The Differentiation-and Proliferation-Inhibitory Effects of Sporamin from Sweet Potato in 3T3-L1 Preadipocytes

The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-LI preadipocytes, providing the theoretical basis for the development of food to treat obesity and diabetes, The isolation and purification of sporamin from sweet potato species 55-2 were performed by ammonium sulphate precipitation in combination with ion-exchange and gel filtration chromatography. With berberine as a positive control, different concentrations ofsporamin （0.000, 0.125, 0.025, 0.250, 0.500, and 1.000 mg·mL^-1 were used to treat 3T3-L1 preadipocytes. Intracellular fat accumulation and the degree of adipogenesis were quantified using Oil Red O staining and colorimetry, Preadipocytes differentiation was measured by 3（4,5-dimethylthiazolyl-2-yl）-2,5-diphenyltetrazolium bromide （MTT） spectrophotometric assay. Two sporamin proteins, which were separated into sporamin A （31 kD） and sporamin B （22 kD）, could be purified by ion-exchange and gel filtration chromatography. After being treated by different concentrations of sporamin, the differentiation of 3T3-L1 preadipocytes was significantly inhibited, compared with the positive control. When the sporamin solution concentration was 0.500 mg mL-1, the accumulation of lipid droplets within the cells was significantly decreased and the optical density （OD） value of the solution from destained Oil Red O reached to 0.35, which was the lowest value （P〈 0.05）. The proliferation of 3T3-L1 preadipocytes was significantly inhibited by treating at higher sporamin concentrations. In addition, the inhibitory effect was more obvious with the prolonged treatment time （P〈 0.05）. The differentiation and proliferation of 3T3-L1 preadipocytes could be inhibited significantly by the addition of higher concentration sporamin. It was, therefore, suggested that the sporamin was potentially effective for weight loss.

Effects of pioglitazone on proliferation and differentiation of human preadipocytes

Objective： To explore the effects of thiazolidinediones （TZDs） pioglitazone on proliferation and differentiation of human preadipocytes. Methods：Omental adipose tissue biopsies were obtained from 15 patients who were undergoing elective open-abdominal surgery. The primary culture and differentiated induction of human preadipocytes were performed, and the human preadipo-cytes were treated with pioglitazone at different concentrations at proper moments. Dynamic morphological changes of the human preadipocytes were observed, and their proliferation and differentiation were assessed with Colorimetric MTT Assay and Oil Red O Staining. Results：After 24 hours and 72 hours with pioglitazone, 0.1 μmol/L （μmol/ml） pioglitazone increased the MTT values of the human preadipocytes by 25.3% and 34.8%,respectively（P 〈 0.05）, while 1 μmol/L pioglitazone by 27.4% and 26.6%（P 〈 0.05）, compared with the control group without pioglitazone. The human preadipocytes with pioglitazone cumulated more adipose in the endochylema than those without pioglitazone obviously. 0.1 μmol/L pioglitazone increased the differentiation degree of the human preadipocytes differentiated for 8-10 days by 44.81% and 1 μmol/L pioglitazone by 53.76%（P 〈 0.05）. Conclusion：Thi- azolidinediones pioglitazone may significantly promote the proliferation and differentiation of the human omental preadipocytes.

Effects of Conjugated Linoleic Acids on Proliferation and Differentiation of Rat Preadipocytes

Conjugated linoleic acids （CLAs） are a generic term for linoleic acid isomers and have a variety of biological functions. After rat preadi- pocytes were incubated in CLAs-supplemented media, their proliferation and differentiation were observed by the cell count and the oil red O stai- ning. The results showed that the CLAs at different concentrations inhibited proliferation of the rat preadipocytes in a time- and dose-dependent manner. And the CLAs greatly decreased intracellular lipid content in mature adipocytes. Moreover, lipogenesis was inhibited by the CLAs in a dose-dependent manner. Therefore, the CLAs inhibit the lipogenesis by reducing the number of preadipocytes and decreasing the intracellular lipid

Effects of Ghrelin on the Proliferation and Differentiation of 3T3-L1 Preadipocytes

The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study. 3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin. Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR. Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining. The mRNA levels of peroxisome proliferator-activated receptor γ （PPARγ） and CAAT/enhancer binding protein （C/EBPa） in the cells at different differentiation stages were detected by RT-PCR. The results showed that ghrelin at concentrations of 10^-7 to 10^-15 mol/L could significantly promote preadipocyte proliferation （P〈0.05）, with the most pronounced effect observed at 10^-11 mol/L （P〈0.01）. Treatment of 3T3-L1 preadipocytes with ghrelin significantly increased the mRNA levels of c-myc and thymidine kinase （P〈0.01）. Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin. Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Ghrelin significantly increased the mRNA levels of PPAR7 and C/EBPα during the differentiation, when compared with control group （P〈0.05）. The mRNA levels of PPARγ and C/EBPα were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin. There were significant difference in the mRNA levels of PPARγ and C/EBPu on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes （P〈0.01）. In conclusion, ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARγ and C/EBPα and therefore enhance the sensitivity of adipocytes against insulin.

Verapamil inhibits 3T3-L1 preadipocyte differentiation

Objective： To investigate the effect of the calcium channel blocker verapamil on adipocyte differentiation and its mechanism of action. Methods： Preadipocytes from 3T3-L1 strain mouse embryos were cultured and differentiated into matured adipocytes in vitro. Verapamil was added to the culture medium in the concentration of 30 μmol/L on Day 0. Cell differentiation was determined by Oil Red O staining and marker gene mRNA expression was evaluated and compared by RT-PCR. The fluo-3/AM probe and laser scanning confocal microscopy were used to measure intracetlular calcium concentrations. Results： （1）The differentiation rate of 3T3-L1 preadipocytes exposed to verapamil was lower than that of untreated cells. （2）Verapamil promoted the retention of pref-1 gene expression. Lipoprotein lipase expression in the verapamil group was significantly lower than that in the control group on Day 4, Day 6 and Day 8 （P 〈 0.05） and resistin expression was significantly lower than that in the control group on Day 6, Day 8 and Day 10 （P 〈 0.05）. Fatty acid synthase expression in the verapamil group was significantly lower than that in the control group from Day 2 （P 〈 0.05）. （3） Intracellular concentrations of calcium [Ca^2＋]i in the verapamil group were significantly decreased compared with those in the control group on Day 2, Day 4 and Day 6 （P 〈 0.05）, while there was no obvious difference between the two groups on Day 0 （P 〉 0.05）. Conclusion： In 3T3-L1 preadipocytes verapamil significantly reduced adipocyte differentiation, down-regulated the mRNA expression of three marker genes for adipocytes differentiation, and prolonged the mRNA expression of an inhibitor of differentiation. The inhibitory effect of verapamil on differentiation may involve its role as a blocker of calcium influx in adipocytes.

A genome-wide landscape of mRNAs,lncRNAs,and circRNAs during subcutaneous adipogenesis in pigs

Background: Preadipocyte differentiation plays a critical role in subcutaneous fat deposition in pigs.However,the roles of different RNAs,such as messenger RNAs(mRNAs),long non-coding RNAs(lncRNAs),and circular RNAs(circRNAs) in the differentiation process of subcutaneous preadipocytes,are still largely unclear.In the present study,a transcriptome analysis,including the analysis of mRNAs,lncRNAs,and circRNAs,during different differentiation stages,namely,day 0(D0),day 2(D2),day 4(D4),and day 8(D8),of subcutaneous preadipocytes from Chinese Erhualian pigs was performed.Results: A total of 1554,470,1344,1777,and 676 differentially expressed(DE) mRNAs,112,58,95,136,and 93 DE lncRNAs,and 902,787,710,932,and 850 DE circRNAs were identified between D2 and D0,between D4 and D2,between D8 and D4,between D4 and D0,and between D8 and D0,respectively.Furthermore,functional enrichment analysis showed that the common DE mRNAs during the entire differentiation process were mainly involved in lipid metabolic and cell differentiation processes.Additionally,co-expression network analysis identified the potential lncRNAs related to adipogenesis,e.g.,MSTRG.131380 and MSTRG.62128.Conclusions: Our study provides new insights of the expression changes of RNAs during adipogenic differentiation,which might contribute to the phenotype of subcutaneous adipogenesis.These results greatly improve our understanding of the molecular mechanisms regulating subcutaneous fat deposition in pigs.

HBP1 inhibits chicken preadipocyte differentiation by activating the STAT3 signaling via directly enhancing JAK2 expression

Obesity presents a serious threat to human health and broiler performance.The expansion of adipose tissue is mainly regulated by the differentiation of preadipocytes.The differentiation of preadipocytes is a complex biological process regulated by a variety of transcription factors and signaling pathways.Previous studies have shown that the transcription factor HMG-box protein 1(HBP1)can regulate the differentiation of mouse 3T3-L1 preadipocytes by activating the Wnt/β-catenin signaling pathway.However,it is unclear whether HBP1 involved in chicken preadipocyte differentiation and which signaling pathways it regulates.The aim of the current study was to explore the biological function and molecular regulatory mechanism of HBP1 in the differentiation of chicken preadipocytes.The expression patterns of chicken HBP1 in abdominal adipose tissue and during preadipocyte differentiation were analyzed by RT-qPCR and Western blot.The preadipocyte stably overexpressing HBP1 or knockout HBP1 and their control cell line were used to analyze the effect of HBP1 on preadipocyte differentiation by oil red O staining,RT-qPCR and Western blot.Cignal 45-Pathway Reporter Array was used to screen the signal pathways that HBP1 regulates in the differentiation of chicken preadipocytes.Chemical inhibitor and siRNA for signal transducer and activator of transcription 3(STAT3)were used to analyze the effect of STAT3 on preadipocyte differentiation.The preadipocyte stably overexpressing HBP1 was transfected by the siRNA of STAT3 or treated with a chemical inhibitor of STAT3 for the rescue experiment.The results of gene expression analysis showed that the expression of HBP1 was related to abdominal fat deposition and preadipocyte differentiation in chickens.The results of function gain and loss experiments indicated that overexpression/knockout of HBP1 in chicken preadipocytes could inhibit/promote(P<0.05)lipid droplet deposition and the expression of adipogenesis-related genes.Mechanismlly,HBP1 activates(P<0.05)the signal transducer and activator of transcription 3(STAT3)signaling pathway by targeting janus kinase 2(JAK2)transcription.The results of functional rescue experiments indicated that STAT3 signaling mediated the regulation of HBP1 on chicken preadipocyte differentiation.In conclusion,HBP1 inhibits chicken preadipocyte differentiation by activating the STAT3 signaling pathway via directly enhancing JAK2 expression.Our findings provided new insights for further analysis of the molecular genetic basis of chicken adipose tissue growth and development.

Higher Cell Viability and Enhanced Sample Quality Following Laser-Assisted Liposuction versus Mechanical Liposuction

Background: Despite the popularity of autologous fat transfer applications, high resorption rates, and consequential volume loss, have been reported. Viable adipocyte content has been defined as a key determinant of fat transfer longevity. Moreover, traces of blood, free oil fat and fibrotic tissue accelerate adipocyte degradation. Objective: To compare the effectiveness of a 1470 nm, radial emitting laser-assisted liposection device to a mechanical liposection device in maintaining adipocyte viability in fat tissue harvests. Methods: Bilateral subcutaneous adipose tissue samples were harvested from ten female patients. Fat was harvested from one side using the LipoLife laser-assisted liposuction device and from the other side with a Byron mechanical aspirator. Samples were visually analyzed and blood:fat ratios and cell viability were determined. Results: Laser-harvested samples separated into two distinct phases, with a negligible blood phase at the bottom (1.1%) and a significant adipose phase at the top (98.9%), containing small, uniform-sized cells, of which 95.7% ± 2.7% proved viable. Mechanically harvested samples separated into blood (18%), adipose (60%) and lipid (22%) phases. The adipose phase contained significant amounts of connective tissue, large adipose tissue fragments, large oil droplets and a mean 79.7% ± 18.3% viable adipocytes. Conclusions: Laser liposuctioning was superior to mechanical liposuctioning, providing both higher cell viability and enhanced sample quality. The 1470 nm diode laser bears the potential of improving long-term clinical outcomes of fat transfer procedures. Improved purity of the harvested sample and heightened preadipocyte content are projected to provide for extended graft longevity.

The composition,function,and regulation of adipose stem and progenitor cells

White adipose tissue(WAT)is a highly plastic organ that plays a central role in regulating whole-body energy metabolism.Adipose stem and progenitor cells(ASPCs)are essential components of the stromal vascular fraction(SVF)of adipose tissue.They give rise to mature adipocytes and play a critical role in maintaining adipose tissue function.However,the molecular heterogeneity and functional diversity of ASPCs are still poorly understood.Recently,single-cell RNA sequencing(scRNA-seq)analysis has identified distinct subtypes of ASPCs in murine and human adipose tissues,providing new insights into the cellular complexity of ASPCs among multiple fat depots.This review summarizes the current knowledge on ASPC populations,including their markers,functions,and regulatory mechanisms.Targeting one or several of these cell populations may ameliorate metabolic disorders by promoting adaptive hyperplastic adipose growth.

Imipramine Inhibits Adipogenic Differentiation in Both 3T3-L1 Preadioocvtes and Mouse Marrow Stromal Cells

Imipramine （IM） has been widely used clinically for the treatment of mental disorders. Its actions on tissues or organs other than the nervous system also need to be understood for its proper clinical use. In this study, the effects of IM on adipogenic differentiation in both 3T3-L1 preadipocytes and mouse marrow stromal cells （MSCs） were investigated. The results showed that fewer adipocytic cells were developed from 3T3-L1 preadipocytes in the presence of 0.001 to 1 μmol/L of IM as compared to control. Similar inhibitory effect was also observed in mouse MSCs. The decrease in the formation of adipocytes was accompanied with significant down-regulation at mRNA expression of the early adipogenic transcription factor, peroxisome proliferator-activated receptor γ2 （PPARγ2）. Western blot analysis further revealed that the protein expression of PPARγ2 was reduced markedly in ceils treated with IM at concentrations of 0.01, 0.1 and 1 μmol/L, suggesting that the suppression on PPAR72 was involved in IM＇s inhibition on MSCs adipogenesis. Moreover, IM at the above concentrations could stimulate the mRNA expression of β2-adrenergic receptor （AR） and β3-AR, which implicated that the effect of IM on adipogenic differentiation was partially mediated by β-ARs. Our results demonstrated for the first time that the conventional antidepressive imipramine exerts accompanied inhibitory effect on adipocyte formation, which may have possible clinical implications.

Identification and validation of novel C/EBPp-regulated genes in preadipocyte proliferation

Background CCAAT/enhancer-binding protein β（C/EBPβ） is required for mitotic clonal expansion （MCE） during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPβ is required for preadipocyte proliferation, and identify new target genes of C/EBPP with chromatin immunoprecipitation （ChlP）-on-chip. Methods Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. Chip was performed at 20 hours after induction with specific anti-C/EBPβ antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.Results A total of 110 candidate genes were identified. BTG3 associated nuclear protein （SMAR1, Banp） and tripartite motif-containing 35 （Hls5, trim35） were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBP3 to the promoter of banp and trim35 was verified by ChlP-PCR. Conclusion C/EBPβ may regulate preadipocyte proliferation through activation of banp and trim35.

Cryo-copolymerization preparation of dextran-hyaluronate based supermacroporous cryogel scaffolds for tissue engineering applications

Dextran-hyaluronate （Dex-HA） based supermacroporous cryogel scaffolds for soft tissue engineering were prepared by free radical cryo-copolymerization of aqueous solutions containing the dextran methacrylate （Dex-MA） and hyaluronate methacrylate （HA-MA） at various macromonomer concentrations under the freezing condition. It was observed that the suitable total concen- tration of macromonomers for the preparation of Dex-HA cryogel scaffold with satisfied properties was 5% （w/w） at the HA-MA concentration of 1% （w/v）, which was then used to produce the test scaffold. The obtained cryogel scaffold with 5% （w/w） macromonomer solution had high water permeability （5.1 × 10 ^-2m2） and high porosity （92.4%）. The pore diameter examined by scanning electron microscopy （SEM） was in a broad range of 50-135 um with the mean pore diameter of 91 um. Furthermore, the cryogel scaffold also had good elastic nature with the elastic modulus of 17.47±1.44 kPa. The culture of 3T3-L1 preadipocyte within the scaffold was investigated and observed by SEM. Cells clustered on the pore walls and grew inside the scaffold indicating the Dex-HA cryogel scaffold could be a promising porous biomaterial for applications in tissue engineering.

Activation of Na^+/H^+ exchange on rat preadipocyte plasma membrane and its role in cell proliferation and differentiation

An in vitro cultured rat perirenal preadipocyte （PA） was established as a model system to investigate the role of the intracellular pH （pHi） and of the Na+/H+ exchanger during PA proliferation and differentiation, pH sensitive probe, 2’ ,7’-bis-（2-carboxyethyl）-5（6）-carboxyfluorescein（BCECF）, was employed to measure the pHi of PA and to determine the Na+/H+ exchange activity. The results showed that there was Na+/H+ exchange activity in the plasma membrane of PA, FCS stimulated DNA synthesis measured by 3H-TdR incorporation, and the activation of Na+/H+ exchanger resulted in phi increase （nearly 0.2 pH unit） within 2 min. Ethyl-isopropyl-amiloride （EIPA）, a specific Na+/H+ exchange inhibitor, inhibited Na+/H+ exchange activity and DNA synthesis. In the absence of serum insulin did not stimulate DNA synthesis but did induce PA differentiation characterized by the appearance of adiposome in the cell and the enhancement of glycerol-3-phosphate dehydrogenase （G3PDHase） activity. Meantime,

Equisetin is an anti-obesity candidate through targeting 11β-HSD1

Obesity is increasingly prevalent globally, searching for therapeutic agents acting on adipose tissue is of great importance. Equisetin(EQST), a meroterpenoid isolated from a marine sponge-derived fungus, has been reported to display antibacterial and antiviral activities. Here, we revealed that EQST displayed anti-obesity effects acting on adipose tissue through inhibiting adipogenesis in vitro and attenuating HFD-induced obesity in mice, doing so without affecting food intake, blood pressure or heart rate.We demonstrated that EQST inhibited the enzyme activity of 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1), a therapeutic target of obesity in adipose tissue. Anti-obesity properties of EQST were all offset by applying excessive 11β-HSD1’s substrates and 11β-HSD1 inhibition through knockdown in vitro or 11β-HSD1 knockout in vivo. In the 11β-HSD1 bypass model constructed by adding excess11β-HSD1 products, EQST’s anti-obesity effects disappeared. Furthermore, EQST directly bond to11β-HSD1 protein and presented remarkable better intensity on 11β-HSD1 inhibition and better efficacy on anti-obesity than known 11β-HSD1 inhibitor. Therefore, EQST can be developed into anti-obesity candidate compound, and this study may provide more clues for developing higher effective 11β-HSD1 inhibitors.

The thermogenic circuit: Regulators of thermogenic competency and differentiation

In mammals,a thermogenic mechanism exists that increases heat production and consumes energy.Recent work has shed light on the cellular and physiological mechanisms that control this thermogenic circuit.Thermogenically active adipocytes,namely brown and closely related beige adipocytes,differentiate from progenitor cells that commit to the thermogenic lineage but can arise from different cellular origins.Thermogenic differentiation shares some features with general adipogenesis,highlighting the critical role that common transcription factors may play in progenitors with divergent fates.However,thermogenic differentiation is also discrete from the common adipogenic program and,excitingly,cells with distinct origins possess thermogenic competency that allows them to differentiate into thermogenically active mature adipocytes.An understanding of this thermogenic differentiation program and the factors that can activate it has led to the development of assays that are able to measure thermogenic activity both indirectly and directly.By combining these assays with appropriate cell models,novel therapeutic approaches to combat obesity and its related metabolic disorders by enhancing the thermogenic circuit can be developed.