. Equine adipocytes were isolated from 21 necropsy cases, regardless of the interval from time after death to establishment in primary ceiling cultures. However, while all cultures produced adipocytes, only 2 attempts to produce long-term equine adipocyte cultures from the subcutaneous rump fat depots were successful and not contaminated. Findings from these experiments indicate that it is possible to collect and culture equine adipocytes from necropsy cases with varying intervals of time of death to culturing provided that the issue of contamination is addressed. Viable cells were produced from tissue with an interval of 38.5 hours as well as 45 minutes. This result encourages the continuation of research using equine necropsy cases as a source of adipose tissue.     

Adipocytes and macrophages secretomes coregulate catecholamine-synthesizing enzymes

Obesity associates with macrophage accumulation in adipose tissue where these infiltrating cells interact with adipocytes and contribute to the systemic chronic metabolic inflammation present in immunometabolic diseases. Tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) are two of the main enzymes of catecholamines (CA) synthesis. Adipocytes and macrophages produce, secrete and respond to CA, but the regulation of their synthesis in the interplay between immune and metabolic systems remains unknown. A model of indirect cell coculture with conditioned medium (CM) from RAW 264.7 macrophages with or without LPS-activation and 3T3-L1 adipocytes and preadipocytes was established to study the effect of cellular secretomes on the expression of the above enzymes. During the adipocyte differentiation process, we found a decrease of TH and PNMT expression. The secretome from LPS-activated macrophages downregulated TH and PNMT expression in preadipocytes, but not in mature adipocytes. Mature adipocytes CM induced a decrease of PNMT levels in RAW 264.7 macrophages. Pre and mature adipocytes showed a similar pattern of TH, PNMT and peroxisome proliferator-activated receptor gamma expression after exposure to pro and anti-inflammatory cytokines. We evidenced macrophages and adipocytes coregulate the expression of CA synthesis enzymes through secretome, with non-inflammatory signaling networks possibly being involved. Mediators released by macrophages seem to equally affect CA production by adipocytes, while adipocytes secretome preferentially affect AD production by macrophages. CA synthesis seems to be more determinant in early stages of adipogenic differentiation. Our results suggest that CA are key signaling molecules in the regulation of immune-metabolic crosstalk within the adipose tissue.     

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.     

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.     

Concise review: adipocyte origins: weighing the possibilities

Adipose tissue is the primary energy reservoir in the body and an important endocrine organ that plays roles in energy homeostasis, feeding, insulin sensitivity, and inflammation. While it was tacitly assumed that fat in different anatomical locations had a common origin and homogenous function, it is now clear that regional differences exist in adipose tissue characteristics and function. This is exemplified by the link between increased deep abdominal or visceral fat, but not peripheral adipose tissue and the metabolic disturbances associated with obesity. Regional differences in fat function are due in large part to distinct adipocyte populations that comprise the different fat depots. Evidence accrued primarily in the last decade indicates that the distinct adipocyte populations are generated by a number of processes during and after development. These include the production of adipocytes from different germ cell layers, the formation of distinct preadipocyte populations from mesenchymal progenitors of mesodermal origin, and the production of adipocytes from hematopoietic stem cells from the bone marrow. This review will examine each of these process and their relevance to normal adipose tissue formation and contribution to obesity-related diseases.     

油酸诱导SW872前脂肪细胞分化及分化过程中促酰化蛋白的分泌

目的探讨油酸对SW872前脂肪细胞的诱导分化作用及SW872细胞分化过程中促酰化蛋白的分泌。方法体外培养SW872细胞,0.6mmol/L油酸作为诱导分化剂刺激SW872前脂肪细胞,油红O染色观察细胞形态的变化及脂滴的聚集,化学比色法测定甘油三酯的总量,ELISA方法测定培养上清中促酰化蛋白的含量。结果SW872细胞分化之前呈成纤维细胞样,胞内无脂滴,甘油三酯的含量较低;加入0.6mmol/L油酸诱导分化刺激72h时,SW872细胞已经完全分化,胞浆中脂滴明显增多,油红O染色可见胞浆中丰富的脂肪染色,甘油三酯的含量是诱导分化前的14倍(P<0.01)。诱导分化之前,SW872细胞分泌ASP的量非常低,几乎不能检测出;诱导分化72h,ASP的含量增加到1.12ng/mg细胞总蛋白质。结论油酸能诱导SW872前脂肪细胞分化为成熟的脂肪细胞;ASP的分泌与脂肪细胞的分化程度密切相关。     

Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-L1 cells

Fucoxanthin is a major carotenoid found in edible seaweed such as Undaria pinnatifida and Hijikia fusiformis. We investigated the suppressive effects of fucoxanthin and its metabolite, fucoxanthinol, on the differentiation of 3T3-L1 preadipocytes to adipocytes. Fucoxanthin inhibited intercellular lipid accumulation during adipocyte differentiation of 3T3-L1 cells. Furthermore, fucoxanthin was converted to fucoxanthinol in 3T3-L1 cells. Fucoxanthinol also exhibited suppressive effects on lipid accumulation and decreased glycerol-3-phosphate dehydrogenase activity, an indicator of adipocyte differentiation. The suppressive effect of fucoxanthinol was stronger than that of fucoxanthin. In addition, in 3T3-L1 cells treated with fucoxanthin and fucoxanthinol, peroxisome proliferator-activated receptor gamma (PPARgamma), which regulates adipogenic gene expression, was down-regulated in a dose-dependent manner. These results suggest that fucoxanthin and fucoxanthinol inhibit the adipocyte differentiation of 3T3-L1 cells through down-regulation of PPARgamma. Fucoxanthinol had stronger suppressive effects than fucoxanthin on adipocyte differentiation in 3T3-L1 cells.     

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.     

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.     

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.     

Preadipocyte seeded PLGA scaffolds for adipose tissue engineering.

Adipose tissue equivalents have not been addressed as yet despite the clinical need in congenital deformities, posttraumatic repair, cancer rehabilitation, and other soft tissue defects. Preadipocytes were successfully harvested from rat epididymal fat pads of Sprague-Dawley and Lewis rats and expanded ex vivo. In vitro cultures demonstrated full differentiation of preadipocytes into mature adipocytes with normal lipogenic activity. The onset of differentiation was well-controlled by regulating preadipocyte confluency. Poly(lactic-co-glycolic) acid (PLGA) polymer disks with 90% porosity, 2.5 mm thick, 12 mm diameter, pore size range of 135-633 microm were fabricated and seeded with preadipocytes at 10(5) cells/mL. Disks in vitro demonstrated fully differentiated mature adipocytes within the pores of the disks. Short-term in vivo experiments were conducted by implanting preseeded disks subcutaneously on the flanks of rats for 2 and 5 weeks. Histologic staining of harvested disks with osmium tetroxide (OsO4) revealed the formation of adipose tissue throughout the disks. Fluorescence labeling of preadipocytes confirmed that formed adipose tissue originated from seeded preadipocytes rather than from possible infiltrating perivascular tissue. This study demonstrates the potential of using primary preadipocytes as a cell source in cell-seeded polymer scaffolds for tissue engineering applications.     

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.     

脂肪细胞在肝细胞胰岛素耐受过程中的作用

目的研究脂肪细胞在不同分化阶段对肝细胞胰岛素抵抗的影响。方法体外诱导3T3-L1脂肪前体细胞分化,细胞内脂滴增加,逐步分化成脂肪细胞。采用不同分化阶段脂肪细胞(未分化0d、中期分化4d、接近完全分化8d)与原代肝细胞共培养。Western印迹法检测共培养后肝细胞内胰岛素信号通路的反应性;葡萄糖同位素标记方法检测肝细胞糖原合成能力。结果以未共培养的肝细胞为对照组,共培养后肝细胞内胰岛素受体底物-2酪氨酸磷酸化(Tyr612)(pIRS-2)水平及Akt磷酸化(Ser473)(pAkt)水平均显著下调;肝糖原合成能力明显降低;与较成熟脂肪细胞共培养后,肝细胞pIRS-2及pAkt水平与其他分化阶段组共培养比较下调明显,肝糖原合成能力随着脂肪细胞的成熟而明显降低。结论脂肪细胞可能诱导肝细胞发生胰岛素抵抗,肝细胞胰岛素信号通路的阻滞程度与脂肪细胞的分化程度呈正相关。