Mitochondrial Energy Metabolism in the Regulation of Thermogenic Brown Fats and Human Metabolic Diseases.

Brown fats specialize in thermogenesis by increasing the utilization of circulating blood glucose and fatty acids. Emerging evidence suggests that brown adipose tissue (BAT) prevents the incidence of obesity-associated metabolic diseases and several types of cancers in humans. Mitochondrial energy metabolism in brown/beige adipocytes regulates both uncoupling protein 1 (UCP1)-dependent and -independent thermogenesis for cold adaptation and the utilization of excess nutrients and energy. Many studies on the quantification of human BAT indicate that mass and activity are inversely correlated with the body mass index (BMI) and visceral adiposity. Repression is caused by obesity-associated positive and negative factors that control adipocyte browning, de novo adipogenesis, mitochondrial energy metabolism, UCP1 expression and activity, and noradrenergic response. Systemic and local factors whose levels vary between lean and obese conditions include growth factors, inflammatory cytokines, neurotransmitters, and metal ions such as selenium and iron. Modulation of obesity-associated repression in human brown fats is a promising strategy to counteract obesity and related metabolic diseases through the activation of thermogenic capacity. In this review, we highlight recent advances in mitochondrial metabolism, thermogenic regulation of brown fats, and human metabolic diseases.

Retinoic acid-related orphan receptor γ (RORγ): a novel participant in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity.

The hepatic circadian clock plays a key role in the daily regulation of glucose metabolism, but the precise molecular mechanisms that coordinate these two biological processes are not fully understood. In this study, we identify a novel connection between the regulation of RORγ by the clock machinery and the diurnal regulation of glucose metabolic networks. We demonstrate that particularly at daytime, mice deficient in RORγ exhibit improved insulin sensitivity and glucose tolerance due to reduced hepatic gluconeogenesis. This is associated with a reduced peak expression of several glucose metabolic genes critical in the control of gluconeogenesis and glycolysis. Genome-wide cistromic profiling, promoter and mutation analysis support the concept that RORγ regulates the transcription of several glucose metabolic genes directly by binding ROREs in their promoter regulatory region. Similar observations were made in liver-specific RORγ-deficient mice suggesting that the changes in glucose homeostasis were directly related to the loss of hepatic RORγ expression. Altogether, our study shows that RORγ regulates several glucose metabolic genes downstream of the hepatic clock and identifies a novel metabolic function for RORγ in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity. The inhibition of the activation of several metabolic gene promoters by an RORγ antagonist suggests that antagonists may provide a novel strategy in the management of metabolic diseases, including type 2 diabetes.

Retinoid acid-related orphan receptor γ, RORγ, participates in diurnal transcriptional regulation of lipid metabolic genes.

The hepatic circadian clock plays a pivotal role in regulating major aspects of energy homeostasis and lipid metabolism. In this study, we show that RORγ robustly regulates the rhythmic expression of several lipid metabolic genes, including the insulin-induced gene 2a, Insig2a, elongation of very long chain fatty acids-like 3, Elovl3 and sterol 12α-hydroxylase, Cyp8b1, by enhancing their expression at ZT20-4. The time-dependent increase in their expression correlates with the rhythmic expression pattern of RORγ. The enhanced recruitment of RORγ to ROREs in their promoter region, increased histone acetylation, and reporter and mutation analysis support the concept that RORγ regulates the transcription of several lipid metabolic genes directly by binding ROREs in their promoter regulatory region. Consistent with the disrupted expression of a number of lipid metabolic genes, loss of RORγ reduced the level of several lipids in liver and blood in a ZT-preferred manner. Particularly the whole-body bile acid pool size was considerably reduced in RORγ(-/-) mice in part through its regulation of several Cyp genes. Similar observations were made in liver-specific RORγ-deficient mice. Altogether, our study indicates that RORγ functions as an important link between the circadian clock and the transcriptional regulation of several metabolic genes.

RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20-hydroxyvitamin D3 [20(OH)D3] and 20,23-dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet-on RORα or RORγ expression vector and a ROR-responsive element (RORE)-LUC reporter, and a mammalian 2-hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD-interacting LXXLL-peptide, were used to study ROR-antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20,23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE-mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL-17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.-Slominski, A. T., Kim, T.-K., Takeda, Y., Janjetovic, Z., Broz˙yna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation.

In this study, we identify Prospero-related homeobox 1 (Prox1) as a novel co-repressor of the retinoic acid-related orphan receptors, RORα and RORγ. Prox1 interacts directly with RORγ and RORα and negatively regulates their transcriptional activity. The AF2 domain of RORs is essential for the interaction, whereas Prox1 interacts with RORs through either its 28 amino acids N-terminal region or its C-terminal prospero-like domain. RORγ antagonists stabilize the interaction between RORγ and Prox1. The homeodomain and the interaction through the prospero-like domain of Prox1 are critical for its repression of ROR transcriptional activity. Chromatin immunoprecipitation analysis demonstrated that in liver, Prox1 is recruited to the ROR response element sites of the clock genes, brain and muscle Arnt-like protein 1 (Bmal1), neuronal PAS domain protein 2 (Npas2) and cryptochrome 1 (Cry1), as part of the same complex as RORs. Knockdown of Prox1 by siRNAs in human hepatoma Huh-7 cells increased the expression of RORγ and several ROR-target genes, along with increased histone acetylation at these ROR response element sites. Chromatin immunoprecipitation sequencing analysis suggests that Prox1 is a potential ROR target gene in liver, which is supported by the regulation of the rhythmic expression of Prox1 by RORγ. Our data suggest that Prox1 is part of a feedback loop that negatively regulates the transcriptional control of clock and metabolic networks by RORs.

RORγ directly regulates the circadian expression of clock genes and downstream targets in vivo.

In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP-Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ-/- liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes.

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with ß-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.

Retinoic acid-related orphan receptor γ directly regulates neuronal PAS domain protein 2 transcription in vivo.

Retinoic acid-related orphan receptors (RORs) and the basic helix-loop-helix-PAS transcription factor Npas2 have been implicated in the control of circadian rhythm. In this study, we demonstrate that RORγ directly regulates Npas2 expression in vivo. Although the rhythmicity of Npas2 mRNA expression was maintained in RORγ(-/-) mice, the peak level of expression was significantly reduced in several tissues, while loss of RORα had little effect. Inversely, overexpression of RORγ in hepatoma Hepa1-6 cells greatly induced the expression of Npas2. RORγ-activated Npas2 transcription directly by binding two ROREs in its proximal promoter. ChIP analysis demonstrated that RORγ was recruited to this promoter in the liver of wild-type mice, but not RORγ-deficient mice. Activation of Npas2 correlated positively with chromatin accessibility and level of H3K9 acetylation. The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317. Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ. Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes.

Inhibitory effects of azole-type fungicides on interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ.

The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. However, it remains unclear whether environmental chemicals, including pesticides, have agonistic and/or antagonistic activity against RORα/γ. In this study, we investigated the RORα/γ activity of several azole-type fungicides, and the effects of these fungicides on the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In the ROR-reporter gene assays, five azole-type fungicides (imibenconazole, triflumizole, hexaconazole, tetraconazole and imazalil) suppressed RORα- and/or RORγ-mediated transcriptional activity as did benzenesulphonamide T0901317, a ROR inverse agonist and a liver X receptor (LXR) agonist. In particular, imibenconazole, triflumizole and hexaconazole showed RORγ inverse agonistic activity at concentrations of 10(-6)M. However, unlike T0901317, these fungicides failed to show any LXRα/ß agonistic activity. Next, five azole-type fungicides, showing ROR inverse agonist activity, were tested on IL-17 mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin. The quantitative RT-PCR analysis revealed that these fungicides suppressed the expression of IL-17 mRNA without effecting RORα and RORγ mRNA levels. In addition, the inhibitory effect of imibenconazole as well as that of T0901317 was absorbed in RORα/γ-knocked down EL4 cells. Taken together, these results suggest that some azole-type fungicides inhibit IL-17 production via RORα/γ. This also provides the first evidence that environmental chemicals can act as modulators of IL-17 expression in immune cells.

Capsaicin directly promotes adipocyte browning in the chemical compound-induced brown adipocytes converted from human dermal fibroblasts.

Human brown fat is a potential therapeutic target for preventing obesity and related metabolic diseases by dissipating energy as heat through uncoupling protein 1 (UCP1). We have previously reported a method to obtain chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts under serum-free conditions. However, pharmacological responses to bioactive molecules have been poorly characterised in ciBAs. This study showed that the treatment with Capsaicin, an agonist of transient receptor potential vanilloid 1, directly activated adipocyte browning such as UCP1 expression, mitochondrial biogenesis, energy consumption rates, and glycerol recycling in ciBAs. Furthermore, genome-wide transcriptome analysis indicated that Capsaicin activated a broad range of metabolic genes including glycerol kinase and glycerol 3-phosphate dehydrogenase 1, which could be associated with the activation of glycerol recycling and triglyceride synthesis. Capsaicin also activated UCP1 expression in immortalised human brown adipocytes but inhibited its expression in mesenchymal stem cell-derived adipocytes. Altogether, ciBAs successfully reflected the direct effects of Capsaicin on adipocyte browning. These findings suggested that ciBAs could serve as a promising cell model for screening of small molecules and dietary bioactive compounds targeting human brown adipocytes.

Chronic Fatty Acid Depletion Induces Uncoupling Protein 1 (UCP1) Expression to Coordinate Mitochondrial Inducible Proton Leak in a Human-Brown-Adipocyte Model.

Thermogenic brown fat contributes to metabolic health in adult humans. Obese conditions are known to repress adipose-tissue browning and its activity. Herein, we found that chronic fatty acid (FA) depletion induced uncoupling protein 1 (UCP1) expression in the chemical-compound-induced brown adipocytes (ciBAs). The ciBAs, converted from human dermal fibroblasts under FA-free conditions, had low intracellular triglyceride levels and strongly activated UCP1 expression. Prolonged treatment with carnitine also reduced triglyceride accumulation and induced UCP1 expression. Transcriptome analysis revealed that the UCP1 induction was accompanied by the activation of lipid metabolic genes. The FA-depleted conditions repressed mitochondrial proton-leak activity and mitochondrial membrane potential (MMP), despite maintaining a high UCP1 expression. The evidence suggested that UCP1 expression was induced to compensate for the proton-leak activity under low MMP. Our study reports a regulatory mechanism underlying UCP1 expression and mitochondrial-energy status in human brown adipocytes under different nutritional conditions.

Transcriptional profiling reveals a role for RORalpha in regulating gene expression in obesity-associated inflammation and hepatic steatosis.

Retinoid-related orphan receptor (ROR)α4 is the major RORα isoform expressed in adipose tissues and liver. In this study we demonstrate that RORα-deficient staggerer mice (RORα(sg/sg)) fed with a high-fat diet (HFD) exhibited reduced adiposity and hepatic triglyceride levels compared with wild-type (WT) littermates and were resistant to the development of hepatic steatosis, adipose-associated inflammation, and insulin resistance. Gene expression profiling showed that many genes involved in triglyceride synthesis and storage, including Cidec, Cidea, and Mogat1, were expressed at much lower levels in liver of RORα(sg/sg) mice. In contrast, overexpression of RORα in mouse hepatoma Hepa1-6 cells significantly increased the expression of genes that were repressed in RORα(sg/sg) liver, including Sult1b1, Adfp, Cidea, and ApoA4. ChIP and promoter analysis suggested that several of these genes were regulated directly by RORα. In addition to reduced lipid accumulation, inflammation was greatly diminished in white adipose tissue (WAT) of RORα(sg/sg) mice fed with an HFD. The infiltration of macrophages and the expression of many immune response and proinflammatory genes, including those encoding various chemo/cytokines, Toll-like receptors, and TNF signaling proteins, were significantly reduced in RORα(sg/sg) WAT. Moreover, RORα(sg/sg) mice fed with an HFD were protected from the development of insulin resistance. RORα(sg/sg) mice consumed more oxygen and produced more carbon dioxide, suggesting increased energy expenditure in this genotype. Our study indicates that RORα plays a critical role in the regulation of several aspects of metabolic syndrome. Therefore, RORα may provide a novel therapeutic target in the management of obesity and associated metabolic diseases.

A circadian neuropeptide PDF in the honeybee, Apis mellifera: cDNA cloning and expression of mRNA.

Pigment-dispersing factor (PDF) is a pacemaker hormone regulating the locomotor rhythm in insects. In the present study, we cloned the cDNAs encoding the Apis PDF precursor protein, and found that there are at least seven different pdf mRNAs yielded by an alternative splicing site and five alternative polyadenylation sites in the 5'UTR and 3'UTR regions. The amino acid sequence of Apis PDF peptide has a characteristic novel amino acid residue, aspargine (Asn), at position 17. Quantitative real-time PCR of total and 5'UTR insertion-type pdf mRNAs revealed, for the first time, that the expression levels change in a circadian manner with a distinct trough at the beginning of night in LD conditions, and at the subjective night under DD conditions. In contrast, the expression level of 5'UTR deletion-type pdf mRNAs was about half of that of the insertion type, and the expression profile failed to show a circadian rhythm. As the expression profile of the total pdf mRNA exhibited a circadian rhythm, transcription regulated at the promoter region was supposed to be controlled by some of the clock components. Whole mount in situ hybridization revealed that 14 lateral neurons at the frontal margin of the optic lobe express these mRNA isoforms. PDF expressing cells examined with a newly produced antibody raised against Apis PDF were also found to have a dense supply of axon terminals in the optic lobes and the central brain.

Transcriptome analysis reveals brown adipogenic reprogramming in chemical compound-induced brown adipocytes converted from human dermal fibroblasts.

Brown adipogenesis contributes to controlling systemic energy balance by enhancing glucose and lipid consumptions. We have previously reported chemical compound-induced brown adipocytes (ciBAs) directly converted from human dermal fibroblasts using a serum-free medium. In this study, genome-wide transcriptional analysis was performed in ciBAs in comparison with the control fibroblasts. A broad range of integrated gene expression was enhanced in functional groups including tricarboxylic acid cycle, electron transfer chain, triglycerides metabolism, fatty acid and glucose metabolism, and adaptive thermogenesis. The results suggested that the chemical conversion underwent metabolic and mitochondrial reprogramming closely associated with functions in brown/beige adipocytes. Moreover, we also compared the transcriptional changes to those of adipocyte browning in adipose tissue-derived mesenchymal stem cells (AdMSCs). Transcriptome analysis indicated that the same sets of metabolic and mitochondria-related genes were similarly changed in the adipocyte browning. Interestingly, ciBAs more expressed Ucp1, while AdMSC-derived adipocytes predominantly expressed Ucp2. UCP1 protein was also more expressed in ciBAs than in AdMSC-derived adipocytes. Based on the evidence that UCP1, but not UCP2, is responsible for adrenergic thermogenesis, ciBAs could be a promising model for human beige adipocytes applicable for basic research, drug development, and clinical uses.

A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes.

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-ß signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-ß pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to ß-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.

Analysis of the Transcriptional Activity of Retinoic Acid-Related Orphan Receptors (RORs) and Inhibition by Inverse Agonists.

Here, we describe several assays to analyze the transcriptional activity of retinoic acid-related orphan receptors (RORs) and the effect of inverse agonists on their activity. One assay measures the effect of an inverse agonist on the transcriptional activation of a luciferase reporter by RORs in a Tet-On cell system. A mammalian two-hybrid assay analyzes the interaction of the ROR ligand binding domain with a coactivator peptide. Two additional assays examine the effect of an inverse agonist on the activation of a luciferase reporter under control of the promoter of the ROR target gene, IL17, and on ROR-mediated activation using a mammalian monohybrid assay.

Chemical compound-based direct reprogramming for future clinical applications.

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy.

Retinoic acid-related Orphan Receptor γ (RORγ): connecting sterol metabolism to regulation of the immune system and autoimmune disease.

Cholesterol and its metabolites are bioactive lipids that interact with and regulate the activity of various proteins and signaling pathways that are implicated in the control of a variety of physiological and pathological processes. Recent studies revealed that retinoic acid-related orphan receptors, RORα and γ, members of the ligand-dependent nuclear receptor superfamily, exhibit quite a wide binding specificity for a number of sterols. Several cholesterol intermediates and metabolites function as natural ligands of RORα and RORγ and act as agonists or inverse agonists. Changes in cholesterol homeostasis that alter the level or type of sterol metabolites in cells, can either enhance or inhibit ROR transcriptional activity that subsequently result in changes in the physiological processes regulated by RORs, including various immune responses and metabolic pathways. Consequently, this might negatively or positively impact pathologies, in which RORs are implicated, such as autoimmune disease, inflammation, metabolic syndrome, cancer, and several neurological disorders. Best studied are the links between cholesterol metabolism, RORγt activity, and their regulation of Th17 differentiation and autoimmune disease. The discovery that Th17-dependent inflammation is significantly attenuated in RORγ-deficient mice in several experimental autoimmune disease models, initiated a search for ROR modulators that led to the identification of a number of small molecular weight RORγ inverse agonists. The inverse agonists suppress Th17 differentiation and IL-17 production and protect against autoimmunity. Together, these studies suggest that RORγt may provide an attractive therapeutic target in the management of several (inflammatory) diseases.

Double-labelled in situ hybridization reveals the lack of co-localization of mRNAs for the circadian neuropeptide PDF and FMRFamide in brains of the flies Musca domestica and Drosophila melanogaster.

Many lines of evidence have suggested that neuropeptides other than pigment-dispersing factor (PDF) are involved in regulating insect circadian rhythms, and FMRFamide-related peptides are additional candidates acting as such neuromodulators. Double-immunolabelling in insect brains with anti-crustacean beta-PDH and anti-FMRFamide antibodies had previously suggested that insect PDF and FMRFamide-like peptides may coexist in the same cells. However, it is critical for this kind of comparative investigations to use antibodies of proven specificity, to eliminate the possibility of both reciprocal cross-reactivity and the detection of unknown peptides. In the present study, we achieved the cDNA cloning of an fmrf mRNA from the housefly Musca domestica, for which co-localization of FMRFamide and PDF peptides was previously suggested. In order to examine the possible co-expression of this gene with the pdf gene, we carried out double-labelled in situ hybridization for simultaneous detection of both pdf and fmrf mRNAs in housefly, Musca brains. The results clearly indicated that they occur in distinctly different cells. This was also proven for the fruit fly Drosophila melanogaster by similar double-labelled in situ hybridization. The results thus revealed no reason to evoke the physiological release of FMRFamide and PDF peptides from the same neurons.

Direct conversion of human fibroblasts to brown adipocytes by small chemical compounds.

Brown adipocytes play an important role in human energy metabolism and prevention of obesity and diabetes. Induced pluripotent stem cells (iPSCs) represent a promising source for brown adipocytes; however, exogenous gene induction is generally required for iPSCs generation, which might cause undesired effects particularly in long-term treatment after transplantation. We have previously reported a cocktail of six small chemical compounds that enables a conversion of human fibroblasts into chemical compound-induced neuronal cells (CiNCs). Here, we report that modified combinations of the chemical compounds and rosiglitazone, a PPARγ agonist, afforded direct conversion of human fibroblasts into brown adipocytes. The chemical compound-induced brown adipocytes (ciBAs) exhibit induction of human brown adipocyte-specific genes such as Ucp1, Ckmt1, Cited1 and other adipocyte-specific genes such as Fabp4, AdipoQ, and Pparγ. Treatment with either isoproterenol or Forskolin further induced the expression of Ucp1, suggesting that ß adrenergic receptor signalling in ciBAs could be functional for induction of thermogenic genes. Moreover, oxygen consumption rates were elevated in ciBAs along with increase of cellular mitochondria. Our findings might provide an easily accessible approach for generating human brown adipocytes from fibroblasts and offer therapeutic potential for the management of obesity, diabetes, and related metabolic disorders.