Undifferentiated Preadipocytes Research Articles

Adipocyte-Derived Paracrine Factors Regulate the In Vitro Development of Bovine Mammary Epithelial Cells.

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell-cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC's migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium.

The Effect of TGFβ1 in Adipocyte on Inflammatory and Fibrotic Markers at Different Stages of Adipocyte Differentiation.

Transforming growth factor beta (TGFβ) is a versatile cytokine. Although a profibrotic role of TGFβ is well established, its effect on tissue inhibitor of metalloproteinase (TIMPs) and inflammatory mediators are incompletely described. This study investigates the profibrotic and pro-inflammatory role of TGFβ1 during adipocyte differentiation. NIH3T3L1 cells were used for the in vitro study and were differentiated by adding a standard differentiation mix either with rosiglitazone (R-Diff) or without (S-Diff). Recombinant TGFβ1 (2 ng/mL) was added to the undifferentiated preadipocyte during the commitment stage and at the terminal differentiation stage. TGFβ1 treatment significantly decreased adiponectin mRNA at both early commitment (>300 fold) and terminal differentiated cells [S-Diff (~33%) or R-Diff (~20%)]. TGFβ1 upregulated collagen VI mRNA and its regulators connective tissue growth factor (CCN2/CTGF), TIMP1 and TIMP3 mRNA levels in undifferentiated preadipocytes and adipocytes at commitment stage. But in the terminal differentiated adipocytes, changes in mRNA and protein of collagen VI and TIMP3 mRNA were not observed despite an increase in CCN2/CTGF, TIMP1 mRNA. Although TGFβ1 upregulated interleukin-6 (IL6) and monocyte chemoattractant protein-1 (MCP1) mRNA at all stages of differentiation, decreased tumor necrosis factor-α (TNFα) mRNA was observed early in adipocyte differentiation. This study highlights the complex role of TGFβ1 on extracellular matrix (ECM) remodeling and inflammatory markers in stimulating both synthetic and inhibitory markers of fibrosis at different stages of adipocyte differentiation.

Analysis of the different characteristics between omental preadipocytes and differentiated white adipocytes using bioinformatics methods

ABSTRACT Obesity is emerging as an epidemiological issue, being associated with the onset and progress of various metabolism-related disorders. Obesity is characterized by the white adipose expansion, which encounters white adipocyte hypertrophy and hyperplasia. White adipocyte hyperplasia is defined as adipogenesis with the increase in the number of the white adipocytes from the preadipocytes. Adipogenesis contributes to distributing excess triglycerides among the smaller newly formed adipocytes, reducing the number of hypertrophic adipocytes and secreting anti-inflammatory factor. Therefore, adipogenesis is emerging as a new therapeutic target for the treatment of obesity. In the present study, for a better understanding of the contribution of the alteration of the omental differentiated white adipocytes to the systemic metabolic disorders, we downloaded the mRNA expression profiles from GEO database GSE1657, 328 differentially expressed genes (DEGs) were screened between the undifferentiated preadipocytes (UNDIF) and omental differentiated white adipocytes (DIF). The contributions of the upregulated and downregulated DEGs to the system were performed via the Gene Ontology (GO) analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Protein–Protein Interaction (PPI) network, respectively. The potential contribution of the whole altered genes in the differentiated white adipocytes was explored with the performance of Gene Set Enrichment Analysis (GSEA), especially on the GO analysis, KEGG analysis, hallmark analysis, oncogenic analysis and related miRNA analysis. The output of the current study will shed light on the new targets for the treatment of obesity and obesity-related disorders.

A fluorinated peptide with high serum- and lipid-tolerence for the delivery of siRNA drugs to treat obesity and metabolic dysfunction

Obesity is the major risk factor for metabolic diseases such as fatty liver, hyperlipidemia and insulin resistance. Beige fat has been recognized as a therapeutic target considering its great potential to burn energy. Since the evolutionary discovery of RNA interference and its utilization for gene knockdown in mammalian cells, a remarkable progress has been achieved in siRNA-based therapeutics. However, efficient delivery of siRNA into adipose tissues or differentiated adipocytes is challenging due to high lipid contents in these tissues. Here, we discovered a highly efficient fluoropolypeptide with excellent serum and lipid tolerance for this purpose from a library of amphiphlic polypeptides. The lead material F13-16 exhibited high gene knockdown efficacies in undifferentiated preadipocytes and differentiated adipocytes, as well as adipose tissues. It successfully delivered a siRNA targeting Tle3, an established suppressor gene for energy expenditure, in beige fat, and thereby ameliorated diet-induced obesity and metabolic disorders by increasing energy expenditure and thermogenic capacity. The results demonstrated that fluoropolypeptide is a useful tool for the delivery of siRNA-based therapeutics into adipocyte/adipose tissues for gene therapy.

Jateorhizine alleviates insulin resistance by promoting adipolysis and glucose uptake in adipocytes

Jateorhizine (Jat) can reduce blood glucose in diabetic mice, but there are few studies on its role in insulin resistance (IR). This study analyzed the effect of Jat on adipocytes, so as to provide an evidence for the clinical application of Jat. MDI was used to differentiate preadipocytes into adipocytes and induce IR cell models. Different concentrations of Jat (1, 5, 10, 20 μmol/L) were added into undifferentiated and differentiated cells. The cell viability was detected using MTT method. Oil red O staining was performed to observe the lipid formation in cells. Adipolysis method was used to detect the release of glycerol in cell culture medium. The level of 2-DG in cells was detected by glucose uptake assay based on insulin treatment. The expression of adipose transcription factors and IRS2/p-PI3K/p-AKT/GLUT4 signaling pathway was analyzed by western blot (WB) analysis. Neither the activity of differentiated nor undifferentiated preadipocytes was affected by the addition of Jat. There was numerous lipid formation in cells induced by MDI, which was decreased visibly by Jat. Jat reduced the expression levels of MDI-induced elevated levels of PPARγ, C/EBPα, FABP4, perilipin and FAS, as well as increased the release of glycerol in adipocytes. Moreover, Jat further enhanced the 2-DG uptake in MDI-induced adipocytes, and activated the IRS2/p-PI3K/p-AKT/GLUT4 signaling pathway. In general, the role of Jat in adipocytes was concentration-dependent. Jat can not only promote adipolysis, but also increase the glucose uptake in adipocytes, which might be a potential therapy for IR.

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function.

One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) are critical lipolytic enzymes, and their phosphorylation generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. Although we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on ATGL and HSL suggests that 14-3-3ζ could also influence mature adipocyte processes like lipolysis. Here we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and free fatty acid (FFA) release. Unexpectedly, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of peroxisome proliferator-activated receptor (PPAR)γ2 protein and expression of mature adipocyte genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated preadipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3ζ was found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.

Elastin-derived peptide VGVAPG decreases differentiation of mouse embryo fibroblast (3T3-L1) cells into adipocytes

ABSTRACT Elastin is a highly elastic protein present in connective tissue. As a result of protease activity, elastin hydrolysis occurs, and during this process, elastin-derived peptides (EDPs) are released. One of the constitutively repeating elastin and EDP building sequences is the hexapeptide VGVAPG. Therefore, the aim of our research was to define the effect of VGVAPG peptide on adipogenesis in a mouse 3T3-L1 cell line. 3T3-L1 cells were differentiated according to a previously described protocol and exposed to increasing concentrations of VGVAPG or VVGPGA peptide. The obtained results showed that VGVAPG peptide does not stimulate reactive oxygen species (ROS) production, caspase-1 activation, and 3T3-L1 cell proliferation. In the second part of the experiments, it was proved that VGVAPG peptide decreased lipid accumulation as measured by oil red O staining, which was confirmed by the profile of increased expression markers of undifferentiated preadipocytes. In our experiments, 10 nM VGVAPG added for differentiating to adipocytes increased the expression of Pref-1, serpin E1, and adiponectin as compared to rosiglitazone (PPARγ agonist)-treated group and simultaneously decreased the expression of VEGF and resistin as compared to the rosiglitazone-treated group. The obtained results show that VGVAPG peptide sustains 3T3 cells in undifferentiated state. Abbreviations: DMSO: dimethyl sulphoxide; EBP: elastin-binding protein; EDPs: elastin-derived peptides; FBS: foetal bovine serum; Glb1: gene for beta-galactosidase; LDL: low-density-lipoprotein; PAI-1 (Serpin E1): plasminogen activator inhibitor-1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor gamma; Pref-1: preadipocyte factor 1; ROS: reactive oxygen species; VEGF-A: vascular endothelial growth factor-A; VGVAPG: Val-Gly-Val-Ala-Pro-Gly; β-Gal: beta-galactosidase; ORO: oil red O; IBMX: 3-isobutyl-1-methylxanthine; H2DCFDA: 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; DMEM: Dulbecco’s Modified Eagle’s Medium; VVGPGA: Val-Val-Gly-Pro-Gly-Ala.

Colocynth (Citrullus colocynthis) seed extracts attenuate adipogenesis by down-regulating PPARγ/ SREBP-1c and C/EBPα in 3T3-L1 cells

Adipogenesis is the overall process of adipocyte differentiation. With a positive energy balance, intracellular accumulation of triglyceride increases through the generation of functional adipocytes. This occurs with adipogenesis of undifferentiated preadipocytes. Citrullus colocynthis (CC), a member of the Cucurbitaceae family, is a desert viny plant native to North Africa. The effect of ethanolic seed extracts of colocynth (SCEE) on adipogenesis was investigated using 3T3-L1 preadipocytes. Cell viability was measured using the MTT assay; triglycerides were stained with Oil Red O and adipogenesis-related gene expressions were quantified using qRT-PCR. Results showed that SCEE helps to inhibit intracellular triglyceride accumulation during adipogenesis without affecting cell viability. Likewise, SCEE not only showed anti-adipogenic activities, essentially during the early stage, but their effects during the middle and late stages were very low. These effects have been confirmed by the down-regulation of C/EBPβ and C/EBPδ at 24, 48 and 72 h. In addition, SCEE treatment in 3T3-L1 cells induced down-regulation of the transcription factors CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator activated receptor γ (PPARγ), and sterol regulatory element-binding protein 1c (SREBP-1c) and their target genes. UPLC-ESI-MS/MS profiling showed 10 bioactive compounds including quinic acid, isovitexin, scoparin, vitexin-2’’-O-rhamnoside, reserpine, digitoxin, triprolidine, naringenin, linoleic acid and oleic acid. Thus, these results suggested that the seeds of colocynth may have beneficial health effects.

Identification of Long Non-Coding RNA-Associated Competing Endogenous RNA Network in the Differentiation of Chicken Preadipocytes.

Emerging evidence indicates that long noncoding RNAs (lncRNAs) play important roles in the regulation of cell differentiation by acting as competing endogenous RNA (ceRNA). However, the regulatory mechanisms of lncRNA and the lncRNA-associated ceRNA network involved in adipogenic differentiation of chicken preadipocytes remain elusive. Here, we first constructed the chicken preadipocyte in vitro induction model. Then, we identified differentially expressed lncRNAs (DELs), miRNAs (DEMis), and mRNAs (DEMs) between differentiated and undifferentiated preadipocytes. Furthermore, we constructed the lncRNA associated ceRNA network by gene expression correlation analysis and target prediction of DELs, DEMis, and DEMs. Finally, we determined twelve candidate lncRNA-miRNA-mRNA interactions from the lncRNA associated ceRNA network. Eight out of the twelve interactions were validated by RT-qPCR, indicating their potential role in the regulation of chicken preadipocytes differentiation. Among the eight interactions, TCONS_00026544-gga-miR-128-1-5p-RASD1, TCONS_00055280-gga-miR-135a-5p-JAM3, TCONS_00055280-gga-miR-135a-5p-GPR133, TCONS_00055280-gga-miR-135a-5p-CLDN1, and TCONS_00055280-gga-miR-135a-5p-TMEM123 may promote adipogenic differentiation of chicken preadipocytes while TCONS_00057272-gga-miR-146a-3p-FOXO6, TCONS_00057242-gga-miR-6615-3p-FOXO6, and TCONS_00057242-gga-miR-6615-3p-ENSGALT00000043224 have the opposite effects. Our results not only provide novel insights into ceRNA roles of lncRNAs in chicken preadipocytes differentiation and but also contribute to a better understanding of chicken fat deposition.

Molecular and expression characteristics of resistin (RETN) and its effects on the differentiation of intramuscular preadipocyte in goat

Resistin (RETN) is a hormone secreted by adipocytes, which plays an important role in glucose and lipid metabolism. The aim of this study is to clone and obtain the full length open reading frame (ORF) of goat RETN gene sequence, and to reveal its molecular and expression characteristics. Simultaneously, we explore its effect on the differentiation of intramuscular preadipocytes in goat. The full length ORF sequence of goat RETN gene was cloned by RT-PCR technique, and bioinformatics analysis was performed though relevant biological softwares. In this study, the expression of RETN mRNA in goat tissues and intramuscular preadipocytes during differentiation was detected by qPCR technique. Furthermore, RNA interference was used to explore the effects of RETN on intramuscular preadipocytes differentiation in goat. The results showed that the cloned goat RETN gene sequence was 428 bp in length, of which the ORF was 330 bp, encoding 109 amino acids. Sequence analysis revealed that it had 12 phosphorylation sites and an O-glycosylation site, and its protein contained a signal peptide sequence. Also, the RETN gene is expressed in goat various analyzed tissues, and the results showed that the expression of RETN gene in lung tissue was higher than that in other analyzed tissues of goat (p < .01). Moreover, the expression level of RETN gene in the goat's intramuscular preadipocytes decreased first and then increased, and reached the highest on the fifth day, which was significantly higher than that of undifferentiated intramuscular preadipocytes (p < .001). After transfecting intramuscular preadipocyte with siRNA, we found that the mRNA level of RETN was significantly down-regulated by 70% and 87% (p < .01). Oil red O staining results showed that the interference of RETN gene can promote the differentiation of intramuscular preadipocytes. After knockdown of RETN with siRNA, the PPARγ, AP2, C/EBPα, C/EBPβ and SREBP1 genes were significantly up-regulated (p < .01). Thus, it can be inferred that RETN inhibits the differentiation of goat intramuscular preadipocytes, probably through regulating the expression of C/EBPα, C/EBPβ, PPARγ, AP2 and SREBP-1 genes.

Alpha-glucosidase inhibitor 1-Deoxynojirimycin promotes beige remodeling of 3T3-L1 preadipocytes via activating AMPK

Obesity is a serious health challenge in the world, and searching effective drugs to cure obesity is of great importance. 1-Deoxynojirimycin (DNJ) is extracted from mulberry leaves and acts as an α-glucosidase inhibitor to lower blood glucose. Recent studies demonstrated that it also has anti-obesity effect, but the mechanisms remain unknown. In our present study, we mainly examined the effects of DNJ on beige remodeling of 3T3-L1 preadipocytes. We observed that DNJ didn't affect the mRNA levels of fatty acid binding protein 4 (aP2), peroxisome proliferator-activated receptor γ (PPARγ), preadipocyte factor-1 (Pref-1) as well as the mitochondrial uncoupling protein 1 (UCP1), PR domain containing protein 16 (PRDM16), transmembrane protein 26 (TMEM26) in undifferentiated preadipocytes. But after inducing 3T3-L1 preadipocytes to differentiation with white or beige adipogenic medium, DNJ significantly reduced aP2, PPARγ and Pref-1 expressions, while up-regulated the expressions of UCP1, PRDM16 and TMEM26, accompanying with decreased lipid deposition. The ratio of p-AMPK/AMPK was up-regulated by DNJ (10 μM) treatment for 10 days, and the effects of DNJ on p-AMPK/AMPK, UCP1 and PRDM16 could be blocked by AMPK inhibitor Compound C. These results demonstrated that hypoglycemic agent DNJ could suppress the adipogenesis during the differentiation of white preadipocytes, and promote the switch of white preadipocytes to beige adipocytes via activating AMPK, which provided new mechanisms for explaining the benefits of DNJ on obesity-related disorders.

A novel role for the Wnt inhibitor APCDD1 in adipocyte differentiation: Implications for diet-induced obesity

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.

Glycolysis is Required for Rapid Adipocyte Thermogenesis Induced by Cold Stress

Adipose tissue responds to environmental cold stimulation by nonshivering thermogenesis as an adaptive mechanism to maintain body temperature. The adipose thermogenic response is in part activated by the sympathetic nervous system which senses cold, but recent studies show that adipocytes can also directly sense cold temperature and respond by initiating thermogenesis. Most studies on adipose thermogenic responses have focused on measuring the upregulation of genes such as the mitochondrial uncoupling protein 1 (UCP1) which are thought to mediate thermogenesis but there is little data on the actual intracellular temperature changes within adipocytes. We therefore used a cell-permeable thermosensitive fluorescent polymer to measure adaptive intracellular temperature changes in human subcutaneous adipocytes as a response to cold exposure. Ratiometric fluorescence measurements in cold-exposed human subcutaneous adipocytes increased 29% (p<0.001) in response to a temperature decline from 34°C to 25°C within two hours as compared to undifferentiated preadipocytes which did not show any intracellular heat generation. The studies also indicated a 20% intracellular temperature increase (p<0.001) in cold-exposed adipocytes when compared to control subcutaneous adipocytes maintained at 34°C. Increases in intracellular temperature occurred within 40 minutes during an external temperature decline from 34°C to 29°C. Chemical inhibition of glucose uptake using 2-deoxyglucose or chemical inhibition of the glycolysis regulatory enzyme PFKFB3 prevented the intracellular temperature increase (p<0.001). Additionally, a ratiometric fluorescent biosensor for ATP indicated an increase in the ATP:ADP ratio in response to cold. Our findings suggest that glycolytic activity is necessary for the cold-induced thermogenic response in adipocytes. By directly measuring the intracellular temperature changes we were able to assess the rapid response to cold. This thermogenic response occurred within minutes of the cold stimulation and does not require de novo transcription or translation of thermogenic genes. Enhancing glucose metabolism in adipocytes may therefore be a novel approach to enhance thermogenesis. Support or Funding InformationSupport for this work was provided by NSF 1253060 and NIH R01 GM094220. Intracellular temperature in adipocytes is altered by external temperature decline. (A) Ratiometric fluorescent intracellular probe measurements are normalized in adipocytes exposed to cold (squares), preadipocytes exposed to cold (open circles), and control cells maintained at constant warm temperature (triangles) over 150 minutes. (B) Cell culture temperature is shown in the cold exposure condition (circles) and control warm only condition (open diamonds).

A study of genes involved in adipocyte differentiation.

With the use of the microarray technique, genes expressed in the late phase of adipocyte differentiation were investigated. These genes play an important role in stimulating adipocyte growth and lipid droplet formation. Therefore, they contribute a great deal to the onset of obesity. With the use of SW872 adipocytes and the microarray technique, genes related to adipocyte differentiation were tested and compared with undifferentiated preadipocytes 14 days after induction. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used for confirmation. More than 21,329 transcriptors were expressed and determined, of which 1326 increased and 687 decreased undifferentiated adipocytes. Among them, 21 were highly expressed by more than 10-fold. With RT-PCR, 12 were confirmed, including apelin, CIDEC, PID1, LYRM1, ADD1, PPARγ2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Furthermore, genes involved in lipid metabolism, signal transduction, DNA replication, redox status and transcription factors were determined as well. Novel genes involved in adipogenesis (e.g., apelin) were detected. A variety of genes were discovered and validated with RT-PCR at the late phase of adipocyte differentiation. This may help us better understand the onset of obesity and the potential role of adipocytes in other organs.

DNA microarray analysis of genes differentially expressed in adipocyte differentiation.

In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a greater than or equal to 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RTPCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR?2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

Differential expression of cyclin G2, cyclin-dependent kinase inhibitor 2C and peripheral myelin protein 22 genes during adipogenesis

Increase of fat cells (FCs) in adipose tissue is attributed to proliferation of preadipocytes or immature adipocytes in the early stage, as well as adipogenic differentiation in the later stage of adipose development. Although both events are involved in the FC increase, they are contrary to each other, because the former requires cell cycle activity, whereas the latter requires cell cycle withdrawal. Therefore, appropriate regulation of cell cycle inhibition is critical to adipogenesis. In order to explore the important cell cycle inhibitors and study their expression in adipogenesis, we adopted a strategy combining the Gene Expression Omnibus (GEO) database available on the NCBI website and the results of quantitative real-time PCR (qPCR) data in porcine adipose tissue. Three cell cycle inhibitors – cyclin G2 (CCNG2), cyclin-dependent kinase inhibitor 2C (CDKN2C) and peripheral myelin protein (PMP22) – were selected for study because they are relatively highly expressed in adipose tissue compared with muscle, heart, lung, liver and kidney in humans and mice based on two GEO DataSets (GDS596 and GDS3142). In the latter analysis, they were found to be more highly expressed in differentiating/ed preadipocytes than in undifferentiated preadipocytes in human and mice as shown respectively by GDS2366 and GDS2743. In addition, GDS2659 also suggested increasing expression of the three cell cycle inhibitors during differentiation of 3T3-L1 cells. Further study with qPCR in Landrace pigs did not confirm the high expression of these genes in adipose tissue compared with other tissues in market-age pigs, but confirmed higher expression of these genes in FCs than in the stromal vascular fraction, as well as increasing expression of these genes during in vitro adipogenic differentiation and in vivo development of adipose tissue. Moreover, the relatively high expression of CCNG2 in adipose tissue of market-age pigs and increasing expression during development of adipose tissue was also confirmed at the protein level by western blot analysis. Based on the analysis of the GEO DataSets and results of qPCR and Western blotting we conclude that all three cell cycle inhibitors may inhibit adipocyte proliferation, but promote adipocyte differentiation and hold a differentiated state by inducing and maintaining cell cycle inhibition. Therefore, their expression in adipose tissue is positively correlated with age and mature FC number. By regulating the expression of these genes, we may be able to control FC number, and, thus, reduce excessive fat tissue in animals and humans.

Abstract B77: Targeting ovarian cancer promoting effects of adipocytes by metformin

Abstract At the time of surgery, 80% of the ovarian cancers are found to be metastasized to the omentum, a large fat pad covering the abdominal organs. Omental metastasis is thought to be due to the promotion of homing, growth and migration of ovarian cancer cells by factors secreted by adipocytes, which include cytokines, adipokines and growth factors. These adipocytes also act as fuel depot providing for the energy needs of fast growing ovarian tumor cells. The objective of our study was to investigate if metformin can modulate or limit the adipocyte mediated tumor-promoting and migrating effects. For this, mouse preadipocyte cells were differentiated in the presence or absence of metformin and various parameters including lipid accumulation, AMPK activation and adipogenesis transcription factors were examined. Conditioned media from undifferentiated and differentiated adipocytes was used to evaluate ID-8 mouse ovarian cancer cell proliferation and migration. Conditioned media from differentiated but not from undifferentiated preadipocytes, increased ID-8 cell proliferation (p&amp;lt;0.05) and migration (p&amp;lt;0.050), which were attenuated in presence of metformin (p&amp;lt;0.05). Metformin treatment inhibited adipogenesis as evident by the reduction of neutral lipid accumulation. Metformin treatment increased AMP-activated protein kinase (AMPK) activity as evident from higher level of phosphorylation of AMPK and its immediate downstream target ACC (Acetyl Co Carboxylase). It also inhibited the key adipogenesis transcription factors (CEBPαa; CEBPβb, SREBP1) during the process of adipocyte diffrenetiation. A targeted Cancer Pathway Finder RT-PCR based gene array (Qiagen) revealed 21 genes that were upreguated and 2 were downregulated more than 2-fold in ID-8 cells in presence of adipocyte conditioned media, which were modulated by metformin. Together, metformin treatment inhibited the adipocyte mediated ovarian cancer cell proliferation and migration. It also blunted the expression of cancer associated genes that were induced by adipocyte influence in cancer cells. This study suggests that metformin could be a therapeutic option for ovarian cancer as it not only targets ovarian cancer but alternatively, also modulates the environmental milieu around it. Citation Format: Ramandeep Rattan, Calvin Tebbe, Jasdeep Chhina, Kalli Sarigiannis, Shailendra Giri, Adnan Munkarah. Targeting ovarian cancer promoting effects of adipocytes by metformin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B77.

Cis-9, trans-11 conjugated linoleic acid is endogenously synthesized from palmitelaidic (C16:1 trans-9) acid in bovine adipocytes1

Palmitelaidic (C16:1 trans-9) acid has been suggested to have beneficial effects on human health, including reduced adiposity. Objectives of this research were to quantify the amounts of palmitelaidic acid in beef samples and determine the effect of palmitelaidic acid supplementation on lipogenesis in bovine preadipocytes and adipocytes in vitro. For the first objective, palmitelaidic acid content of LM samples from steers finished on forage or concentrate systems was determined. Palmitelaidic acid in LM samples from forage-finished beef ranged from 10 to 17 mg/100 g of muscle corresponding to 0.52% to 0.65% of total fatty acids. Forage species grazed during finishing, and animal age at harvest also altered palmitelaidic acid concentrations and contents in the LM of forage-finished beef. Palmitelaidic acid concentration of concentrate-finished beef was lower (P < 0.05; 0.25% vs. 0.56%); however, because of increased (P < 0.05) total fatty acid content with concentrate finishing, amount of palmitelaidic acid was similar (P > 0.05) to beef from steers finished on pearl millet and greater (P < 0.05) than those finished on alfalfa. For the second objective, undifferentiated preadipocytes and differentiated adipocytes were supplemented with 0 to 300 μM of palmitelaidic acid. Palmitelaidic acid supplementation reduced (P < 0.05) cell viability of undifferentiated preadipocytes at greater levels (150 and 300 μM) but did not affect (P > 0.05) the viability of differentiated adipocytes. In preadipocytes, palmitelaidic acid increased (P < 0.05) palmitelaidic and trans-11 vaccenic (C18:1 trans-11) acids at high levels of supplementation (300 μM). In adipocytes, palmitelaidic acid supplementation increased (P < 0.05) palmitelaidic acid, trans-11 vaccenic acid, and total fatty acid content. In addition, cis-9, trans-11 CLA also increased (P < 0.05) with palmitelaidic acid supplementation in adipocytes. These results indicate that palmitelaidic acid can be elongated in both preadipocytes and adipocytes and desaturated in adipocytes to generate trans-11 vaccenic acid and cis-9, trans-11 CLA, respectively. Beef products are a source of palmitelaidic acid in the human diet, which can be elongated and desaturated to produce trans-11 vaccenic acid and cis-9, trans-11 CLA.

A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

BackgroundSweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells.Methodology/Principal FindingsIn undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects.Conclusions3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.

Targeting AMP-activated protein kinase in adipocytes to modulate obesity-related adipokine production associated with insulin resistance and breast cancer cell proliferation.

BackgroundAdipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance.AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer.MethodsHuman preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 μg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation.ResultsConditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05).ConclusionsAdipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast cancer development and progression.