Adipocyte Cell Model Research Articles

Unveiling the molecular mechanisms of adzuki bean (Vignaangularis) derived bioactive phytochemicals in combating obesity

Obesity is a global chronic metabolic disorder, and our previous research studied the anti-obesity effect of the adzuki bean and identified some phytochemicals, including flavonoids and saponins. However, it is still unclear which phytochemical derived from adzuki bean play a crucial role in the anti-obesity effect and its mechanisms. This research is the first study to reveal the anti-obesity phytochemicals in adzuki bean and its molecular mechanisms. The active compounds of adzuki beans were identified from previous research of our research team and TCMSP database. A total of 13 active phytochemicals and 205 anti-obesity targets were identified. The protein-protein interaction, network analysis, gene ontology, and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the Wnt signaling pathway, PI3K/Akt signaling pathway, and MAPK signaling pathway may be involved in anti-obesity effect of adzuki bean. Through PPI analysis, TNF, SRC, IL6, VEGFA, HRAS, and JUN were identified as core targets and further utilized for molecular docking. The molecular docking analysis shows that core active compounds can bind to core anti-obesity targets. Based on the above results, ADMET analysis, and further molecular simulation, vitexin-4″-O-glucoside has the highest potential for the anti-obesity effect and was further validated by Nile red staining and ORO staining in the in vitro adipocyte cell model. Furthermore, the vitexin-4″-O-glucoside achieves anti-obesity effect through regulating the Wnt/β-catenin signaling pathway and its downstream transcription factor C/EBPα and PPARγ. This may provide a solid foundation for the clinical application and further research of adzuki beans in treating obesity.

Anti-Obesity Effects of Adzuki Bean Saponins in Improving Lipid Metabolism Through Reducing Oxidative Stress and Alleviating Mitochondrial Abnormality by Activating the PI3K/Akt/GSK3β/β-Catenin Signaling Pathway.

Obesity is a chronic and complex disease defined by the excessive deposition of fat and is highly associated with oxidative stress. Adzuki bean saponins (ABS) showed anti-obesity activity in our previous in vivo study; however, the active saponins of adzuki beans and potential mechanisms are still unclear. This research aims to elucidate the anti-obesity effects of ABS in improving lipid metabolism and oxidative stress, exploring the effective ingredients and potential molecular mechanisms through UHPLC-QE-MS analysis, network pharmacology, bioinformatics, and in vitro experiments both in the 3T3-L1 cell line and HepG2 cell line. The results indicate that ABS can improve intracellular lipid accumulation, adipogenesis, oxidative stress, and mitochondrial damage caused by lipid accumulation including ROS generation, abnormal mitochondrial membrane potential, and ATP disorder. Fifteen saponin components were identified with the UHPLC-QE-MS analysis. The network pharmacology and bioinformatics analyses indicated that the PI3K/Akt signaling pathway is associated with the bioactive effect of ABS. Through Western blotting and immunofluorescence analysis, the anti-obesity effect of ABS is achieved through regulation of the PI3K/Akt/GSK3β/β-catenin signaling pathway and activation of downstream transcription factor c-Myc in the lipid accumulation cell model, and regulation of β-catenin signaling and inhibition of downstream transcription factor C/EBPα in the adipocyte cell model. These results illustrate the biological activity of ABS in improving fat metabolism and oxidative stress by restoring mitochondrial function through β-catenin signaling, the PI3K/Akt/GSK3β/β-catenin signaling pathway, laying the foundation for its further development.

Identifying G protein-coupled receptors involved in adipose tissue function using the innovative RNA-seq database FATTLAS

G protein-coupled receptors (GPCRs) modulate the function of adipose tissue (AT) in general and of adipocytes, specifically. Although it is well-established that GPCRs are widely expressed in AT, their repertoire as well as their regulation and function in (patho)physiological conditions (e.g., obesity) is not fully resolved. Here, we established FATTLAS, an interactive public database, for improved access and analysis of RNA-seq data of mouse and human AT. After extracting the GPCRome of non-obese and obese individuals, highly expressed and differentially regulated GPCRs were identified. Exemplarily, we describe four receptors (GPR146, MRGPRF, FZD5, PTGER2) and analyzed their functions in a (pre)adipocyte cell model. Besides all receptors being involved in adipogenesis, MRGPRF is essential for adipocyte viability and regulates cAMP levels, while GPR146 modulates adipocyte lipolysis via constitutive activation of Gi proteins. Taken together, by implementing and using FATTLAS we describe four hitherto unrecognized GPCRs associated with AT function and adipogenesis.

Convolvulus pluricaulis Choisy's Extraction, Chemical Characterization and Evaluation of the Potential Effects on Glycaemic Balance in a 3T3-L1 Adipocyte Cell Model.

Convolvulus pluricaulis (CP) is a common Indian herb, largely employed in Ayurvedic medicine and known for its neuroprotective and neuroinflammatory action. Its effectiveness against several pathologic/sub-pathologic conditions is widely accepted, but it is not yet completely chemically characterized. In recent years, several researchers have pointed out the involvement of CP and other Convolvulaceae in lipidic and glucidic metabolism, particularly in the control of hyperlipidaemia and diabetic conditions. In this scenario, the aim of the study was to chemically characterize the medium polarity part of the CP whole plant and its fractions and to shed light on their biological activity in adipocyte differentiation using the 3T3-L1 cell model. Our results demonstrated that the CP extract and fractions could upregulate the adipocyte differentiation through the modulation of the nuclear receptor PPARγ (Peroxisome Proliferator-Activated Receptor γ), broadly recognized as a key regulator of adipocyte differentiation, and the glucose transporter GLUT-4, which is fundamental for cellular glucose uptake and for metabolism control. CP also showed the ability to exert an anti-inflammatory effect, downregulating cytokines such as Rantes, MCP-1, KC, eotaxin, and GM-CSF, which are deeply involved in insulin resistance and glucose intolerance. Taken together, these data suggest that CP could exert a potential beneficial effect on glycemia and could be employed as an anti-diabetic adjuvant or, in any case, a means to better control glucose homeostasis.

Parathyroid hormone receptor stimulation induces human adipocyte lipolysis and browning.

Activation of brown adipose tissue is a promising strategy to treat and prevent obesity and obesity-related disorders. Activation of uncoupling protein 1 (UCP1) leads to uncoupled respiration and dissipation of stored energy as heat. Induction of UCP1-rich adipocytes in white adipose tissue, a process known as 'browning', serves as an alternative strategy to increase whole body uncoupling capacity. Here, we aim to assess the association between parathyroid hormone (PTH) receptor expression and UCP1 expression in human adipose tissues and to study PTH effects on human white and brown adipocyte lipolysis and UCP1 expression. A descriptive study of human neck adipose tissue biopsies substantiated by an interventional study on human neck-derived adipose tissue cell models. Thermogenic markers and PTH receptor gene expression are assessed in human neck adipose tissue biopsies and are related to individual health records. PTH-initiated lipolysis and thermogenic gene induction are assessed in cultured human white and brown adipocyte cell models. PTH receptor involvement is investigated by PTH receptor silencing. PTH receptor gene expression correlates with UCP1 gene expression in the deep-neck adipose tissue in humans. In cell models, PTH receptor stimulation increases lipolysis and stimulates gene transcription of multiple thermogenic markers. Silencing of the PTH receptor attenuates the effects of PTH indicating a direct PTH effect via this receptor. PTH 1 receptor stimulation by PTH may play a role in human adipose tissue metabolism by affecting lipolysis and thermogenic capacity.

Vesicles Shed by Pathological Murine Adipocytes Spread Pathology: Characterization and Functional Role of Insulin Resistant/Hypertrophied Adiposomes.

Extracellular vesicles (EVs) have recently emerged as a relevant way of cell to cell communication, and its analysis has become an indirect approach to assess the cell/tissue of origin status. However, the knowledge about their nature and role on metabolic diseases is still very scarce. We have established an insulin resistant (IR) and two lipid (palmitic/oleic) hypertrophied adipocyte cell models to isolate EVs to perform a protein cargo qualitative and quantitative Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH) analysis by mass spectrometry. Our results show a high proportion of obesity and IR-related proteins in pathological EVs; thus, we propose a panel of potential obese adipose tissue EV-biomarkers. Among those, lipid hypertrophied vesicles are characterized by ceruloplasmin, mimecan, and perilipin 1 adipokines, and those from the IR by the striking presence of the adiposity and IR related transforming growth factor-beta-induced protein ig-h3 (TFGBI). Interestingly, functional assays show that IR and hypertrophied adipocytes induce differentiation/hypertrophy and IR in healthy adipocytes through secreted EVs. Finally, we demonstrate that lipid atrophied adipocytes shed EVs promote macrophage inflammation by stimulating IL-6 and TNFα expression. Thus, we conclude that pathological adipocytes release vesicles containing representative protein cargo of the cell of origin that are able to induce metabolic alterations on healthy cells probably exacerbating the disease once established.

The Potential Synergistic Modulation of AMPK by Lippia citriodora Compounds as a Target in Metabolic Disorders.

Lippia citriodora (LC) represents a complex plant-derived source of polyphenols and iridoids that has shown beneficial properties against obesity-related metabolic disorders. The complete extract and its major compound, verbascoside, have shown AMPK-activating capacity in cell and animal models. In this work, we aimed to elucidate the contribution of the different compounds present in the LC extract on the AMPK activation capacity of the whole extract. Semipreparative reversed-phase high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RP-HPLC-ESI-TOF-MS) was used to identify the major compounds with bioassay-guided fractionation in an adipocyte cell model for the measurement of AMPK activity. Twenty-two compounds were identified and purified almost to homogeneity in 16 fractions, and three compounds, namely verbascoside, luteolin-7-diglucuronide and loganic acid, showed the highest AMPK-activating capacity. The synergy study using the checkerboard and fractional inhibitory concentration index (FICI) methods exhibited synergistic behavior between loganic acid and luteolin-7-diglucuronide. Molecular docking experiments revealed that these three compounds might act as direct agonists of AMPK, binding to the AMP binding sites of the gamma subunit and/or the different sites of the interaction zones between the gamma and beta subunits. Although our findings conclude that the bioactivity of the extract is mainly due to verbascoside, the synergy found between loganic acid and luteolin-7-diglucuronide deserves further research aimed to develop optimized combinations of polyphenols as a new nutritional strategy against obesity-related metabolic disorders.

ITRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS

The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

Peanut Sprout Extracts Attenuate Triglyceride Accumulation by Promoting Mitochondrial Fatty Acid Oxidation in Adipocytes.

Peanut sprouts (PS), which are germinated peanut seeds, have recently been reported to have anti-oxidant, anti-inflammatory, and anti-obesity effects. However, the underlying mechanisms by which PS modulates lipid metabolism are largely unknown. To address this question, serial doses of PS extract (PSE) were added to 3T3-L1 cells during adipocyte differentiation. PSE (25 µg/mL) significantly attenuated adipogenesis by inhibiting lipid accumulation in addition to reducing the level of adipogenic protein and gene expression with the activation of AMP-activated protein kinase (AMPK). Other adipocyte cell models such as mouse embryonic fibroblasts C3H10T1/2 and primary adipocytes also confirmed the anti-adipogenic properties of PSE. Next, we investigated whether PSE attenuated lipid accumulation in mature adipocytes. We found that PSE significantly suppressed lipogenic gene expression, while fatty acid (FA) oxidation genes were upregulated. Augmentation of FA oxidation by PSE in mature 3T3-L1 adipocytes was confirmed via a radiolabeled-FA oxidation rate experiment by measuring the conversion of [3H]-oleic acid (OA) to [3H]-H2O. Furthermore, PSE enhanced the mitochondrial oxygen consumption rate (OCR), especially maximal respiration, and beige adipocyte formation in adipocytes. In summary, PSE was effective in reducing lipid accumulation in 3T3-L1 adipocytes through mitochondrial fatty acid oxidation involved in AMPK and mitochondrial activation.

Differential effects of a combination of Hibiscus sabdariffa and Lippia citriodora polyphenols in overweight/obese subjects: A randomized controlled trial

Plant-polyphenols have shown the capacity to ameliorate obesity-induced metabolic disturbances, both in cell and animal models, where most therapeutic approaches have failed. On the basis of previous research, a dietary supplement containing 500 mg of a combination of polyphenolic extracts from Lippia citriodora L. and Hibiscus sabdariffa L. (LC-HS), in the context of an equilibrated isocaloric diet, was evaluated in a double blind, placebo-controlled and randomized trial in 56 obese/overweight subjects for two months. Compared to controls, the consumption of the LC-HS polyphenols showed significant improvements in body weight, abdominal circumference of overweight subjects (−6.79 ± 0.80 cm in overweight LC-HS group vs −1.85 ± 0.83 cm in controls, p < 0.001) and body fat % (−1.33 ± 0.15% in overweight LC-HS group vs −0.66 ± 0.17% in controls, p < 0.05). Heart rate and systolic blood pressure also presented significant improvements in overweight LC-HS participants. However, changes were more modest in obese subjects. Further, LC-HS extract significantly reduced lipid content and increased AMPK activity in a hypertrophied adipocyte cell model. Therefore, consumption of 500 mg/day of LC-HS extracts enriched in polyphenols for two months in the context of an isocaloric diet by overweight subjects decreased symptoms associated to obesity-related diseases. Modulation of fat metabolism in adipose tissue, probably mediated by AMPK activation, is proposed as a molecular target to be explored in future research.

Lipogenesis inhibition and adipogenesis regulation via PPARγ pathway in 3T3-L1 cells by Zingiber cassumunar Roxb. rhizome extracts

Zingiber cassumunar (ZC) is used by tribal people in northern Thailand in traditional remedies for anti-obesity and in food recipes. Extracts from this plant have been studied for several pharmacological effects including anti-obesity, but with no clear evidence on cellular mechanism of activity. This study aim to investigate the lipolytic and anti-adipogenic activity of crude extracts from ZC on in vitro cultures of the mouse adipocyte cell-model, 3T3-L1. Dry rhizome powder was extracted with absolute ethanol and boiled-water. On the exposed pre-adipocytes to the extracts, cytotoxicity was not detected by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Lipid content and glycerol release were assessed using Oil Red-O and a commercial Adipolysis Assay kits respectively. The extracts exhibited no significant lipolytic activity on the exposed mature-adipocytes, in serial dilutions ranging from 1 to 800 µg/ml. However, anti-lipogenic activity was presented. All extracts significantly reduced the lipid content of exposed differentiating-adipocytes. This anti-lipogenic activity was confirmed by the expression of selected genes, determined by using real-time PCR techniques, in four groups namely: adipocyte differentiation genes, glucose uptake genes, lipid metabolism genes and fatty acid oxidation genes. 1H NMR spectrum of the extracts exhibited the prominent olefinic protons of phenylbutanoids, the group of compounds previously proved with several bioactivities. This study provided evidences of mechanisms that apparently verify the traditional use of ZC to prevent obesity.

Adipose Tissue Transferrin and Insulin Resistance

Iron is an important micronutrient involved in several metabolic processes, and excessive iron is a risk factor for insulin resistance (IR) and diabetes. To define the genetic regulation of iron metabolism and its role in IR, we used gene expression, genotype, and insulin sensitivity data from an African American cohort (AAGMEx, N=256). Among the genes in a manually curated list of 62 transcripts representing iron homeostasis genes, expression of 32 in adipose tissue showed significant correlation with SI (p&amp;lt;0.01). The expression levels of transferrin (TF, β= 0.29, p=7.84 x 10-6) and ferritin heavy chain 1 (FTH1, β= -1.2, p=3.75 x 10-13) in adipose tissue were the most positively and negatively associated with SI, respectively. Observations were replicated in independent adipose tissue data sets from individuals of European ancestry (ARCA, N=99; METSIM, N=720). Among these SI-associated transcripts, the strongest cis-regulatory genetic variant (cis-eSNP) was for TF (rs6785596, β= 0.962, p=7.84 x 10-18) in adipose, but not in muscle or liver. To identify the role of TF in adipocytes, we downregulated its expression in a human adipocyte cell model (SGBS). Gene specific shRNA knockdown of TF caused differential expression (log2FC +/-0.4, probability&amp;gt;70%) of 465 genes, involved in mitochondrial function (CPT1B, UCP2), glucose transport (GLUT4), Wnt-pathway/insulin sensitivity (SFRP4, SFRP1), chemokine activity/cell-cell interaction (CXCL1, CXCL12, ICAM1), and genes with possible roles in obesity (CES1, RARRES2). Knockdown of TF mRNA in differentiated SGBS cells impaired mitochondrial respiration (49±10% reduction of maximal oxygen consumption rate) and caused a 34±4% reduction in maximal insulin-stimulated glucose uptake (p&amp;lt;0.05). In summary, genetic regulation of transferrin expression in adipose tissue plays a novel role in regulating insulin sensitivity. Disclosure D. McClain: None. N.K. Sharma: None. F. Lorenzo: None. S. Jain: None. C.D. Langefeld: None. M.E. Comeau: None. L. Salaye: None. S.K. Das: None.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. These results support BAT as a putative metformin target.

Antihyperlipidemic effect of a Rhamnus alaternus leaf extract in Triton-induced hyperlipidemic rats and human HepG2 cells

The Mediterranean buckthorn, Rhamnus alaternus L., is a plant used in traditional medicine in Mediterranean countries. We aimed at characterizing its phenolic compounds and explore potential antihyperlipidemic activity of this plant. The profile of phenolic compounds in R. alaternus leaf crude methanolic extract (CME) and its liquid-liquid extraction-derived fractions were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS2). Effects of CME on: circulating lipids in rats with Triton WR-1339-induced hyperlipidemia, intracellular lipid accumulation and expression of genes of fatty acid metabolism in human hepatoma HepG2 cells, and adipogenesis in the 3T3-L1 murine adipocyte cell model were assessed. The HPLC/ESI-MS2 analytical profile revealed a total of fifteen compounds, of which eleven were identified. Oral CME administration decreased blood levels of cholesterol and triacylglycerols in hyperlipidemic rats (by 60% and 70%, respectively, at 200 mg CME/kg). In HepG2 cells, CME exposure dose-dependently decreased intracellular lipids and up-regulated gene expression of carnitine palmitoyltransferase 1 involved in fatty acid oxidation. In the 3T3-L1 model, CME favored preadipocyte proliferation and adipogenesis, pointing to positive effects on adipose tissue expandability. These results suggest novel uses of R. alaternus by showing that its leaves are rich in flavonoids and flavonoid derivatives with an antihyperlipidemic effect in vivo and in hepatic cells.

Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin-Stimulated Glucose Uptake.

ScopeWe investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness.Methods and resultsEVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin‐stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin‐stimulated 2‐deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin‐mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2‐deoxyglucose uptake in adipocytes (p = 0.0159).ConclusionEVs released by stressed adipocytes impair insulin action in neighboring adipocytes.

Differential expression of Lp-PLA2 in obesity and type 2 diabetes and the influence of lipids

Aims/hypothesisLipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulatory macrophage-derived factor that increases with obesity and leads to a higher risk of cardiovascular disease (CVD). Despite this, its role in adipose tissue and the adipocyte is unknown. Therefore, the aims of this study were to clarify the expression of Lp-PLA2 in relation to different adipose tissue depots and type 2 diabetes, and ascertain whether markers of obesity and type 2 diabetes correlate with circulating Lp-PLA2. A final aim was to evaluate the effect of cholesterol on cellular Lp-PLA2 in an in vitro adipocyte model.MethodsAnalysis of anthropometric and biochemical variables from a cohort of lean (age 44.4 ± 6.2 years; BMI 22.15 ± 1.8 kg/m2, n = 23), overweight (age 45.4 ± 12.3 years; BMI 26.99 ± 1.5 kg/m2, n = 24), obese (age 49.0 ± 9.1 years; BMI 33.74 ± 3.3 kg/m2, n = 32) and type 2 diabetic women (age 53.0 ± 6.13 years; BMI 35.08 ± 8.6 kg/m2, n = 35), as part of an ethically approved study. Gene and protein expression of PLA2 and its isoforms were assessed in adipose tissue samples, with serum analysis undertaken to assess circulating Lp-PLA2 and its association with cardiometabolic risk markers. A human adipocyte cell model, Chub-S7, was used to address the intracellular change in Lp-PLA2 in adipocytes.ResultsLp-PLA2 and calcium-independent PLA2 (iPLA2) isoforms were altered by adiposity, as shown by microarray analysis (p < 0.05). Type 2 diabetes status was also observed to significantly alter gene and protein levels of Lp-PLA2 in abdominal subcutaneous (AbdSc) (p < 0.01), but not omental, adipose tissue. Furthermore, multivariate stepwise regression analysis of circulating Lp-PLA2 and metabolic markers revealed that the greatest predictor of Lp-PLA2 in non-diabetic individuals was LDL-cholesterol (p = 0.004). Additionally, in people with type 2 diabetes, oxidised LDL (oxLDL), triacylglycerols and HDL-cholesterol appeared important predictors, accounting for 59.7% of the variance (p < 0.001). Subsequent in vitro studies determined human adipocytes to be a source of Lp-PLA2, as confirmed by mRNA expression, protein levels and immunochemistry. Further in vitro experiments revealed that treatment with LDL-cholesterol or oxLDL resulted in significant upregulation of Lp-PLA2, while inhibition of Lp-PLA2 reduced oxLDL production by 19.8% (p < 0.05).Conclusions/interpretationOur study suggests adipose tissue and adipocytes are active sources of Lp-PLA2, with differential regulation by fat depot and metabolic state. Moreover, levels of circulating Lp-PLA2 appear to be influenced by unfavourable lipid profiles in type 2 diabetes, which may occur in part through regulation of LDL-cholesterol and oxLDL metabolism in adipocytes.

A thrifty variant in CREBRF strongly influences body mass index in Samoans.

Samoans are a unique founder population with a high prevalence of obesity, making them well suited for identifying new genetic contributors to obesity. We conducted a genome-wide association study (GWAS) in 3,072 Samoans, discovered a variant, rs12513649, strongly associated with body mass index (BMI) (P = 5.3 × 10(-14)), and replicated the association in 2,102 additional Samoans (P = 1.2 × 10(-9)). Targeted sequencing identified a strongly associated missense variant, rs373863828 (p.Arg457Gln), in CREBRF (meta P = 1.4 × 10(-20)). Although this variant is extremely rare in other populations, it is common in Samoans (frequency of 0.259), with an effect size much larger than that of any other known common BMI risk variant (1.36-1.45 kg/m(2) per copy of the risk-associated allele). In comparison to wild-type CREBRF, the Arg457Gln variant when overexpressed selectively decreased energy use and increased fat storage in an adipocyte cell model. These data, in combination with evidence of positive selection of the allele encoding p.Arg457Gln, support a 'thrifty' variant hypothesis as a factor in human obesity.

Adipose Tissue Free Fatty Acid Storage In Vivo: Effects of Insulin Versus Niacin as a Control for Suppression of Lipolysis.

Insulin stimulates the translocation fatty acid transport protein 1 (FATP1) to plasma membrane, and thus greater free fatty acid (FFA) uptake, in adipocyte cell models. Whether insulin stimulates greater FFA clearance into adipose tissue in vivo is unknown. We tested this hypothesis by comparing direct FFA storage in subcutaneous adipose tissue during insulin versus niacin-medicated suppression of lipolysis. We measured direct FFA storage in abdominal and femoral subcutaneous fat in 10 and 11 adults, respectively, during euglycemic hyperinsulinemia or after oral niacin to suppress FFA compared with 11 saline control experiments. Direct palmitate storage was assessed using a [U-13C]palmitate infusion to measure palmitate kinetics and an intravenous palmitate radiotracer bolus/timed biopsy. Plasma palmitate concentrations and flux were suppressed to 23 ± 3 and 26 ± 5 µmol ⋅ L−1 (P = 0.91) and 44 ± 4 and 39 ± 5 µmol ⋅ min−1 (P = 0.41) in the insulin and niacin groups, respectively, much less (P < 0.001) than the saline control group (102 ± 8 and 104 ± 12 µmol ⋅ min−1, respectively). In the insulin, niacin, and saline groups, abdominal palmitate storage rates were 0.25 ± 0.05 vs. 0.25 ± 0.07 vs. 0.32 ± 0.05 µmol ⋅ kg adipose lipid−1 ⋅ min−1, respectively (P = NS), and femoral adipose storage rates were 0.19 ± 0.06 vs. 0.20 ± 0.05 vs. 0.31 ± 0.05 µmol ⋅ kg adipose lipid−1 ⋅ min−1, respectively (P = NS). In conclusion, insulin does not increase FFA storage in adipose tissue compared with niacin, which suppresses lipolysis via a different pathway.

Increased adipogenesis of human adipose-derived stem cells on polycaprolactone fiber matrices.

With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells - differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics – studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.

A Gold Coordination Compound as a Chemical Probe to Unravel Aquaporin‐7 Function

Aquaporins (AQPs) are membrane water/glycerol channels that are involved in many physiological functions. Aquaporin-based modulators are predicted to have potential utility in the treatment of several diseases, as well as chemical tools to assess AQPs function in biological systems. We recently reported gold(III) compounds as human AQP3 inhibitors, with Auphen as the most potent of the series. In this work, we assessed the modulation of aquaporin-7 (AQP7) expressed in an adipocyte cell model and show that Auphen significantly inhibits mouse and human AQP7. By homology modeling and molecular docking it was possible to identify the thioether groups of methionine residues, in particular Met47, as likely candidates for binding to the gold(III) complex. Our data point to Auphen as a useful chemical tool to detect AQP7 function. It might constitute a basis to develop inhibitors with improved affinity towards different aquaglyceroporin isoforms.