Differentiation Of Human Preadipocytes Research Articles

Endothelin 3/EDNRB signaling induces thermogenic differentiation of white adipose tissue

Thermogenic adipose tissue, consisting of brown and beige fat, regulates nutrient utilization and energy metabolism. Human brown fat is relatively scarce and decreases with obesity and aging. Hence, inducing thermogenic differentiation of white fat offers an attractive way to enhance whole-body metabolic capacity. Here, we show the role of endothelin 3 (EDN3) and endothelin receptor type B (EDNRB) in promoting the browning of white adipose tissue (WAT). EDNRB overexpression stimulates thermogenic differentiation of human white preadipocytes through cAMP-EPAC1-ERK activation. In mice, cold induces the expression of EDN3 and EDNRB in WAT. Deletion of EDNRB in adipose progenitor cells impairs cold-induced beige adipocyte formation in WAT, leading to excessive weight gain, glucose intolerance, and insulin resistance upon high-fat feeding. Injection of EDN3 into WAT promotes browning and improved whole-body glucose metabolism. The findings shed light on the mechanism of WAT browning and offer potential therapeutics for obesity and metabolic disorders.

SAT652 The Expression Of The Immunoproteasome Subunit Psmb9 Is Related To Obesity And The Differentiation Of Human Preadipocytes

Abstract Disclosure: Y. Cui: None. X. Zhang: None. L. You: None. H. Zhong: None. X. Cui: None. C. Ji: None. Obesity and its associated chronic diseases, such as type 2 diabetes mellitus, hypertension, and coronary artery disease, afflicting adults and children globally. PSMB9, a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides, is hypothesized to be associated with obesity. To investigate this association, we examined PSMB9 expression in primary mature adipocytes from obese and normal individuals. LC-MS/MS-PRM was used to quantify PSMB9 content in primary mature adipocytes, revealing a negative correlation between PSMB9 content and obesity while positively correlated with high-density lipoprotein levels. Furthermore, we examined the effect of PSMB9 on human preadipocyte differentiation. During the early stages of differentiation, the expression of PSMB9 was at its highest level, decreasing as preadipocytes matured. Overexpression of PSMB9 significantly inhibited preadipocyte differentiation, resulting in reduced lipid droplets and decreased expression levels of classical differentiation markers such as FABP4, C/EBPα, and PPARγ. These findings suggest that PSMB9 could be a potential therapeutic target for preventing and treating obesity and metabolic disorders. In conclusion, our study provides insights into the role of the immunoproteasome in obesity and highlights the importance of further exploration of this pathway. Presentation: Saturday, June 17, 2023

Human Preadipocytes Differentiated under Hypoxia following PCB126 Exposure during Proliferation: Effects on Differentiation, Glucose Uptake and Adipokine Profile.

Persistent organic pollutants (POPs) accumulation and hypoxia are two factors proposed to adversely alter adipose tissue (AT) functions in the context of excess adiposity. Studies have shown that preadipocytes exposure to dioxin and dioxin-like POPs have the greatest deleterious impact on rodent and immortalized human preadipocyte differentiation, but evidence on human preadipocytes is lacking. Additionally, hypoxia is known to strongly interfere with the dioxin-response pathway. Therefore, we tested the effects of pre-differentiation polychlorinated biphenyl (PCB)126 exposure at 10 µM for 3 days and subsequent differentiation under hypoxia on human subcutaneous adipocytes (hSA) differentiation, glucose uptake and expression of selected metabolism- and inflammation-related genes. Pre-differentiation PCB126 exposure lowered the adenosine triphosphate (ATP) content, glucose uptake and leptin expression of mature adipocytes but had limited effects on differentiation under normoxia (21% O2). Under hypoxia (3% O2), preadipocytes ability to differentiate was significantly reduced as reflected by significant decreased lipid accumulation and downregulation of key adipocyte genes such as peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin. Hypoxia increased glucose uptake and glucose transporter 1 (GLUT1) expression but abolished the adipocytes insulin response and GLUT4 expression. The expression of pro-inflammatory adipokine interleukin-6 (IL-6) was slightly increased by both PCB126 and hypoxia, while IL-8 expression was significantly increased only following the PCB126-hypoxia sequence. These observations suggest that PCB126 does not affect human preadipocyte differentiation, but does affect the subsequent adipocytes population, as reflected by lower ATP levels and absolute glucose uptake. On the other hand, PCB126 and hypoxia exert additive effects on AT inflammation, an important player in the development of chronic diseases such as type 2 diabetes and cardiovascular diseases.

Cross-tissue omics analysis discovers ten adipose genes encoding secreted proteins in obesity-related non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a fast-growing, underdiagnosed, epidemic. We hypothesise that obesity-related inflammation compromises adipose tissue functions, preventing efficient fat storage, and thus driving ectopic fat accumulation into the liver. To identify adipose-based mechanisms and potential serum biomarker candidates (SBCs) for NAFLD, we utilise dual-tissue RNA-sequencing (RNA-seq) data in adipose tissue and liver, paired with histology-based NAFLD diagnosis, from the same individuals in a cohort of obese individuals. We first scan for genes that are differentially expressed (DE) for NAFLD in obese individuals' subcutaneous adipose tissue but not in their liver; encode proteins secreted to serum; and show preferential adipose expression. Then the identified genes are filtered to key adipose-origin NAFLD genes by best subset analysis, knockdown experiments during human preadipocyte differentiation, recombinant protein treatment experiments in human liver HepG2 cells, and genetic analysis. We discover a set of genes, including 10 SBCs, that may modulate NAFLD pathogenesis by impacting adipose tissue function. Based on best subset analysis, we further follow-up on two SBCs CCDC80 and SOD3 by knockdown in human preadipocytes and subsequent differentiation experiments, which show that they modulate crucial adipogenesis genes, LPL, SREBPF1, and LEP. We also show that treatment of the liver HepG2 cells with the CCDC80 and SOD3 recombinant proteins impacts genes related to steatosis and lipid processing, including PPARA, NFE2L2, and RNF128. Finally, utilizing the adipose NAFLD DE gene cis-regulatory variants associated with serum triglycerides (TGs) in extensive genome-wide association studies (GWASs), we demonstrate a unidirectional effect of serum TGs on NAFLD with Mendelian Randomization (MR) analysis. We also demonstrate that a single SNP regulating one of the SBC genes, rs2845885, produces a significant MR result by itself. This supports the conclusion that genetically regulated adipose expression of the NAFLD DE genes may contribute to NAFLD through changes in serum TG levels. Our results from the dual-tissue transcriptomics screening improve the understanding of obesity-related NAFLD by providing a targeted set of 10 adipose tissue-active genes as new serum biomarker candidates for the currently grossly underdiagnosed fatty liver disease. The work was supported by NIH grants R01HG010505 and R01DK132775. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The KOBS study (J. P.) was supported by the Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2019), and the Academy of Finland grant (Contract no. 138006). This study was funded by the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant No. 802825 to M. U. K.). K. H. P. was funded by the Academy of Finland (grant numbers 272376, 266286, 314383, and 335443), the Finnish Medical Foundation, Gyllenberg Foundation, Novo Nordisk Foundation (grant numbers NNF10OC1013354, NNF17OC0027232, and NNF20OC0060547), Finnish Diabetes Research Foundation, Finnish Foundation for Cardiovascular Research, University of Helsinki, and Helsinki University Hospital and Government Research Funds. I. S. was funded by the Instrumentarium Science Foundation. Personal grants to U. T. A. were received from the Matti and Vappu Maukonen Foundation, Ella och Georg Ehrnrooths Stiftelse and the Finnish Foundation for Cardiovascular Research.

Optical diffraction tomography and Raman spectroscopy reveal distinct cellular phenotypes during white and brown adipocyte differentiation

White adipose tissue (WAT) and brown adipose tissue (BAT) are the primary types of fats in humans, and they play prominent roles in energy storage and thermogenesis, respectively. While the mechanisms of terminal adipogenesis are well understood, much remains unknown about the early stages of adipogenic differentiation. Label-free approaches, such as optical diffraction tomography (ODT) and Raman spectroscopy, offer the ability to retrieve morphological and molecular information at the single cell level without the negative effects of photobleaching and system perturbation due to introduction of fluorophores. In this study, we employed 3D ODT and Raman spectroscopy to gain deeper insights into the early stages of differentiation of human white preadipocytes (HWPs) and human brown preadipocytes (HBPs). We utilized ODT to retrieve morphological information, including cell dry mass and lipid mass, and Raman spectroscopy to obtain molecular information about lipids. Our findings reveal that HWPs and HBPs undergo dynamic and differential changes during the differentiation process. Notably, we found that HBPs accumulated lipids more rapidly and had a higher lipid mass than HWPs. Additionally, both cell types experienced an increase and subsequent decrease in cell dry mass during the first seven days, followed by an increase after day 7, which we attribute to the transformation of adipogenic precursors in the early stages. Finally, HBPs had higher lipid unsaturation levels than HWPs for the same differentiation timepoints. The insights gained from our study provide crucial contributions towards the advancement of new therapies for obesity and related diseases.

Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes.

Obesity is one of the most prevalent chronic metabolic diseases, and induction of apoptosis in preadipocytes and adipocytes is a potential strategy to treat obesity. Celastrol represents one of the most robust anti-obesity phytochemicals so far, yet its direct binding target remains elusive. Here, we determined that celastrol could induce apoptosis in preadipocytes via mitochondrial mediated pathway. Further study clarified that celastrol inhibited the fusion of autophagosome and lysosome to prohibit autophagy, leading to cell apoptosis. By conducting virtual screening and genetic manipulation, we verified that overexpression of VAMP7 and RAB7 could block the effects of celastrol on inhibiting autophagy and inducing apoptosis. The Surface Plasmon Resonance study confirmed the direct binding of celastrol with VAMP7 and RAB7. The functional study illustrated the inhibition of RAB7 GTPase activity after celastrol treatment. Moreover, celastrol induced comparable apoptosis in murine epididymal adipose tissue, human preadipocytes and adipocytes, but not in human hepatocytes. An inhibitory effect on differentiation of human primary visceral preadipocytes was also observed. In conclusion, celastrol exhibited inhibitory effect of autophagy via direct binding with VAMP7 and RAB7, leading to an increase in preadipocytes apoptosis. These results advance our understanding in the potential application of celastrol in treating obesity.

Methionine sulfoxide suppresses adipogenic differentiation by regulating the mitogen-activated protein kinase signaling pathway.

In this study, methionine sulfoxide (MetO) was identified as an active metabolite that suppresses adipogenesis after screening obese individuals versus the normal population. MetO suppressed the gene and protein expression of CCAAT/enhancer binding protein (C/EBP) α, adipocyte fatty acid binding protein 4 (FABP4), and the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) during human preadipocyte (HPA) differentiation. Adipogenesis decreased following MetO treatment; however, the preadipocyte number, proliferation, and apoptosis were unaffected. The activity of phosphorylated extracellular signal-related kinase (P-ERK) of the mitogen-activated protein kinase (MAPK) pathway was significantly inhibited in HPA after MetO treatment. Furthermore, treatment of preadipocytes with the selective P-ERK1/2 agonist Ro 67-7476 abolished the effect of MetO against adipogenesis suggesting that MetO function is dependent on the MAPK pathway. The mechanistic insights of adipogenesis suppression by MetO presented in this study shows its potential as an antiobesity drug.

Arnica montana L. extract containing 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin accelerates growth and differentiation of human subcutaneous preadipocytes and leads volumizing of skin.

An important factor in the aging of the face is a reduction in the volume of adipose tissue. This reduction in adipose tissue contributes to decreased skin elasticity, which is also part of the aging process. Overall, these lead to wrinkle formation. Fat injection is a common means of addressing this issue and is used to reduce the effects of aging on the face and to increase the fullness of the lips and breasts. However, fat injection is an invasive surgical procedure. This study aimed to discover novel cosmetic ingredients that increase the volume of subcutaneous (pre)adipocytes to create the appearance of more youthful skin. We focused on the number of subcutaneous preadipocytes and the accumulation of lipid droplets. To discover natural ingredients that increase both of these, extracts of 380 natural products were prepared and screened for their effects on both growth and differentiation (i.e., lipid droplet accumulation) of human subcutaneous preadipocytes. One extract was found to have the desired effects, and this was further studied to determine the active compounds. We then evaluated its efficacy in a human clinical study. We found that Arnica montana L. flower extract (AFE) accelerates both the growth and the differentiation of human subcutaneous preadipocytes. AFE was found to significantly increase the volume of adipocyte spheroids. The active compounds 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin were found to be responsible for the effects of AFE on preadipocytes. In a human clinical study, gels containing 1% AFE successfully enhanced the volume of the lips and face with reduction of wrinkles with no adverse reactions. This is the first report to demonstrate that AFE and the included compounds, 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin, act on preadipocytes. AFE would be ideal for use in products that plump the face to reduce wrinkles and create a more youthful appearance.

Untapped Pharmaceutical Potential of 4,5,4'-Trihydroxy-3,3'-dimethoxybibenzyl for Regulating Obesity: A Cell-Based Study with a Focus on Terminal Differentiation in Adipogenesis.

Obesity and its global prevalence has become a threat to human health, while its pharmacotherapy via the application of natural products is still underdeveloped. Here, we probed how 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (TDB) derived from an orchid (Dendrobium ellipsophyllum) could exert its roles on the differentiation and function of murine (3T3-L1) and human (PCS-210-010) pre-adipocytes and offer some implications to modulate obesity. Cytotoxic effects of TDB on adipocytes were 2-fold lower than those detected with pre-adipocytes, and no significant difference was detected in cytotoxic profiles between both cell lineages. TDB in a dose-dependent manner decreased cellular lipid accumulation and enhanced lipolysis of both cell lines assessed at early differentiation and during maturation. Underlining molecular mechanisms proved that TBD paused the cell cycle progression by regulating inducers and inhibitors in mitotic clonal expansion, leading to growth arrest of pre-adipocytes at the G0/G1 phase. The compound also governed adipocyte differentiation by repressing expressions of crucial adipogenic regulators and effectors through deactivating the AKT/GSK-3β signaling pathway and activating the AMPK-ACC pathway. To this end, TDB has shown its pharmaceutical potential for modulating adipocyte development and function, and it would be a promising candidate for further assessments as a therapeutic agent to defeat obesity.

Amniotic growth factors enhanced human pre-adipocyte cell viability and differentiation under hypoxia.

One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods are time‐consuming, complex, and demand significant technical skill. In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin‐ferulic acid (Gtn‐FA) hydrogel with variable oxygen concentration and treated with amniotic membrane‐derived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O2 concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy. The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT‐qPCR. The expression of genes: Pref‐1, C/EBP β, C/EBP α, PPAR‐ƴ, SLC2A4, and VEGF‐A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre‐adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR‐ƴ (p < .05), SLC2A4, and VEGF‐A (p < .05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis.

Excess glucocorticoid exposure contributes to adipose tissue fibrosis which involves macrophage interaction with adipose precursor cells

Chronic exposure to elevated glucocorticoid levels, as seen in patients with Cushing’s syndrome, can induce adipose tissue fibrosis. Macrophages play a pivotal role in adipose tissue remodelling. We used the synthetic glucocorticoid analogue dexamethasone to address glucocorticoid effects on adipose tissue fibrosis, in particular involving macrophage to preadipocyte communication. We analysed the direct effects of dexamethasone at a supra-physiological level, 0.3 µM, on gene expression of pro-fibrotic markers in human subcutaneous adipose tissue. The effects of dexamethasone on the differentiation of human SGBS preadipocytes were assessed in the presence or absence of THP1-macrophages or macrophage-conditioned medium. We measured the expression of different pro-fibrotic factors, including α-smooth muscle actin gene (ACTA2) and protein (α-SMA). Dexamethasone increased the expression of pro-fibrotic genes, e.g. CTGF, COL6A3, FN1, in adipose tissue. Macrophages abolished preadipocyte differentiation and increased the expression of the ACTA2 gene and α-SMA protein in preadipocytes after differentiation. Exposure to dexamethasone during differentiation reduced adipogenesis in preadipocytes, and elevated the expression of pro-fibrotic genes. Moreover, dexamethasone added together with macrophages further increased ACTA2 and α-SMA expression in preadipocytes, making them more myofibroblast-like. Cells differentiated in the presence of conditioned media from macrophages pretreated with or without dexamethasone had a higher expression of profibrotic genes compared to control cells. Our data suggest that macrophages promote adipose tissue fibrosis by directly interfering with preadipocyte differentiation and stimulating gene expression of pro-fibrotic factors. Excess glucocorticoid exposure also has pro-fibrotic effect on adipose tissue, but this requires the presence of macrophages.

Activation of the aryl hydrocarbon receptor by clozapine induces preadipocyte differentiation and contributes to endothelial dysfunction

Background:The superior therapeutic benefit of clozapine is often associated with metabolic disruptions as obesity, insulin resistance, tachycardia, higher blood pressure, and even hypertension.Aims:These adverse vascular/ metabolic events under clozapine are similar to those caused by polycyclic aromatic hydrocarbons (PAHs), and clozapine shows structural similarity to well-known ligands of the aryl hydrocarbon receptor (AhR). Therefore, we speculated that the side effects caused by clozapine might rely on AhR signaling.Methods:We examined clozapine-induced AhR activation by luciferase reporter assays in hepatoma HepG2 cells and we proved upregulation of the prototypical AhR target gene Cyp1A1 by realtime-PCR (RT-PCR) analysis and enzyme activity. Next we studied the physiological role of AhR in clozapine’s effects on human preadipocyte differentiation and on vasodilatation by myography in wild-type and AhR-/- mice.Results:In contrast to other antipsychotic drugs (APDs), clozapine triggered AhR activation and Cyp1A1 expression in HepG2 cells and adipocytes. Clozapine induced adipogenesis via AhR signaling. After PGF2α-induced constriction of mouse aortic rings, clozapine strongly reduced the maximal vasorelaxation under acetylcholine in rings from wild-type mice, but only slightly in rings from AhR-/- mice. The reduction was also prevented by pretreatment with the AhR antagonist CH-223191.Conclusion:Identification of clozapine as a ligand for the AhR opens new perspectives to explain common clozapine therapy-associated adverse effects at the molecular level.

Alternatives for the worse: Molecular insights into adverse effects of bisphenol a and substitutes during human adipocyte differentiation.

Bisphenol A (BPA), which is used in a variety of consumer-related plastic products, was reported to cause adverse effects, including disruption of adipocyte differentiation, interference with obesity mechanisms, and impairment of insulin- and glucose homeostasis. Substitute compounds are increasingly emerging but are not sufficiently investigated.We aimed to investigate the mode of action of BPA and four of its substitutes during the differentiation of human preadipocytes to adipocytes and their molecular interaction with peroxisome proliferator-activated receptor γ (PPARγ), a pivotal regulator of adipogenesis.Binding and effective biological activation of PPARγ were investigated by surface plasmon resonance and reporter gene assay, respectively. Human preadipocytes were continuously exposed to BPA, BPS, BPB, BPF, BPAF, and the PPARγ-antagonist GW9662. After 12days of differentiation, lipid production was quantified via Oil Red O staining, and global protein profiles were assessed using LC-MS/MS-based proteomics. All tested bisphenols bound to human PPARγ with similar efficacy as the natural ligand 15d-PGJ2in vitroand provoked an antagonistic effect on PPARγ in the reporter gene assay at non-cytotoxic concentrations. During the differentiation of human preadipocytes, all bisphenols decreased lipid production. Global proteomics displayed a down-regulation of adipogenesis and metabolic pathways, similar to GW9662. Interestingly, pro-inflammatory pathways were up-regulated, MCP1 release was increased, and adiponectin decreased. pAKT/AKT ratios revealed significantly reduced insulin sensitivity by BPA, BPB, and BPS upon insulin stimulation.Thus, our results show that not only BPA but also its substitutes disrupt crucial metabolic functions and insulin signaling in adipocytes under low, environmentally relevant concentrations. This effect, mediated through inhibition of PPARγ, may promote hypertrophy of adipose tissue and increase the risk of developing metabolic syndrome, including insulin resistance.

Serotonin 5-HT2A receptor activity mediates adipocyte differentiation through control of adipogenic gene expression

Serotonin 5-HT2 receptors are expressed in many tissues and play important roles in biological processes. Although the 5-HT2A receptor is primarily known for its role in central nervous system, it is also expressed in peripheral tissues. We have found that 5-HT2A receptor antagonists inhibit human subcutaneous primary adipocyte differentiation. We also show that siRNA knockdown of the 5-HT2A receptor blocks differentiation. Using gene expression analysis in combination with receptor antagonists we found that activity of 5-HT2A receptors is necessary very early in the differentiation process to mediate expression of adipogenic genes, including peroxisome proliferator-activated receptor gamma (ppar-γ), adipocyte protein 2 (aP2), adiponectin, and serine/threonine-protein kinase 1 (sgk1). We show here for the first time that 5-HT2A receptor activity is necessary for differentiation of human primary subcutaneous preadipocytes to adipocytes, and that 5-HT2A receptor activity mediates key genes related to adipogenesis during this process. Importantly, this work contributes to a greater understanding of the adipocyte differentiation process, as well as to the role of 5-HT2A receptors in peripheral tissues, and may be relevant to the development of novel therapeutic strategies targeting this receptor for the treatment of obesity related diseases.

Extracellular cystine influences human preadipocyte differentiation and correlates with fat mass in healthy adults

Plasma cysteine is associated with human obesity, but it is unknown whether this is mediated by reduced, disulfide (cystine and mixed-disulfides) or protein-bound (bCys) fractions. We investigated which cysteine fractions are associated with adiposity in vivo and if a relevant fraction influences human adipogenesis in vitro. In the current study, plasma cysteine fractions were correlated with body fat mass in 35 adults. Strong positive correlations with fat mass were observed for cystine and mixed disulfides (r ≥ 0.61, P < 0.001), but not the quantitatively major form, bCys. Primary human preadipocytes were differentiated in media containing cystine concentrations varying from 10–50 μM, a range similar to that in plasma. Increasing extracellular cystine (10–50 μM) enhanced mRNA expression of PPARG2 (to sixfold), PPARG1, PLIN1, SCD1 and CDO1 (P = 0.042– < 0.001). Adipocyte lipid accumulation and lipid-droplet size showed dose-dependent increases from lowest to highest cystine concentrations (P < 0.001), and the malonedialdehyde/total antioxidant capacity increased, suggesting increased oxidative stress. In conclusion, increased cystine concentrations, within the physiological range, are positively associated with both fat mass in healthy adults and human adipogenic differentiation in vitro. The potential role of cystine as a modifiable factor regulating human adipocyte turnover and metabolism deserves further study.

Chronic and Intermittent Hyperglycemia Modulates Expression of Key Molecules of PI3K/AKT Pathway in Differentiating Human Visceral Adipocytes.

Background: Due to its prominence in the regulation of metabolism and inflammation, adipose tissue is a major target to investigate alterations in insulin action. This hormone activates PI3K/AKT pathway which is essential for glucose homeostasis, cell differentiation, and proliferation in insulin-sensitive tissues, like adipose tissue. The aim of this work was to evaluate the impact of chronic and intermittent high glucose on the expression of biomolecules of insulin signaling pathway during the differentiation and maturation of human visceral preadipocytes. Methods: Human visceral preadipocytes (HPA-V) cells were treated with high glucose (30 mM)during the proliferation and/or differentiation and/or maturation stage. The level of mRNA (by Real-Time PCR) and protein (by Elisa tests) expression of IRS1, PI3K, PTEN, AKT2, and GLUT4 was examined after each culture stage. Furthermore, we investigated whether miR-29a-3p, miR-143-3p, miR-152-3p, miR-186-5p, miR-370-3p, and miR-374b-5p may affect the expression of biomolecules of the insulin signaling pathway. Results: Both chronic and intermittent hyperglycemia affects insulin signaling in visceral pre/adipocytes by upregulation of analyzed PI3K/AKT pathway molecules. Both mRNA and protein expression level is more dependent on stage-specific events than the length of the period of high glucose exposure. What is more, miRs expression changes seem to be involved in PI3K/AKT expression regulation in response to hyperglycemic stimulation.

Src family kinases involved in the differentiation of human preadipocytes

BackgroundObesity is characterized by the excess accumulation of white adipose tissue (WAT). Src family kinases (SFKs) are non-receptor tyrosine kinases consisting of eight members (SRC, FYN, YES1, HCK, LCK, LYN, FGR and BLK) that have been studied extensively in mammalian cells. Although individual members in murine cells provide some clues that are associated with the regulation of adipogenesis, the specific role of this family in adipocyte differentiation has rarely been assessed, especially in human adipocytes. MethodsHerein, we first explored the expression profiles of SFKs during human preadipocyte differentiation. Then, we used the pyrazolo-pyrimidinyl-amine compound PP1, a potent SFK inhibitor, to evaluate the function of SFKs during adipocyte differentiation. Furthermore, we adopted a loss-of-function strategy with siRNAs to determine the role of FGR in adipocyte differentiation. ResultsHere, we found that SRC, FYN, YES1, LYN and FGR were expressed in human preadipocytes and induced after the initiation of differentiation. Furthermore, the SFK inhibitor PP1 suppressed adipocyte differentiation. We also found that PP1 significantly suppressed the SFK activity in preadipocytes and decreased the expression of adipogenic genes in early and late differentiation. Given that FGR exhibited the most expression enhancement in mature adipocytes, we focused on FGR and found that its knockdown reduced lipid accumulation and adipogenic gene expression. ConclusionsCollectively, these findings suggest that SFKs, especially FGR, are involved in the differentiation of human preadipocytes. Our results lay a foundation for further understanding the role of SFKs in adipocyte differentiation and provide new clues for anti-obesity therapies.

Pathogenic Microenvironment from Diabetic-Obese Visceral and Subcutaneous Adipocytes Activating Differentiation of Human Healthy Preadipocytes Increases Intracellular Fat, Effect of the Apocarotenoid Crocetin.

In diabetes mellitus type 2 (DM2), developed obesity is referred to as diabesity. Implementation of a healthy diet, such as the Mediterranean, prevents diabesity. Saffron is frequently used in this diet because of its bioactive components, such as crocetin (CCT), exhibit healthful properties. It is well known that obesity, defined as an excessive accumulation of fat, leads to cardiometabolic pathology through adiposopathy or hypertrophic growth of adipose tissue (AT).This is related to an impaired adipogenic process or death of adipocytes by obesogenic signals. We aimed to evaluate the effect of the pathogenic microenvironment and CCT, activating differentiation of healthy preadipocytes (PA). For this, we used human cryopreserved PA from visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) depots obtained from healthy and obese-DM2 donors. We studied the effect of a metabolically detrimental (diabesogenic) environment, generated by obese-DM2 adipocytes from VAT (VdDM) or SAT (SdDM), on the viability and accumulation of intracellular fat of adipocytes differentiated from healthy PA, in the presence or absence of CCT (1 or 10 μM). Intracellular fat was quantified by Oil Red O staining. Cytotoxicity was measured using the MTT assay. Our results showed that diabesogenic conditions induce cytotoxicity and provide a proadipogenic environment only for visceral PA. CCT at 10 μM acted as an antiadipogenic and cytoprotective compound.

Lysophosphatidic Acid Receptor Agonism: Discovery of Potent Nonlipid Benzofuran Ethanolamine Structures

Lysophosphatidic acid (LPA) is the natural ligand for two phylogenetically distinct families of receptors (LPA1-3, LPA4-6) whose pathways control a variety of physiologic and pathophysiological responses. Identifying the benefit of balanced activation/repression of LPA receptors has always been a challenge because of the high lability of LPA and the limited availability of selective and/or stable agonists. In this study, we document the discovery of small benzofuran ethanolamine derivatives (called CpX and CpY) behaving as LPA1-3 agonists. Initially found as rabbit urethra contracting agents, their elusive receptors were identified from [35S]GTPγS-binding and β-arrestin2 recruitment investigations and then confirmed by [3H]CpX binding studies (urethra, hLPA1-2 membranes). Both compounds induced a calcium response in hLPA1-3 cells within a range of 0.4-1.5-log lower potency as compared with LPA. The contractions of rabbit urethra strips induced by these compounds perfectly matched binding affinities with values reaching the two-digit nanomolar level. The antagonist, KI16425, dose-dependently antagonized CpX-induced contractions in agreement with its affinity profile (LPA1≥LPA3>>LPA2). The most potent agonist, CpY, doubled intraurethral pressure in anesthetized female rats at 3 µg/kg i.v. Alternatively, CpX was shown to inhibit human preadipocyte differentiation, a process totally reversed by KI16425. Together with original molecular docking data, these findings clearly established these molecules as potent agonists of LPA1-3 and consolidated the pivotal role of LPA1 in urethra/prostate contraction as well as in fat cell development. The discovery of these unique and less labile LPA1-3 agonists would offer new avenues to investigate the roles of LPA receptors. SIGNIFICANCE STATEMENT: We report the identification of benzofuran ethanolamine derivatives behaving as potent selective nonlipid LPA1-3 agonists and shown to alter urethra muscle contraction or preadipocyte differentiation. Unique at this level of potency, selectivity, and especially stability, compared with lysophosphatidic acid, they represent more appropriate tools for investigating the physiological roles of lysophosphatidic acid receptors and starting point for optimization of drug candidates for therapeutic applications.

Soluble Epoxide Hydrolase 2 Expression Is Elevated in Obese Humans and Decreased by Physical Activity.

Epoxide hydrolase 2 (EPHX2) is an emerging therapeutic target in several immunometabolic disorders. EPHX2 metabolizes anti-inflammatory epoxyeicosatrienoic acids into pro-inflammatory diols. The contribution of EPHX2 activity to human obesity remains unexplored. We compared the expression of EPHX2 between lean and obese humans (n = 20 each) in subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs) using RT-PCR, Western Blot analysis, immunohistochemistry, and confocal microscopy before and after a 3-month physical activity regimen. We also assessed EPHX2 levels during preadipocyte differentiation in humans and mice. EPHX2 mRNA and protein expression were significantly elevated in obese subjects, with concomitant elevated endoplasmic reticulum (ER) stress components (the 78-kDa glucose-regulated protein; GRP78, and the Activating transcription factor 6; ATF6) and inflammatory markers (Tumor necrosis factor-α; TNFα, and Interleukin 6; IL6) as compared to controls (p < 0.05). EPHX2 mRNA levels strongly correlated with adiposity markers. In obese individuals, physical activity attenuated EPHX2 expression levels in both the SAT and PBMCs, with a parallel decrease in ER stress and inflammation markers. EPHX2 expression was also elevated during differentiation of both human primary and 3T3-L1 mouse preadipocytes. Mediators of cellular stress (palmitate, homocysteine, and macrophage culture medium) also increased EPHX2 expression in 3T3-L1 preadipocytes. Our findings suggest that EPHX2 upregulation is linked to ER stress in adiposity and that physical activity may attenuate metabolic stress by reducing EPHX2 expression.