Human Preadipocytes Research Articles

Adipogenic Activity of Oligomeric Hexafluoropropylene Oxide (Perfluorooctanoic Acid Alternative) through Peroxisome Proliferator-Activated Receptor γ Pathway.

Hexafluoropropylene oxide trimer acid (HFPO-TA) and hexafluoropropylene oxide dimer acid (HFPO-DA) have been used as perfluorooctanoic acid (PFOA) alternatives in the fluoropolymer industry for years. Their widespread environmental distribution, high bioaccumulation capability, and human exposure have caused great concern. Nevertheless, their potential toxicity and health risk remain largely unknown. In the present study, we compared potential disruption effects of HFPO-TA, HFPO-DA, and PFOA on peroxisome proliferator-activated receptor γ (PPARγ) via the investigation of receptor binding, receptor activity, and cell adipogenesis effects. The receptor binding experiment showed HFPO-TA exhibited 4.8-7.5 folds higher binding affinity with PPARγ than PFOA, whereas HFPO-DA exhibited weaker binding affinity than PFOA. They also showed agonistic activity toward PPARγ signaling pathway in HEK 293 cells in the order of HFPO-TA > PFOA > HFPO-DA. Molecular docking simulation indicated HFPO-TA formed more hydrogen bonds than PFOA, whereas HFPO-DA formed fewer hydrogen bonds than PFOA. HFPO-TA promoted adipogenic differentiation and lipid accumulation in both mouse and human preadipocytes with potency higher than PFOA. Adipogenesis in human preadipocytes is a more sensitive end point than mouse preadipocytes. Collectively, HFPO-TA exerts higher binding affinity, agonistic activity, and adipogenesis activity than PFOA. The potential health risk of HFPO-TA should be of concern.

A novel peptide suppresses adipogenic differentiation through activation of the AMPK pathway

Obesity rates have risen rapidly over the past several decades and obesity is now a global public health challenge. The reduction of excessive adipogenesis is thought to be an effective intervention for obesity and obesity-related metabolic diseases such as type 2 diabetes. In this study, a novel peptide PDBSN was identified that functions to suppress adipogenesis. In both human preadipocytes and mouse adipose-derived stem cells (ADSCs), PDBSN exhibited a suppressive effect on the accumulation of lipids and the expression of genes as well as their corresponding proteins (CCAAT/enhancer binding protein (C/EBP)β, C/EBPα and nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ)) relevant to adipogenic cell differentiation. Although adipogenesis decreased, the preadipocyte number and proliferation were not influenced by the PDBSN treatment. Apoptosis and the cell cycle were also determined to not have a role in the action of PDBSN. Mechanistically, the activity of the AMPK (adenosine 5ʹ-monophosphate-activated protein kinase) pathway was markedly increased upon PDBSN treatment. Moreover, treatment of preadipocytes with compound C, a selective AMPK inhibitor, abolished the effect of PDBSN in anti-adipogenesis, suggesting that the function of PDBSN relied on the AMPK pathway. These results suggest an effective role for PDBSN in suppressing adipogenesis and show potential for anti-obesity drug discovery.

1\u03b1,25(OH)2D3 attenuates IL-6 and IL-1\u03b2-mediated inflammatory responses in macrophage conditioned medium-stimulated human white preadipocytes by modulating p44/42 MAPK and NF-\u03baB signaling pathways

BackgroundMetabolic syndrome is characterized by macrophage infiltration and inflammatory responses—metaflammation in adipose tissue. IL-6 and IL-1β could mediate the inflammatory responses in macrophage stimulated-preadipocytes by modulating MAPK and NF-κB pathways. To test this hypothesis we used antibodies to block IL-6 and IL-1β action in macrophage conditioned medium (MacCM)-stimulated human white preadipocytes. Moreover, as interventions that prevent this could potentially be used to treat or prevent metabolic syndrome, and 1α,25(OH)2D3 has previously been reported to exert an anti-inflammatory action on macrophage-stimulated adipocytes, in this study we also investigated whether 1α,25(OH)2D3 could attenuate inflammatory responses in MacCM-stimulated preadipocytes, and explored the potential anti-inflammatory mechanisms.MethodsHuman white preadipocytes were cultured with 25% MacCM for 24 h to elicit inflammatory responses. This was confirmed by measuring the concentrations and mRNA levels of major pro-inflammatory factors [IL-1β, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cell expressed and secreted (RANTES)] by ELISA and qPCR, respectively. IL-6 and IL-1β actions were blocked using IL-6 antibody (300 ng/ml) and IL-1β antibody (15 μg/ml), respectively. Potential anti-inflammatory effects of 1α,25(OH)2D3 were investigated by pre-treatment and treatment of 1α,25(OH)2D3 (0.01 to 10 nM) for 48 h in MacCM-stimulated preadipocytes. In parallel, western blotting was used to determine inflammatory signaling molecules including relA of the NF-κB pathway and p44/42 MAPK modified during these processes.ResultsMacCM enhanced the secretion and gene expression of IL-1β, IL-6, IL-8, MCP-1 and RANTES by increasing the phosphorylation levels of relA and p44/42 MAPK in preadipocytes, whereas blocking IL-6 and IL-1β action inhibited the inflammatory responses by decreasing p44/42 MAPK and relA phosphorylation, respectively. Furthermore, 10 nM of 1α,25(OH)2D3 generally inhibited the IL-6 and IL-1β-mediated inflammatory responses, and reduced both p44/42 MAPK and relA phosphorylation in MacCM-stimulated preadipocytes.Conclusions1α,25(OH)2D3 attenuates IL-6 and IL-1β-mediated inflammatory responses, probably by inhibiting p44/42 MAPK and relA phosphorylation in MacCM-stimulated human white preadipocytes.

Membrane metallo-endopeptidase (Neprilysin) regulates inflammatory response and insulin signaling in white preadipocytes

ObjectiveAccumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties. MethodsWe performed RNA sequencing on human subcutaneous and visceral preadipocytes and array gene expression profiling in murine subcutaneous and visceral preadipocytes. We conducted several insulin signaling and inflammatory response experiments on different cellular states of MME expression. ResultsMME in white preadipocytes is expressed at a higher level in subcutaneous compared to visceral WAT and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in subcutaneous preadipocytes increased the inflammatory response to substance P and amyloid β aggregates. This associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor. ConclusionMME is a novel regulator of the insulin receptor in adipose tissue. Given the clinical significance of both chronic inflammation and insulin sensitivity in metabolic disease, these results show a potentially new target to increase insulin sensitivity and decrease inflammatory susceptibility.

Adipose-on-a-chip: a dynamic microphysiological in vitro model of the human adipose for immune-metabolic analysis in type II diabetes.

Infiltration of immune cells into adipose tissue is associated with chronic low-grade inflammation in obese individuals. To better understand the crosstalk between immune cells and adipocytes, in vivo-like in vitro models are required. Conventionally transwell culture plates are used for studying the adipocyte-immune cell interaction; however, the static culture nature of this approach falls short of closely recapitulating the physiological environment. Here we present a compartmentalized microfluidic co-culture system which provides a constant-rate of nutrient supply as well as waste removal, resembling the microvascular networks of the in vivo environment. Human adipocytes and U937 cells were co-cultured in close proximity in an enclosed system. The porous barrier between the adjacent compartments comprises an array of microchannels, which enables paracrine interaction between cells in adjacent compartments and improved perfusion-based long term cell feeding. Human pre-adipocytes were fully differentiated into adipocytes on the chip and remained viable for several weeks. Upon co-culturing with immune cells, adipocytes showed a tendency to develop insulin resistance. The immune-metabolic correlation has been studied by monitoring adiponectin and IL-6 expression, as well as glucose uptake upon treatment with insulin. Our microfluidic system can be potentially used to develop physiologically relevant adipose tissue models to study obesity-associated diseases such as insulin resistance and type 2 diabetes and therefore, facilitate drug development to treat these diseases.

Promotion of Human Epidermal Keratinocyte Expansion in Feeder Cell Co-culture.

Co-culture of human epidermal keratinocytes with mouse 3T3-J2 feeder cells, developed by Green and colleagues, has been used worldwide to generate skin autografts since the early 1980s. In addition, co-culture with 3T3-J2 cells has served as a fundamental tool in skin stem cell biology as it allows the evaluation of self-renewal capacity of epidermal stem cells. This chapter describes a recent improvement in the Green method to promote further the expansion of human epidermal keratinocytes utilizing a small-molecule inhibitor of TGF-β signaling. This new protocol enables more rapid expansion of human epidermal keratinocytes in co-culture with not only 3T3-J2 cells but also other feeder cells including human dermal fibroblasts and human preadipocytes, two major alternatives to 3T3-J2 cells, with a long-term goal of developing customized skin autografts.

Synthesis and Antiproliferative Activities of Triphenylmethanol Conjugates of Leuprorelin

Leuprorelin® (LEP) is an FDA drug for breast cancer and prostate cancer treatment. There are several reported adverse effects such as transient hypertension, excessive salivation, and increased dysuria during treatment with LEP. In this study, the efficacy and toxicity of LEP were modified by using a drug delivery system to adjust the physicochemical properties. In this regard, Leuprorelin® conjugates of triphenylmethanol derivatives (TPMs) were synthesized as prodrugs. Comparative antiproliferative assays showed that LEP-TPMs conjugates had significantly higher antiproliferative activities than the corresponding non-covalent physical mixtures of the TPMs and LEP against human invasive ductal carcinoma (BT-549), human prostate carcinoma (PC3), human lung cancer (A549) and mouse pre-adipocytes (3T3-L1) cells.

An ameloblastin C-terminus variant is present in human adipose tissue

ObjectiveTranscriptional regulatory elements in the ameloblastin (AMBN) promoter indicate that adipogenesis may influence its expression. The objective here was to investigate if AMBN is expressed in adipose tissue, and have a role during differentiation of adipocytes. DesignAMBN expression was examined in adipose tissue and adipocytes by real-time PCR and ELISA. Distribution of ameloblastin was investigated by immunofluorescence in sections of human subcutaneous adipose tissue. The effect of recombinant proteins resembling AMBN and its processed products on proliferation of primary human pre-adipocytes and murine 3T3-L1 cell lines was measured by [3H]-thymidine incorporation. The effect on adipocyte differentiation was evaluated by the expression profile of the adipogenic markers PPARγ and leptin, and the content of lipids droplets (Oil-Red-O staining). ResultsAMBN was found to be expressed in human adipose tissue, human primary adipocytes, and in 3T3-L1 cells. The C-terminus of the AMBN protein and a 45 bp shorter splice variant was identified in human subcutaneous adipose tissue. The expression of AMBN was found to increase four-fold during differentiation of 3T3-L1 cells. Administration of recombinant AMBN reduced the proliferation, and enhanced the expression of PPARγ and leptin in 3T3-L1 and human pre-adipocytes, respectively. ConclusionsThe AMBN C-terminus variant was identified in adipocytes. This variant may be encoded from a short splice variant. Increased expression of AMBN during adipogenesis and its effect on adipogenic factors suggests that AMBN also has a role in adipocyte development.

Interleukin-1β and prostaglandin-synthesizing enzymes as modulators of human omental and subcutaneous adipose tissue function

IL-1β stimulates expression of prostaglandin (PG)-synthesizing enzymes cyclooxygenase (COX)-2 and aldo-keto reductase (AKR)1B1 in human preadipocytes. We aimed to examine the impact of IL-1β, COX-2 and AKR1B1 on markers of human visceral and subcutaneous adipose tissue function, and to assess whether PG synthesis by these enzymes mediates IL-1β effects. Omental and subcutaneous fat samples were obtained from bariatric surgery patients. PG release and expression of inflammatory and adipogenic markers were assessed in explants treated with COX-2 inhibitor NS-398 or AKR1B1 inhibitor Statil, with or without IL-1β. Preadipocyte differentiation experiments were also performed. IL-1β decreased expression of PPARγ in both fat depots compared to control and increased expression of NF-κB1, IL-6, CCL-5, ICAM-1 and VEGFA, especially in visceral fat for IL-6, CCL-5 and VEGFA. Adding Statil or NS-398 to IL-1β blunted PGF2α and PGE2 release, but did not alter IL-1β effects on adipose tissue function markers. IL-1β down-regulated adipocyte differentiation whereas NS-398 alone increased this process. However, NS-398 did not prevent IL-1β inhibition of adipogenesis. We conclude that IL-1β induces a pro-inflammatory response in human adipose tissues, particularly in visceral fat, and acts independently of concomitant PG release. IL-1β and COX-2 appear to be critical determinants of adipose tissue pathophysiologic remodeling in obesity.

Exosomal proteins constitute an essential part of the human adipose tissue secretome

Adipose tissue is an endocrine organ, secreting various adipokines, either directly or via extracellular vesicles, including exosomes. Exosomes are vesicles of 40–150 nm size that represent a novel concept of biomolecule release. We purified exosomes from isolated primary human preadipocytes differentiated to mature adipocytes. The analyses of these exosomal preparations by LC-MS identified 884 proteins, so called exoadipokines. The comparison of exoadipokines with previously identified human exosome-associated proteins in ExoCarta database show an overlap of 817 proteins, but also revealed 67 proteins not assigned to human exosomes, yet. We further compared all exoadipokines to our previously reported reference secretome of human adipose tissue (http://diabesityprot.org/), finding 212 common proteins, whereas 672 proteins were specific for the exosomal fraction. Bioinformatic analyses revealed that the 212 common proteins can be assigned to all major functions of adipose tissue secreted proteins e.g. molecules involved in fibrotic processes or inflammation. In contrast, the exosome-specific proteins were rather assigned to signaling pathways and membrane-mediated processes. In conclusion, the isolation of exosomes allows to further specify the functionality of adipokines and exoadipokines as part of the adipocyte secretome in signaling and interorgan crosstalk.

Differential open chromatin profile and transcriptomic signature define depot-specific human subcutaneous preadipocytes: primary outcomes

BackgroundIncreased lower body fat is associated with reduced cardiometabolic risk. The molecular basis for depot-specific differences in gluteofemoral (GF) compared with abdominal (A) subcutaneous adipocyte function is poorly understood. In the current report, we used a combination of Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq), RNA-seq, and chromatin immunoprecipitation (ChIP)-qPCR analyses that provide evidence that depot-specific gene expression patterns are associated with differential epigenetic chromatin signatures.MethodsPreadipocytes cultured from A and GF adipose tissue obtained from premenopausal apple-shaped women were used to perform transcriptome analysis by RNA-seq and assess accessible chromatin regions by ATAC-seq. We measured mRNA expression and performed ChIP-qPCR experiments for histone modifications of active (H3K4me3) and repressed chromatin (H3K27me3) regions respectively on the promoter regions of differentially expressed genes.ResultsRNA-seq experiments revealed an A-fat and GF-fat selective gene expression signature, with 126 genes upregulated in abdominal preadipocytes and 90 genes upregulated in GF cells. ATAC-seq identified almost 10-times more A-specific chromatin-accessible regions. Using a combined analysis of ATAC-seq and global gene expression data, we identified 74 of the 126 abdominal-specific genes (59%) with A-specific accessible chromatin sites within 200 kb of the transcription start site (TSS), including HOXA3, HOXA5, IL8, IL1b, and IL6. Interestingly, only 14 of the 90 GF-specific genes (15%) had GF-specific accessible chromatin sites within 200 kb of the corresponding TSS, including HOXC13 and HOTAIR, whereas 25 of them (28%) had abdominal-specific accessible chromatin sites. ChIP-qPCR experiments confirmed that the active H3K4me3 chromatin mark was significantly enriched at the promoter regions of HOXA5 and HOXA3 genes in abdominal preadipocytes, while H3K27me3 was less abundant relative to chromatin from GF. This is consistent with their A-fat specific gene expression pattern. Conversely, analysis of the promoter regions of the GF specific HOTAIR and HOXC13 genes exhibited high H3K4me3 and low H3K27me3 levels in GF chromatin compared to A chromatin.ConclusionsGlobal transcriptome and open chromatin analyses of depot-specific preadipocytes identified their gene expression signature and differential open chromatin profile. Interestingly, A-fat-specific open chromatin regions can be observed in the proximity of GF-fat genes, but not vice versa.Trial registrationClinicaltrials.gov, NCT01745471. Registered 5 December 2012.

Elevated Carbohydrate Response Element-Binding Protein Beta (ChREBPβ) and Thioredoxin Interacting Protein (TXNIP) Levels in Human Adipocytes Differentiated in High Glucose Concentrations.

Obesity and type 2 diabetes often coexist. The effect of hyperglycemia on adipose tissue is, therefore, of interest. Although studies have shown that high glucose (HG) concentrations do not inhibit adipocyte differentiation, the resulting adipocyte phenotype has not been investigated. In particular, the levels of the glucose-responsive transcription factor carbohydrate-responsive response element binding protein (ChREBP) isoforms have not been assessed. Human preadipocytes were differentiated into adipocytes in either normal glucose (NG) or HG conditions. RNA and protein analyses were used to measure the expression of ChREBP isoforms, thioredoxin interacting protein (TXNIP) and lipogenic genes. Insulin-stimulated glucose uptake was measured. HG- vs. NG-differentiated adipocytes expressed more ChREBPβ and more TXNIP at the mRNA and protein levels. There was no change in lipogenic gene expression. HG- vs. NG-differentiated adipocytes displayed an inhibition of insulin-stimulated glucose uptake. HG-differentiated human adipocytes have distinct molecular differences and are insulin resistant. More studies are warranted to investigate potential mechanisms linking changes in ChREBPβ and TXNIP to insulin responsiveness.

Genome-wide methylation analysis identifies ELOVL5 as an epigenetic biomarker for the risk of type 2 diabetes mellitus

Genome-wide DNA methylation has been implicated in complex human diseases. Here, we identified epigenetic biomarkers for type 2 diabetes (T2D) underlying obesogenic environments. In a blood-based DNA methylation analysis of 11 monozygotic twins (MZTW) discordant for T2D, we discovered genetically independent candidate methylation sites. In a follow-up replication study (17 MZTW pairs) for external validation, we replicated the T2D-association at a novel CpG signal in the ELOVL fatty acid elongase 5 (ELOVL5) gene specific to T2D-discordant MZTW. For concordant DNA methylation signatures in tissues, we further confirmed that a CpG site (cg18681426) was associated with adipogenic differentiation between human preadipocytes and adipocytes isolated from the same biopsy sample. In addition, the ELOVL5 gene was significantly differentially expressed in adipose tissues from unrelated T2D patients and in human pancreatic islets. Our results demonstrate that blood-derived DNA methylation is associated with T2D risk as a proxy for cumulative epigenetic status in human adipose and pancreatic tissues. Moreover, ELOVL5 expression was increased in cellular and mouse models of induced obesity-related diabetes. These findings may provide new insights into epigenetic architecture by uncovering methylation-based biomarkers.

Impact of X-ray Exposure on the Proliferation and Differentiation of Human Pre-Adipocytes.

Radiotherapy is a widely used treatment option for cancer patients as well as for patients with musculoskeletal disorders. Adipocytes, the dominant cell type of adipose tissue, are known to constitute an active part of the tumor microenvironment. Moreover, adipocytes support inflammatory processes and cartilage degradation in chronic inflammatory diseases, i.e., rheumatoid and osteoarthritis. Since the production of inflammatory factors is linked to their differentiation stages, we set out to explore the radiation response of pre-adipocytes that may influence their inflammatory potential and differentiation capacity. This is the first study investigating the effects of X-ray irradiation on the proliferation and differentiation capacity of human primary pre-adipocytes, in comparison to Simpson–Golabi–Behmel Syndrome (SGBS) pre-adipocytes, an often-used in vitro model of human primary pre-adipocytes. Our results demonstrate a dose-dependent reduction of the proliferation capacity for both cell strains, whereas the potential for differentiation was mostly unaffected by irradiation. The expression of markers of adipogenic development, such as transcription factors (PPARγ, C/EBPα and C/EBPβ), as well as the release of adipokines (visfatin, adiponectin and leptin) were not significantly changed upon irradiation. However, after irradiation with high X-ray doses, an increased lipid accumulation was observed, which suggests a radiation-induced response of adipocytes related to inflammation. Our results indicate that pre-adipocytes are radio-resistant, and it remains to be elucidated whether this holds true for the overall inflammatory response of adipocytes upon irradiation.

Adipose tissue TSH as a new modulator of human adipocyte mitochondrial function.

Recent studies indicate a possible role of TSH/TSHR signalling axis on adipogenesis and adipose tissue physiology. Here, we aimed to investigate the relationship between adipose tissue TSHB and adipose tissue physiology-related gene expression. Subcutaneous and visceral adipose tissue TSHB gene expression was analysed in two independent cohorts [Cohort1 (N = 96) and Cohort2 (N = 45)] and after bariatric surgery-induced weight loss [Cohort3 (N = 22)]. Adipose tissue TSH protein expression was also analysed in a subgroup of participants from Cohort 1 (N = 16). The effects of recombinant TSH on human subcutaneous preadipocytes and adipocytes were investigated. In cohort 1, both visceral and subcutaneous adipose tissue TSHB gene expression was positively correlated with the expression of mitochondrial function (PPARGC1A, ISCA2, CISD1, SIRT1, NFE2L2, NRF1) and fatty acid mobilization (CAV1, ENGL1), but not with adipogenic-related genes. Of note, adipose tissue TSH protein levels were also associated with some of these markers of mitochondrial function and fatty acid mobilization. These associations were replicated in cohort 2. Bariatric surgery-induced weight loss resulted in increased subcutaneous adipose tissue TSHB in parallel to increased PPARGC1A. In human subcutaneous adipocytes, rh-TSH administration led to increased mitochondrial respiratory capacity in parallel to increased mitochondrial function- and adipogenic-related gene expression, but no significant effects were observed during differentiation of human preadipocytes. These data point to a possible role of adipose tissue TSH in the maintenance of adipocyte mitochondrial function.

Activation of brown adipocytes by placental growth factor

Gestational diabetes mellitus (GDM) is a type of diabetes and occurs during pregnancy. Brown adipose tissue (BAT) improves glucose homeostasis and mitigates insulin resistance, however, its activity is reduced in GDM. Placenta growth factor (PlGF) is an angiogenic factor produced by placental trophoblasts. Nevertheless, whether and how PlGF could affect BAT function in GDM are not defined. To investigate this question, 91 non-diabetic pregnant participants and 73 GDM patients were recruited to Gynaecology and Obstetrics Centre in Lu He hospital. Serum levels of PlGF were quantified by ELISA. Skin temperature was measured by far infrared thermography in the supraclavicular region where classical BATs were located. The direct effect of PlGF on BAT function was explored using the established human preadipocyte differentiation system. Thereby, we demonstrated that serum levels of PlGF were lower in GDM patients compared with controls, which was accompanied by decreased skin temperature in the supraclavicular region. By qPCR and western blot, mRNA and protein expression of UCP1 and OXPHOS were elevated in differentiated adipocytes treated with PlGF. PlGF stimulated mitochondrion transcription and increased copy number of mitochondrial. When subjected for respirometry, PlGF-treated differentiated adipocytes showed higher oxygen consumption rates than controls. PlGF induced AMPK phosphorylation and blockade of AMPK phosphorylation blunted UCP1 and OXPHOS expression in differentiated adipocytes. PlGF administration reduced cholesterol and triglyceride content in the liver and improved insulin sensitivity in db mice compared with control. In Conclusion, PlGF could activate BAT function. Downregulation of PlGF might contribute to the reduced BAT activity in GDM.

IL-6-mediated cross-talk between human preadipocytes and ductal carcinoma in situ in breast cancer progression

BackgroundThe function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model.MethodsGFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody.ResultsIn the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition.ConclusionsOur findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Autophagy mediates calcium-sensing receptor-induced TNFα production in human preadipocytes

Obesity is a major current public health problem worldwide due to the severe co-morbid conditions that this disease entails. The development of obesity-related cardiometabolic disorders is in direct association with adipose tissue inflammation that leads to its functional impairment. Activation of the Calcium-Sensing Receptor (CaSR) in adipose tissue contributes to inflammation and adipose dysfunction. Autophagy, a process of cell component degradation, is closely related to inflammation in many diseases, however, whether autophagy is associated with CaSR-induced inflammation remains unknown. Using LS14 and SW872 preadipose cell lines as well as primary human preadipocytes, we show that CaSR activation with the allosteric activator cinacalcet induces autophagosome formation. Cinacalcet-induced LC3II content elevation was precluded by knockdown of the CaSR and enhanced by CaSR overexpression, indicating a specific effect. Autophagy inhibition using 3-methyladenine prevented CaSR-induced TNFα production, indicating that autophagy contributes to CaSR-induced inflammation in human preadipocytes. Our results suggest that modulation of CaSR-induced autophagy is an attractive target in obese inflamed adipose tissue, to prevent the development of diseases triggered by adipose dysfunction. We describe a novel mechanism and possible new target to modulate and prevent adipose inflammation and hence the resulting disease-generating adipose tissue dysfunction.

Effects of Genistein on Differentiation and Viability of Human Visceral Adipocytes.

Obesity can lead to pathological growth of adipocytes by inducing inflammation and oxidative stress. Genistein could be a potential candidate for the treatment of obesity due to its antioxidant properties. Specific kits were used to examine the effects of genistein vs adiponectin on human visceral pre-adipocytes differentiation, cell viability, mitochondrial membrane potential, and oxidative stress in pre-adipocytes and in white/brown adipocytes. Western Blot was performed to examine changes in protein activation/expression. Genistein increased human visceral pre-adipocytes differentiation and browning, and caused a dose-related improvement of cell viability and mitochondrial membrane potential. Similar effects were observed in brown adipocytes and in white adipocytes, although in white cells the increase of cell viability was inversely related to the dose. Moreover, genistein potentiated AMP-activated protein kinase (AMPK)/mitofusin2 activation/expression in pre-adipocytes and white/brown adipocytes and protected them from the effects of hydrogen peroxide. The effects caused by genistein were similar to those of adiponectin. The results obtained showed that genistein increases human visceral pre-adipocytes differentiation and browning, protected against oxidative stress in pre-adipocytes and white/brown adipocytes through mechanisms related to AMPK-signalling and the keeping of mitochondrial function.

An inhibitor of 11-\u03b2 hydroxysteroid dehydrogenase type 1 (PF915275) alleviates nonylphenol-induced hyperadrenalism and adiposity in rat and human cells

BackgroundNonylphenol (NP) is an environmental endocrine-disrupting chemical (EDC) detected in human cord blood and milk. NP exposure in developmental periods results in hyperadrenalism and increasing 11β-hydroxysteroid dehydrogenase I (11β-HSD1) activity in an adult rat model. Alleviating 11β-HSD1 activity is therefore a logical and common way to treat hyperadrenalism. PF915275 (PF; 4′-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide) is a selective inhibitor for 11β-HSD1. This study aimed to determine whether PF915275 could alleviate the hyperadrenalism induced by NP. In addition to a rat model, the effects of NP and PF915275 were measured in human preadipocytes.MethodsFor the in vivo rat model, female adult rats exposed to NP during the developmental period were divided into two treatment groups, with one receiving oral DMSO solution and the other receiving PF915275 once per day for 4 weeks. After the final treatment, the rats from each group were sacrificed for analysis. For the in vitro human model, human preadipocytes received 2 regimens of NP treatment. One treatment regimen occurred before differentiation (to mimic the sensitive developmental period; P exposure), and the other included continuous exposure from preadipocytes to fully differentiated adipocytes (to mimic the growing and adult periods, respectively; C exposure). Protein and RNA were extracted from rat tissues and the preadipocytes for western blot and real-time PCR analysis.ResultsIn the rat model, PF915275 alleviated NP-induced effects by interfering with adipogenesis pathways, including enhancing PPARα expression, decreasing PPARγ expression, and reducing both 11β-HSD1 protein and mRNA expression levels. Additionally, PF915275 reduced the effects of the adrenal corticoid synthesis pathway by reducing StAR expression and 11β-hydroxylase and aldosterone synthase activities. With short-term exposure, NP enhanced PPARγ and FASN mRNA expression levels and reduced PPARα expression, whereas PF915275 alleviated these effects. With C exposure, the NP-induced accumulation of intracellular lipids was reduced by PF915275 treatment, which was mediated by decreased PPARγ mRNA and protein expression levels and increased PPARα protein expression.ConclusionsThe effects of NP and PF915275 treatment in both rat and human cell models are similar. Rats may be an appropriate model to study the effects of NP in humans, especially during the developmental period.