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.     

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.     

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.

     

Lactoferrin gene knockdown leads to similar effects to iron chelation in human adipocytes

In human and mice adipose tissue, lactoferrin (LTF) has been found to be associated with increased adipogenesis and decreased inflammatory markers. Here, we aimed to investigate the effects of LTF knockdown (KD) in human adipocyte differentiation. In addition, the effects of exogenous LTF administration and iron chelation [using deferoxamine (DFO, 10 μM)] were tested. In both subcutaneous and visceral pre‐adipocytes, LTF KD led to decrease significantly adipogenic, lipogenic and insulin signalling‐related gene expression and a significant increase in the gene expression of inflammatory mediators. Human lactoferrin (hLf, 1 μM) administration led to recover adipocyte differentiation in LTF KD pre‐adipocytes. Interestingly, iron chelation triggered similar effects to LTF KD, decreasing significantly adipogenic gene expressions. Of note, DFO (10 μM) and hLf (1 and 10 μM) co‐administration led to a dose‐dependent recovery of adipocyte differentiation. These new data reveal that endogenous LTF biosynthesis during human adipocyte differentiation is essential to achieve this process, possibly, modulating adipocyte iron homoeostasis. hLf administration might be a useful therapeutic target in obesity‐associated adipose tissue dysfunction.     

Cysteine deprivation prevents induction of peroxisome proliferator-activated receptor gamma-2 and adipose differentiation of 3T3-L1 cells

Plasma cysteine is strongly associated with body fat mass in human cohorts and diets low in cysteine prevents fat accumulation in mice. It is unclear if plasma cysteine affects fat development or if fat accumulation raises plasma cysteine. To determine if cysteine affects adipogenesis, we differentiated 3T3-L1 preadipocytes in medium with reduced cysteine. Cells incubated in media with 10–20 μM cysteine exhibited reduced capacity to differentiate into triacylglycerol-storing mature adipocytes compared with cells incubated with 50 μM cysteine. Low cysteine severely reduced expression of peroxisome proliferator-activated receptor gamma2 (Pparγ2) and its target genes perlipin1 (Plin1) and fatty acid binding protein-4 (Fabp4). Expression of stearoyl-CoA desaturase-1 (Scd1), known to be repressed with cysteine depletion, was also reduced with low cysteine. Medium depletion of the essential amino acids leucine, valine, and isoleucine had only a modest effect on adipocyte specific gene expression and differentiation. Stimulation with the PPARγ agonist BRL-49653 or addition of a hydrogen sulfide donor enhanced differentiation of 3T3-L1 cells cultured in low cysteine. This demonstrates that the ability to induce PPARγ expression is preserved when cells are cultured in low cysteine. It therefore appears that cysteine depletion inhibits adipogenesis by specifically affecting molecular pathways required for induction of PPARγ expression, rather than through a general reduction of global protein synthesis. In conclusion, we show that low extracellular cysteine reduces adipocyte differentiation by interfering with PPARγ2 and PPARγ target gene expression. Our results provide further evidence for the hypothesis that plasma cysteine is a casual determinant for body fat mass.     

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.     

Resveratrol,a naturally occurring diphenolic compound,affects lipogenesis,lipolysis and the antilipolytic action of insulin in isolated rat adipocytes

Resveratrol is a naturally occurring diphenolic compound exerting numerous beneficial effects in the organism. The present study demonstrated its short-term, direct influence on lipogenesis, lipolysis and the antilipolytic action of insulin in freshly isolated rat adipocytes. In fat cells incubated for 90 min with 125 and 250 μM resveratrol (but not with 62.5 μM resveratrol), basal and insulin-induced lipogenesis from glucose was significantly reduced. The antilipogenic effect was accompanied by a significant diminution of CO₂ release and enhanced production of lactate. The inhibition of glucose conversion to lipids found in the presence of resveratrol was not attenuated by activator of protein kinase C. However, acetate conversion to lipids appeared to be insensitive to resveratrol.In adipocytes incubated for 90 min with epinephrine, 10 and 100 μM resveratrol significantly enhanced lipolysis, especially at lower concentrations of the hormone. However, the lipolytic response to dibutyryl-cAMP, a direct activator of protein kinase A, was unchanged. Further studies demonstrated that, in cells stimulated with epinephrine, 1, 10 and 100 μM resveratrol significantly enhanced glycerol release despite the presence of insulin or H-89, an inhibitor of protein kinase A. The influence of resveratrol on epinephrine-induced lipolysis and on the antilipolytic action of insulin was not abated by the blocking of estrogen receptor and was accompanied by a significant (with the exception of 1 μM resveratrol in experiment with insulin) increase in cAMP in adipocytes. It was also revealed that resveratrol did not change the proportion between glycerol and fatty acids released from adipocytes exposed to epinephrine.Results of the present study revealed that resveratrol reduced glucose conversion to lipids in adipocytes, probably due to disturbed mitochondrial metabolism of the sugar. Moreover, resveratrol increased epinephrine-induced lipolysis. This effect was found also in the presence of insulin and resulted from the synergistic action of resveratrol and epinephrine. The obtained results provided evidence that resveratrol affects lipogenesis and lipolysis in adipocytes contributing to reduced lipid accumulation in these cells.     

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.     

Effect of clenbuterol on apoptosis,adipogenesis, and lipolysis in adipocytes

Clenbuterol, a beta₂-adrenergic receptor (β₂-AR) selective agonist, has been shown to decrease body fat in animals and can induce apoptosis in adipose tissue in mice. We hypothesized that direct actions of a β-adrenergic receptor agonist on adipocytes could trigger the observed apoptotic effect. The hypothesis was inspected by investigating the direct effect of clenbuterol on apoptosis, adipogenesis, and lipolysis in vitro using the 3T3-L1 cell line and rat primary adipocytes. Cells were treated with 10⁻⁹ to 10⁻⁵ M clenbuterol depending on the experiments. There was no apoptotic effect of clenbuterol both in 3T3-L1 cells and rat primary adipocytes. Adipogenesis monitored by Oil Red O staining and AdipoRed™ assay was modestly decreased by clenbuterol treatment (p < 0.05). In fully differentiated primary adipocytes, clenbuterol increased basal lipolysis compared with the control (p < 0.01). In summary, direct stimulation of β₂-AR by clenbuterol does not cause apoptosis in adipocytes, despite a direct lipolytic stimulation and attenuation of adipogenesis.     

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.     

Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone Fiber Matrices

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.     

Effects of fermented rooibos (Aspalathus linearis) on adipocyte differentiation

Rooibos (Aspalathus linearis) contains a rich complement of polyphenols, including flavonoids, considered to be largely responsible for its health promoting effects, including combatting obesity. The purpose of this study was to examine the effect of fermented rooibos hot water soluble solids on in vitro adipocyte differentiation by using differentiating 3T3-L1 adipocytes. Hot water soluble solids were obtained when preparing an infusion of fermented rooibos at “cup-of-tea” strength. The major phenolic compounds (>5 mg/g) were isoorientin, orientin, quercetin-3-O-robinobioside and enolic phenylpyruvic acid-2-O-β-d-glucoside. Treatment of 3T3-L1 adipocytes with 10 μg/ml and 100 μg/ml of the rooibos soluble solids inhibited intracellular lipid accumulation by 22% (p < 0.01) and 15% (p < 0.05), respectively. Inhibition of adipogenesis was accompanied by decreased messenger RNA (mRNA) expression of PPARγ, PPARα, SREBF1 and FASN. Western blot analysis exhibited decreased PPARα, SREBF1 and AMPK protein expression. Impeded glycerol release into the culture medium was observed after rooibos treatment. None of the concentrations of rooibos hot water soluble solids was cytotoxic, in terms of ATP content. Interestingly, the higher concentration of hot water soluble solids increased ATP concentrations which were associated with increased basal glucose uptake. Decreased leptin secretion was observed after rooibos treatment. Our data show that hot water soluble solids from fermented rooibos inhibit adipogenesis and affect adipocyte metabolism, suggesting its potential in preventing obesity.     

Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis

Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.     

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.     

The influence of preadipocyte differentiation capacity on lipolysis in human mature adipocytes.

The ability of catecholamines to maximally stimulate adipocyte lipolysis (lipolytic capacity) is decreased in obesity. It is not known whether the lipolytic capacity is determined by the ability of adipocytes to differentiate. The aim of the study was to investigate if lipolytic capacity is related to preadipocyte differentiation and if the latter can predict lipolysis in mature adipocytes. IN VITRO experiments were performed on differentiating preadipocytes and isolated mature adipocytes from human subcutaneous adipose tissue. In preadipocytes, noradrenaline-induced lipolysis increased significantly until terminal differentiation (day 12). However, changes in the expression of genes involved in lipolysis (hormone sensitive lipase, adipocyte triglyceride lipase, the alpha2-and beta1-adrenoceptors, perilipin, and fatty acid binding protein) reached a plateau much earlier during differentiation (day 8). A significant positive correlation between lipolysis in differentiated preadipocytes and mature adipocytes was observed for noradrenaline (r=0.5, p<0.01). The late differentiation capacity of preadipocytes measured as glycerol-3-phosphate dehydrogenase activity was positively correlated with noradrenaline-induced lipolysis in preadipocytes (r=0.51, p<0.005) and mature fat cells (r=0.35, p<0.05). In conclusion, intrinsic properties related to terminal differentiation determine the ability of catecholamines to maximally stimulate lipolysis in fat cells. The inability to undergo full differentiation might in part explain the low lipolytic capacity of fat cells among the obese.