Ankrd26 gene disruption enhances adipogenesis of mouse embryonic fibroblasts

We previously reported that partial disruption of the Ankrd26 gene in mice leads to hyperphagia and leptin-resistant obesity. To determine whether the Ankrd26 mutation can affect the development of adipocytes, we studied mouse embryo fibroblasts (MEFs) from the mutant mice. We found that Ankrd26(-/-) MEFs have a higher rate of spontaneous adipogenesis than normal MEFs and that adipocyte formation is greatly increased when the cells are induced with troglitazone alone or with a mixture of troglitazone, insulin, dexamethasone, and methylisobutylxanthine. Increased adipogenesis was detected as an increase in lipid droplet formation and in the expression of several markers of adipogenesis. There was an increase in expression of early stage adipogenesis genes such as Krox20, KLF5, C/EBPβ, C/EBPδ, and late stage adipogenesis regulators KLF15, C/EBPα, PPARγ, and aP2. There was also an increase in adipocyte stem cell markers CD34 and Sca-1 and preadipocyte markers Gata2 and Pref-1, indicating an increase in both stem cells and progenitor cells in the mutant MEFs. Furthermore, ERK was found constitutively activated in Anrd26(-/-) MEFs, and the addition of MEK inhibitors to mutant cells blocked ERK activation, decreased adipogenesis induction, and significantly reduced expression of C/EBPδ, KLF15, PPARγ2, CD34, and Pref-1 genes. We conclude that Ankrd26 gene disruption promotes adipocyte differentiation at both the progenitor commitment and differentiation steps and that ERK activation plays a role in this process.     

Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK‐dependent PPARγ expression and phosphorylation

Mesenchymal stem cells (MSCs) provide us an excellent cellular model to uncover the molecular mechanisms underlying adipogenic differentiation of adult stem cells. PPARγ had been considered as an important molecular marker of cells undergoing adipogenic differentiation. Here, we demonstrated that expression and phosphorylation of PPARγ could be found in bone marrow–derived MSCs cultured in expansion medium without any adipogenic additives (dexamethasone, IBMX, insulin or indomethacin). Then, PPARγ was dephosphorylated in MSCs during the process of adipogenic differentiation. We then found that inhibition of MEK activation by specific inhibitor (PD98059) counteracted the PPARγ expression and phosphorylation. However, expression and phosphorylation of PPARγ did not present in MSCs cultured in medium with lower serum concentration. When these MSCs differentiated into adipocytes, no phosphorylation could be detected to accompany the expression of PPARγ. Moreover, exposure of MSCs to higher concentration of serum induced stronger PPARγ expression, and subsequently enhanced their adipogenesis. These data suggested that activation of the MEK/ERK signalling pathway by high serum concentration promoted PPARγ expression and phosphorylation, and subsequently enhanced adipogenic differentiation of MSCs.     

EMF acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

The use of electromagnetic fields (EMFs) to treat nonunion fractures developed from observations in the mid‐1900s. Whether EMF directly regulates the bone marrow mesenchymal stem cells (MSCs), differentiating into osteoblasts or adipocytes, remains unknown. In the present study, we investigated the roles of sinusoidal EMF of 15 Hz, 1 mT in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that EMF promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. EMF increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast‐specific mRNA expression of RUNX2, ALP, BMP2, DLX5, and BSP. In contrast, EMF decreased adipogenesis and inhibited adipocyte‐specific mRNA expression of adipsin, AP‐2, and PPARγ2, and also inhibited protein expression of PPARγ2. These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is influenced by EMF. Bioelectromagnetics 31:277–285, 2010. © 2009 Wiley‐Liss, Inc.     

Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 μM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.     

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.     

The Role of Calcium in Differentiation of Human Adipose-Derived Stem Cells to Adipocytes

Differentiation process of mesenchymal stem cells (MSCs) into adipocyte is involved in obesity. Multiple factors such as Ca²⁺ play important roles in different stages of this process. Because of the complicated roles of Ca²⁺ in adipogenesis, the aim of present investigation was to study the influx and efflux of Ca²⁺ into and out of the cells during adipogenesis. Adipose-derived MSCs were used to differentiate into adipocytes. MSCs were exposed to 2.5 mM Ca²⁺ or 1.8 mM Ca²⁺ plus calcium ionophore, A23187, for 3 days. Lipid staining, triglycerides (TG) content, and glyceraldehyde phosphate dehydrogenase (GAPDH) activity were evaluated to confirm the efficiency of the differentiation. Gene expression of GLUT4, PPARγ2, RAR-α, and calreticulin, as well as the protein levels of GLUT4 and PPARγ2 were determined. Ca²⁺ and in particular Ca²⁺ plus A23187 significantly lowered the efficiency of differentiation accompanied by decrease in intracellular TG deposits, GAPDH activity and alleviation of gene, and protein levels of GLUT4 and PPARγ2. While calreticulin and RAR-α were remarkably upregulated in A23187 group. This study showed the inhibitory effects of calcium in adipogenesis. Additionally, it indicated the greater inhibitory effect of calreticulin and RAR-α in controlling adipogenesis by higher levels of calcium.     

Pref-1 (preadipocyte factor 1) activates the MEK/extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation

Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore, treatment of Pref-1 null MEF with Pref-1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation primarily by preventing peroxisome proliferator-activated receptor gamma2 induction. However, in the presence of PD98059 or depletion of ERK1/2, exogenous Pref-1 cannot inhibit adipocyte differentiation in Pref-1 null MEF. We conclude that Pref-1 activates MEK/ERK signaling, which is required for Pref-1 inhibition of adipogenesis.     

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.     

Skp2 promotes adipocyte differentiation via a p27-independent mechanism in primary mouse embryonic fibroblasts

Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, a principal target of the SCF^Skp2 complex. Genetic ablation of p27^Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27^Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2^-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2^−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27^Kip1 expression.     

The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line

Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR γ and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC₅₀ at about 1 nM. The induction medium up-regulated expression of PPARγ and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression patterns of peroxisome proliferator-activated receptor gamma 1 versus gamma 2, and their association with intramuscular fat in goat tissues

Intramuscular fat (IMF) shortage causes the lack of juiciness and tenderness of goat meat, while peroxisome proliferator-activated receptor gamma 1 (PPARγ1) and gamma 2 (PPARγ2) play key roles in lipid metabolism. Nevertheless, their expression patterns and the relationship with IMF have been poorly exposed. Using quantitative polymerase chain reaction (qPCR), classical Soxhlet extraction, and in situ hybridization, we demonstrated that among 13 goat tissues, expression of PPARγ1 was dramatically higher than that of PPARγ2 except for lung. We further demonstrated the expression patterns of PPARγ1 and PPARγ2 and their negative association with intramuscular fat content in three goat muscles with kids growing. Meanwhile, PPARγ expression was located in the connective tissues. These results suggest that PPARγ1 is rather active for most tissues of goat, and closely related with the muscular fat metabolism during early postnatal life, but a more direct proof remains to be provided.