Introduction

The treatment of the ever growing number of obese patients is a life long challenge and specifically life style interventions and drug treatment, although often successful in the short run, have failed to convincingly demonstrate long term weight and health improvements. For some years, orlistat was available as the only weight loss drug that has been vigorously tested for efficacy and safety and with approval for long term treatment. Several other weight loss drugs had been withdrawn from marketing authorization due to unacceptable side effects. Recently, lorcaserin and phentermine/topiramate combinations have been approved for marketing authorization in the U. S V体育ios版. , but these drugs have yet to demonstrate an acceptable long-term safety profile. Bariatric surgery on the other hand has demonstrated long-term safety and efficacy with most procedures, but is definitely not the solution for any obese patient. Furthermore, even massive weight loss does not reduce the number of adipocytes, but only their size [1]. Considering these problems, reduction of adipocyte number by (induced) apoptosis may complement other therapeutic options [2].

Furthermore, the role of apoptosis is important when the potential of adipose tissue explants and liposuction material is considered for plastic surgery approaches. In free fat grafts, apoptosis along with necrosis are responsible for long term volume reduction [3]. Some drugs used in this context (e. g. local anesthetics) may reduce graft viability by causing preadipocity necrosis [4, 5]. With more than 90 % of the volume, adipocytes represent the major cell type in lipoaspirate material. About 1 % of lipoaspirate adipocytes show signs of necrosis and 20 % undergo apoptosis [6]. These findings demonstrate the importance of understanding the process of adipocyte apoptosis for successful fat grafting VSports最新版本. Eto et al. analysed human adipose-derived stromal cells (ASCs), adipocytes, and endothelial cells in vitro in response to ischemia. Within 24 h, adipocytes underwent apoptosis (annexin positive/propidium iodide negative) or necrosis (propidium iodide positive), and endothelial cells lost adherence to the culture dish rather early. In contrast, ASCs remained viable up to 72 h under ischemic conditions [7]. In summary, the goal would be to keep fat cell apoptosis on a low level to ensure successful soft tissue reconstruction when fat tissue transplants are employed. This is especially true when applying autologous lipofilling because a maximum survival of implanted fat cells and ASCs is necessary, as surviving fat cells and differentiated ASCs play a role in fat graft survival [7]. Apoptotic pathways have been sufficiently studied in various tissues, but the knowledge about apoptotic pathways in adipocytes is surprisingly scarce.

A general overview of apoptotic pathways (V体育官网)

Apoptosis as programmed cell death is a central mechanism of cell homeostasis V体育平台登录. This physiological process allows to delete selected cells in an efficient fashion [8]. A defined sequence of morphological and biochemical events appears during apoptosis such as membrane blebbing, shrinkage of the cell, aggregation of chromatin and the appearance of apoptotic bodies [8–10]. Definite cellular mechanisms mediating this process have not been clearly identified until now, but it is known that the molecular sequence of apoptosis is a cascade of enzymatic activation of proteins. The initiation of apoptosis is possible by many mechanisms and pathways and a number of important regulators have been identified [8]. The extrinsic pathway starts with special TNF-receptors, also called ‘death receptors’. These cell surface receptors, e. g. tumor necrosis factor receptor 1 (TNFR1) or Fas, bind specific ligands and start the apoptotic cascade [8]. Pro-caspase 8 molecules are assembled in the vincinity of the death receptors by binding to their adaptor proteins and start an autocatalytic process that activates a growing number of caspase 8 molecules which activate other caspases later on [8].

The intrinsic pathway contains proteins such as second mitochondria-derived activator of caspase (SMAC) or cytochrome c [11]. All pathways result in the activation of substrate specific cysteinproteases, also called caspases. They are the effector molecules of apoptosis and 14 different caspases have been identified as yet [12]. Caspases either work as effectors or initiators. The active subunits of caspases are called cleaved caspases. Intracellular occurrence of cleaved caspases delivers information about the activated apoptotic pathways. Mitochondrion-dependent and death receptor-dependent apoptotic pathways are known to activate the same effector caspases, but are starting with different initiator caspases. Caspase 8 is found in the extrinsic cell death pathway [13, 14]. Activated caspase 8 is able to cleave and activate downstream caspases such as caspase 3 and to trigger cytochrome c release [15]. Caspase 9 is important in the mitochondrion-induced intrinsic apoptosis pathway. Mitochondria seem to play an important role in the apoptotic cascade, generally by stopping energy metabolism and electron transport [8] VSports注册入口. Upon apoptotic stimulation, cytochrome c is released from mitochondria and activates caspase 9 by self-cleavage at Asp315 [16–18] or by binding apoptotic protease activating factor 1 (Apaf-1) [8]. Cleaved caspase 9 further processes other caspase members, including caspase 3 to initiate the apoptotic cascade [16–18].

Apoptotic pathways in adipose tissue

Immunofluorescence examination of rat fat tissue demonstrated that apoptotic pathways via mitochondria, endoplasmatic reticulum and death receptors were activated, and cleaved caspases 8, 9 and caspase 12 were detected. Cleaved caspase 3 as an effector of the common apoptotic pathway was detected as well [9]. In another animal study, depot specific response were demonstrated when rats were treated with oleoyl-estrone: whereas subcutaneous and retroperitoneal adipose tissue was resistant against oleoyl-estrone induced apoptosis, mesenteric adipose tissue responded with a strong induction of Bid, Bax, caspase 3 and caspase 8, and periovaric adipose tissue with a strong induction of Bax and caspase 3 [19] V体育官网入口.

Other regulators like Bcl-2 and Bcl-XL were found in adipose tissue. These act as cell death antagonists and prevent apoptosis and stabilize cell membranes VSports在线直播. Adipose differentiation may also be regulated by transactivation of adipose-specific genes [8]. Nuclear factor (NFκB) and peroxisome proliferators-activated receptor γ (PPARγ) have been described to play a role in an apoptotic signal transduction pathway in adipocytes [20]. Depending on the metabolic situation, PPARγ may play an pro-apoptotic or anti-apoptotic role [8]. In primary cultured subcutaneous human preadipocytes and adipocytes, several apoptotic and anti-apoptotic pathways have been studied [21]. Death receptors CD95, TNFR1 and TNF-related apoptosis-inducing ligand receptors 1 and 2 are all expressed on human adipose cells and are activated by ligands such as TNF-α. In these cells, IGF-1 represents an important hormone with antiapoptotic activity. Downregulation of FLIP (FLICE-like inhibitory protein) appears also to be involved in human fat cell apoptosis, in sensitizing cells to CD95-mediated apoptotic stimuli [22].

These studies clearly demonstrate that the apoptotic molecular machinery is present in rodent and human adipocytes and not different in general from other cell types. The question however is how apoptosis is induced under specific situations. Direct effects of macrophage accumulation and local adipose tissue inflammation on adipocytes may result in increased secretion of proapoptotic molecules from macrophages [23]. These in vitro findings were extended to adipose tissue explants from subcutaneous and visceral adipose tissue. A close correlation was found in these explants between CD11c expression as a marker of macrophage infiltration and the number of apoptotic mature adipocytes [23]. On the other hand, macrophage infiltration of adipose tissue is partly mediated by adipocyte necrosis and concomitant secretion of chemotactic molecules [24]. Considering those two aspects, the chicken or the egg causality dilemma is present when the relationship between adipocyte death and macrophage infiltration is considered and most likely both situations may occur in vivo V体育2025版.

Apoptosis and autophagy are upregulated in human visceral adipose tissue of patients with type 2 diabetes. Ghrelin plays a role in the control of apoptosis and autophagy in human adipocytes VSports. Ghrelin and ghrelin O-acyltransferase (GOAT) are produced in omental and subcutaneous adipose tissue. In human omental adipocytes acylated and desacyl ghrelin reduces TNF-α-induced activation of caspase-8 and caspase-3 and therefore apoptosis [25].

An unusual apoptotic stimulus was recently identified when the weight loss activities of conjugated linoleic acid isomers were investigated. Conjugated linoleic acid is recommended by some advertisers as dietary supplement to lower body weight. Direct treatment of human Simpson Golabi Behmel Syndrome preadipocytes (derived from a patient with SGBS) reduced their proliferation capacity, and decreased the amount of lipid storage during adipogenesis. Cells of all differentiation stages, preadipocytes, differentiating preadipocytes and adipocytes, responded with increased frequency of apoptosis, as assessed by hypodiploid DNA, to higher concentrations of conjugated linoleic acid in the culture medium [26].

Induced adipose tissue apoptosis in animal models (VSports)

The transgenic FAT-ATTAC mouse is a model carrying a myristoylated caspase 8-FKBP fusion protein. Treatment with a chemical dimerization compound activates caspase 8 and results in fat cell apoptosis. The FAT-ATTAC mouse model allows studying apoptosis in a development-specific manner as well as in diet-induced obesity models. The model is also reversible, because cessation of dimerizer treatment allows adipose tissue to regenerate [27]. Two weeks after apoptosis induction, mice showed elements of severe lipodystrophy, e.g. glucose intolerance, ectopic lipid storage, and strongly reduced adipokine plasma concentrations. On the other hand, when fat apoptosis was induced in obese mice, a significantly lower level of systemic inflammation was detected, demonstrating the role of accumulated adipose tissue for the chronic inflammation state induced by obesity [28]. Induction of lipodystrophic metabolic changes by targeted fat cell ablation is of course not a wanted treatment option in humans, but interestingly the ablation of adipocytes in a more modest model of obesity demonstrated that the subsequent infiltration of adipose tissue depots by macrophages was predominantly by anti-inflammatory M2 type macrophages. Thus, a modest degree of targeted adipocyte ablation may represent a future treatment option [29].

Histological evaluation of adipose tissue reveals a high degree of vascularisation: multiple capillaries are in close contact with every adipocyte, suggesting the importance of blood vessels for maintenance of the tissue mass [30]. Consequently, a nonspecific angiogenesis inhibitor prevented the development of obesity in mice [31]. Targeting existing blood vessels in white fat should result in adipose tissue ablation. Kolonin et al. analysed targeted delivery of a chimeric peptide containing a proapoptotic sequence to the adipose tissue vasculature of obese mice. They identified prohibitin as the vascular receptor for the peptide ligand in white fat tissue. Prohibitin is expressed in blood vessels of human white fat. Targeting a proapoptotic peptide to prohibitin in the adipose vasculature causes resorption of established white adipose tissue and normalization of metabolism [32].

Apoptosis signaling in adipocytes and cancer cells

Inhibition of apoptotic mechanisms results in initiation and progression of tumor cells during tumorigenesis. Defects in apoptotic signaling pathways have been detected in many cancer types. For example, Fas ligand is only expressed in a low level in normal cells, but upregulation of Fas ligand was found in many tumor tissues. Furthermore, downregulation or upregulation of caspases in tumor cells have been detected in several tumor tissues [33].

Doxorubicin, cisplatin, and sunitinib treatment of the liposarcoma cell line SW872 resulted in a dose-dependent inhibition of both liposarcoma and adipose-derived cell proliferation, whereas curcumin dose-dependently inhibited liposarcoma cells only, but did not affect adipose-derived cells. Caspase-3 and caspase-8 activity increased after curcumin treatment, whereas no changes in either Fas or FADD protein expression occurred after curcumin treatment. Wang et al. [34] concluded that curcumin-mediated ER stress via inhibition of SERCA2 activity caused increasing expressions of CHOP and its transcription target death receptor 5 (TRAIL-R2) that in turn stimulated caspase-3 and caspase-8 apoptotic pathways in SW872 cells in vitro and in vivo.

The efficacy of drug or radiation treatment partly relies on apoptosis induction in tumor cells. Consecutively, however, the development of treatment resistance represents a severe clinical problem and one key factor in the development of treatment resistance is the progressive inability of tumor cells to undergo apoptosis [35].

Epidemiologic studies suggest an association of cancer development with obesity, but the molecular mechanisms remain unknown. Cells from fat tissue may be recruited to tumor tissue and then contribute to survival of malignant cells and tumor growth. Zhang et al. [36] described a direct effect of excess white adipose tissue on the promotion of tumor growth. E.g. adipose stromal cells contain a pool of perivascular adipocyte progenitor cells that stimulated tumor growth when transplanted into mice. Zhang et al. compared circulating and tumor-infiltrating cell populations in lean and obese mice and demonstrated a six-fold increase of ASC frequency in the systemic circulation of mice with cancer. After recruitment into tumors, these ASCs were either incorporated into blood vessels as pericytes, or differentiated into adipocytes in an obesity-dependent manner. Thus, ASCs can be recruited from endogenous adipose tissue by tumor tissue to potentiate the supportive properties of the tumor microenvironment [36]. There is also evidence that multipotent MSCs from visceral adipose tissue depots promote endometrial tumor growth more potently than MSCs from subcutaneous adipose tissue depots [37]. Recent data implicate that systemic and paracrine factors secreted from adipose tissue promote cancer development and progression in obese patients. This adipocyte-cancer cell link is supported by local pro-tumorigenic effects of adipocytes in the microenvironment of at least some cancers types [38].

Clinical implications of fat cell apoptosis

Ritonavir as one of the older anti-HIV protease inhibitors induced apoptosis in a significant number of in vitro differentiated SGBS adipocytes which may explain the fat distribution changes and lipodystrophyic phenotypes observed with effective anti-HIV treatment [39]. In a case report on a young girl with autoimmune lipodystrophy the authors reported that regression of adipose tissue was associated with lymphohistiocytic inflammation and increased TNF-α concentrations. TNF-α and INF-γ stimulated CD95 expression and enhanced CD95-death inducing signalling complex formation in cultured subcutaneous adipocytes, thus suggesting a CD95-related pathway for the loss of adipocytes in this patient [40]. Public interest recently focused on resveratrol. If not from red wine, resveratrol may also be used as a dietary supplement in the form of capsules. In human subcutaneous primary fat cells and SGBS cells, resveratrol enhanced TRAIl and CD95-induced apoptotic events, most likely through increased Bax activation, loss of mitochondrial membrane potential, cytochrome c release and activation of caspases. These events were dependent on the intrinsic apoptotic pathway because Bcl-2 overexpression and caspase inhibitors were able to prevent resveratrol-induced apoptosis in human adipose cells [41]. Furthermore green tea is able to activate apoptosis signalling in adipocytes. Health-benefiting effects of green tea have been attributed to catechins like epigallocatechin gallate. Lee et al. demonstrated that experimental diet in mice supplemented with epigallocatechin-3-gallate (EGCG) results in reduction of various adipose tissue depots and weight loss in a dose-dependent manner. In epididymal white adipose tissue of EGCG-fed mice, mRNA levels of adipogenic genes (PPARγ, CEBPs, SREBP-1c, aP2, lipoprotein lipase and fatty acid synthase) were decreased and adipokines such as leptin, resistin and adipsin were also significantly reduced. Lee et al. [42] concluded that the effects of EGCG might be at least partially mediated via regulation of the expression of multiple genes involved in adipogenesis, lipolysis, fatty acid oxidation and thermogenesis in white adipose tissue.

Phosphorylated c-Jun NH2-terminal kinase (JNK) is a crucial mediator between metabolic stress and insulin resistance and apoptosis in insulin target tissues. In adipose tissue of patients with non-alcoholic fatty liver disease, protein kinase B (Akt) phosphorylation was significantly impaired compared to patients with simple steatosis. Total JNK decreased from steatosis to non-alcoholic fatty liver disease in muscle and adipose tissues. Thus, increased JNK phosphorylation may explain increased insulin resistance and adipose apoptosis in NASH, compared with simple steatosis. Apoptosis and insulin resistance increase with more severe non-alcoholic fatty liver disease stages in morbidly obese patients [43].

Conclusions

Human adipocytes and preadipocytes express numerous molecules of the intrinsic and extrinsic apoptotic pathways. First studies on depot differences suggest that probably visceral adipocytes are more prone to apoptosis than subcutaneous adipocytes, but this notion is far from being conclusive due to the paucity of experimental data. Whereas obesity is associated with a certain degree of apoptosis in adipose tissues that is related to the grade of local inflammation, that mechanism is not sufficient to control body fat mass. Mechanisms of apoptosis induction have been studied in mouse 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. Thus 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. This notion has become even more evident since the reevaluation of brown adipose tissue in humans and its possible pathophysiological role in the development or possibly also treatment of obesity [44]. The specific susceptibility of brown adipocytes or the so called brite or beige adipocytes (a mixed phenotype between white and brown adipocytes) to apoptotic stimuli has not been studied so far. Thus, induced apoptosis may also lead to brown adipose tissue dysfunction as another unwanted metabolic effect.

On the other hand, the prevention of fat cell apoptosis in fat flap transplants or autologous lipofilling procedures has not been studied in sufficient detail yet. A confirmed antiapoptotic signal in fat cells is IGF-1. Erythropoeitin as well has been reported to have a positive effect on fat cell survival [45]. Treatment of autologous adipocytes used for lipofilling procedures with appropriate substances may result in more satisfactory long-term outcomes. Lipoaspirate material is very useful to treat soft-tissue defects. Adipocytes are the major cell type in lipoaspirate material that make up more than 90 % of the graft volume. In clinical fat transplantation, numerous transplanted adipocytes die and are replaced by new ones [7]. Adipose-derived stem cells constitute a minor fraction and make up <1 % of the graft volume, but they have great potential for regeneration that may compensate graft volume loss [6]. Thus in the future, a more promising approach with respect to graft survival and function may be to stimulate stem cell differentiation in a strictly local manner.