Preadipocyte Apoptosis Research Articles

Pentoxifylline Induces Lipolysis and Apoptosis of Human Preadipocytes, Keratinocytes and Fibroblasts In Vitro

Pentoxifylline (PTX) has been used for the local reduction of fat tissue in the clinical setting. However, its safety and efficacy have not been proven. The aim of this study was to evaluate the effects of PTX on cell lines established from fat tissue. Newly cultured human preadipocytes and adipocytes from subcutaneous abdominal fat in addition to purchased human lung fibroblasts and keratinocytes were treated with PTX at different concentrations. Cell viability was determined using the Cell counting kit (CCK)-8 assay and lipolysis was evaluated using an Elisa kit. DNA fragmentation, Western blot analysis, Hoechst and Propidium Iodide (PI) staining and fluorescence activated cell scanning analysis were performed to confirm apoptosis. The viability of adipocytes, preadipocytes, keratinocytes and fibroblasts was markedly decreased at concentrations of PTX above 20 mM. Apoptosis was induced at concentrations of PTX over 40 mM in all cell lines. Lipolysis was increased by 60% at concentrations of PTX of 20 mM compared to the control. In conclusion, the results of this study showed that 20 mM of PTX induced lipolysis. At concentrations over 20 mM, PTX reduced the viability of all cells studied including: adipocytes, preadipocytes, fibroblasts and keratinocytes, in a non-specific manner.

Identification of a novel proapoptotic function of resveratrol in fat cells: SIRT1‐independent sensitization to TRAIL‐induced apoptosis

The phytochemical resveratrol has recently gained attention for its protection against metabolic disease and for extension of life span, which have been linked to its metabolic effects and SIRT1 activation in fat cells. However, little is known about the effect of resveratrol on fat cell apoptosis. Here, we identify a novel, SIRT1-independent mechanism by which resveratrol regulates fat cell numbers. We demonstrate for the first time that resveratrol enhances TNF-related apoptosis-inducing ligand (TRAIL)- or CD95-induced apoptosis of human preadipocytes in a highly synergistic manner (EC(50) at 72 h: resveratrol, >300 microM; TRAIL, >100 ng/ml; combination: 30 microM resveratrol and 10 ng/ml TRAIL, combination index 0.4). Similar results in primary human preadipocytes prepared from subcutaneous white adipose tissue and mature human adipocytes underline the relevance to human physiology. Mechanistic studies reveal that resveratrol inhibits PI3K-driven phosphorylation of Akt, leading to increased Bax activation, loss of mitochondrial membrane potential, cytochrome c release, and caspase-dependent apoptosis. The synergistic interaction of resveratrol and TRAIL depends on the intrinsic apoptosis pathway and caspases, since Bcl-2 overexpression and the caspase inhibitor zVAD.fmk inhibit apoptosis, whereas knockdown of SIRT1 by RNA interference has no effect. The discovery of this novel activity of resveratrol significantly advances the knowledge of fat tissue regulation by resveratrol and has important implications for its use in metabolic and age-related diseases.

Serum-borne factors in cancer patients with advanced cachexia: influence on adipose cells

Background: The clinical syndrome cancer cachexia is recognized by a considerable weight loss being out of proportion to any reduction in energy intake. The underlying mechanisms are not completely known, but the marked weight loss is attributable to depletion of adipose tissue as well as skeletal muscle mass. Enhanced lipolysis in adipocytes, apoptosis of preadipocytes may be important for loss of adipose tissue. Results: Sera from cachectic cancer patients induced apoptosis in cultured human preadipocytes at a higher rate than sera from non-cachectic cancer patients (control group). There was a tendency towards increased mRNA levels of the pro-apoptotic Bcl-2 gene Bax after incubation of preadipocytes with cachectic sera. Moreover, the mRNA levels of anti-apoptotic Bcl-XL and pro-apoptotic Bcl-XS were increased and decreased, respectively, as compared to incubation with control sera. However, lipolysis was not enhanced in cultured human adipocytes after incubation with sera from cachectic cancer patients as compared to non-cachectic cancer patients. Methods: Serum samples from cachectic cancer patients (n=8) and non-cachectic cancer patients (n=6) were collected. Human SGBS (Simpson-Golabi-Behmel syndrome) preadipocytes and differentiated adipocytes were incubated in the presence of serum from cachectic and non-cachectic (control) cancer patients. Induction of apoptosis and necrosis was examined by cell staining with Hoechst 342 (HO342) and propidium iodide (PI), respectively. Expression of pro- and anti-apoptotic Bcl-2 genes was measured by quantitative RT-PCR. Lipolysis was monitored by measuring the release of radiolabeled fatty acids. Conclusion: Our in vitro data suggest that apoptosis of preadipocytes can be increased by serum-borne factors in cancer cachexia. Death or survival of preadipocytes may depend on the balance of pro- and anti-apoptotic mediators. Further studies of patients with cancer cachexia will be needed to reveal if the disease involves loss of adipose tissue due to apoptosis of preadipocytes. We could not show that serum-borne factors associated with cachexia have a major impact on lipolysis in cultured human adipocytes. Adipobiology 2009; 1: 57-66.

Effect of retinoid X receptor α (RXRα) on apoptosis in primary cultured porcine pre-adipocytes

AbstractUsing semi-quantitative reverse transcriptase polymerase chain reaction (semi-qRT-PCR), cell transfection, Hoechst 33342, acridine orange (AO) and flow cytometry methods, apoptotic changes in porcine pre-adipocytes treated with 9-cis retinoic acid (9-cisRA)–retinoid X receptor α (RXRα) ligand, transfected with pEnhanced green fluorescent proteins C2–RXRα (pEGFPC2–RXRα) or RXRα–small interfering RNA (RXRα–siRNA), respectively, were investigated. Observations of morphologic changes showed changes in cell structure, cellular shrinkage and condensation of chromatin, pyknosis of the nucleus into a compact body, complete fragmentation within the nuclear membrane, continuous budding and shedding, and, finally conversion of the cell into several apoptotic bodies wrapped in membranes of different sizes. Compared with the control group, the apoptotic rate was significantly lowered in the 10 nmol/l 9-cisRA-treated group, and significantly higher in the 10 μmol/l 9-cisRA-treated group (both P<0.05). Gene expression of RXRα was significantly up-regulated and down-regulated when cultured pre-adipocytes were transfected with pEGFPC2–RXRα or RXRα–siRNA (P<0.05), respectively; the apoptotic rate decreased in the pEGFPC2–RXRα group and significantly increased in the RXRα–siRNA group when compared with a control group (P<0.05). This implies that RXRα inhibits the apoptosis of porcine pre-adipocytes.

Mitochondrial dysfunction is induced by the overexpression of UCP4 in 3T3-L1 adipocytes

Uncoupling proteins (UCPs) belong to a superfamily of mitochondrial transporters that uncouple ATP synthesis from electron transport. We have previously shown that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. Overexpression of UCP4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes. In this work, we further characterized the effect of UCP4 on mitochondrial function in mature 3T3-L1 adipocytes. Transmission electron microscopy (TEM) showed that adipocytes overexpressing UCP4 displayed condensed mitochondria with twisted, condensed, and unclear cristae. Moreover, the loss of the mitochondrial membrane potential and intramitochondrial calcium was found. The adipocytes overexpressing UCP4 also showed decreased mitochondrial copy number (mtDNA) and lower mRNA expression of key factors in mitochondrial biogenesis, including PGC-1alpha and mtTFA. NRF-1 and ERRbeta levels were down-regulated, while NRF-2 levels were upregulated. In addition, UCP4 overexpression impaired mitochondrial fusion and fission, as indicated by decreased mitofusin mfn1, mfn2, and mitofission DRP1. When it came to total adipocytes, the UCP4 overexpressing adipocytes showed higher production reactive oxygen species and diminished levels of intracellular ATP. Furthermore, overexpression of UCP4 brought about impaired insulin sensitivity in adipocytes. UCP4 plays an important role in mitochondrial function and adipocyte insulin resistance. Its function deserves further attention.

Chlorella Methanol Extract Reduces Lipid Accumulation in and Increases the Number of Apoptotic 3T3‐L1 Cells

Obesity is a fast-growing problem that is reaching pandemic proportions. Chlorella has many biological merits for promoting health, including detoxification, boosting the immune system, and even reversing cancer. In this study, we found that methanol extract of Chlorella reduces lipid accumulation in 3T3-L1 adipocytes. It has been postulated that these antiobesity effects could be a result of reducing adipogenesis. First, the MTT assay indicated that Chlorella significantly inhibited cell growth of 3T3-L1 preadipocytes. The accumulation of triacylglycerol in 3T3-L1 adipocytes decreased in cells treated with Chlorella versus those in untreated cells by Oil Red O staining. In parallel, Chlorella showed a significant dose-dependent increase in lactate dehydrogenase activity in culture medium of differentiated 3T3-L1 adipocytes. Second, we investigated the effects of Chlorella on the induction of apoptosis by fluorescence-activated cell sorting analysis. Chlorella showed that apoptotic cells increased in a time- and dose-dependent manner in cell apoptosis analysis by propidium iodide (PI) staining. Treatment with Chlorella decreased the number of normal cells and increased the number of apoptotic cells in a dose-dependent manner in annexin V-fluorescein isothiocyanate/PI double staining. Therefore, Chlorella is expected to efficiently reduce adipogenesis in 3T3-L1 adipocytes and to induce apoptosis in 3T3-L1 preadipocytes.

Preadipocyte apoptosis is prevented by macrophage-conditioned medium in a PDGF-dependent manner

Obesity is associated with macrophage accumulation and inflammation in adipose tissue. Macrophage-secreted factors have been reported to inhibit the differentiation of preadipocytes into adipocytes and to modulate adipogenic extracellular matrix gene expression. To enlarge our understanding of macrophages and the scope of their interactions with preadipocytes, we investigated their effect on preadipocyte survival. Acute exposure of 3T3-L1 preadipocytes to J774A.1 macrophage-conditioned medium (MacCM) stimulated platelet-derived growth factor receptor (PDGFR) tyrosine phosphorylation by 4.1-fold. There were significant increases in the phosphocontent of downstream PDGFR targets Akt and ERK1/2 (5.3-fold and 2.4-fold, respectively) that were inhibited by PDGF immunoneutralization or by the selective PDGFR inhibitor imatinib. Serum-free J774A.1-MacCM or RAW264.7-MacCM completely prevented 3T3-L1 preadipocyte apoptosis normally induced by serum deprivation. Addition of PDGF alone to serum-free control medium was sufficient to prevent 3T3-L1 preadipocyte apoptosis. Inhibition of PDGFR activation by MacCM, either by addition of imatinib or by PDGF immunodepletion of MacCM, effectively disrupted the prosurvival effect. In summary, our data indicate that MacCM promotes preadipocyte survival in a PDGF-dependent manner.

SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.

Ajoene exerts potent effects in 3T3‐L1 adipocytes by inhibiting adipogenesis and inducing apoptosis

This paper describes effects of several sulfur-containing compounds from garlic on the cell viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. In both preadipocytes and mature adipocytes, 100 and 200 microM ajoene significantly decreased cell viability and increased apoptosis. The effect on apoptosis was further confirmed with Hoechst staining. In contrast, diallyl sulfide, diallyl disulfide, diallyl trisulfide, deoxyalliin, and allyl methyl sulfide had no significant effect on cell viability or apoptosis in either preadipocytes or mature adipocytes. In maturing preadipocytes ajoene significantly decreased lipid accumulation in a dose-dependent manner and these results were further confirmed by a decrease in lipid droplet number and lipid content through Oil Red O staining. There was no significant change in lipid accumulation in maturing preadipocytes treated with other garlic derivatives. Thus, despite the same source of origin, garlic, ajoene was the only one with potent effects on cell viability, apoptosis and adipogenesis in 3T3-L1 adipocytes.

LYRM1, a novel gene promotes proliferation and inhibits apoptosis of preadipocytes

To characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects. RT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1 expression is widely distributed, with the highest levels of expression occurring in adipose tissue. A fusion protein of LYRM1 and green fluorescent protein as well as western blot analysis were used to identify the subcellular localization of LYRM1 in the nucleus. Based on Oil red O staining and the expression profile of specific differentiation markers, ectopic LYRM1 expression was not found to significantly affect adipogenesis. MTT assays and cell cycle analysis showed that LYRM1 promotes preadipocyte proliferation, and data from annexin V-FITC and caspase-3 activity assays further determined that LYRM1 can inhibit apoptosis of preadipocytes. By increasing cell proliferation and lowering the rate of apoptosis, LYRM1 has the potential to modulate the size of the preadipocyte pool and influence adipose tissue homeostasis.

Naringenin and Hesperetin Induce Growth Arrest, Apoptosis, and Cytoplasmic Fat Deposit in Human Preadipocytes

Citrus flavonoids are reported to be promising bioactive compounds against hyperlipidemia and lipid biosynthesis. However, the mechanism of the lipid lowering effect by flavonoids remains unknown. The present study examines the effect of some flavanones on the adipocytic conversion of the human preadipocyte cell line, AML-I. Among four structure-related flavanones including naringenin, naringenin-7-rhamnoglucoside (naringin), hesperetin, and hesperetin-7-rhamnoglucoside (hesperidin), the aglycones such as naringenin and hesperetin exhibited the growth arrest of AML-I cells. When the cells were examined by Annexin V-FITC staining method, it was noticed that growth arrest was induced by apoptotic cell death. In the study of apoptosis-related protein in the naringenin-treated cells, anti-apoptotic proteins such as p-Akt, NF-kappaB, and Bcl-2 were decreased, and pro-apoptotic protein Bad was accumulated by Western blot analysis. Interestingly, exposure of AML-I cells to naringenin or hesperetin during short-term cultures increased cytoplasmic lipid droplets by Sudan Black B staining. Furthermore, expression of fatty acid synthase (FAS) and peroxisome proliferator activated receptor (PPAR)-gamma was enhanced in naringenin-treated cells. These data suggest that apoptosis by flavanones does not inhibit the adipocytic conversion of AML-I preadipocytes. The result also indicates that adipocyte may not be a direct target for the lipid-lowering activity of the flavanones.

Activation of Liver X Receptors Prevents Statin-induced Death of 3T3-L1 Preadipocytes

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.

Gallic Acid Induces Apoptosis in 3T3-L1 Pre-adipocytes via a Fas- and Mitochondrial-Mediated Pathway

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound. Our previous studies have shown that some phenolic acids such as gallic acid inhibit cell growth and induce apoptosis in 3T3-L1 pre-adipocytes. However, the molecular mechanism of gallic acid in the induction of cell apoptosis is still unclear. In this study, we investigated the effect of gallic acid on the apoptotic pathway in 3T3-L1 pre-adipocytes. Western blot data revealed that gallic acid stimulated an increase in the protein expression of Fas, FasL, and p53. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was changed by gallic acid treatment. Gallic acid released mitochondrial cytochrome c into the cytosol and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase. Pretreatment with a general caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) prevented gallic acid from inhibiting cell viability in 3T3-L1 pre-adipocytes. The data also indicated that treatment with gallic acid inhibited histone deacetylase activity in 3T3-L1 pre-adipocytes. These results demonstrate that gallic acid induces apoptosis in 3T3-L1 pre-adipocytes through the Fas and mitochondrial pathway. The induction of cell apoptosis by gallic acid may prove to be a pivotal mechanism for decreased pre-adipocyte proliferation.

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

Although fatty acids enhance preadipocyte differentiation in the presence of adequate hormone cocktails, little is known regarding their effects in the absence of these hormones. We have now shown that palmitate, a common long-chain saturated fatty acid, induced apoptosis in both mouse 3T3-L1 and rat primary preadipocytes grown in a normal serum-containing medium. Treatment of preadipocytes with palmitate induced multiple endoplasmic reticulum (ER) stress responses, evidenced by increased protein content of CHOP and GRP78 and splicing of XBP-1 mRNA, as well as altered phosphorylation of eIF2alpha and increased phosphorylation of JNK and Erk1/2. Intriguingly, palmitate induced an early activation of Akt but diminished both Akt activation and its protein mass after prolonged incubation (>6 h). In association with these changes, palmitate reduced expression of beta-catenin and its downstream target, c-Myc and cyclin D1, two key prosurvival proteins. Overexpression of constitutively active Akt did not block the apoptotic effect of palmitate. Cotreatment with unsaturated fatty acids (oleate, linoleate) or with LiCl (a glycogen synthase kinase-3beta inhibitor) attenuated the palmitate-induced apoptosis. Subsequent analysis suggested that the unsaturated fatty acids probably counteracted palmitate by reducing, not eliminating, ER stress, whereas LiCl probably improved viability by activating the Wnt signaling pathway. Cotreatment of palmitate with a standard adipogenic hormone cocktail also abolished the apoptotic effect and promoted adipocyte differentiation. Collectively, our results suggest that palmitate causes multiple cellular stresses that may lead to apoptosis in preadipocytes in the absence of adipogenic stimuli, highlighting the importance of exogenous hormones in directing cell fate in response to increased fatty acid influx.

Engineered Adipose Tissue Formation Enhanced by Basic Fibroblast Growth Factor and a Mechanically Stable Environment

Engineered adipose tissue can be used in plastic and reconstructive surgery to augment soft tissue lost due to mastectomy or lumpectomy. The three-dimensional space provided by a scaffold capable of withstanding in vivo compressive forces and neovascularization may promote engineered adipose tissue formation. The objective of this study was to determine whether voluminous adipose tissue can be engineered by combining a mechanically stable environment with basic fibroblast growth factor (bFGF). Mechanical support structures, fabricated from biodegradable synthetic polymers, were placed into subcutaneous pockets of athymic mice. Human preadipocytes, containing fibrin matrix, with (group 1) or without (group 2) bFGF were injected into the space created by the support structures. Additionally, human preadipocytes containing fibrin matrix, with (group 3) or without (group 4) bFGF, were injected into subcutaneous spaces without support structures. Six weeks after implantation, the original implant volume was approximately maintained in groups 1 and 2, whereas groups 3 and 4 showed significant implant shrinkage. Adipogenesis and angiogenesis were more extensive in the group 1 than any other group. The fraction of human nuclear antigen-positive adipocytes in the implant was highest in group 1. Mouse adipocyte-specific genes were also expressed in the implants, again at the highest levels in group 1. Implanted preadipocyte apoptosis was significantly reduced in the groups treated with bFGF (groups 1 and 3) as opposed to those without (groups 2 and 4). This study demonstrates that combining a mechanically stable environment with bFGF can promote voluminous adipose tissue regeneration. This adipogenesis was likely promoted by the mechanically stable three-dimensional space, enhanced neovascularization, implanted cell survival, and host adipogenic cell migration. The method described in this study could be useful to augment adipose tissue used in plastic and reconstructive surgery.

Conjugated Linoleic Acids Promote Human Fat Cell Apoptosis

Conjugated linoleic acids (CLAs) are conjugated dienoic isomers of linoleic acid. Some isomers have been shown to reduce fat mass in animal and cell culture models. However, controversial results were obtained in studies of supplementation of CLAs in human subjects. In order to get more insights into the direct effects of CLAs on human fat cells, we have studied the influence of cis-9, trans-11 CLA and trans-10, cis-12 CLA on the biology of human SGBS preadipocytes and adipocytes. Both CLA isomers equally inhibited the proliferation of preadipocytes in a dose-dependent manner. Continuous treatment with 1-10 microM trans-10, cis-12 CLA, and to a weaker extent cis-9, trans-11 CLA, inhibited accumulation of lipids during adipogenic differentiation. Treatment with higher doses of CLA induced apoptosis in preadipocytes, in differentiating cells, and adipocytes. The trans-10, cis-12 isomer had a higher apoptotic potency in adipocytes than cis-9, trans-11 CLA. Taken together, the treatment of human preadipocytes and adipocytes with physiological relevant concentrations of CLAs resulted in an impairment of proliferation and differentiation and induction of apoptosis. The trans-10, cis-12 isomer was more potent than the cis-9, trans-11 isomer. Further clinical studies are needed to evaluate the effects of CLAs on human fat mass and metabolism in vivo.

Effects of Capsaicin on Induction of Apoptosis and Inhibition of Adipogenesis in 3T3-L1 Cells

Currently, at the beginning of the 21st century, obesity has become the leading metabolic disease in the world. It is a serious health problem in industrialized countries. Previous research has suggested that decreased preadipocyte differentiation and proliferation and decreased lipogenesis are mechanisms to reduce obesity. In the present study, the effects of capsaicin on the induction of apoptosis and inhibition of lipid accumulation in 3T3-L1 preadipocytes and adipocytes were investigated. The results demonstrated that capsaicin decreased cell population growth of 3T3-L1 preadipocytes, assessed with the MTT assay. Flow cytometric analysis of 3T3-L1 preadipocytes exposed to capsaicin showed that apoptotic cells increased in a time- and dose-dependent manner. Treatment with capsaicin decreased the number of normal cells and increased the number of early apoptotic and late apoptotic cells in a dose-dependent manner. The treatment of cells with capsaicin caused the loss of mitochondria membrane potential (delta psi m). The induction of apoptosis in 3T3-L1 preadipocytes by capsaicin was mediated through the activation of caspase-3, Bax, and Bak, and then through the cleavage of PARP and the down-regulation of Bcl-2. Moreover, capsaicin significantly decreased the amount of intracellular triglycerides and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 adipocytes. Capsaicin also inhibited the expression of PPARgamma, C/EBPalpha, and leptin, but induced up-regulation of adiponectin at the protein level. These results demonstrate that capsaicin efficiently induces apoptosis and inhibits adipogenesis in 3T3-L1 preadipocytes and adipocytes.

Docosahexaenoic acid inhibits adipocyte differentiation and induces apoptosis in 3T3-L1 preadipocytes.

Docosahexaenoic acid (DHA, C22:6), a (n-3) fatty acid in fish oil, has been shown to decrease body fat and fat accumulation in rodents. We investigated the direct effect of DHA on cell growth, differentiation, apoptosis, and lipolysis using 3T3-L1 adipocytes. Cells were treated with 25-200 mumol/L DHA containing 0.2 mmol/L alpha-tocopherol or bovine serum albumin vehicle as a control. Proliferation of preconfluent preadipocytes was not affected by the DHA treatment. When added to postconfluent preadipocytes, all concentrations of DHA inhibited differentiation-associated mitotic clonal expansion (P < 0.01). Postconfluent preadipocytes demonstrated apoptosis after 48 h with 100 mumol/L DHA and after 24 and 48 h with 200 mumol/L DHA (P < 0.01). Differentiation was examined by Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity after DHA treatment for 6 d. DHA decreased mean droplet size and percent lipid area in a dose-dependent manner (P < 0.01). GPDH activity was also decreased by DHA treatment (P < 0.01). In fully differentiated adipocytes, DHA increased basal lipolysis compared with the control (P < 0.01). These results demonstrate that DHA may exert its antiobesity effect by inhibiting differentiation to adipocytes, inducing apoptosis in postconfluent preadipocytes and promoting lipolysis.

Committed subcutaneous preadipocytes are reduced in human obesity

The aim of this study was to test whether the availability of committed preadipocytes in abdominal and femoral subcutaneous adipose tissue varies with obesity and body fat distribution. Body composition, fat cell size, committed preadipocytes and macrophages were measured in subcutaneous abdominal and femoral adipose depots of 17 lean, 16 upper-body-obese (UBO) and 13 lower-body-obese (LBO) women. Preadipocytes and macrophages were identified by simultaneous staining with the respective markers aP2 and CD68. In a subset of samples we measured preadipocyte proliferation, differentiation and susceptibility to apoptosis. Abdominal adipocytes were smaller in lean than in obese women. Committed preadipocytes represented a greater fraction of stromovascular cells in lean than in obese women but were similar between UBO and LBO women (abdomen: approximately 30 +/- 3 vs approximately 17 +/- 2%; thigh: approximately 30 +/- 3 vs approximately 17 +/- 2%). Preliminary data suggested that preadipocyte kinetics were similar in LBO and lean women, whereas preadipocytes of UBO women differentiated less and were more susceptible to apoptotic stimuli. The fraction of stromovascular cells that were macrophages was greater in both depots in obese women (UBO and LBO) than in normal-weight women, but the difference was not statistically significant. The proportion of subcutaneous adipose tissue stromovascular cells that are committed preadipocytes is reduced with obesity. This could be due to greater recruitment of preadipocytes to adipogenesis or greater preadipocyte apoptosis, depending upon the obesity phenotype. These data are consistent with the concept that body fat distribution may be regulated partly through differences in adipogenesis.

Induction of cell apoptosis in 3T3‐L1 pre‐adipocytes by flavonoids is associated with their antioxidant activity

Obesity is biologically characterized at the cellular level by an increase in the number and size of adipocytes differentiated from fibroblastic pre-adipocytes in adipose tissue. In this study, we focused on the relationship between the influence of flavonoids on cell population growth and their antioxidant activity. The results showed that the inhibition of flavonoids (naringenin, rutin, hesperidin, resveratrol, naringin and quercetin) on 3T3-L1 pre-adipocytes was 28.3, 8.1, 11.1, 33.2, 5.6 and 71.5%, respectively. In oxygen radical absorbance capacity (ORAC) assay, quercetin had the highest ORAC(ROO) value among the six flavonoids tested. Apoptosis assays showed that quercetin increased apoptotic cells in time- and dose-dependent manner. Treatment of cells with quercetin decreased the mitochondrial membrane potential in the courses of time and dose. The cell apoptosis/necrosis assay showed that quercetin increased the number of apoptotic cells, but not necrotic cells. Quercetin treatment of cells caused a significant time- and dose-dependent increase in the caspase-3 activity. Western analysis indicated that treatment of quercetin markedly down-regulated PARP and Bcl-2 proteins, and activated caspase-3, Bax, and Bak proteins. These results indicate that quercetin efficiently inhibits cell population growth and induction of apoptosis in 3T3-L1 pre-adipocytes.