The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

Phytochemicals and regulation of the adipocyte life cycle

Natural products have potential for inducing apoptosis, inhibiting adipogenesis and stimulating lipolysis in adipocytes. The objective of this review is to discuss the adipocyte life cycle and various dietary bioactives that target different stages of adipocyte life cycle. Different stages of adipocyte development include preadipocytes, maturing preadipocytes and mature adipocytes. Various dietary bioactives like genistein, conjugated linoleic acid (CLA), docosahexaenoic acid, epigallocatechin gallate, quercetin, resveratrol and ajoene affect adipocytes during specific stages of development, resulting in either inhibition of adipogenesis or induction of apoptosis. Although numerous molecular targets that can be used for both treatment and prevention of obesity have been identified, targeted monotherapy has resulted in lack of success. Thus, targeting several signal transduction pathways simultaneously with multiple natural products to achieve additive or synergistic effects might be an appropriate approach to address obesity. We have previously reported two such combinations, namely, ajoene+CLA and vitamin D+genistein. CLA enhanced ajoene-induced apoptosis in mature 3T3-L1 adipocytes by synergistically increasing the expression of several proapoptotic factors. Similarly, genistein potentiated vitamin D's inhibition of adipogenesis and induction of apoptosis in maturing preadipocytes by an enhanced expression of VDR (vitamin D receptor) protein. These two examples indicate that combination therapy employing compounds that target different stages of the adipocyte life cycle might prove beneficial for decreasing adipose tissue volume by inducing apoptosis or by inhibiting adipogenesis or both.     

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.     

Inhibition of adipogenesis and induction of apoptosis and lipolysis by stem bromelain in 3T3-L1 adipocytes

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein G(i)α(1), as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.     

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.     

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.     

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.     

Different mechanisms of cis-9,trans-11- and trans-10,cis-12- conjugated linoleic acid affecting lipid metabolism in 3T3-L1 cells

Conjugated linoleic acid (CLA) has been shown to reduce body fat mass in various experimental animals. It is valuable to identify its influence on enzymes involved in energy expenditure, apoptosis, fatty acid oxidation and lipolysis. We investigated isomer-specific effects of high dose, long treatment of CLA (75.4 μmol/L, 8 days) on protein and gene expression of these enzymes in cultured 3T3-L1 cells. Proteomics identified significant up- or down-regulation of 52 proteins by either CLA isomer. Protein and gene expression of uncoupling protein (UCP) 1, UCP3, perilipin and peroxisome proliferator-activated receptor (PPAR) α increased whereas UCP2 reduced for both CLA isomers. And eight-day treatment of trans-10,cis-12 CLA, but not cis-9,trans-11 CLA, significantly up-regulated protein and mRNA levels of PKA (P<.05), CPT-1 and TNF-α (P<.01). Compared to protein expression, both isomers did not significantly influence the mRNA expression of HSL, ATGL, ACO and leptin. In conclusion, high-dose, long treatment of cis-9,trans-11 CLA did not promote apoptosis, fatty acid oxidation and lipolysis in adipocytes, but may induce an increase in energy expenditure. trans-10,cis-12 CLA exhibited greater influence on lipid metabolism, stimulated adipocyte energy expenditure, apoptosis and fatty acid oxidation, but its effect on lipolysis was not obvious.     

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.     

用成熟脂肪建立一种新的猪前体脂肪细胞培养模型

用去分化的成熟脂肪细胞建立一种新的具有再增殖和再分化能力的猪前体脂肪细胞模型. 用“天花板” 培养法分离、培养1～3日龄仔猪皮下成熟脂肪细胞, 显微镜下观察细胞形态变化并计数, 流式细胞术检测细胞周期;油红O染色法检测脂肪细胞分化率, RT-PCR分析前体脂肪细胞标志基因Pref-1及成熟脂肪细胞关键转录因子PPARγ和C/EBPα等mRNA表达情况. 发现刚贴壁的细胞为单室脂滴成熟脂肪细胞, 油红O染色完全阳性; 14d后这种成熟脂肪细胞完全去分化为无脂滴的纤维状细胞, 并表达前体脂肪细胞标志基因Pref-1, 油红O染色阴性. 这种去分化的前体脂肪细胞在成脂诱导剂作用下,可重新分化为成熟的脂肪细胞. 结果证实,成熟脂肪细胞去分化后的前体脂肪细胞可重新增殖、分化为成熟脂肪细胞, 是一种新的有效的前体脂肪细胞模型.     

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.     

Enhanced effects of xanthohumol plus honokiol on apoptosis in 3T3-L1 adipocytes

Objective: To study the effects of xanthohumol (XN), a flavonoid found in hops (Humulus lupulus) and honokiol (HK), a lignan isolated from Magnolia officinalis, alone and in combination, on apoptotic signaling in 3T3‐L1 adipocytes. Methods and Procedures: 3T3‐L1 mature adipocytes were incubated with various concentrations of XN and HK alone and in combination. Viability and apoptosis were quantified using an MTS‐based cell viability assay and single‐stranded DNA assay, respectively. Expression of apoptosis related proteins including cleaved poly(ADP‐ribose) polymerase (PARP), cytochrome c, Bcl‐2, caspase‐3/7, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt was analyzed by western blotting. Results: Combinations of XN and HK significantly decreased viability and induced apoptosis in a dose‐dependent manner and more than the additive responses to XN and HK alone. Western blot analysis showed an increase in cleaved PARP and cytochrome c release and decrease in expression of Bcl‐2 protein by XN plus HK, whereas XN and HK individually had no effect. Furthermore, the combination of XN and HK activated PTEN and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. Discussion: We demonstrated that although XN and HK showed little or no effect as individual compounds, in combination (XN plus HK) they showed enhanced activity in inducing apoptosis via the cytochrome c/caspase‐3/PARP and PTEN/Akt pathways in 3T3‐L1 adipocytes.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

Effect of TAT‐obestatin on proliferation,differentiation, apoptosis and lipolysis in 3T3‐L1 preadipocytes

It has been reported that obestatin regulates adipocyte metabolism via receptors on the cell surface. We wondered whether obestatin can interact with intracellular components that activated signalling pathways in adipocytes. Because obestatin (human) only presents one lysine (at position 10), which cannot penetrate the cell membrane, therefore, we used a cell‐permeable peptide TAT (49‐57) as a vector to carry obestatin across the cell membrane. The goal of this study was to further understand the function of obestatin after penetrating the cell membrane. Our results showed that TAT‐obestatin could cross the 3T3‐L1 cell membrane in the absence of cytotoxicity. TAT‐obestatin showed no effect on the proliferation of 3T3‐L1 preadipocytes. In contrast, obestatin significantly stimulated proliferation at a dose of 10^‐11 M and 10^‐13 M. In addition, TAT‐obestatin demonstrated a more potent inhibitory effect on cell apoptosis induced by serum starvation than that of obestatin. During the progress of adipocyte differentiation, TAT‐obestatin and obestatin had no effect on adipogenesis. In the lipolysis assay, TAT‐obestatin significantly increased glycerol and free fatty acid release from 3T3‐L1 adipocytes after 3 h treatment but showed no significant effect on lipolysis after 24 h and 48 h of treatment. In contrast, obestatin (10^‐7 M) had no effect on glycerol release after 3, 24 and 48 h of treatment. The difference between the effect of TAT‐obestatin and obestatin on adipocytes metabolism indicated that TAT‐obestatin may trigger intracellular signalling as well as signalling at the cell membrane. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Gene expression profiles analyzed by DNA sequencing of cDNA clones constructed from porcine preadipocytes and adipocytes

As a step towards understanding the molecular mechanism of adipogenesis in pigs, preadipocytes purified from the back fat of 1 day-old female piglets were used for in vitro culture. Normalized cDNA libraries were constructed with 1.6×10⁷ and 1.1×10⁷ independent clones from preadipocyte and mature adipocyte mRNAs, respectively. Polymerase chain reaction (PCR) result using primers T3 and T7 (universal primer) confirmed the presence of the insert in the vector. Sequencing of 2,112 randomly selected clones from each cDNA library identified 217 clusters, 1,169 singletons, and 216 contigs in preadipocytes and 231 clusters, 1,100 singletons, and 233 contigs in mature adipocytes. Expressed sequence tag (EST) identified 24 genes with known annotation highly expressed in adipocytes and 21 in preadipocytes by at least four EST number. Among those 45 genes, when analyzed by real time RT-PCR, 76% of the gene showed significant difference between preadipocytes and mature adipocytes. Highly expressed genes in mature adipocytes were related to adipogenesis, extracellular matrix control and oncogenes, whereas cytoskeleton-related genes were down-regulated. An interesting similarity found during gene profile studies indicated a correlation between cancer and adipogenesis.