Nutritional and metabolic control of germ cell fate through O-GlcNAc regulation.

Fate determination of primordial germ cells (PGCs) is regulated in a multi-layered manner, involving signaling pathways, epigenetic mechanisms, and transcriptional control. Chemical modification of macromolecules, including epigenetics, is expected to be closely related with metabolic mechanisms but the detailed molecular machinery linking these two layers remains poorly understood. Here, we show that the hexosamine biosynthetic pathway controls PGC fate determination via O-linked ß-N-acetylglucosamine (O-GlcNAc) modification. Consistent with this model, reduction of carbohydrate metabolism via a maternal ketogenic diet that decreases O-GlcNAcylation levels causes repression of PGC formation in vivo. Moreover, maternal ketogenic diet intake until mid-gestation affects the number of ovarian germ cells in newborn pups. Taken together, we show that nutritional and metabolic mechanisms play a previously unappreciated role in PGC fate determination.

Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.

The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single "master regulator" of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.

A case of convergent evolution: Several viral and bacterial pathogens hijack RSK kinases through a common linear motif.

Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.

Retinoids rescue ceruloplasmin secretion and alleviate oxidative stress in Wilson's disease-specific hepatocytes.

Wilson's disease (WD) is a copper metabolic disorder caused by a defective ATP7B function. Conventional therapies cause severe side effects and significant variation in efficacy, according to cohort studies. Thus, exploring new therapeutic approaches to prevent progression to liver failure is urgent. To study the physiology and pathology of WD, immortalized cell lines and rodent WD models have been used conventionally; however, a large gap remains among different species as well as in genetic backgrounds among individuals. We generated induced pluripotent stem cells (iPSCs) from four WD patients carrying compound heterozygous mutations in the ATP7B gene. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Although the expression of ATP7B protein varied among WD-specific hepatocytes differentiated from these iPSCs, the expression and secretion of ceruloplasmin (Cp), a downstream copper carrier in plasma, were consistently decreased in WD patient-derived and ATP7B-deficient hepatocytes. A transcriptome analysis detected abnormalities in the retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Drug screening using WD patient-derived hepatocytes identified retinoids as promising candidates for rescuing Cp secretion. All-trans retinoic acid also alleviates reactive oxygen species production induced by lipid accumulation in WD-specific hepatocytes treated with oleic acid. These patient-derived iPSC-based hepatic models function as effective platforms for the development of potential therapeutics for hepatic steatosis in WD and other fatty liver diseases.

Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review.

PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-ß cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells.

Xenograft of human pluripotent stem cell-derived cardiac lineage cells on zebrafish embryo heart.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are a promising cell source for regenerative medicine and drug discovery. However, the use of animal models for studying human cardiomyocytes derived from hiPSCs in vivo is limited and challenging. Given the shared properties between humans and zebrafish, their ethical advantages over mammalian models, and their immature immune system that is rejection-free against xenografted human cells, zebrafish provide a suitable alternative model for xenograft studies. We microinjected fluorescence-labeled cardiac lineage cells derived from hiPSCs, specifically mesoderm or cardiac mesoderm cells, into the yolk and the area proximal to the outflow tract of the linear heart at 24 hours post-fertilization (hpf). The cells injected into the yolk survived and did not migrate to other tissues. In contrast, the cells injected contiguous with the outflow tract of the linear heart migrated into the pericardial cavity and heart. After 1 day post injection (1 dpi, 22-24 hpi), the injected cells migrated into the pericardial cavity and heart. Importantly, we observed heartbeat-like movements of some injected cells in the zebrafish heart after 1 dpi. These results suggested successful xenografting of hiPSC-derived cardiac lineage cells into the zebrafish embryo heart. Thus, we developed a valuable tool using zebrafish embryos as a model organism for investigating the molecular and cellular mechanisms involved in the grafting process. This is essential in developing cell transplantation-based cardiac therapeutics as well as for drug testing, notably contributing to advancements in the field of cardio-medicine.

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming.

Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of (odd-skipped related 2 [Osr2]) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of transforming growth factor-ß (TGF-ß) signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-ß signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.

Metabolic pathways regulating the development and non-genomic heritable traits of germ cells.

Metabolism is an important cellular process necessary not only for producing energy and building blocks for cells, but also for regulating various cell functions, including intracellular signaling, epigenomic effects, and transcription. The regulatory roles of metabolism have been extensively studied in somatic cells, including stem cells and cancer cells, but data regarding germ cells are limited. Because germ cells produce individuals of subsequent generations, understanding the role of metabolism and its regulatory functions in germ cells is important. Although limited information concerning the specific role of metabolism in germ cells is available, recent advances in related research have revealed specific metabolic states of undifferentiated germ cells in embryos as well as in germ cells undergoing oogenesis and spermatogenesis. Studies have also elucidated the functions of some metabolic pathways associated with germ cell development and the non-genomic heritable machinery of germ cells. In this review, we summarized all the available knowledge on the characteristic metabolic pathways in germ cells, focusing on their regulatory functions, while discussing the issues that need to be addressed to enhance the understanding of germ cell metabolism.

SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells.

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not-e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased ß3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMP signaling.

In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. Setdb1-deficient embryos exhibit drastic reduction of nascent PGCs. Dppa2, Otx2 and Utf1 are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by Setdb1 knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by Setdb1 knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to Dppa2, Otx2 and Utf1, and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.

Abnormal early folliculogenesis due to impeded pyruvate metabolism in mouse oocytes†.

Fetal ovarian germ cells show characteristic energy metabolism status, such as enhanced mitochondrial metabolism as well as glycolysis, but their roles in early folliculogenesis are unclear. We show here that inhibition of pyruvate uptake to mitochondria by UK5099 in organ cultures of fetal mouse ovaries resulted in repressed early folliculogenesis without affecting energy production, survival of oocytes, or meiosis. In addition, the abnormal folliculogenesis by UK5099 was partially rescued by α-ketoglutarate and succinate, intermediate metabolites in the TCA cycle, suggesting the importance of those metabolites. The expression of TGFß-related genes Gdf9 and Bmp15 in ovarian germ cells, which are crucial for folliculogenesis, was downregulated by UK5099, and the addition of recombinant GDF9 partially rescued the abnormal folliculogenesis induced by UK5099. We also found that early folliculogenesis was similarly repressed, as in the culture, in the ovaries of a germ cell-specific knockout of Mpc2, which encodes a mitochondria pyruvate carrier that is targeted by UK5099. These results suggest that insufficient Gdf9 expression induced by abnormal pyruvate metabolism in oocytes results in early follicular dysgenesis, which is a possible cause of defective folliculogenesis in humans.

OPC-167832, a Novel Carbostyril Derivative with Potent Antituberculosis Activity as a DprE1 Inhibitor.

There is an urgent need for new, potent antituberculosis (anti-TB) drugs with novel mechanisms of action that can be included in new regimens to shorten the treatment period for TB. After screening a library of carbostyrils, we optimized 3,4-dihydrocarbostyril derivatives and identified OPC-167832 as having potent antituberculosis activity. The MICs of the compound for Mycobacterium tuberculosis ranged from 0.00024 to 0.002 µg/ml. It had bactericidal activity against both growing and intracellular bacilli, and the frequency of spontaneous resistance for M. tuberculosis H37Rv was less than 1.91 × 10-7 It did not show antagonistic effects with other anti-TB agents in an in vitro checkerboard assay. Whole-genome and targeted sequencing of isolates resistant to OPC-167832 identified decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), an essential enzyme for cell wall biosynthesis, as the target of the compound, and further studies demonstrated inhibition of DprE1 enzymatic activity by OPC-167832. In a mouse model of chronic TB, OPC-167832 showed potent bactericidal activities starting at a dose of 0.625 mg/kg of body weight. Further, it exhibited significant combination effects in 2-drug combinations with delamanid, bedaquiline, or levofloxacin. Finally, 3- or 4-drug regimens comprised of delamanid and OPC-167832 as the core along with bedaquiline, moxifloxacin, or linezolid showed efficacy in reducing the bacterial burden and preventing relapse superior to that of the standard treatment regimen. In summary, these results suggest that OPC-167832 is a novel and potent anti-TB agent, and regimens containing OPC-167832 and new or repurposed anti-TB drugs may have the potential to shorten the duration of treatment for TB.

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†.

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells.

Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of 'chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease.

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells.

Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.

Characterization of PC12 Cell Subclones with Different Sensitivities to Programmed Thermal Stimulation.

Neuritogenesis is the process underling nervous system regeneration; however, optimal extracellular signals that can promote neuronal regenerative activities require further investigation. Previously, we developed a novel method for inducing neuronal differentiation in rat PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) with a heating plate. Based on neurogenic sensitivity to TRTS, PC12 cells were classified as either hyper- or hyposensitive. In this study, we aimed to investigate the mechanism of hyposensitivity by establishing two PC12-derived subclones according to TRTS sensitivity during differentiation: PC12-P1F1, a hypersensitive subclone, and PC12-P1D10, a hyposensitive subclone. To characterize these subclones, cell size and neuritogenesis were evaluated in subclones treated with nerve growth factor (NGF), bone morphogenetic protein (BMP), or various TRTS. No significant differences in cell size were observed among the parental cells and subclones. BMP4- or TRTS-induced neuritogenesis was increased in PC12-P1F1 cells compared to that in the parental cells, while no neuritogenesis was observed in PC12-P1D10 cells. In contrast, NGF-induced neuritogenesis was observed in all three cell lines. Furthermore, a BMP inhibitor, LDN-193189, considerably inhibited TRTS-induced neuritogenesis. These results suggest that the BMP pathway might be required for TRTS-induced neuritogenesis, demonstrating the useful aspects of these novel subclones for TRTS research.

Pluripotent Stem Cell Heterogeneity.

Pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, show heterogeneity with respect to their pluripotency, self-renewal ability, and other traits. PSC heterogeneity may exist among cell lines, among cells within a line, and among temporal states of individual cells. Both genetic and epigenetic factors can cause heterogeneity among cell lines. Heterogeneity among cells within a cell line may arise during long-term culturing even when a PSC cell line is derived from a single cell. Moreover, the expression levels of genes and proteins in PSCs fluctuate continuously at a frequency ranging from a few hours to a few days. Such heterogeneity decreases the reproducibility of research. Thus, methods related to the detection, reduction, and control of heterogeneity in experiments involving human PSCs need to be developed. Further, the presupposition that PSCs are highly heterogeneous should be taken into account by all researchers not only when they plan their own studies but also when they review the studies of other researchers in this field.

Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program.

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

Association between Beta3-Adrenergic Receptor Trp64Arg Polymorphism and Fat Preference in Healthy Young Japanese Women.

Beta3-adrenergic receptor (ADRB3) is a mediator of catecholamine-stimulated lipolysis in humans. The Trp64Arg polymorphism with T/C transition in the ADRB3 gene has been considered to reduce lipolysis and metabolic expenditure. Here, we investigated the hitherto unknown role of the Trp64Arg substitution on food preference among healthy young adults (mean age, 24.3; n = 53, including 25 men). Preference toward four food types (bitter, sour, salty, or sweet) and greasy (high-fat) foods was examined using a self-reported questionnaire. There was no noticeable sex-difference in food preference. Incidentally, only among female subjects, the genotype frequencies of the Trp64Arg polymorphism were in accordance with the Hardy-Weinberg equilibrium. Consequently, female subjects were divided into two groups for further analyses: 18 subjects with TT genotype (Trp64Trp) (wild-type group) and 10 subjects with TC genotype (Trp64Arg) (heterozygous group). No significant difference was observed in preference for four food types between the groups. However, when sweet foods were divided into high-fat and low-fat subgroups, food preference for high-fat sweet foods in heterozygous group was significantly higher than that in wild-type group. Moreover, when subjects were divided into two classes based on preference for greasy foods (like, n = 16 or dislike, n = 12), the preference degree in heterozygous group who liked high-fat foods (n = 5) was significantly higher than that in wild-type group (n = 11), suggesting that the Trp64Arg substitution might genetically enhance high-fat food preference. Thus, understanding the relationship between ADRB3 Trp64Arg substitution and fat preference will be valuable for obesity prevention.