Supraclavicular brown adipocytes originate from Tbx1+ myoprogenitors.

Brown adipose tissue (BAT) dissipates energy as heat, contributing to temperature control, energy expenditure, and systemic homeostasis. In adult humans, BAT mainly exists in supraclavicular areas and its prevalence is associated with cardiometabolic health. However, the developmental origin of supraclavicular BAT remains unknown. Here, using genetic cell marking in mice, we demonstrate that supraclavicular brown adipocytes do not develop from the Pax3+/Myf5+ epaxial dermomyotome that gives rise to interscapular BAT (iBAT). Instead, the Tbx1+ lineage that specifies the pharyngeal mesoderm marks the majority of supraclavicular brown adipocytes. Tbx1Cre-mediated ablation of peroxisome proliferator-activated receptor gamma (PPARγ) or PR/SET Domain 16 (PRDM16), components of the transcriptional complex for brown fat determination, leads to supraclavicular BAT paucity or dysfunction, thus rendering mice more sensitive to cold exposure. Moreover, human deep neck BAT expresses higher levels of the TBX1 gene than subcutaneous neck white adipocytes. Taken together, our observations reveal location-specific developmental origins of BAT depots and call attention to Tbx1+ lineage cells when investigating human relevant supraclavicular BAT.

Interleukin-6 released from differentiating human beige adipocytes improves browning.

Brown and beige adipocytes contribute significantly to the regulation of whole body energy expenditure and systemic metabolic homeostasis not exclusively by thermogenesis through mitochondrial uncoupling. Several studies have provided evidence in rodents that brown and beige adipocytes produce a set of adipokines ("batokines") which regulate local tissue homeostasis and have beneficial effects on physiological functions of the entire body. We observed elevated secretion of Interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1, but not tumor necrosis factor alpha (TNFα) or IL-1ß pro-inflammatory cytokines, by ex vivo differentiating human beige adipocytes (induced by either PPARγ agonist or irisin) compared to white. Higher levels of IL-6, IL-8 and MCP-1 were released from human deep neck adipose tissue biopsies (enriched in browning cells) than from subcutaneous ones. IL-6 was produced in a sustained manner and mostly by the adipocytes and not by the undifferentiated progenitors. Continuous blocking of IL-6 receptor by specific antibody during beige differentiation resulted in downregulation of brown marker genes and increased morphological changes that are characteristic of white adipocytes. The data suggest that beige adipocytes adjust their production of IL-6 to reach an optimal level for differentiation in the medium enhancing browning in an autocrine manner.

Thermogenic Activation Downregulates High Mitophagy Rate in Human Masked and Mature Beige Adipocytes.

Thermogenic brown and beige adipocytes oxidize metabolic substrates producing heat, mainly by the mitochondrial uncoupling protein UCP1, and can thus counteract obesity. Masked beige adipocytes possess white adipocyte-like morphology, but can be made thermogenic by adrenergic stimuli. We investigated the regulation of mitophagy upon thermogenic activation of human masked and mature beige adipocytes. Human primary abdominal subcutaneous adipose-derived stromal cells (hASCs) and Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes were differentiated to white and beige adipocytes, then their cAMP-induced thermogenic potential was assessed by detecting increased expressions of UCP1, mitochondrial DNA content and respiratory chain complex subunits. cAMP increased the thermogenic potential of white adipocytes similarly to beige ones, indicating the presence of a masked beige population. In unstimulated conditions, a high autophagic flux and mitophagy rates (demonstrated by LC3 punctae and TOM20 co-immunostaining) were observed in white adipocytes, while these were lower in beige adipocytes. Silencing and gene expression experiments showed that the ongoing mitophagy was Parkin-independent. cAMP treatment led to the downregulation of mitophagy through PKA in both types of adipocytes, resulting in more fragmented mitochondria and increased UCP1 levels. Our data indicates that mitophagy is repressed upon encountering a short-term adrenergic stimulus, as a fast regulatory mechanism to provide high mitochondrial content for thermogenesis.

Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation.

Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice.

During cold-exposure 'beige' adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2+/+ and TG2-/- mice had the same amount of WAT and BAT, we found that TG2+/+ animals could tolerate acute cold exposure for 4h, whereas TG2-/- mice only for 3h. Both TG2-/- and TG2+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3h treatment; however, TG2-/- mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of 'beige' marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2-/- mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2-/- mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.

Triamcinolone regulated apopto-phagocytic gene expression patterns in the clearance of dying retinal pigment epithelial cells. A key role of Mertk in the enhanced phagocytosis.

BACKGROUND: The apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes have relevance to development of age-related macular degeneration (AMD). METHODS: ARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDM engulfing anoikic cells. RESULTS: The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDM engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDM engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoikic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC-treated HMDMs. CONCLUSIONS: Specific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. GENERAL SIGNIFICANCE: The use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy.

Comparative Analysis of Volatile Compounds and Biochemical Activity of Curcuma xanthorrhiza Roxb. Essential Oil Extracted from Distinct Shaded Plants.

The application of shade during plants' growth significantly alters the biochemical compounds of the essential oil (EO). We aimed to analyze the effect of shade on the volatile compounds and biochemical activities of EO extracted from Curcuma xanthorrhiza Roxb. (C. xanthorrhiza) plants. Four shading conditions were applied: no shading (S0), 25% (S25), 50% (S50), and 75% shade (S75). The volatile compounds of EO extracted from each shaded plant were analyzed by gas chromatography-mass spectrometry. The antioxidant, antibacterial, and antiproliferative activities of EO were also investigated. We found that shade application significantly reduced the C. xanthorrhiza EO yield but increased its aroma and bioactive compound concentration. α-curcumene, xanthorrhizol, α-cedrene, epicurzerenone, and germacrone were found in EO extracted from all conditions. However, ß-bisabolol, curzerene, curcuphenol, and γ-himachalene were only detected in the EO of S75 plants. The EO of the shaded plants also showed higher antioxidant activity as compared to unshaded ones. In addition, the EO extracted from S75 exerted higher antiproliferative activity on HeLa cells as compared to S0. The EO extracted from S0 and S25 showed higher antibacterial activity against Gram-positive bacteria than kanamycin. Our results suggest that shade applications alter the composition of the extractable volatile compounds in C. xanthorrhiza, which may result in beneficial changes in the biochemical activity of the EO.

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages.

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This "engulfment synapse" can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.

Extracellular thiamine concentration influences thermogenic competency of differentiating neck area-derived human adipocytes.

Introduction: Brown adipose tissue (BAT) dissipates energy in the form of heat majorly via the mitochondrial uncoupling protein 1 (UCP1). The activation of BAT, which is enriched in the neck area and contains brown and beige adipocytes in humans, was considered as a potential therapeutic target to treat obesity. Therefore, finding novel agents that can stimulate the differentiation and recruitment of brown or beige thermogenic adipocytes are important subjects for investigation. The current study investigated how the availability of extracellular thiamine (vitamin B1), an essential cofactor of mitochondrial enzyme complexes that catalyze key steps in the catabolism of nutrients, affects the expression of thermogenic marker genes and proteins and subsequent functional parameters during ex vivo adipocyte differentiation. Methods: We differentiated primary human adipogenic progenitors that were cultivated from subcutaneous (SC) or deep neck (DN) adipose tissues in the presence of gradually increasing thiamine concentrations during their 14-day differentiation program. mRNA and protein expression of thermogenic genes were analyzed by RT-qPCR and western blot, respectively. Cellular respiration including stimulated maximal and proton-leak respiration was measured by Seahorse analysis. Results: Higher thiamine levels resulted in increased expression of thiamine transporter 1 and 2 both at mRNA and protein levels in human neck area-derived adipocytes. Gradually increasing concentrations of thiamine led to increased basal, cAMP-stimulated, and proton-leak respiration along with elevated mitochondrial biogenesis of the differentiated adipocytes. The extracellular thiamine availability during adipogenesis determined the expression levels of UCP1, PGC1a, CKMT2, and other browning-related genes and proteins in primary SC and DN-derived adipocytes in a concentration-dependent manner. Providing abundant amounts of thiamine further increased the thermogenic competency of the adipocytes. Discussion: Case studies in humans reported that thiamine deficiency was found in patients with type 2 diabetes and obesity. Our study raises the possibility of a novel strategy with long-term thiamine supplementation, which can enhance the thermogenic competency of differentiating neck area-derived adipocytes for preventing or combating obesity.

Availability of abundant thiamine determines efficiency of thermogenic activation in human neck area derived adipocytes.

Brown/beige adipocytes express uncoupling protein-1 (UCP1) that enables them to dissipate energy as heat. Systematic activation of this process can alleviate obesity. Human brown adipose tissues are interspersed in distinct anatomical regions including deep neck. We found that UCP1 enriched adipocytes differentiated from precursors of this depot highly expressed ThTr2 transporter of thiamine and consumed thiamine during thermogenic activation of these adipocytes by cAMP which mimics adrenergic stimulation. Inhibition of ThTr2 led to lower thiamine consumption with decreased proton leak respiration reflecting reduced uncoupling. In the absence of thiamine, cAMP-induced uncoupling was diminished but restored by thiamine addition reaching the highest levels at thiamine concentrations larger than present in human blood plasma. Thiamine is converted to thiamine pyrophosphate (TPP) in cells; the addition of TPP to permeabilized adipocytes increased uncoupling fueled by TPP-dependent pyruvate dehydrogenase. ThTr2 inhibition also hampered cAMP-dependent induction of UCP1, PGC1a, and other browning marker genes, and thermogenic induction of these genes was potentiated by thiamine in a concentration-dependent manner. Our study reveals the importance of amply supplied thiamine during thermogenic activation in human adipocytes which provides TPP for TPP-dependent enzymes not fully saturated with this cofactor and by potentiating the induction of thermogenic genes.

Human abdominal subcutaneous-derived active beige adipocytes carrying FTO rs1421085 obesity-risk alleles exert lower thermogenic capacity.

Introduction: White adipocytes store lipids, have a large lipid droplet and few mitochondria. Brown and beige adipocytes, which produce heat, are characterized by high expression of uncoupling protein (UCP) 1, multilocular lipid droplets, and large amounts of mitochondria. The rs1421085 T-to-C single-nucleotide polymorphism (SNP) of the human FTO gene interrupts a conserved motif for ARID5B repressor, resulting in adipocyte type shift from beige to white. Methods: We obtained abdominal subcutaneous adipose tissue from donors carrying FTO rs1421085 TT (risk-free) or CC (obesity-risk) genotypes, isolated and differentiated their preadipocytes into beige adipocytes (driven by the PPARγ agonist rosiglitazone for 14 days), and activated them with dibutyryl-cAMP for 4 hours. Then, either the same culture conditions were applied for additional 14 days (active beige adipocytes) or it was replaced by a white differentiation medium (inactive beige adipocytes). White adipocytes were differentiated by their medium for 28 days. Results and Discussion: RNA-sequencing was performed to investigate the gene expression pattern of adipocytes carrying different FTO alleles and found that active beige adipocytes had higher brown adipocyte content and browning capacity compared to white or inactive beige ones when the cells were obtained from risk-free TT but not from obesity-risk CC genotype carriers. Active beige adipocytes carrying FTO CC had lower thermogenic gene (e.g., UCP1, PM20D1, CIDEA) expression and thermogenesis measured by proton leak respiration as compared to TT carriers. In addition, active beige adipocytes with CC alleles exerted lower expression of ASC-1 neutral amino acid transporter (encoded by SLC7A10) and less consumption of Ala, Ser, Cys, and Gly as compared to risk-free carriers. We did not observe any influence of the FTO rs1421085 SNP on white and inactive beige adipocytes highlighting its exclusive and critical effect when adipocytes were activated for thermogenesis.

Corrigendum: Human abdominal subcutaneous-derived active beige adipocytes carrying FTO rs1421085 obesity-risk alleles exert lower thermogenic capacity.

[This corrects the article DOI: 10.3389/fcell.2023.1155673.].

Effects of Methods and Durations of Extraction on Total Flavonoid and Phenolic Contents and Antioxidant Activity of Java Cardamom (Amomum compactum Soland Ex Maton) Fruit.

Free radicals contribute to the pathophysiology of degenerative diseases which increase mortality globally, including mortality in Indonesia. Amomum compactum Soland. Ex Maton fruit from the Zingiberaceae family, also known as Java cardamom, contains secondary metabolites that have high antioxidant activities. The antioxidant activity of the methanol extract of Java cardamom fruit correlates with its flavonoid and phenolic compound contents, which can be affected by different methods and durations of extraction. This study aimed to measure and compare the effects of extraction methods and durations on total flavonoid and phenolic contents (TFCs and TPCs) and subsequent antioxidant activities by the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, ferric reducing antioxidant power (FRAP), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and cupric ion reducing antioxidant capacity (CUPRAC) assays. Methanol extracts of Java cardamom were produced by continuous shaking (CSE), microwave-assisted (MAE), or ultrasonic-assisted extractions (UAE) for three different durations. CSE for 360 min resulted in the highest TFCs (3.202 mg Quercetin Equivalent/g dry weight), while the highest TPCs (1.263 mg Gallic Acid Equivalent/g dry weight) were obtained by MAE for 3 min. Out of the investigated methods, MAE for 3 min resulted in the highest antioxidant activity results for the extracts. We conclude that the polyphenolic antioxidant yield of Java cardamom depends on two parameters: the method and the duration of extraction.

Mitophagy Mediates the Beige to White Transition of Human Primary Subcutaneous Adipocytes Ex Vivo.

Brown and beige adipocytes have multilocular lipid droplets, express uncoupling protein (UCP) 1, and promote energy expenditure. In rodents, when the stimulus of browning subsides, parkin-dependent mitophagy is activated and dormant beige adipocytes persist. In humans, however, the molecular events during the beige to white transition have not been studied in detail. In this study, human primary subcutaneous abdominal preadipocytes were differentiated to beige for 14 days, then either the beige culture conditions were applied for an additional 14 days or it was replaced by a white medium. Control white adipocytes were differentiated by their specific cocktail for 28 days. Peroxisome proliferator-activated receptor γ-driven beige differentiation resulted in increased mitochondrial biogenesis, UCP1 expression, fragmentation, and respiration as compared to white. Morphology, UCP1 content, mitochondrial fragmentation, and basal respiration of the adipocytes that underwent transition, along with the induction of mitophagy, were similar to control white adipocytes. However, white converted beige adipocytes had a stronger responsiveness to dibutyril-cAMP, which mimics adrenergic stimulus, than the control white ones. Gene expression patterns showed that the removal of mitochondria in transitioning adipocytes may involve both parkin-dependent and -independent pathways. Preventing the entry of beige adipocytes into white transition can be a feasible way to maintain elevated thermogenesis and energy expenditure.

Influence of Single Nucleotide Polymorphism of ENPP1 and ADIPOQ on Insulin Resistance and Obesity: A Case-Control Study in a Javanese Population.

Single nucleotide polymorphisms (SNPs) in obesity-related genes, such as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and adiponectin (ADIPOQ), potentially increase the risk of insulin resistance, the most common metabolic dysregulation related to obesity. We investigated the association of ENPP1 SNP K121Q (rs1044498) with insulin resistance and ADIPOQ SNP + 267G > T (rs1501299) with circulating adiponectin levels in a case-control study involving 55 obese and 55 lean Javanese people residing in Yogyakarta, Indonesia. Allele frequency was determined by a chi squared test or Fisher's exact test with an expected value less than 0.05. Odds ratios and 95% confidence intervals were estimated by regression logistic analysis. The presence of the Q121 allele of ENPP1 resulted in significantly higher fasting glucose, fasting insulin levels, and HOMA-IR, as compared to homozygous K121 carriers. The risk of insulin resistance was elevated in obese individuals carrying Q121 instead of homozygous K121. Adiponectin level was significantly lower in the obese group as compared to the lean group. Obese individuals carrying homozygous protective alleles (TT) of ADIPOQ tended to have lower adiponectin levels as compared to GT and GG carriers, however, we did not find statistically significant effects of the +276G > T SNP of the ADIPOQ gene on the plasma adiponectin levels or on the development of obesity.

BMP7 Increases UCP1-Dependent and Independent Thermogenesis with a Unique Gene Expression Program in Human Neck Area Derived Adipocytes.

White adipocytes contribute to energy storage, accumulating lipid droplets, whereas brown and beige adipocytes mainly function in dissipating energy as heat primarily via the action of uncoupling protein 1 (UCP1). Bone morphogenic protein 7 (BMP7) was shown to drive brown adipocyte differentiation in murine interscapular adipose tissue. Here, we performed global RNA-sequencing and functional assays on adipocytes obtained from subcutaneous (SC) and deep-neck (DN) depots of human neck and differentiated with or without BMP7. We found that BMP7 did not influence differentiation but upregulated browning markers, including UCP1 mRNA and protein in SC and DN derived adipocytes. BMP7 also enhanced mitochondrial DNA content, levels of oxidative phosphorylation complex subunits, along with PGC1α and p-CREB upregulation, and fragmentation of mitochondria. Furthermore, both UCP1-dependent proton leak and UCP1-independent, creatine-driven substrate cycle coupled thermogenesis were augmented upon BMP7 addition. The gene expression analysis also shed light on the possible role of genes unrelated to thermogenesis thus far, including ACAN, CRYAB, and ID1, which were among the highest upregulated ones by BMP7 treatment in both types of adipocytes. Together, our study shows that BMP7 strongly upregulates thermogenesis in human neck area derived adipocytes, along with genes, which might have a supporting role in energy expenditure.

ASC-1 transporter-dependent amino acid uptake is required for the efficient thermogenic response of human adipocytes to adrenergic stimulation.

Brown and beige adipocytes dissipate energy by uncoupling protein 1 (UCP1)-dependent and UCP1-independent thermogenesis, which may be utilized to develop treatments against obesity. We have found that mRNA and protein expression of the alanine/serine/cysteine transporter-1 (ASC-1) was induced during adipocyte differentiation of human brown-prone deep neck and beige-competent subcutaneous neck progenitors, and SGBS preadipocytes. cAMP stimulation of differentiated adipocytes led to elevated uptake of serine, cysteine, and glycine, in parallel with increased oxygen consumption, augmented UCP1-dependent proton leak, increased creatine-driven substrate cycle-coupled respiration, and upregulation of thermogenesis marker genes and several respiratory complex subunits; these outcomes were impeded in the presence of the specific ASC-1 inhibitor, BMS-466442. Our data suggest that ASC-1-dependent consumption of serine, cysteine, and glycine is required for efficient thermogenic stimulation of human adipocytes.

Irisin Stimulates the Release of CXCL1 From Differentiating Human Subcutaneous and Deep-Neck Derived Adipocytes via Upregulation of NFκB Pathway.

Thermogenic brown and beige adipocytes might open up new strategies in combating obesity. Recent studies in rodents and humans have indicated that these adipocytes release cytokines, termed "batokines". Irisin was discovered as a polypeptide regulator of beige adipocytes released by myocytes, primarily during exercise. We performed global RNA sequencing on adipocytes derived from human subcutaneous and deep-neck precursors, which were differentiated in the presence or absence of irisin. Irisin did not exert an effect on the expression of characteristic thermogenic genes, while upregulated genes belonging to various cytokine signaling pathways. Out of the several upregulated cytokines, CXCL1, the highest upregulated, was released throughout the entire differentiation period, and predominantly by differentiated adipocytes. Deep-neck area tissue biopsies also showed a significant release of CXCL1 during 24 h irisin treatment. Gene expression data indicated upregulation of the NFκB pathway upon irisin treatment, which was validated by an increase of p50 and decrease of IκBα protein level, respectively. Continuous blocking of the NFκB pathway, using a cell permeable inhibitor of NFκB nuclear translocation, significantly reduced CXCL1 release. The released CXCL1 exerted a positive effect on the adhesion of endothelial cells. Together, our findings demonstrate that irisin stimulates the release of a novel adipokine, CXCL1, via upregulation of NFκB pathway in neck area derived adipocytes, which might play an important role in improving tissue vascularization.

FTO Intronic SNP Strongly Influences Human Neck Adipocyte Browning Determined by Tissue and PPARγ Specific Regulation: A Transcriptome Analysis.

Brown adipocytes, abundant in deep-neck (DN) area in humans, are thermogenic with anti-obesity potential. FTO pro-obesity rs1421085 T-to-C single-nucleotide polymorphism (SNP) shifts differentiation program towards white adipocytes in subcutaneous fat. Human adipose-derived stromal cells were obtained from subcutaneous neck (SC) and DN fat of nine donors, of which 3-3 carried risk-free (T/T), heterozygous or obesity-risk (C/C) FTO genotypes. They were differentiated to white and brown (long-term Peroxisome proliferator-activated receptor gamma (PPARγ) stimulation) adipocytes; then, global RNA sequencing was performed and differentially expressed genes (DEGs) were compared. DN and SC progenitors had similar adipocyte differentiation potential but differed in DEGs. DN adipocytes displayed higher browning features according to ProFAT or BATLAS scores and characteristic DEG patterns revealing associated pathways which were highly expressed (thermogenesis, interferon, cytokine, and retinoic acid, with UCP1 and BMP4 as prominent network stabilizers) or downregulated (particularly extracellular matrix remodeling) compared to SC ones. Part of DEGs in either DN or SC browning was PPARγ-dependent. Presence of the FTO obesity-risk allele suppressed the expression of mitochondrial and thermogenesis genes with a striking resemblance between affected pathways and those appearing in ProFAT and BATLAS, underlining the importance of metabolic and mitochondrial pathways in thermogenesis. Among overlapping regulatory influences that determine browning and thermogenic potential of neck adipocytes, FTO genetic background has a thus far not recognized prominence.

Differentiating SGBS adipocytes respond to PPARγ stimulation, irisin and BMP7 by functional browning and beige characteristics.

Brown and beige adipocytes are enriched in mitochondria with uncoupling protein-1 (UCP1) to generate heat instead of ATP contributing to healthy energy balance. There are few human cellular models to reveal regulatory networks in adipocyte browning and key targets for enhancing thermogenesis in obesity. The Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte line has been a useful tool to study human adipocyte biology. Here we report that SGBS cells, which are comparable to subcutaneous adipose-derived stem cells, carry an FTO risk allele. Upon sustained PPARγ stimulation or irisin (a myokine released in response to exercise) treatment, SGBS cells differentiated into beige adipocytes exhibiting multilocular lipid droplets, high UCP1 content with induction of typical browning genes (Cidea, Elovl3) and the beige marker Tbx1. The autocrine mediator BMP7 led to moderate browning with the upregulation of the classical brown marker Zic1 instead of Tbx1. Thermogenesis potential resulted from PPARγ stimulation, irisin and BMP7 can be activated in UCP1-dependent and the beige specific, creatine phosphate cycle mediated way. The beige phenotype, maintained under long-term (28 days) conditions, was partially reversed by withdrawal of PPARγ ligand. Thus, SGBS cells can serve as a cellular model for both white and sustainable beige adipocyte differentiation and function.