Enhanced pro-apoptotic and anti-adipogenic effects of genistein plus guggulsterone in 3T3-L1 adipocytes

Genistein (G), an isoflavone, and guggulsterone (GS), the active substance in guggulipid, have been reported to possess therapeutic effects for obesity. In the present study, we investigated the effects of combinations of G plus GS on apoptosis and adipogenesis in 3T3-L1 cells. In mature adipocytes, G and GS individually caused apoptosis, but combination of G plus GS significantly increased apoptosis, more than either compound alone. Furthermore, G plus GS caused a greater increase in procaspase-3 cleavage, Bax expression, cytochrome c release, and proteolytic cleavage of PARP than either compound alone. In maturing preadipocytes G and GS each suppressed lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. These results suggest that combination of genistein and guggulsterone may exert anti-obesity effects by inhibiting adipogenesis and inducing apoptosis in adipocytes.     

Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3‐L1 Adipocytes

Objective: Green tea catechins have been shown to promote loss of body fat and to inhibit growth of many cancer cell types by inducing apoptosis. The objective of this study was to determine whether epigallocatechin gallate (EGCG), the primary green tea catechin, could act directly on adipocytes to inhibit adipogenesis and induce apoptosis. Research Methods and Procedures: Mouse 3T3‐L1 preadipocytes and mature adipocytes were used. To test the effect of EGCG on viability, cells were incubated for 3, 6, 12, or 24 hours with 0, 50, 100, or 200 μM EGCG. Viability was quantitated by MTS assay. To determine the effect of EGCG on apoptosis, adipocytes were incubated for 24 hours with 0 to 200 μM EGCG, then stained with annexin V and propidium iodide and analyzed by laser scanning cytometry. Both preadipocytes and adipocytes were also analyzed for apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labeling assay. To determine the effect of EGCG on adipogenesis, maturing preadipocytes were incubated during the 6‐day induction period with 0 to 200 μM EGCG, then stained with Oil‐Red‐O and analyzed for lipid content. Results: EGCG had no effect on either viability or apoptosis of preconfluent preadipocytes. EGCG also did not affect viability of mature adipocytes; however, EGCG increased apoptosis in mature adipocytes, as demonstrated by both laser scanning cytometry and terminal deoxynucleotidyl transferase dUTP nick‐end labeling assays. Furthermore, EGCG dose‐dependently inhibited lipid accumulation in maturing preadipocytes. Discussion: These results demonstrate that EGCG can act directly to inhibit differentiation of preadipocytes and to induce apoptosis of mature adipocytes and, thus, could be an important adjunct in the treatment of obesity.     

Oroxylin A,a constituent of Oroxylum indicum inhibits adipogenesis and induces apoptosis in 3T3-L1 cells

Oroxylin A (OA) is a flavonoid found in Oroxylum indicum, a medicinal plant with multiple biological activities. This study was taken up to investigate the effect of OA, on adipogenesis, lipolysis and apoptosis in 3T3 L1 cells. Pre-adipocytes were treated with 10–40 μM OA on various days of adipogenesis treatment schedule. Mature adipocytes were treated with OA for lipolysis and apoptosis studies. In maturing pre-adipocytes, 10 μM OA suppressed intracellular lipid accumulation by 42.19% which was confirmed by lipidTox imaging of cells. In addition, OA decreased the nuclear translocation of PPARγ and mRNA expression of its downstream genes (FAS and LPL) along with adiponectin secretion. In mature adipocytes, 40 μM of OA decreased cell viability by 30% of control. Annexin V/PI staining showed induction of apoptosis which was further confirmed by enhanced levels of pro-apoptotic proteins Bax, cyt c, AIF and chromatin condensation. OA enhanced TNF-α secretion, lipolysis and decreased Akt phosphorylation in mature adipocytes. Findings suggest that OA possibly exerts its anti-obesity effect by affecting adipocyte life cycle at critical points of differentiation and maturity. When we compared the potency of OA with non-methoxylated flavonoids morin, naringenin and kaempferol on adipocyte life cycle OA was far more potent. Thus, study clearly indicates a new role for oroxylin A as regulator of adipocyte life cycle. In addition, study also suggested a specific role of methoxylated group in exerting lipolysis and cytotoxic effects in mature adipocytes.     

Enhanced effects of 1,25(OH)(2)D(3) plus genistein on adipogenesis and apoptosis in 3T3-L1 adipocytes

Objective: To investigate the ability of 1,25(OH)₂D₃ (D) and genistein (G), alone and in combination, to inhibit adipogenesis and induce apoptosis in 3T3‐L1 adipocytes. Methods and Procedures: 3T3‐L1 preadipocytes and mature adipocytes were incubated with various concentrations of D and G, alone and in combination, for 48 h. Viability was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. Post‐confluent preadipocytes were incubated with D and G for up to 6 days during adipogenesis and lipid content was quantified by Nile Red dye; apoptosis was quantified by measurement of single‐stranded DNA. Expression of adipocyte‐specific proteins and VDR was analyzed by western blotting. Results: Combining D and G did not cause an enhanced effect on cell viability in either preadipocytes or mature adipocytes. In maturing preadipocytes, D at 0.5 nmol/l (D0.5) increased apoptosis by 47 ± 10.25% (P < 0.05) and inhibited lipid accumulation by 28 ± 10% (P < 0.001), while G at 25 μmol/l (G25) had no significant effect. However, D+G caused an enhanced apoptosis by 136 ± 12.6% (P < 0.001) and enhanced inhibition of lipid accumulation by 82.46 ± 2.95% (P < 0.001). Similarly, D0.5 alone decreased adipose‐specific gene 422 (aP2) expression to 34.2 ± 2.3% and increased VDR expression levels by 41.8 ± 11% (P < 0.001), but G25 showed no effect. However, D0.5+G25 decreased aP2 expression to 52 ± 4.2% (P < 0.05) and increased VDR expression levels by 131 ± 14.5% (P < 0.0001). Discussion: These findings suggest that combining 1,25(OH)₂D₃ with genistein results in an enhanced inhibition of lipid accumulation and induction of apoptosis in maturing 3T3‐L1 preadipocytes.     

Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase

Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovascular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure. We have investigated the effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein (20-200muM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG, and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further implying AMPK as a prime target of obesity control.     

Hedgehog信号通路与脂肪细胞发育育

TGFβ、Wnt、FGF和Hedgehog（Hh)等信号通路是参与胚胎发育的关键信号通路.从果蝇到人类,Hh信号通路广泛存在并高度保守,在多种器官的发育过程中发挥重要作用. 脂肪细胞发育的过程包括多潜能干细胞向前脂肪细胞定向和脂肪细胞终末分化两个阶段.近年来,Hh信号通路在脂肪细胞发育过程中的作用逐渐成为研究热点.越来越多的研究表明,Hh信号通路抑制脂肪细胞发育.本文将对Hh信号通路抑制脂肪细胞发育的作用以及其发挥作用的阶段进行综述,并分析将该信号通路作为靶点治疗肥胖症及相关疾病的可行性.     

LRP1 Receptor Controls Adipogenesis and Is Up-Regulated In Human and Mouse Obese Adipose Tissue

The cell surface low-density lipoprotein receptor-related protein 1, LRP1, plays a major role in lipid metabolism. The question that remains open concerns the function of LRP1 in adipogenesis. Here, we show that LRP1 is highly expressed in murine preadipocytes as well as in primary culture of human adipocytes. Moreover, LRP1 remains abundantly synthesised during mouse and human adipocyte differentiation. We demonstrate that LRP1 silencing in 3T3F442A murine preadipocytes significantly inhibits the expression of PPARγ, HSL and aP2 adipocyte differentiation markers after adipogenesis induction, and leads to lipid-depleted cells. We further show that the absence of lipids in LRP1-silenced preadipocytes is not caused by lipolysis induction. In addition, we provide the first evidences that LRP1 is significantly up-regulated in obese C57BI6/J mouse adipocytes and obese human adipose tissues. Interestingly, silencing of LRP1 in fully-differentiated adipocytes also reduces cellular lipid level and is associated with an increase of basal lipolysis. However, the ability of mature adipocytes to induce lipolysis is independent of LRP1 expression. Altogether, our findings highlight the dual role of LRP1 in the control of adipogenesis and lipid homeostasis, and suggest that LRP1 may be an important therapeutic target in obesity.     

Protein tyrosine phosphatase 1B inhibits adipocyte differentiation and mediates TNFα action in obesity

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of systemic glucose and insulin homeostasis; however, its exact role in adipocytes is poorly understood. This study was to elucidate the role of PTP1B in adipocyte differentiation and its implication in obesity. During differentiation of 3T3-L1 white preadipocytes, PTP1B decreased progressively with adipocyte maturation. Lentivirus-mediated PTP1B overexpression in preadipocytes delayed adipocyte differentiation, shown as lack of mature adipocytes, low level of lipid accumulation, and down-regulation of main markers (PPARγ2, SREBP-1c, FAS and LPL). In contrast, lentivirus-mediated PTP1B knockdown accelerated adipocyte differentiation, demonstrated as full of mature adipocytes, high level of lipid accumulation, and up-regulation of main markers. Dominant-negative inhibition on endogenous PTP1B by lentivirus-mediated overexpression of PTP1B double mutant in Tyr-46 and Asp-181 residues (LV-D/A-Y/F) also stimulated adipogenesis, more efficient than PTP1B knockdown. Diet-induced obesity mice exhibited an up-regulation of PTP1B and TNFα accompanied by a down-regulation of PPARγ2 in white adipose tissue. TNFα recombinant protein impeded PTP1B reduction and inhibited adipocyte differentiation in vitro; this inhibitory effect was prevented by LV-D/A-Y/F. Moreover, PTP1B inhibitor treatment improved adipogenesis and suppressed TNFα in adipose tissue of obese mice. All together, PTP1B negatively regulates adipocyte development and may mediate TNFα action to impair adipocyte differentiation in obesity. Our study provides novel evidence for the importance of PTP1B in obesity and for the potential application of PTP1B inhibitors.     

Regulation of adipocyte differentiation by PEGylated all-trans retinoic acid: reduced cytotoxicity and attenuated lipid accumulation

Obesity is major risk factor for many disorders, including diabetes, hypertension and heart disease. Unfortunately, there is a dearth of therapeutic agents available to clinicians for the treatment of obesity. The principal aim of this study was to investigate whether PEGylated all-trans retinoic acid (PRA) can have favorable stability and biological activity in 3T3-L1 preadipocytes as an antiobesity drug. Here, we found that PRA inhibits the process of adipogenesis, including survival of adipocytes and differentiation to mature adipocytes. The results showed that RA nanoparticles (NPs) were prepared by PEGylation; below 200 nm, PRA-NPs were obtained. Moreover, PRA decreased glycerol-3-phosphate dehydrogenase activity in 3T3-L1 preadipocytes by acting with major adipocyte marker proteins such as PPARgamma2, C/EBPalpha and aP2 modulators. Apoptosis, in addition, increased as the level of RA increased from 10 to 20 microM, whereas PRA reduced apoptosis with increasing concentrations. Our data suggest that PRA-NP has potential as an antiobesity drug carrier due to its small particle size and PEGylated core-shell structure. In addition, our results suggest that PRA inhibits the process of adipogenesis and may be developed to treat obesity. Based on these results, PRA is suitable for adipocyte studies, and an enhanced effect of PRA with adipocyte differentiation offers a challenging approach for pharmaceutical applications.     

Differential effects of flavonoids on 3T3-L1 adipogenesis and lipolysis

Flavonoids, polyphenolic compounds that exist widelyin plants, inhibit cell proliferation and increase cell differentiation in many cancerous and noncancerous cell lines. Because terminal differentiation of preadipocytes to adipocytes depends on proliferation of both pre- and postconfluent preadipocytes, we predicted that flavonoids would inhibit adipogenesis in the 3T3-L1 preadipocyte cellline. The flavonoids genistein and naringenin inhibited proliferation of preconfluent preadipocytes in a time- and dose-dependent manner. When added to 2-day postconfluent preadipocytes at the induction ofdifferentiation, genistein inhibited mitotic clonal expansion, triglyceride accumulation, and peroxisome proliferator-activated receptor- expression, but naringenin had no effect. Theantiadipogenic effect of genistein was not due to inhibition of insulinreceptor subtrate-1 tyrosine phosphorylation. When added 3 days afterinduction of differentiation, neither flavonoid inhibiteddifferentiation. In fully differentiated adipocytes, genisteinincreased basal and epinephrine-induced lipolysis, but naringenin hadno significant effects. These data demonstrate that genistein andnaringenin, despite structural similarity, have differential effects onadipogenesis and adipocyte lipid metabolism.

     

Gene expression of isoformic enzymes in arachidonate cyclooxygenase pathway and the regulation by tumor necrosis factor alpha during life cycle of adipocytes

Adipocytes serve not only as a storage depot of fats but also as endocrine cells secreting adipocytokines including tumor necrosis factor alpha (TNFalpha). Using preadipogenic 3T3-L1 cells, we attempt to determine the response of adipocytes at different stages of the life cycle to TNFalpha with respect to the gene expression of the arachidonate cyclooxygenase (COX) pathway and the role of endogenous prostaglandins (PGs). The gene expression analysis of the COX pathway revealed the marked increase in mRNA and protein levels of COX-2 in response to TNFalpha in preadipocytes, whereas COX-1 was expressed constitutively. Moreover, the cells at different cycle stages exhibited the specific gene expression of isoformic enzymes of prostaglandin (PG) synthases for PGs of the D(2), E(2), and F(2alpha) series upon exposure to TNFalpha. The treatment of preadipocytes with TNFalpha along with calcium ionophore A23187 resulted in the stimulated formation of PGE(2) and PGF(2alpha), attenuating the apoptotic cell death induced by TNFalpha alone. The response of adipocytes to synthesize these PGs declined during the differentiation and maturation phases. The cells during the differentiation phase were the most sensitive to TNFalpha in terms of the decrease in adipogenesis without the mediation of endogenous PGs. TNFalpha was also effective in suppressing adipogenesis during the maturation process. Taken together, TNFalpha can control cell number of preadipocytes as well as the size of fat storage in mature adipocytes. The action of TNFalpha on preadipocytes can be modulated by the production of endogenous PGs through the induction of COX-2.     

A Role for Bone Morphogenetic Protein-4 in Adipocyte Development

Obesity is characterized by increases in the number of mature adipocytes. Nascent adipocytes arise from mesenchymal stem cells (MSCs) by a multi-step process — MSCs are recruited to the adipocyte lineage forming determined preadipocytes, these committed progenitors proliferate, undergo growth arrest, and finally differentiate into mature adipocytes. Although the genetic mechanisms that control the differentiation of preadipocytes into mature adipocytes are understood to a large extent, the earliest events in adipogenesis — especially the commitment of MSCs into preadipocytes — are largely unknown. Recently, bone morphogenetic protein-4 (BMP-4) has been implicated in the commitment of pluripotent MSCs to the adipocyte lineage by two independent lines of investigation. First, growth-arrested 10T1/2 cells do not normally respond to a hormonal cocktail that causes various growth-arrested preadipocyte cell lines to differentiate into adipocytes, but if 10T1/2 cells are first treated with BMP-4 they will respond to these hormonal inducers by undergoing terminal adipocyte differentiation. Second, a preadipocyte cell line, A33 cells, derived from 10T1/2 cells after exposing the cells to the DNA methyltransferase inhibitor 5-azacytidine was shown to express BMP-4, and this endogenous BMP-4 expression is required for acquisition of the preadipocyte phenotype of these cells. A role for the BMP-4 signaling pathway in adipogenesis is discussed.     

An Evi1-C/EBPβ complex controls peroxisome proliferator-activated receptor γ2 gene expression to initiate white fat cell differentiation

Fibroblastic preadipocyte cells are recruited to differentiate into new adipocytes during the formation and hyperplastic growth of white adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, is expressed at low levels in preadipocytes, and its levels increase dramatically and rapidly during the differentiation process. However, the mechanisms controlling the dynamic and selective expression of PPARγ in the adipocyte lineage remain largely unknown. We show here that the zinc finger protein Evi1 increases in preadipocytes at the onset of differentiation prior to increases in PPARγ levels. Evi1 expression converts nonadipogenic cells into adipocytes via an increase in the predifferentiation levels of PPARγ2, the adipose-selective isoform of PPARγ. Conversely, loss of Evi1 in preadipocytes blocks the induction of PPARγ2 and suppresses adipocyte differentiation. Evi1 binds with C/EBPβ to regulatory sites in the Pparγ locus at early stages of adipocyte differentiation, coincident with the induction of Pparγ2 expression. These results indicate that Evi1 is a key regulator of adipogenic competency.     

Enhancement of ajoene-induced apoptosis by conjugated linoleic acid in 3T3-L1 adipocytes

Ajoene has been shown to induce apoptosis in 3T3-L1 adipocytes. In this report the effects on apoptosis of combinations of ajoene and trans-10, cis-12 conjugated linoleic acid (t10,c12CLA) in 3T3-L1 adipocytes were investigated. Although t10,c12CLA alone had no effect, ajoene plus t10,c12CLA reduced cell viability more than ajoene alone at 24 h (59.1 vs. 85.9% of control, respectively; p<0.05). Compared to treatment with t10,c12CLA, ajoene increased apoptosis 218% after 24 h (p<0.01), whereas ajoene plus t10,c12CLA increased apoptosis 122% over that caused by ajoene alone (p<0.01). Immunoblotting analysis also indicated that ajoene plus t10,c12CLA caused a greater increase in phosphorylation of c-Jun N-terminal kinase (JNK) and Bax expression and a greater release of mitochondrial proteins (cytochrome c, AIF) than additive responses to each compound alone. Ajoene plus t10,c12CLA also increased ROS production more than that resulting from ajoene treatment alone (264 vs 204% after 40 min, respectively; p<0.01). Furthermore, the antioxidant NAC prevented ROS generation and apoptosis by ajoene plus t10,c12CLA. Interestingly, the combination of ajoene and t10,c12CLA increased NF-κB activation and decreased the level of phosphorylated Akt more than each compound alone. Altogether, our observations indicate that t10,c12CLA potentiates the effect of ajoene on apoptosis in 3T3-L1 adipocytes. This work was supported by the Georgia Research Alliance, AptoTec, Inc., and by the Georgia Research Alliance Eminent Scholar endowment held by CAB.     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.