Overgrowth of fat autotransplanted for correction of localized steroid-induced atrophy

A case is presented in which regional subcutaneous atrophy in the lateral thigh after steroid injection was treated with autologous fat grafting. More than 1 year after treatment, an increase in the volume of the transplanted site necessitated a secondary procedure to debulk the area. We believe that this is a case in which the transplanted adipocytes (or preadipocytes) survived and subsequently (for an unclear reason) increased significantly in volume. This case report supports the cell survival theory of fat autotransplantation. Animal studies using radioisotope-labeled lipocytes might provide further understanding of the mechanisms of fat graft "survival."     

Bioactivation of free-fat transfers: a potential new approach to improving graft survival.

A new approach to free-fat autotransplantation resorption was evaluated experimentally in a rat animal model. Bioactive fat grafts were created by the addition of basic fibroblast growth factor delivered by dextran beads to the grafts and compared with free fat alone, free fat plus beads, and free fat plus beads and a control solution in the same animal. The grafts were assessed by weight and histology at 1 and 12 months postoperatively in 40 animals. A graded response in weight retention was observed at 1 and 12 months, with the growth factor-treated grafts exhibiting near complete weight maintenance after 1 year. All other bead-containing grafts had an intermediate response, with free fat alone averaging more than one-half graft weight loss after 1 year. Histologically, the bead-containing grafts had good fibroblastic ingrowth, but extensive intercellular collagen formation and the occurrence of small-sized adipocytes among the larger adipocytes were seen only in the growth factor-treated grafts. These findings indicate that graft manipulations that affect the preadipocyte cells of the graft or fibroblastic components of the recipient site, either through polypeptide stimulation or surface charge attraction, may offer a viable approach to postoperative fat-graft volume maintenance.     

The effect of interleukin-8 on the viability of injected adipose tissue in nude mice

Adipose tissue injection as a free graft for the correction of soft-tissue defects is a widespread procedure in plastic surgery. The main problem in achieving long-term soft-tissue augmentation is partial absorption of the injected fat and hence the need for overcorrection and re-injection. The purpose of this study was to improve the viability of the injected fat by the use of interleukin-8. The rationale for the use of interleukin-8 was its abilities to accelerate angiogenesis and attract inflammatory cells and fibroblasts, providing the injected adipocytes more feeding vessels and a well-established graft bed to enhance their viability. Human adipose tissue, obtained by suction-assisted lipectomy, was re-injected into the subcutis in the scalp of nude mice. Interleukin-8 (0.25 ng) was injected subcutaneously to the scalp as a preparation of the recipient site 24 hours before the fat injection and was added to the fat graft itself (25 ng per 1 cc of injected fat). In the control group, pure fat without interleukin-8 was injected and no interleukin-8 was added for the preparation of the recipient site. One cubic centimeter of fat was injected in each animal in both the study and control groups. There were 10 animals in each group. The animals were euthanized 15 weeks after the procedure. Graft weight and volume were measured and histologic evaluation was performed. In addition, triglyceride content and adipose cell sizes were measured as parameters for fat cells viability. Histologic analysis demonstrated significantly less cyst formation in the group treated with interleukin-8. No significant differences were found between the groups with regard to graft weight and volume or the other histologic parameters investigated. No significant differences were demonstrated in adipose cell sizes and their triglyceride content. In conclusion, less cyst formation, indicating improved quality of the injected fat, can be obtained by the addition of interleukin-8. Further studies of various dosages of interleukin-8 and their long-term effect are required before these encouraging results could be applied clinically.     

Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue

We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro. This work was supported by grants from the Japan Ministry of Education, Science, Sports, and Culture (no. 19580348) and from MEXT. HAITEKU (2007–2011).     

CD36 is important for adipocyte recruitment and affects lipolysis

Objective: The scavenger receptor CD36 facilitates the cellular uptake of long‐chain fatty acids. As CD36‐deficiency attenuates the development of high fat diet (HFD)‐induced obesity, the role of CD36‐deficiency in preadipocyte recruitment and adipocyte function was set out to characterize. Design and Methods: Fat cell size and number were determined in gonadal, visceral, and subcutaneous adipose tissue of CD36^?/? and WT mice after 6 weeks on HFD. Basal lipolysis and insulin‐inhibited lipolysis were investigated in gonadal adipose tissue. Results: CD36^?/? mice showed a reduction in adipocyte size in all fat pads. Gonadal adipose tissue also showed a lower total number of adipocytes because of a lower number of very small adipocytes (diameter <50 μm). This was accompanied by an increased pool of preadipocytes, which suggests that CD36‐deficiency reduces the capacity of preadipocytes to become adipocytes. Regarding lipolysis, in adipose tissue from CD36^?/? mice, cAMP levels were increased and both basal and 8‐bromo‐cAMP stimulated lipolysis were higher. However, insulin‐mediated inhibition of lipolysis was more potent in CD36^?/? mice. Conclusions: These results indicate that during fat depot expansion, CD36‐deficiency negatively affects preadipocyte recruitment and that in mature adipocytes, CD36‐deficiency is associated with increased basal lipolysis and insulin responsiveness.     

Conditionally immortalized white preadipocytes: a novel adipocyte model

This study describes a novel approach to generate conditionally immortalized preadipocyte cell lines from white adipose tissue (IMWAT) that can be induced to differentiate into white adipocytes even after expansion in culture. Such adipocytes express markers of white fat such as peroxisome proliferator-activated receptor gamma and aP2 but not brown fat markers, have an intact insulin signaling pathway, and express proinflammatory cytokines. They can be readily transduced with adenoviral vectors, allowing them to be used to investigate the consequences of the depletion of specific adipocyte factors using short hairpin RNA. This approach has been used to study the effect of reduced expression of the nuclear receptor corepressor receptor interacting protein 140 (RIP140), a regulator of adipocyte function. The depletion of RIP140 results in changes in metabolic gene expression that resemble those in adipose tissue of the RIP140 null mouse. Thus, IMWAT cells provide a novel model for adipocytes that are derived from preadipocytes rather than fibroblasts and provide an alternative system to primary preadipocytes for the investigation of adipocyte function.     

Reconstituted basement membrane potentiates in vivo adipogenesis of 3T3-F442A cells

Adipocytes forming fat pad in vivo are surrounded by well developed basement membranes. Synthesis of basement membrane is enhanced during in vitro differentiation of preadipocyte line. In order to know the role of basement membrane in adipogenesis in vivo, we injected 3T3-F442A preadipocytes subcutaneously into nude mice together with or without the reconstituted basement membrane, Matrigel. Histological sections of the fat pads newly formed by injecting the cell alone showed dense population of immature adipocytes and microvessels within 2 weeks and they matured rapidly. In contrast, injection of the cells together with Matrigel showed sparse adipocytes after 2 weeks and they matured slowly over the period of 6 weeks. Quantification of the process by measuring the weight, DNA content, triglyceride content and glycerophosphate dehydrogenase (GPDH) activity of the fat pads showed that injection of the cell alone resulted in early maturation of adipose tissue with fewer adipocytes while the presence of Matrigel decelerated but potentiated the maturation of adipose tissue with 2 fold contents of DNA, triglyceride and GPDH activity. We thus showed that reconstituted basement membrane (Matrigel) supported the survival and maturation of adipocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Necdin controls proliferation of white adipocyte progenitor cells

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development.     

Analysis of lipocyte viability after liposuction

Free fat grafts from liposuction aspirate can be used as donor material for soft-tissue augmentation. The purpose of this study was to attempt to identify a subpopulation of adipose cells within liposuction aspirate with the greatest viability and, it is hoped, a greater chance for increased survival after transplantation. Liposuction samples were obtained from 20 individuals (16 women, four men; age range, 27 to 49 years). These samples were then centrifuged at 50 g. At 2-minute intervals, specimens from three different areas (superficial, middle, deep) were obtained from each specimen. After collagenase degradation, the specimens were stained with trypan blue, and the number of viable cells were counted. The bottom (deepest) layer consistently contained the highest number of viable cells after centrifugation: 250 percent more viable cells when compared with the top layer (p < 0.0001) and 140 percent more viable cells when compared with the middle layer (p < 0.0002). Centrifugation beyond 2 minutes did not increase the number or proportion of viable adipocytes. When using aspirated fat from liposuction for soft-tissue augmentation, centrifugation for 2 minutes at 50 g will stratify the adipocytes, with more viable cells being found at the deepest layer. Using only this bottom portion of the fat layer for transplantation will yield a fat graft with a greater number of viable adipocytes, potentially improving fat graft survival and decreased fat graft resorption.     

Cellular changes during cold acclimatation in adipose tissues

Cold exposure is a well-known physiological stimulus that activates the sympathetic nervous system and induces brown adipose tissue (BAT) hyperplasia. The effects of cold exposure or cold acclimatation have been extensively studied in interscapular BAT (IBAT). However, it has been recently shown that brown adipocytes are present in adipose deposits considered as white fat such as periovarian (PO) fat pad. We have investigated the kinetic of brown precursor recruitment in adipose tissues using DNA measurement and specific marker expression. In IBAT, cold exposure induces proliferation of precursor cells and differentiation into preadipocytes characterized by the expression of A2COL6, a marker specific to early steps of the differentiation process. A chronic stimulation of the tissue is necessary to observe the full effect. In PO fat pad, no proliferation can be detected, whereas differentiation of brown preadipocytes and maybe phenotypic conversion of white adipocytes seems to be promoted. In conclusion, these data demonstrated that 1) the same stimulus (cold exposure) does not induce the same response in terms of preadipocyte proliferation and differentiation in periovarian and brown adipose tissues, although both contain brown adipocytes, and 2) preadipocyte recruitment in adipose tissues after cold exposure depends on the predominant type of fat cells. © 1996 Wiley-Liss, Inc.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

The vascular endothelium of the adipose tissue gives rise to both white and brown fat cells

Adipose tissue expansion involves the enlargement of existing adipocytes, the formation of new cells from committed preadipocytes, and the coordinated development of the tissue vascular network. Here we find that murine endothelial cells (ECs) of classic white and brown fat depots share ultrastructural characteristics with pericytes, which are pluripotent and can potentially give rise to preadipocytes. Lineage tracing experiments using the VE-cadherin promoter reveal localization of reporter genes in ECs and also in preadipocytes and adipocytes of white and brown fat depots. Furthermore, capillary sprouts from human adipose tissue, which have predominantly EC characteristics, are found to express Zfp423, a recently identified marker of preadipocyte determination. In response to PPARγ activation, endothelial characteristics of sprouting cells are progressively lost, and cells form structurally and biochemically defined adipocytes. Together these data support an endothelial origin of murine and human adipocytes, suggesting a model for how adipogenesis and angiogenesis are coordinated during adipose tissue expansion.     

Pref-1 and ADSF/resistin: two secreted factors inhibiting adipose tissue development.

Adipose tissue is the source of a wide array of factors of great biological significance that are involved in many aspects of organism physiology, including appetite control and peripheral metabolism. Here, we describe two secreted factors from adipose tissue that inhibit adipogenesis. Pref-1 is a preadipocyte secreted factor synthesized as a transmembrane protein that undergoes proteolitic cleavage to generate two distinct soluble forms. In vitro assays have demonstrated that only the large soluble form of Pref-1 is biologically active and inhibits adipocyte differentiation. In vivo, mice lacking Pref-1 expression show accelerated fat deposition, perinatal mortality and growth retardation as well as distinct skeletal malformations, highlighting the importance of Pref-1 during mouse development in addition to its role in adipose tissue development. ADSF/resistin is secreted by adipocytes and inhibits adipose cells differentiation in vitro. Its function is still unclear, but its expression and high circulating levels have been associated with an impairment of insulin action. The findings show that Pref-1 and possibly ADSF/resistin secretion control fat cell differentiation and adipose tissue development.     

Apoptotic pathways in adipose tissue

To treat the ever growing number of obese patients, reduction of adipocyte number by apoptosis may complement other therapeutic options. On the other hand in free fat grafts, apoptosis along with necrosis is responsible for long term volume reduction. To ensure successful soft tissue reconstruction it is mandatory to keep apoptosis on a low level in adipocytes, adipose-derived stromal cells and others cells of the fat graft. Apoptotic pathways have been sufficiently studied in various tissues, but the knowledge about apoptotic pathways in adipocytes is surprisingly scarce. Current knowledge about apoptotic pathways in adipose tissue is elaborately reflected in this review as well as the association of cancer with obesity. Possibilities to induce and reduce adipose tissue apoptosis in animal models are discussed as well as clinical implications of fat cell apoptosis. Mechanisms of apoptosis induction have been studied in animal models and suggest that a tight control of apoptosis induction is necessary because otherwise detrimental metabolic effects of fat mass loss will occur that may mimic lipodystrophic diseases. At present, targeted induction of adipocyte apoptosis appears to be of some concern related to increased blood lipid concentrations, ectopic lipid storage and other detrimental metabolic effects. Treatment of autologous adipocytes used for lipofilling procedures with appropriate substances may result in more satisfactory long-term outcomes as well as stimulation of stem cell differentiation in a strictly local manner.     

Androgens and body fat distribution

An important sex difference in body fat distribution is generally observed. Men are usually characterized by the android type of obesity, with accumulation of fat in the abdominal region, whereas women often display the gynoid type of obesity, with a greater proportion of their body fat in the gluteal-femoral region. Accordingly, the amount of fat located inside the abdominal cavity (intra-abdominal or visceral adipose tissue) is twice as high in men compared to women. This sex difference has been shown to explain a major portion of the differing metabolic profiles and cardiovascular disease risk in men and women. Association studies have shown that circulating androgens are negatively associated with intra-abdominal fat accumulation in men, which explains an important portion of the link between low androgens and features of the metabolic syndrome. In women, the low circulating sex hormone-binding globulin (SHBG) levels found in abdominal obesity may indirectly indicate that elevated free androgens are related to increased visceral fat accumulation. However, data on non SHBG-bound and total androgens are not unanimous and difficult to interpret for total androgens. These studies focusing on plasma levels of sex hormones indirectly suggest that androgens may alter adipose tissue mass in a depot-specific manner. This could occur through site-specific modulation of preadipocyte proliferation and/or differentiation as well as lipid synthesis and/or lipolysis in mature adipocytes. Recent results on the effects of androgens in cultured adipocytes and adipose tissue have been inconsistent, but may indicate decreased adipogenesis and increased lipolysis upon androgen treatment. Finally, adipose tissue has been shown to express several steroidogenic and steroid-inactivating enzymes. Their mere presence in fat indirectly supports the notion of a highly complex enzymatic system modulating steroid action on a local basis. Recent data obtained in both men and women suggest that enzymes from the aldoketoreductase 1C family are very active and may be important modulators of androgen action in adipose tissue.     

脂肪细胞的分化及调控

越来越多的研究结果表明脂肪组织不仅仅是被动的能量储存器官 ,而且是能够分泌多种激素类物质的内分泌器官 ;脂肪细胞分化及其调控失常与人类多种疾病如肥胖症、糖尿病、脂肪肝、高脂血症及乳腺癌等密切相关。对脂肪细胞分化机制及其调控的研究 ,不但对于探讨上述重大生命和疾病过程具有重要理论意义 ,而且对于上述疾病的预防与治疗 ,特别是对于在细胞和分子水平上筛选针对上述疾病的药物 ,也具有实际意义。本文从脂肪细胞的起源、前脂肪细胞向脂肪细胞的分化过程、脂肪细胞分化的调控 ,以及对脂肪细胞分化研究应注意的问题等进行了综述 ,以期对脂肪细胞分化及其调控进行全面总结     

Insulin-like growth factor I mediates high-fat diet-induced adipogenesis in Osborne-Mendel rats

To determine if high-fat (HF) diet-induced changes in adipose tissue cellularity are associated with the presence of paracrine growth factor(s) that alter preadipocyte proliferation, Osborne-Mendel rats were fed either a HF (76% energy) or a low-fat (LF, 12% energy) diet for 85 days. HF-fed rats had greater (P < 0.05) fat pad size, total fat cell number, number of small (30-70 microm) and large (80-140 microm) adipocytes, and percentage of 100- to 140-microm adipocytes compared with LF-fed rats. Preadipocytes in primary cell culture treated with inguinal adipose tissue conditioned medium (ATCM) prepared from HF-fed rats had greater (P < 0.05) proliferation compared with cultures treated with ATCM from LF-fed rats. Proliferative capacity of ATCM prepared from HF-fed rats was attenuated after the stripping of the medium of insulin-like growth factor I using an immunomagnetic bead separation system. These data are consistent with the concept that insulin-like growth factor I is involved in the paracrine regulation of adipogenesis.     

Level of Preadipocyte Growth Factor in Rat Adipose Tissue which Specifically Permits the Proliferation of Preadipocytes Is Affected by Restricted Energy Intake

We previously reported the presence of a protein growth factor in rat adipose tissue which specifically permits the proliferation of 3T3-L1 and Obl771 preadipocytes [Biochem. Biophys. Res. Commun. 1990;171:905–912, ref. 1] and which is hereinafter referred to as PAGF (preadipocyte growth factor). In this study, the effects of long-term restricted energy intake on the PAGF activity in rat epididymal and perirenal adipose tissue toward 3T3-L1 preadipocytes were investigated. When rats were subjected to restricted energy intake for three weeks, PAGF activity increased with energy intake. The body weight, epididymal and perirenal fat depot weights and glycerol 3-phosphate dehydrogenase activity also increased with the energy intake, whereas the lactate dehydrogenase activity remained almost constant in all energy intake groups. These results suggest that the PAGF in fat depots functions in response to energy intake and contributes to the de novo formation of adipocytes and the growth of adipose tissue. This factor may provide a useful tool for further elucidation of the relationship between energy storage in adipose tissue and adipose tissue development.     

Increased matrix metalloproteinase-3 mRNA expression in visceral fat in mice implanted with cultured preadipocytes

Using preadipocyte implantation methods, we recently demonstrated that adipocytes in the visceral area change their function, as the expression of tumor necrosis factor-alpha (TNF-alpha) increases, thereby causing insulin resistance. In order to clarify the mechanism for changes in the function of adipocytes in visceral area, we examined the mRNA expression profiles in visceral fat tissue specimens. Four weeks after cell implantation, we performed a microarray analysis using the RNA of fat tissue specimens implanted either with 3T3-L1 cells or PBS alone. Sixty-three genes were thus isolated and the expression of matrix metalloproteinase-3 (MMP-3) mRNA was found to dramatically increase in the fat tissue specimens. The neutralization of MMP-3 protein inhibited adipogenesis and the free fatty acid-induced TNF-alpha secretion in 3T3-L1 adipocytes. These results suggest a potential role of MMP-3, which promotes the TNF-alpha secretion, thus contributing to the disturbance of the functions in the adipocytes which accumulate in the visceral area.