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.

Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Cross-Linking Mass Spectrometry on P-Glycoprotein.

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp's conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp's solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.

Adenosine A2A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages.

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A2A receptors (A2AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the A2AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein. The NPC1 protein was identified to interact with the C-terminal tail of A2AR in RAW 264.7 and IPMФ cells by two independent and parallel proteomic approaches. The interaction between the NPC1 protein and the full-length A2AR was further validated in HEK-293 cells that permanently express the receptor and RAW264.7 cells that endogenously express A2AR. A2AR activation reduces the expression of NPC1 mRNA and protein density in LPS-activated mouse IPMФ cells. Additionally, stimulation of A2AR negatively regulates the cell surface expression of NPC1 in LPS-stimulated macrophages. Furthermore, stimulation of A2AR also altered the density of lysosome-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1), two endosomal markers associated with the NPC1 protein. Collectively, these results suggested a putative A2AR-mediated regulation of NPC1 protein function in macrophages, potentially relevant for the Niemann-Pick type C disease when mutations in NPC1 protein result in the accumulation of cholesterol and other lipids in lysosomes.

Tissue Transglutaminase Knock-Out Preadipocytes and Beige Cells of Epididymal Fat Origin Possess Decreased Mitochondrial Functions Required for Thermogenesis.

Beige adipocytes with thermogenic function are activated during cold exposure in white adipose tissue through the process of browning. These cells, similar to brown adipocytes, dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). Recently, we have shown that tissue transglutaminase (TG2) knock-out mice have decreased cold tolerance in parallel with lower utilization of their epididymal adipose tissue and reduced browning. To learn more about the thermogenic function of this fat depot, we isolated preadipocytes from the epididymal adipose tissue of wild-type and TG2 knock-out mice and differentiated them in the beige direction. Although differentiation of TG2 knock-out preadipocytes is phenotypically similar to the wild-type cells, the mitochondria of the knock-out beige cells have multiple impairments including an altered electron transport system generating lower electrochemical potential difference, reduced oxygen consumption, lower UCP1 protein content, and a higher portion of fragmented mitochondria. Most of these differences are present in preadipocytes as well, and the differentiation process cannot overcome the functional disadvantages completely. TG2 knock-out beige adipocytes produce more iodothyronine deiodinase 3 (DIO3) which may inactivate thyroid hormones required for the establishment of optimal mitochondrial function. The TG2 knock-out preadipocytes and beige cells are both hypometabolic as compared with the wild-type controls which may also be explained by the lower expression of solute carrier proteins SLC25A45, SLC25A47, and SLC25A42 which transport acylcarnitine, Co-A, and amino acids into the mitochondrial matrix. As a consequence, the mitochondria in TG2 knock-out beige adipocytes probably cannot reach the energy-producing threshold required for normal thermogenic functions, which may contribute to the decreased cold tolerance of TG2 knock-out mice.

Nucleosome destabilization by polyamines.

The roles and molecular interactions of polyamines (PAs) in the nucleus are not fully understood. Here their effect on nucleosome stability, a key regulatory factor in eukaryotic gene control, is reported, as measured in agarose embedded nuclei of H2B-GFP expressor HeLa cells. Nucleosome stability was assessed by quantitative microscopy [1,2] in situ, in close to native state of chromatin, preserving the nucleosome constrained topology of the genomic DNA. A robust destabilizing effect was observed in the millimolar concentration range in the case of spermine, spermidine as well as putrescine, which was strongly pH and salt concentration-dependent, and remained significant also at neutral pH. The integrity of genomic DNA was not affected by PA treatment, excluding DNA break-elicited topological relaxation as a factor in destabilization. The binding of PAs to DNA was demonstrated by the displacement of ethidium bromide, both from deproteinized nuclear halos and from plasmid DNA. The possibility that DNA methylation patterns may be influenced by PA levels is contemplated in the context of gene expression and DNA methylation correlations identified in the NCI-60 panel-based CellMiner database: methylated loci in subsets of high-ODC1 cell lines and the dependence of PER3 DNA methylation on PA metabolism.

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.

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.

Isocyanide Substitution in Acridine Orange Shifts DNA Damage-Mediated Phototoxicity to Permeabilization of the Lysosomal Membrane in Cancer Cells.

In cancer therapy, immunogenic cell death eliminates tumor cells more efficiently than conventional apoptosis. During photodynamic therapy (PDT), some photosensitizer (PS) targeting lysosomes divert apoptosis to the immunologically more relevant necrosis-like cell death. Acridine orange (AO) is a PS targeting lysosome. We synthesized a new compound, 3-N,N-dimethylamino-6-isocyanoacridine (DM), a modified AO, aiming to target lysosomes better. To compare DM and AO, we studied optical properties, toxicity, cell internalization, and phototoxicity. In addition, light-mediated effects were monitored by the recently developed QUINESIn method on nuclei, and membrane stability, morphology, and function of lysosomes utilizing fluorescent probes by imaging cytometry in single cells. DM proved to be a better lysosomal marker at 405 nm excitation and lysed lysosomes more efficiently. AO injured DNA and histones more extensively than DM. Remarkably, DM's optical properties helped visualize shockwaves of nuclear DNA released from cells during the PDT. The asymmetric polar modification of the AO leads to a new compound, DM, which has increased efficacy in targeting and disrupting lysosomes. Suitable AO modification may boost adaptive immune response making PDT more efficient.

Analysis of pathological and biological features of small renal masses based on the tumor size / Kis méretu vesedaganatok patológiai és biológiai jellemzoinek elemzése a tumorméret alapján.

Összefoglaló. Bevezetés: A kis méretu vesedaganatok között lényegesen gyakoribbak a benignus elváltozások, és a kis malignus tumorok biológiai tulajdonságai is kedvezobbek, mint a nagyobb daganatokéi. Célkituzés: Szerzok a kis méretu vesetumorok tulajdonságait vizsgáltuk különbözo alcsoportokban. Módszer: 2000. január 1. és 2015. január 1. között 1272 beteg esetén végeztünk mutétet vesedaganat miatt. Közülük 496 betegnek volt kis méretu vesetumora. A betegek átlagéletkora 59 ± 12 év volt. A betegeket a tumorméret alapján három csoportba osztottuk. Az 1. csoportban a daganat mérete ≤4 cm, a 2. csoportban ≤3 cm és a 3. csoportban ≤2 cm volt. Eredmények: Az eltávolított daganat nagysága átlagosan 29 ± 8 mm volt. A szövettan 418 esetben (84%) malignus, míg 78 alkalommal (16%) benignus elváltozást mutatott. A 2 cm-nél kisebb daganatoknál malignitás csak az esetek 73,2%-ában fordult elo. A malignus és a benignus tumorok méretében szignifikáns eltérés volt (p = 0,008). Rosszul differenciált daganat - grade 3. és 4. - az esetek 10,8%-ában, 14,4%-ában, illetve 20,7%-ában volt jelen, amikor a tumorméret kisebb mint 2 cm, 2,1-3 cm, illetve 3,1-4,0 cm volt. A vesecarcinomáknál az átlagosan 10 éves utánkövetési ido alatt progresszió az esetek 5,5%-ában fordult elo. Következtetés: A kis méretu vesetumor az összes vesedaganat 39%-át tette ki. Ezek nagy része malignus volt, és benignus elváltozás az esetek 16%-ában fordult elo. A malignitás elofordulása a 2 cm-nél kisebb tumoroknál volt a legalacsonyabb. A tumorméret szoros összefüggést mutatott a malignitás gyakoriságával és a daganat differenciáltságával. A kedvezo patológiai és biológiai eredmények alapján a 2 cm alatti daganatoknál felmerül annak lehetosége, hogy esetükben az aktív követés vagy minimálisan invazív kezelés alkalmazása kerüljön elotérbe. Orv Hetil. 2021; 162(42): 1693-1697. INTRODUCTION: The incidence of benign lesions is more common in small renal masses (SRMs) and biological behavior of small malignancies is better compared to larger ones. OBJECTIVE: The authors measured the characteristics of SRMs in different subgroups. METHOD: From January 1, 2000 to January 1, 2015, 1272 patients underwent surgery for renal tumors. In 496 of the 1272 cases, the patients had SRMs. The mean age of the patients was 59 ± 12 years. Based on the sizes, the SRMs were divided into three groups. The sizes of the renal tumors were ≤4 cm in Group 1, ≤3 cm in Group 2 and ≤2 cm in Group 3. RESULTS: The mean diameter of the removed SRMs was 29 ± 8 mm. Histology confirmed renal cell carcinoma in 418 cases (84%), while benign tumor was present in 78 patients (16%). However, with the tumor size ≤2 cm, malignancy was detected in 73.2% of the cases. There was a significant difference in the sizes of the malignant and the benign masses (p = 0.008). Grade 3 or 4 tumors were present in 10.8%, 14.4% and 20.7% when the tumor size was ≤2 cm, 2.1 to 3 cm, and 3.1 to 4 cm in diameter, respectively. During the mean 10-year follow-up period, tumor progression was detected only in 5.5% of malignancies. CONCLUSION: In 39% of all cases, the patients had SRMs. The majority of SRMs were malignant, and benign lesion occurred only in 16% of the cases. The incidence of malignant tumors was the lowest when the size of SRMs was ≤2 cm. The size of the tumor was highly associated with probability of malignancy and tumor grading. Based on the favorable pathological and biological results in tumors below 2 cm, active surveillance or minimally invasive treatment could be the preferred management. Orv Hetil. 2021; 162(42): 1693-1697.

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.

Author Correction: Interactions of Cisplatin and Daunorubicin at the Chromatin Level.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interactions of Cisplatin and Daunorubicin at the Chromatin Level.

Unexpectedly, the widely used anticancer agents Cisplatin (Cis-Pt) and Daunorubicin (Dauno) exhibited cell type- and concentration-dependent synergy or antagonism in vitro. We attempted to interpret these effects in terms of the changes elicited by the drugs in the chromatin, the target held primarily responsible for the cytotoxicity of both agents. We measured the effect of Cis-Pt on the levels of Dauno in different cell compartments, the effect of Cis-Pt on Dauno-induced nucleosome eviction, and assessed the influence of Dauno on DNA platination in flow- and laser scanning cytometry as well as in laser ablation-inductively coupled plasma-mass spectrometry assays. We show that the two drugs antagonize each other through a decrease of interstrand crosslinks upon co-treatment with Dauno, and also via the diminished Dauno uptake in the presence of Cis-Pt, and both effects are observed already at low Dauno concentrations. At high Dauno concentrations synergy becomes dominant because histone eviction by Dauno intercalation into the DNA is enhanced in the presence of co-treatment with Cis-Pt. These interactions may have an impact on the efficacy of combination treatment protocols, considering the long retention time of DNA adducts formed by both agents.

Loss of transglutaminase 2 sensitizes for diet-induced obesity-related inflammation and insulin resistance due to enhanced macrophage c-Src signaling.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes clearance of apoptotic cells (efferocytosis) acting as integrin ß3 coreceptor. Accumulating evidence indicates that defective efferocytosis contributes to the development of chronic inflammatory diseases. Obesity is characterized by the accumulation of dead adipocytes and inflammatory macrophages in the adipose tissue leading to obesity-related metabolic syndrome. Here, we report that loss of TG2 from bone marrow-derived cells sensitizes for high fat diet (HFD)-induced pathologies. We find that metabolically activated TG2 null macrophages express more phospho-Src and integrin ß3, unexpectedly clear dying adipocytes more efficiently via lysosomal exocytosis, but produce more pro-inflammatory cytokines than the wild type ones. Anti-inflammatory treatment with an LXR agonist reverts the HFD-induced phenotype in mice lacking TG2 in bone marrow-derived cells with less hepatic steatosis than in wild type mice proving enhanced lipid clearance. Thus it is interesting to speculate whether LXR agonist treatment together with enhancing lysosomal exocytosis could be a beneficial therapeutic strategy in obesity.

Amino-isocyanoacridines: Novel, Tunable Solvatochromic Fluorophores as Physiological pH Probes.

Amino-isocyanoacridines (ICAAcs), as first members of their class, turned out to be a novel, multifunctional acridine orange (AO) type dye family with a number of additional favorable properties. They have enhanced solvatochromic emission range, low quantum yields (ΦF = 2.9-0.4%) in water, reduced basicity (pKa = 7.05-7.58), and their optical behavior could be fine-tuned by complexation with Ag(I) ions, too. Based on both their vibronic absorption and the charge transfer bands, ICAAcs can be applied as stable pH-probes with great precision (2-3% error) in the physiological pH range of 6-8 using UV-vis and fluorescence detection. The dyes are also able to sense pH change in different microenvironments, such as the Stern layer, as it was demonstrated on sodium lauryl sulfate micelles. The optical behavior of the ICAAc derivatives is discussed based on high-level quantum chemical calculations. All three dyes are well-applicable with conventional epifluorescence imaging. Furthermore, at the blue excitation, diMICAAc is optimally suited as a whole-cell probe for both the conventional microscopic and the laser-illumination studies, like flow- and imaging cytometric, or confocal laser-scanning microscopic examinations.

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.

Macrophages engulf apoptotic and primary necrotic thymocytes through similar phosphatidylserine-dependent mechanisms.

One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat- or H2O2-killed necrotic and apoptotic thymocytes by mouse bone marrow-derived macrophages (BMDMs) in vitro and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDMs. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (a) blocking phosphatidylserine on the surface of dying cells; (b) inhibition of Mer tyrosine kinase, Tim-4, integrin ß3 receptor signaling, or Ras-related C3 botulinum toxin substrate 1 activity; or (c) using BMDMs deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDMs enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.

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.

MICAN, a new fluorophore for vital and non-vital staining of human cells.

Fluorescence time-lapse microscopy is in connection with the invasive properties of fluorochrome applied, and with the toxicity of the excitation energy and wavelength of the dye itself. Experiments with the newly synthesized fluorescent dye 1-N-methylamino-5-isocyanonaphthalene (MICAN) served to test its cytotoxicity on human HaCaT keratinocyte cell cultures. Experiments related to staining capability were performed with paraformaldehyde (PFA) fixed cells and observed with fluorescence microscope. It was assumed that the fluorophore 1-amino-5-isocyanonaphthalene (ICAN) and especially its N-methylamino derivative MICAN, containing condensed aromatic rings could serve as a nonselective fluorescent dye capable to stain cellular structures of fixed, living, damaged and dead cells. This notion was confirmed by the MICAN staining of cytoplasmic proteins primarily rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SEM) and less efficiently nuclear proteins suggesting the involvement of staining of subcellular structures involved in protein synthesis. MICAN was not only well tolerated by living cells but turned out to be a strong heterochromatin and RER staining agent. This led to the development of a MICAN staining protocol for native and living samples. Relative to other fluorescent dyes, MICAN is not only useful but also cost-effective. Toxicology tests were performed using 30, 10, 5, 0.5 µg/ml MICAN concentrations. Time-lapse videomicroscopy at near-infrared (NIR) illumination has been used for the examination of MICAN effect on cell division. It was found that MICAN as a vital stain had no significant harmful effect on HaCaT cells. MICAN turned out to be a non-toxic, highly quantum-efficient vital stain with minimal, or no photobleaching, and can be applied to co-stain with propidium-iodide due the strong spectral separation.