A cell transcriptomic profile p ovides insights into adipocytes of porcine mammary gland across development

Background Studying the composition and developmental mechanisms in mammary gland is crucial for healthy growth of newborns. The mammary gland is inherently heterogeneous, and its physiological function dependents on the gene expression of multiple cell types. Most studies focused on epithelial cells, disregarding the role of neighboring adipocytes.Results Here, we constructed the largest transcriptomic dataset of porcine mammary gland cells thus far. The dataset captured 126,829 high-quality nuclei from physiological mammary glands across five developmental stages(d 90 of gestation, G90;d 0 after lactation, L0;d 20 after lactation, L20;2 d post natural involution, PI2;7 d post natural involution, PI7). Seven cell types were identified, including epithelial cells, adipocytes, endothelial cells, fibroblasts cells, immune cells, myoepithelial cells and precursor cells. Our data indicate that mammary glands at different developmental stages have distinct phenotypic and transcriptional signatures. During late gestation(G90), the differentiation and proliferation of adipocytes were inhibited. Meanwhile, partly epithelial cells were completely differentiated. Pseudo-time analysis showed that epithelial cells undergo three stages to achieve lactation, including cellular differentiation, hormone sensing, and metabolic activation. During lactation(L0 and L20), adipocytes area accounts for less than 0.5% of mammary glands. To maintain their own survival, the adipocyte exhibited a poorly differentiated state and a proliferative capacity. Epithelial cells initiate lactation upon hormonal stimulation. After fulfilling lactation mission, their undergo physiological death under high intensity lactation. Interestingly, the physiological dead cells seem to be actively cleared by immune cells via CCL21-ACKR4 pathway. This biological process may be an important mechanism for maintaining homeostasis of the mammary gland. During natural involution(PI2 and PI7), epithelial cell populations dedifferentiate into mesenchymal stem cells to maintain the lactation potential of mammary glands for the next lactation cycle.Conclusion The molecular mechanisms of dedifferentiation, proliferation and redifferentiation of adipocytes and epithelial cells were revealed from late pregnancy to natural involution. This cell transcriptomic profile constitutes an essential reference for future studies in the development and remodeling of the mammary gland at different stages.

The flavonoid glycoside vaccarin inhibits adipogenesis and stimulates lipolysis via Hedgehog signaling in 3T3-L1 adipocytes

Vaccarin,a flavonoid glycoside isolated from Vaccaria segetalis,is non-toxic to 3T3-L1 cells up to concentrations of 200μM.Accordingly,we investigated the effects of this natural product on adipogenesis and lipolysis in 3T3-L1 adipocytes.Our results revealed that vaccarin significantly inhibited lipid accumulation by suppressing the adipogenesis-related transcription factors peroxisome proliferator-activated receptorγ(PPARγ)and the CCAAT/enhancer-binding proteinα(C/EBPα).Specifically,lipid accumulation decreased by up to 27.7±2.7%when 3T3-L1 adipocytes were treated with a 10μM concentration of vaccarin.Mechanistic studies showed that the compound inhibited adipogenesis through activation of the Hedgehog(Hh)signaling pathway and so restoring Smo and Gli1 expression at an early stage of differentiation.In mature 3T3-L1 cells,vaccarin significantly increased the secretion of glycerol into the surrounding medium and thus indicating that it accelerated the degradation of triglycerides.In addition,vaccarin,was shown to enhance lipolysis through stimulation of the transcription levels of lipoprotein lipase,monoglycerides lipase,adipose triacylglyceride lipase,hormone-sensitive lipase and adipose differentiated-related protein.All told,vaccarin suppressed lipid accumulation and enhanced lipolysis during adipocyte differentiation by restoring Hh signaling.As such,it is a phytochemical capable of halting adipocyte hyperplasia and,thereby,ameliorating the effects of obesity.

Lacticaseibacillus paracasei K56 inhibits lipid accumulation in adipocytes by promoting lipolysis

Probiotics crosstalk immunity to improve host glycolipid metabolism,which is a new strategy for obesity.This study aimed to explore the functions of Lacticaseibacillus paracasei K56(K56),in regulating the lipid metabolism of adipocytes and its immune regulation mechanisms.We co-cultured RAW264.7 macrophages with K56,and the K56-stimulated RAW264.7-conditioned medium(K56-CM)was collected and treated with 3T3-L1 pre-adipocytes.The expression of lipid metabolism-related markers of adipocytes,and the content of cytokines in CMs were detected.The results demonstrated that K56-CM promoted the expression of peroxisome proliferator-activated receptor-γ(PPAR-γ),CCAAT/enhancer binding proteinα(C/EBPα),and other adipogenesis-related markers,which resulted in the upregulation of lipolysis markers,such as hormone-sensitive lipase(HSL),adipose triglyceride lipase(ATGL).The activation of lipolysis enhanced the expression of fatty acidβ-oxidation-related and mitochondrial biogenesis-related markers and reduced lipid accumulation in adipocytes.The glycolipid metabolism pattern of K56 may be due to its immunomodulatory characteristics,which stimulated macrophages to secrete fewer TNF-α,thereby promoting the expression of lipolysis-related markers,and TNF-αsynergized with lipase to promote lipolysis.It has not been reported that L.paracasei modulated lipid metabolism via the lipolysis pathway,which suggested that K56 may regulate glycolipid metabolism of the host by maintaining immune homeostasis.

Identification of porcine fast/slow myogenic exosomes and their regulatory effects on lipid accumulation in intramuscular adipocytes

Background Pork quality is affected by the type of muscle fibers, which is closely related to meat color, tenderness and juiciness. Exosomes are tiny vesicles with a diameter of approximately 30–150 nm that are secreted by cells and taken up by recipient cells to mediate communication. Exosome-mediated muscle-fat tissue crosstalk is a newly discovered mechanism that may have an important effect on intramuscular fat deposition and with that on meat quality. Various of adipose tissue-derived exosomes have been discovered and identified, but the identification and function of muscle exosomes, especially porcine fast/slow myotube exosomes, remain unclear. Here, we first isolated and identified exosomes secreted from porcine extensor digitorum longus(EDL) and soleus(SOL), which represent fast and slow muscle, respectively, and further explored their effects on lipid accumulation in longissimus dorsi adipocytes.Results Porcine SOL-derived exosomes(SOL-EXO) and EDL-derived exosomes(EDL-EXO) were first identified and their average particle sizes were approximately 84 nm with double-membrane disc-shapes as observed via transmission electron microscopy and scanning electron microscopy. Moreover, the intramuscular fat content of the SOL was greater than that of the EDL at 180 days of age, because SOL intramuscular adipocytes had a stronger lipid-accumulating capacity than those of the EDL. Raman spectral analysis revealed that SOL-EXO protein content was much greater than that of EDL-EXO. Proteomic sequencing identified 72 proteins that were significantly differentially expressed between SOL-EXO and EDL-EXO, 31 of which were downregulated and 41 of which were upregulated in SOL-EXO.Conclusions Our findings suggest that muscle-fat tissue interactions occur partly via SOL-EXO promoting adipogenic activity of intramuscular adipocytes.

Silicon dioxide nanoparticles inhibit the effects of cold exposure on metabolism and inflammatory responses in brown adipocytes

Objective:Nanoparticles(NPs)in haze are potentially hazardous to health,which is more severe in the winter.Brown adipose tissue(BAT)plays important roles in obesity,insulin resistance,and diabetes.Though the toxicology of NPs has been intensively studied,few studies have been reported on the antagonistic effects between Silicon dioxide(SiO_(2))NPs and cold exposure in brown adipocytes.Materials and methods:We evaluated changes by quantitative real-time reverse-transcriptase polymerase chain reaction(qRT-PCR)on metabolism genes,plasticity genes and the inflammatory responses genes in brown adipocytes in vitro.Results:The expression of adipogenic genes PRDM16,Dio2,PGC-1αand UCP1 was upregulated upon cold exposure(P<0.05),but downregulated by SiO_(2) NPs(P<0.05).The results demonstrated that there was antagonistic effect between SiO_(2) NPs and cold exposure on the plasticity genes and metabolism genes in brown adipocytes,where the main effects of SiO_(2) NPs or cold exposure on the plasticity genes and metabolism genes were significant(P<0.05).Moreover,the levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor(TNF)-αwere upregulated by SiO_(2) NPs or cold exposure(P<0.05).The factorial analysis indicated that there was also antagonistic effect between SiO_(2) NPs and cold exposure on the toxic effects in brown adipocytes,in which the main effects of cold exposure and/or SiO_(2) NPs on the toxic effects were significant(P<0.05).Conclusion:SiO_(2) NPs inhibit the effect of cold exposure on metabolic genes and inflammatory responses genes in brown adipocytes.

Boiogito Increases the Synthesis and Secretion of Adiponectin by Promoting Differentiation in Cultured Human Adipocytes

Boiogito (BOT) ameliorates insulin resistance and diabetes in several animal models;however, the underlying mechanisms for these in vivo effects remain unclear. Thiazolidine derivatives, which are peroxisome proliferator-activated receptor γ (PPARγ) agonists for the treatment of type II diabetes, promote adiponectin production by inducing adipocyte differentiation, thereby reducing insulin resistance. This study aimed to evaluate the effect of BOT on adipocyte differentiation using cultured human visceral preadipocytes (HVPAds) compared with the thiazolidine derivative troglitazone (TRG). We investigated the effects of BOT (0.125 - 1 mg/mL) and TRG (10 μM) on the differentiation of adipocytes treated with or without tumor necrosis factor-α (TNF-α: 5 ng/mL). On day 14 of culture, the following adipocyte differentiation marker levels were measured: intracellular lipids, extracellular (i.e., medium) adiponectin, and intracellular differentiation-related genes (PPARγ, CCAAT/enhancer binding protein, adiponectin, differentiation cluster 36, glucose transporter type 4). BOT and TRG increased factors associated with differentiation including lipid, adiponectin, and differentiation-related gene expression levels compared with the controls. The increases in these differentiation markers were inhibited by the PPARγ antagonist GW9662 (20 μM). Furthermore, TNF-α decreased all differentiation marker levels. The decreases in differentiation markers were inhibited by BOT and TRG;however, these inhibitory effects were blocked by GW9662. The results suggest that BOT increases the synthesis and secretion of adiponectin by promoting differentiation similar to TRG. This study is the first to demonstrate that adipocyte differentiation-promoting activity is a mechanism for the beneficial effects of BOT on diabetes and insulin resistance.

Role of tissue microenvironment resident adipocytes in colon cancer

Colorectal cancer(CRC) is a multifactorial disease characterized by several genetic and epigenetic alterations occurring in epithelial cells. It is increasingly recognized that tumour progression is also regulated by tumour microenvironment(TME). The bidirectional cross-talk between tumour resident adipocytes and cancer cells within TME has been proposed as active contributor to carcinogenesis. Tumour resident adipocytes exhibit an activated phenotype characterized by increased secretion of pro-tumorigenic factors(angiogenic/inflammatory/immune) which contribute to cancer cell proliferation, invasion, neoangiogenesis, evasion of immune surveillance and therapy resistance. Furthermore, adipocytes represent a fuel rich source for increasing energy demand of rapidly proliferating tumour cells. Interestingly, a relationship between obesity and molecular variants in CRC has recently been identified. Whether adipose tissue promotes cancer progression in subsets of molecular phenotypes or whether local tissue adipocytes are involved in inactivation of tumour suppressor genes and/or activation of oncogenes still needs to be explored. This editorial highlights the major findings related to crosstalk between adipocytes and colon cancer cells and how local paracrine interactions may promote cancer progression. Furthermore, we provide future strategies in studying colonic TME which could provide insights in bidirectional cross-talk mechanisms between adipocytes and colonic epithelial cells. This could enable to decipher critical signalling pathways of both early colonic carcinogenesis and cancer progression.

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

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue(WAT) or brown adipose tissue(BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brownlike adipocytes were discovered in WAT. These brownlike adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expressionpattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation(adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.

Anti-diabetic effects of Caulerpa lentillifera:stimulation of insulin secretion in pancreatic β-cells and enhancement of glucose uptake in adipocytes

Objective:To evaluate anti-diabetic effect of Caulrpa kntillifera(C.lentillifera).Methods:The inhibitory effect of C.lentillifera extract on dipeptidyl peptidase-IV and a-glucosidase enzyme was measured in a cell free system.Then,interleukin-1βand interferon-γinduced cell death and insulin secretion were measured in rat insulinoma(RIN)cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA kit,respectively.Glucose uptake and glucose transporter expression were measured by fluorometry and western blotting,using 3T3-Ll adipocytes.Results:C.lentillifera extract significantly decreased dipeptidyl peptidase-IV and a-glucosidase enzyme activities,and effectively inhibited cell death and iNOS expression in interleukin-1βand interfcron-γinduced RIK cells.Furthermore,C.lntillifera extract significantly enhanced insulin secretion in RTN cells and glucose transporter expression and glucose uptake in 3T3-L1adipocytes.Conclusions:Thus,our results suggest that C.lentillifera could be used as a potential antidiabetic agenl.

Human fetal mesenchymal stem cells differentiate into brown and white adipocytes： a role for ERRα in human UCP1 expression

We investigated the ability of fetal mesenchymal stem cells （fMSCs） to differentiate into brown and white adi- pocytes and compared the expression of a number of marker genes and key regulatory factors. We showed that the expression of key adipocyte regulators and markers during differentiation is similar to that in other human and mu- rine adipocyte models, including induction of PPARy2 and FABP4. Notably, we found that the preadipocyte marker, Pref-1, is induced early in differentiation and then declines markedly as the process continues, suggesting that fMSCs first acquire preadipocyte characteristics as they commit to the adipogenic lineage, prior to their differentiation into mature adipocytes. After adipogenic induction, some stem cell isolates differentiated into cells resembling brown adi- pocytes and others into white adipocytes. Detailed investigation of one isolate showed that the novel brown fat-deter- mining factor PRDM16 is expressed both before and after differentiation. Importantly, these cells exhibited elevated basal UCP-1 expression, which was dependent on the activity of the orphan nuclear receptor ERRa, highlighting a novel role for ERRa in human brown fat. Thus fMSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. They also offer invaluable in- sights into the characteristics of human brown fat.

Monascus pilosus-fermented black soybean inhibits lipid accumulation in adipocytes and in high-fat diet-induced obese mice

Objective:To explore the anti-obesity effects and the mechanism of action of Monascus pilosus(M.pilosus)-fermented black soybean(MFBS)extracts(MFBSE)and MFBS powders(MFBSP)in adipocytes and high-fat diet(HFD)-induced obese mice,respectively.Methods:Black soybean was fermented with M.pilosus,and the main constituents in MFBS were analyzed by HPLC analysis.In vitro,MFBSE were examined for anti-adipogenic effects using Oil-Red O staining.In vivo,mice were fed a normal-fat diet(NFD)control,HFD control or HFD containing 1 g/kg MFBSP for 12 weeks,and then body weight gain and tissues weight measured.Real-time PCR and western blot assay were used to determine the mechanism of anti-adipogenic effects.Results:MFBSE inhibited lipid accumulation in 3T3-L1 adipocytes without exerting cell cytotoxicity.MFBSP treatment in HFD-fed mice significantly decreased the body weight gain compared with the HFD control mice.MFBSE and MFBSP treatment resulted in significantly lower mRNA levels of adipogenesis-related genes,such as peroxisome proliferator-activated receptorγ(PPARγ),fatty acid-binding protein 4(FABP4),and fatty acid synthase(FAS),in adipocytes and in white adipose tissue(WAT)of HFD-induced obese mice.Conclusions:These results suggest that the anti-obesity effects of MFBS are elicited by regulating the expression of adipogenesis-related genes in adipocytes and WAT of HFDinduced obese mice.

Comprehensive evaluation of the metabolic effects of porcine CRTC3 overexpression on subcutaneous adipocytes with metabolomic and transcriptomic analyses

Background:Meat quality is largely driven by fat deposition,which is regulated by several genes and signaling pathways.The cyclic adenosine monophosphate(cAMP)-regulated transcriptional coactivator 3(CRTC3)is a coactivator of cAMP response element binding protein(CREB)that mediates the function of protein kinase A(PKA)signaling pathway and is involved in various biological processes including lipid and energy metabolism.However,the effects of CRTC3 on the metabolome and transcriptome of porcine subcutaneous adipocytes have not been studied yet.Here,we tested whether porcine CRTC3 expression would be related to fat deposition in Heigai pigs(a local fatty breed in China)and Duroc×Landrace×Yorkshire(DLY,a lean breed)pigs in vivo.The effects of adenovirus-induced CRTC3 overexpression on the metabolomic and transcriptomic profiles of subcutaneous adipocytes were also determined in vitro by performing mass spectrometry-based metabolomics combined with RNA sequencing(RNA-seq).Results:Porcine CRTC3 expression is associated with fat deposition in vivo.In addition,CRTC3 overexpression increased lipid accumulation and the expression of mature adipocyte-related genes in cultured porcine subcutaneous adipocytes.According to the metabolomic analysis,CRTC3 overexpression induced significant changes in adipocyte lipid,amino acid and nucleotide metabolites in vitro.The RNA-seq analysis suggested that CRTC3 overexpression alters the expression of genes and pathways involved in adipogenesis,fatty acid metabolism and glycerophospholipid metabolism in vitro.Conclusions:We identified significant alterations in the metabolite composition and the expression of genes and pathways involved in lipid metabolism in CRTC3-overexpressing adipocytes.Our results suggest that CRTC3 might play an important regulatory role in lipid metabolism and thus affects lipid accumulation in porcine subcutaneous adipocytes.

Chemical composition and glucose uptake effect on 3T3-L1 adipocytes of Ligustrum lucidum Ait. flowers

Ligustri lucidi Fructus is a traditional Chinese medicine and possesses various bioactivities,including hypoglycemic effect.Ligustrum lucidum Ait flowers are poorly investigated.Thus,we hypothesized that L.lucidum flowers also could have hypoglycemic effect.Chemical composition and glucose uptake effect of L.lucidum flowers on 3T3-L1 adipocytes were investigated.In this study,the components of L.lucidum flowers were investigated by various chromatographic and spectroscopic methods and the effects of L.lucidum flowers on the induction of glucose uptake were investigated by 3T3-L1 adipocytes.Seven compounds were isolated and identified from L.lucidum flowers,including ursolic acid(1),kaempferol-7-O-α-Lrhamnoside(2,KR),β-sitosterol(3),β-daucosterol(4),kaempferitrin(5,KF),10-hydroxy oleuropein(6,HO),and kaempferol-3-O-β-D-glucopyranoside-7-α-L-rhamnopyranoside(7,KGR).The results of glucose uptake showed that total extract(TE),KR,KF and HO from L.lucidum flowers significantly enhanced glucose uptake.This study demonstrated that compounds 2,5-7 were isolated for the first time from this plant and compounds 2 and 7 were isolated for the first time in genus Ligustrum,L.lucidum flowers,KR,KF and HO may possess potential hypoglycemic effect.

High doses of catecholamines activate glucose transport in human adipocytes independently from adrenoceptor stimulation or vanadium addition

BACKGROUND When combined with vanadium salts,catecholamines strongly activate glucose uptake in rat and mouse adipocytes.AIM To test whether catecholamines activate glucose transport in human adipocytes.METHODS The uptake of 2-deoxyglucose(2-DG)was measured in adipocytes isolated from pieces of abdominal subcutaneous tissue removed from women undergoing reconstructive surgery.Pharmacological approaches with amine oxidase inhibitors,adrenoreceptor agonists and antioxidants were performed to unravel the mechanisms of action of noradrenaline or adrenaline(also named epinephrine).RESULTS In human adipocytes,45-min incubation with 100μmol/L adrenaline or noradrenaline activated 2-DG uptake up to more than one-third of the maximal response to insulin.This stimulation was not reproduced with millimolar doses of dopamine or serotonin and was not enhanced by addition of vanadate to the incubation medium.Among various natural amines and adrenergic agonists tested,no other molecule was more efficient than adrenaline and noradrenaline in stimulating 2-DG uptake.The effect of the catecholamines was not impaired by pargyline and semicarbazide,contrarily to that of benzylamine or methylamine,which are recognized substrates of semicarbazide-sensitive amine oxidase.Hydrogen peroxide at 1 mmol/L activated hexose uptake but not pyrocatechol or benzoquinone,and only the former was potentiated by vanadate.Catalase and the phosphoinositide 3-kinase inhibitor wortmannin inhibited adrenaline-induced activation of 2-DG uptake.CONCLUSION High doses of catecholamines exert insulin-like actions on glucose transport in human adipocytes.At submillimolar doses,vanadium did not enhance this catecholamine activation of glucose transport.Consequently,this dismantles our previous suggestion to combine the metal ion with catecholamines to improve the benefit/risk ratio of vanadium-based antidiabetic approaches.

Human induced pluripotent stem cells: A new source for brown and white adipocytes

Mesenchymal stem cells(MSCs) derived from human induced pluripotent stem cells(hiPSCs) provide a novel source for generating adipocytes, thus opening new avenues for fundamental research and clinical medicine. We present the adipogenic potential of hiPSCs and the various methods to derive hiPSC-MSCs. We discuss the main characteristic of hiPSC-MSCs, which is their low adipogenic capacity as compared to adult-MSCs. Finally, we propose several hypotheses to explanation this feature, underlying a potential critical role of the micro-environment. We favour the hypothesis that the range of factors or culture conditions required to induce adipocyte differentiation of MSCs derived from adult tissues and from embryonic-like cells could differ.

Short-term effects of obestatin on hexose uptake and triacylglycerol breakdown in human subcutaneous adipocytes

AIM To study complete dose-dependent effects of obestatin on lipolytic and glucose transport activities in human adipocyte preparations highly responsive to insulin.METHODS Adipocytes were prepared by liberase digestion from subcutaneous abdominal adipose tissue obtained from overweight subjects undergoing plastic surgery. The index of lipolytic activity was the glycerol released in the incubation medium, while glucose transport was assessed by [~3H]-2-deoxyglucose uptake assay.RESULTS When tested from 0.1 nmol/L to 1 μmol/L, obestatin did not stimulate glycerol release; it did not inhibit the lipolytic effect of isoprenaline and did not alter the insulin antilipolytic effect. Obestatin hardly activated glucose transport at 1 μmol/L only. Moreover, the obestatin stimulation effect was clearly lower than the threefold increase induced by insulin 100 nmol/L.CONCLUSION Low doses of obestatin cannot directly influence lipolysis and glucose uptake in human fat cells.

Effect of Ichnocarpus frutescens (L.) R.Br.hexane extract on preadipocytes viability and lipid accumulation in 3T3-L1 cells

Objective:To investigate the crude extracts of Ichnacarpus frutescens(I.frutescens) for antiobesity effect.Methods:Leaves of I.frutescens were sequentially extracted with hexane,ethyl acetate, and methanol and their effect on viability of 3T3-L1 preadipocytes were evaluated.Based on this the apoptosis on preadipocytes was confirmed by DMA fragmentation and LDH(Lactate dehydrogenase) leakage assays.Anti-adipogenesis was performed by oil red O(ORO) staining and free glycerol release in the medium of differentiated adipocytes.Results:The hexane extract of I.frutescens(IFHE) inhibited cell viability in a time- and dose-related manner.An increased release of LDH.as a marker of membrane integrity,was observed at a dose of 200μg/mL The discontinuous DMA fragments on agarose gel electrophoresis showed the apoptotic effect of the IFHE.Morphological observations of cells stained with ORG showed a decrease in cellular lipid content at the concentrations tested compared to the induced control cells.In the experiment of lipolytic activity,treatment with IFHE enhanced glycerol secretion with the rates of approximately 28%.55%,and 46%at the concentrations of 100,200 and 300μg/mL,respectively.Conclusions: The observed properties clearly revealed the medicinal property of I.frulescens in the treatment of obesity.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

Photoactivation of GLUT4 translocation promotes glucose uptake via PI3-K/Akt2 signaling in 3T3-L1 adipocytes

Insulin resistance is a hallmark of the metabolic syndrome and type 2 diabetes.Dysfunction of PI-3K/Akt signaling was involved in insulin resistance.Glucose transporter 4(GLUT4)is a keyfactor for glucose uptake in muscle and adipose tissues,which is closely regulated by Pi-3K/Aktsignaling in response to insulin treatment.Low-power laser irradiation(LPLI)has been shown toregulate various physiological processes and induce the synthesis or release of multiple moleculessuch as growth factors,which(especially red and near infrared light)is mainly through theactivation of mitochondrial respiratory chain and the initiation of intracellular signaling path-ways.Nevertheless,it is unclear whether LPLI could promote glucose uptake through activationof PI-3K/Akt/GLUT4 signaling in 3T3L-1 adipocytes.In this study,we investigated how LPLIpromoted glucose uptake through activation of PI-3K/Akt/GLUT4 signaling path way.Here,we showed that GLUT4 was localized to the Golgi apparatus and translocated from cytoplasm tocytomembrane upon LPLI treatment in 3T3L-1 adipocytes,which enhanced glucose uptake.Moreover,we found that glucose uptake was mediated by the PI3-K/Akt2 signaling,but notAkt1 upon LPLI treatment with Akt isoforms gene silence and PI3-K/Akt inhibitors.Collec-tively,our results indicate that PI3-K/Akt2/GLUT4 signaling act as the key regulators forimprovement of glucose uptake under LPLI treatment in 3T3L-i adipocytes.More importantly,our findings suggest that activation of PI3-K/Akt2/GLUT4 signaling by LPLI may provideguidance in practical applications for promotion of glucose uptake in insulin-resistant adiposetissue.