The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

The Inhibition of TREK-1 K+ Channels via Multiple Compounds Contained in the Six Kamikihito Components, Potentially Stimulating Oxytocin Neuron Pathways.

Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.

Effect of mitochondrial quantity and quality controls in white adipose tissue on healthy lifespan: Essential roles of GH/IGF-1-independent pathways in caloric restriction-mediated metabolic remodeling.

Long-term caloric restriction is a conventional and reproducible dietary intervention to improve whole body metabolism, suppress age-related pathophysiology, and extend lifespan. The beneficial actions of caloric restriction are widely accepted to be regulated in both growth hormone/insulin-like growth factor 1-dependent and -independent manners. Although growth hormone/insulin-like growth factor 1-dependent regulatory mechanisms are well described, those occurring independent of growth hormone/insulin-like growth factor 1 are poorly understood. In this review, we focus on molecular mechanisms of caloric restriction regulated in a growth hormone/insulin-like growth factor 1-independent manner. Caloric restriction increases mitochondrial quantity and improves mitochondrial quality by activating an axis involving sterol regulatory element binding protein-c/peroxisome proliferator-activated receptor γ coactivator-1α/mitochondrial intermediate peptidase in a growth hormone/insulin-like growth factor 1-independent manner, particularly in white adipose tissue. Fibroblast growth factor 21 is also involved in this axis. Moreover, the axis may be regulated by lower leptin signaling. Thus, caloric restriction appears to induce beneficial actions partially by regulating mitochondrial quantity and quality in white adipose tissue in a growth hormone/insulin-like growth factor 1-independent manner.

Long-Term Dietary Taurine Lowers Plasma Levels of Cholesterol and Bile Acids.

Cholesterol is an essential lipid in vertebrates, but excess blood cholesterol promotes atherosclerosis. In the liver, cholesterol is metabolized to bile acids by cytochrome P450, family 7, subfamily a, polypeptide 1 (CYP7A1), the transcription of which is negatively regulated by the ERK pathway. Fibroblast growth factor 21 (FGF21), a hepatokine, induces ERK phosphorylation and suppresses Cyp7a1 transcription. Taurine, a sulfur-containing amino acid, reportedly promotes cholesterol metabolism and lowers blood and hepatic cholesterol levels. However, the influence of long-term feeding of taurine on cholesterol levels and metabolism remains unclear. Here, to evaluate the more chronic effects of taurine on cholesterol levels, we analyzed mice fed a taurine-rich diet for 14-16 weeks. Long-term feeding of taurine lowered plasma cholesterol and bile acids without significantly changing other metabolic parameters, but hardly affected these levels in the liver. Moreover, taurine upregulated Cyp7a1 levels, while downregulated phosphorylated ERK and Fgf21 levels in the liver. Likewise, taurine-treated Hepa1-6 cells, a mouse hepatocyte line, exhibited downregulated Fgf21 levels and upregulated promoter activity of Cyp7a1. These results indicate that taurine promotes cholesterol metabolism by suppressing the FGF21/ERK pathway followed by upregulating Cyp7a1 expression. Collectively, this study shows that long-term feeding of taurine lowers both plasma cholesterol and bile acids, reinforcing that taurine effectively prevents hypercholesterolemia.

Mitochondrial Unfolded Protein Responses in White Adipose Tissue: Lipoatrophy, Whole-Body Metabolism and Lifespan.

The mitochondrial unfolded protein response (UPRmt) is a stress response mediated by the expression of genes such as chaperones, proteases, and mitokines to maintain mitochondrial proteostasis. Certain genetically modified mice, which defect mitochondrial proteins specifically in adipocytes, developed atrophy of the white adipose tissue, resisted diet-induced obesity, and had altered whole-body metabolism. UPRmt, which has beneficial functions for living organisms, is termed "mitohormesis", but its specific characteristics and detailed regulatory mechanism have not been elucidated to date. In this review, we discuss the function of UPRmt in adipose atrophy (lipoatrophy), whole-body metabolism, and lifespan based on the concept of mitohormesis.

Contribution of PGC-1α to Obesity- and Caloric Restriction-Related Physiological Changes in White Adipose Tissue.

Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) regulates mitochondrial DNA replication and mitochondrial gene expression by interacting with several transcription factors. White adipose tissue (WAT) mainly comprises adipocytes that store triglycerides as an energy resource and secrete adipokines. The characteristics of WAT vary in response to systemic and chronic metabolic alterations, including obesity or caloric restriction. Despite a small amount of mitochondria in white adipocytes, accumulated evidence suggests that mitochondria are strongly related to adipocyte-specific functions, such as adipogenesis and lipogenesis, as well as oxidative metabolism for energy supply. Therefore, PGC-1α is expected to play an important role in WAT. In this review, we provide an overview of the involvement of mitochondria and PGC-1α with obesity- and caloric restriction-related physiological changes in adipocytes and WAT.

A novel method for evaluating activity of transient receptor potential channels using a cellular dielectric spectroscopy.

Cellular dielectric spectroscopy (CDS) is a novel technology enabling pharmacological evaluation of multiple receptor types with a label-free cell-based assay. We evaluated activities of a family of ligand-gated channels, transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels by an electrical impedance-based biosensor (CellKey™ system) using CDS. Measures of both potency (EC50) and efficacy (Emax) of these agonists with CellKey™ were almost identical to those made using the traditional Ca2+ influx assay in TRPV1- or TRPA1-expressing cells, suggesting that CellKey™ is a simpler and easier means of evaluating TRP activities.

Hyperglycemia contributes to the development of Leydig cell hyperplasia in male Spontaneously Diabetic Torii rats.

Spontaneously Diabetic Torii (SDT) rats are a well-known animal model of non-obese type 2 diabetes mellitus. Although this animal model has been studied extensively over the last decade, the incidence rates of Leydig cell hyperplasia and tumors in this model have not been reported. In this study, pathophysiological analyses of the testes were performed on male SDT rats, to understand the effect of insulin treatment on the development of Leydig cell hyperplasia and tumors and the expression of integrins and extracellular matrix proteins. Testicular Leydig cell hyperplasia and tumors were observed in SDT rats at 64 weeks of age but were rarely identified in Sprague-Dawley (SD) rats of the same age. Insulin treatment decreased plasma glucose and HbA1c levels, and interestingly, decreased the number of hyperplastic Leydig cell foci and Leydig cell tumors in treated animals. A similar reduction in the expression of Ki67 in these Leydig cell foci was also observed. In addition, insulin treatment decreased the expression of integrin α5, integrin ß1, integrin αvß3, fibronectin, and vitronectin in hyperplastic Leydig cell foci. These results suggest that insulin might decrease the incidence of Leydig cell hyperplasia by reducing Leydig cell proliferation and the expression of integrins and extracellular matrix proteins through the reduction of serum glucose concentrations in these animals.

Identification of WWP1 as an obesity-associated E3 ubiquitin ligase with a protective role against oxidative stress in adipocytes.

White adipose tissue (WAT) is not only the main tissue for energy storage but also an endocrine organ that secretes adipokines. Obesity is the most common metabolic disorder and is related to alterations in WAT characteristics, such as chronic inflammation and increasing oxidative stress. WW domain containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type ubiquitin E3 ligase that has been implicated in various pathologies. In the present study, we found that WWP1 was upregulated in obese WAT in a p53-dependent manner. To investigate the functions of WWP1 in adipocytes, a proteome analysis of WWP1 overexpression (OE) and knockdown (KD) 3T3-L1 cells was performed. This analysis showed a positive correlation between WWP1 expression and the abundance of several antioxidative proteins. Thus, we measured reactive oxygen species (ROS) in WWP1 OE and KD cells. Consistent with the proteome results, WWP1 OE reduced ROS levels, whereas KD increased them. These findings indicate that WWP1 is an obesity-inducible E3 ubiquitin ligase that can protect against obesity-associated oxidative stress in WAT.

Association between Lysosomal Dysfunction and Obesity-Related Pathology: A Key Knowledge to Prevent Metabolic Syndrome.

Obesity causes various health problems, such as type 2 diabetes, non-alcoholic fatty liver disease, and cardio- and cerebrovascular diseases. Metabolic organs, particularly white adipose tissue (WAT) and liver, are deeply involved in obesity. WAT contains many adipocytes with energy storage capacity and secretes adipokines depending on the obesity state, while liver plays pivotal roles in glucose and lipid metabolism. This review outlines and underscores the relationship between obesity and lysosomal functions, including lysosome biogenesis, maturation and activity of lysosomal proteases in WAT and liver. It has been revealed that obesity-induced abnormalities of lysosomal proteases contribute to inflammation and cellular senescence in adipocytes. Previous reports have demonstrated obesity-induced ectopic lipid accumulation in liver is associated with abnormality of lysosomal proteases as well as other lysosomal enzymes. These studies demonstrate that lysosomal dysfunction in WAT and liver underlies part of the obesity-related pathology, raising the possibility that strategies to modulate lysosomal function may be effective in preventing or treating the metabolic syndrome.

Carboplatin Enhances the Activity of Human Transient Receptor Potential Ankyrin 1 through the Cyclic AMP-Protein Kinase A-A-Kinase Anchoring Protein (AKAP) Pathways.

Carboplatin, an anticancer drug, often causes chemotherapy-induced peripheral neuropathy (PN). Transient receptor potential ankyrin 1 (TRPA1), a non-selective cation channel, is a polymodal nociceptor expressed in sensory neurons. TRPA1 is not only involved in pain transmission, but also in allodynia or hyperalgesia development. However, the effects of TRPA1 on carboplatin-induced PN is unclear. We revealed that carboplatin induced mechanical allodynia and cold hyperalgesia, and the pains observed in carboplatin-induced PN models were significantly suppressed by the TRPA1 antagonist HC-030031 without a change in the level of TRPA1 protein. In cells expressing human TRPA, carboplatin had no effects on changes in intracellular Ca2+ concentration ([Ca2+]i); however, carboplatin pretreatment enhanced the increase in [Ca2+]i induced by the TRPA1 agonist, allyl isothiocyanate (AITC). These effects were suppressed by an inhibitor of protein kinase A (PKA). The PKA activator forskolin enhanced AITC-induced increase in [Ca2+]i and carboplatin itself increased intracellular cyclic adenosine monophosphate (cAMP) levels. Moreover, inhibition of A-kinase anchoring protein (AKAP) significantly decreased the carboplatin-induced enhancement of [Ca2+]i induced by AITC and improved carboplatin-induced mechanical allodynia and cold hyperalgesia. These results suggested that carboplatin induced mechanical allodynia and cold hyperalgesia by increasing sensitivity to TRPA1 via the cAMP-PKA-AKAP pathway.

A novel mouse model for tracking the fate of CXCR5-expressing T cells.

The germinal center (GC) reaction, a critical process in the humoral immune response, requires follicular helper T (Tfh) cells. Tfh cells express the master transcription factor Bcl6 and chemokine receptor CXCR5, which enable them to migrate from the T cell zone to B cell follicles and interact with GC B cells. However, CXCR5 is downregulated when Tfh cells become memory cells. Therefore, it is difficult to track Tfh cells continuously in vivo. In this study, we generated a mouse strain, Cxcr5CreERT2R26Tomato, in which the fluorescent protein tdTomato is inducibly expressed in CXCR5+ cells by tamoxifen administration. After the oral administration of tamoxifen, most Tfh cells in Peyer's patches (PP) from Cxcr5CreERT2R26Tomato mice were tdTomato+. To track antigen-specific Tfh cells in vivo, OVA-specific OT-II T cells derived from Cxcr5CreERT2R26Tomato mice were transferred to wild-type mice, and the recipient mice were immunized with OVA followed by tamoxifen administration. CXCR5+ T cells became tdTomato+ and were mainly located in B cell follicles and GC areas 8 days after immunization. Four weeks after immunization, tdTomato+ OT-II T cells migrated from B cell follicles to the T-B border area and T cell zone after CXCR5 downregulation and CCR7 upregulation. These results indicate that Cxcr5CreERT2R26Tomato mice are a useful tool for studying the cell fate of differentiated Tfh cells in vivo and therefore have implications for the development of therapeutic strategies for infectious and autoimmune diseases.

The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes.

BACKGROUND: C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified. METHODS: We performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database. RESULTS: We obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC. CONCLUSIONS: Our study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.

Taurine is an amino acid with the ability to activate autophagy in adipocytes.

Alterations in adipocyte characteristics are highly implicated in the pathology of obesity. In a recent article, we demonstrated that high-fat diet-induced obesity impairs lysosomal function, thereby suppressing autophagy in mice white adipose tissue. Taurine, an amino acid naturally contained in the normal diet and existing ubiquitously in tissues, has been reported to improve insulin resistance and chronic inflammation in animal models, but underlying mechanisms remain unclear. From these findings, we hypothesized that improvement of obese pathology by taurine may be mediated through recovery of autophagy. In matured 3T3-L1 mouse adipocytes, treatment with taurine-promoted autophagy. Moreover, taurine-induced nuclear translocation of transcription factor EB (TFEB), a master regulator of autophagy- and lysosome-related factors. As this translocation is regulated by several kinase pathways, including extracellular signal-related kinase 1 and 2 (ERK1/2) and mechanistic target of rapamycin protein kinase complex 1 (MTORC1), we examined related signaling elements. Consequently, taurine-reduced phosphorylation levels of ERK1/2 but did not alter the phosphorylation of MTORC1 pathway-associated adenosine monophosphate-activated protein kinase or ribosomal protein S6 kinase. Taken together, these results suggest that taurine may enhance TFEB nuclear translocation through ERK1/2 to accelerate autophagy. The effect discovered in this study may represent a novel mechanism for the improvement of obesity-related pathology by taurine.

SREBP-1c-Dependent Metabolic Remodeling of White Adipose Tissue by Caloric Restriction.

Caloric restriction (CR) delays the onset of many age-related pathophysiological changes and extends lifespan. White adipose tissue (WAT) is not only a major tissue for energy storage, but also an endocrine tissue that secretes various adipokines. Recent reports have demonstrated that alterations in the characteristics of WAT can impact whole-body metabolism and lifespan. Hence, we hypothesized that functional alterations in WAT may play important roles in the beneficial effects of CR. Previously, using microarray analysis of WAT from CR rats, we found that CR enhances fatty acid (FA) biosynthesis, and identified sterol regulatory element-binding protein 1c (SREBP-1c), a master regulator of FA synthesis, as a mediator of CR. These findings were validated by showing that CR failed to upregulate factors involved in FA biosynthesis and to extend longevity in SREBP-1c knockout mice. Furthermore, we revealed that SREBP-1c is implicated in CR-associated mitochondrial activation through the upregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Notably, these CR-associated phenotypes were observed only in WAT. We conclude that CR induces SREBP-1c-dependent metabolic remodeling, including the enhancement of FA biosynthesis and mitochondrial activation, via PGC-1α in WAT, resulting in beneficial effects.

Differential Metabolic Responses to Adipose Atrophy Associated with Cancer Cachexia and Caloric Restriction in Rats and the Effect of Rikkunshito in Cancer Cachexia.

Despite the similar phenotypes, including weight loss, reduction of food intake, and lower adiposity, associated with caloric restriction (CR) and cancer cachexia (CC), CC is a progressive wasting syndrome, while mild CR improves whole body metabolism. In the present study, we compared adipose metabolic changes in a novel rat model of CC, mild CR (70% of the food intake of control rats, which is similar to the food consumption of CC rats), and severe CR (30% of the food intake of controls). We show that CC and severe CR are associated with much smaller adipocytes with significantly lower mitochondrial DNA content; but, that mild CR is not. CC and both mild and severe CR similarly upregulated proteins involved in lipolysis. CC also downregulated proteins involved in fatty acid biosynthesis, but mild CR upregulated these. These findings suggest that CC might impair de novo fatty acid biosynthesis and reduce mitochondrial biogenesis, similar to severe CR. We also found that rikkunshito, a traditional Japanese herbal medicine, does not ameliorate the enhanced lipolysis and mitochondrial impairment, but rather, rescues de novo fatty acid biosynthesis, suggesting that rikkunshito administration might have partially similar effects to mild CR.

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases.

BACKGROUND: CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles. METHODS AND RESULTS: In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at (296)SNLD(299) and (348)TCPD(351), and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified (320)EPRT(323) as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at (320)EPRT(323). Additionally, the cleavage catalyzed by the (320)EPRT(323) protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment. CONCLUSION: CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the "(320)EPRT(323) protease(s)." Furthermore, (320)EPRT(323) cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage. GENERAL SIGNIFICANCE: CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.

New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line.

Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

Inhibitory effect of p53 on mitochondrial content and function during adipogenesis.

The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.

History of the G protein-coupled receptor (GPCR) assays from traditional to a state-of-the-art biosensor assay.

The G protein-coupled receptors (GPCRs) form the largest and the most versatile superfamily that share a seven-transmembrane-spanning architecture. GPCR-signaling is involved in vision, taste, olfaction, sympathetic/parasympathetic nervous functions, metabolism, and immune regulation, indicating that GPCRs are extremely important therapeutic targets for various diseases. Cellular dielectric spectroscopy (CDS) is a novel technology that employs a label-free, real-time and cell-based assay approach for the comprehensive pharmacological evaluation of cells that exogenously or endogenously express GPCRs. Among the biosensors that use CDS technology, the CellKey™ system not only detects the activation of GPCRs but also distinguishes between signals through different subtypes of the Gα protein (Gs, Gi/o, and Gq). In this review, we discuss the traditional assays and then introduce the principles by which the CellKey™ system evaluates GPCR activation, followed by a perspective on the advantages and future prospects of this system.