CREG1 stimulates AMPK phosphorylation and glucose uptake in skeletal muscle cells.

The cellular repressor of adenovirus early region 1A-stimulated gene 1 (CREG1) is a secreted glycoprotein involved in cell differentiation and energy metabolism. It also binds to insulin-like growth factor 2 receptor (IGF2R), a protein implicated in muscle regeneration. However, whether CREG1 regulates the regeneration and metabolism of skeletal muscles via IGF2R remains unclear. This study investigates the role of CREG1 in skeletal muscle regeneration and glucose uptake in C2C12 myotubes and a cardiotoxin (CTX)-induced mouse skeletal muscle regeneration model. CTX-treated skeletal muscle showed significantly higher levels of IGF2R, CREG1, phospho-AMPKα Thr172, and GLUT4 proteins. Similarly, treatment of myotubes with CREG1 also stimulated AMPKα phosphorylation and GLUT4 expression. CREG1-induced AMPKα phosphorylation and 2DG uptake in myotubes were suppressed by IGF2R knockdown and Compound C, an AMPK inhibitor. These results suggest that CREG1 stimulates glucose uptake in skeletal muscles partially through AMPK activation. Hence, CREG1 plays an essential role in muscle regeneration by affecting glucose metabolism in skeletal muscles.

CREG1 improves diet-induced obesity via uncoupling protein 1-dependent manner in mice.

Thermogenic brown and beige adipocytes express uncoupling protein 1 (UCP1) and stimulate energy metabolism, protecting against obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Cellular repressor of E1A-stimulated genes 1 (CREG1) can stimulate thermogenic fat formation, induce UCP1, and reduce diet-induced obesity (DIO) in mice at normal room temperature. In this study, we investigated the effect of CREG1 administration and the importance of UCP1 in DIO inhibition under thermoneutral conditions at 30°C, which attenuate thermogenic fat formation. Interestingly, subcutaneous administration of recombinant CREG1 protein via an osmotic pump in C57BL/6J mice for four weeks increased UCP1 expression in interscapular brown adipose tissue (IBAT), inhibited visceral white fat hypertrophy with partial browning, and reduced DIO compared to that in PBS-treated mice. The mRNA expression of energy metabolism-related genes was significantly increased in the IBAT of CREG1-treated mice compared to that in PBS-treated mice. In contrast, adipocyte-specific overexpression of CREG1 failed to improve DIO in UCP1-knockout mice at thermoneutrality. Our results indicate the therapeutic potential of CREG1 administration for obesity under thermogenic fat-attenuating conditions and highlight the indispensable role of UCP1 in the DIO-inhibitory effect of CREG1.

The effect of Dunaliella tertiolecta supplementation on diet-induced obesity in UCP1-deficient mice.

We previously demonstrated that dietary supplementation with Dunaliella tertiolecta (DT) increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) and improves diet-induced obesity (DIO) in C57BL/6 J mice at thermoneutrality (30 °C). Here, we investigated whether DT improves DIO in a thermoneutral UCP1-deficient (KO) animal. KO mice were fed a high-fat diet supplemented with DT for 12 weeks. Compared to control group without DT, body weight was significantly reduced in DT group with no difference in food intake. Dunaliella tertiolecta-supplemented mice exhibited lower adiposity and well-maintained multilocular morphology in BAT, in which a significant increase in gene expression of PR domain containing 16 was detected in DT group compared to control group. Moreover, increase in UCP2 level and/or decrease in ribosomal protein S6 phosphorylation were detected in adipose tissues of DT group relative to control group. These results suggest that DT supplementation improves DIO by stimulating UCP1-independent energy dissipation at thermoneutrality.

Deletion of UCP1 in Tg2576 Mice Increases Body Temperature and Exacerbates Alzheimer's Disease-Related Pathologies.

We previously demonstrated that the Alzheimer's disease (AD)-like model mice, Tg2576, housed at a high ambient temperature of 30 °C for 13 months, exhibited increased body temperature, which increased amyloid-ß (Aß) levels and tau stability, leading to tau phosphorylation and ultimately inducing memory impairment. Here, we aimed to exclude the possible effect of environmental factors associated with the difference in ambient temperature (23 °C vs. 30 °C) and to further clarify the effects of elevated body temperature on AD-like pathologies. We generated uncoupling protein 1 (UCP1) deletion in Tg2576 mice, Tg2576/UCP1-/-, because UCP1 deletion mice show a sustained rise in body temperature at normal room temperature. As expected, the body temperature in Tg2576/UCP1-/- mice was higher than that in Tg2576/ UCP1+/+ mice at 23 °C, which was accompanied by upregulated Aß levels due to increased ß-secretase (BACE1) and decreased neprilysin (NEP) protein levels in the brains of Tg2576/UCP1-/- mice compared with those in the Tg2576/ UCP1+/+ mice. Elevated body temperature also increased total tau levels, leading to enhanced phosphorylation, heat shock protein induction, and activated tau kinases. Furthermore, elevated body temperature enhanced glial activation and decreased synaptic protein levels in the brain. Taken together, these findings demonstrate that elevated body temperatures exacerbate AD-like pathologies.

High temperature promotes amyloid ß-protein production and γ-secretase complex formation via Hsp90.

Alzheimer's disease (AD) is characterized by neuronal loss and accumulation of ß-amyloid-protein (Aß) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25-40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aß production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aß production. Here, we report temperature-dependent regulation of Aß production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aß production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aß production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aß production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.

Effects of CREG1 on Age-Associated Metabolic Phenotypes and Renal Senescence in Mice.

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.

CREG1 stimulates brown adipocyte formation and ameliorates diet-induced obesity in mice.

Increased formation of brown and beige adipocytes is critical for adaptive thermogenesis to maintain homeothermy in cold or to circumvent diet-induced obesity (DIO). Cellular repressor of adenovirus early region 1A-stimulated genes 1 (CREG1) exhibits the ability to stimulate brown adipogenesis, including the induction of uncoupling protein 1 (UCP1), in vitro. Thus, we aimed to clarify whether CREG1 promotes brown adipocyte formation and inhibits DIO at the whole-animal level. In mouse brown adipose tissue (BAT), CREG1 expression was markedly increased in cold but was decreased under thermoneutrality, suggesting CREG1 involvement in BAT thermogenesis. Moreover, in BAT and white adipose tissue, expression of UCP1 and fibroblast growth factor-21 and browning were both significantly higher in adipocyte P2-Creg1-transgenic (Tg) mice than in wild-type (WT) littermates. Following stimulation with a ß3-adrenergic agonist, energy consumption was elevated in the Tg mice, which showed increased resistance to DIO and improvement of obesity-associated complications including fatty liver relative to WT mice. The CREG1 stimulatory effect on brown adipogenesis was confirmed in Tg-BAT primary cultures. It was also found that CREG1 binds to retinoid X receptor α, which interacts with thyroid hormone receptor for brown adipogenesis. Our findings demonstrate that CREG1 stimulates brown adipocyte formation and browning, ameliorating obesity and its related pathology in vivo.-Hashimoto, M., Kusudo, T., Takeuchi, T., Kataoka, N., Mukai, T., Yamashita, H. CREG1 stimulates brown adipocyte formation and ameliorates diet-induced obesity in mice.

Brown adipocytes and ß3-stimulant-induced brown-like adipocytes contribute to the prevention of renal crystal formation.

According to recent studies, kidney stones are associated with metabolic syndrome. We focused on brown adipocytes and ß3-stimulant-induced brown-like adipocytes to investigate how these adipocytes influence kidney stone disease. For the interscapular brown adipose tissue (iBAT) removal experiment, mice were subjected to either iBAT removal or sham operation (X-BAT group or sham group), and, after 3 wk, renal crystal deposition was induced by intra-abdominal injection of glyoxylate (GOX) for 6 days. For the ß3-stimulant experiment, mice were administered intra-abdominal injections of the ß3-stimulant (ß3-group) or saline (control group) for 6 days. Thereafter, renal crystal deposition was induced by intra-abdominal injection of GOX for 6 days. iBAT removal decreased the expression of Sod1 and increased that of chemokine (C-C motif) ligand 2 (Ccl2), EGF module-containing mucin-like receptor 1 (Emr1), and tumor necrosis factor (Tnf) in the kidneys. Renal crystal deposition was 2.06-fold higher in the X-BAT group than in the sham group. The ß3-stimulant caused differentiation of white adipocytes into brown-like adipocytes. In the kidneys of the ß3-group, the expression of Ccl2 and Emr1 decreased and that of Sod1 increased. Renal crystal deposition was 0.17-fold lower in the ß3-group than in the control group. In summary, iBAT removal promoted kidney inflammation and renal crystal formation. ß3-Stimulant-induced brown-like adipocytes reduced inflammation and improved antioxidant action in the kidneys, which suppressed renal crystal formation. This is the first report on the therapeutic role of brown and brown-like adipocytes for kidney stone formation.

Trends and quality of randomized controlled trials on acupuncture conducted in Japan by decade from the 1960s to the 2010s: a systematic review.

BACKGROUND: More new randomized controlled trials (RCTs) on acupuncture have been published in Japan since our last updated systematic review (2010). This systematic review aimed to evaluate the quality of RCTs on acupuncture conducted in Japan and understand the decade-wise changes in the methodological characteristics of the relevant RCTs. METHODS: The literature search was performed using Ichushi Web, the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed and our team's compilation of relevant papers. We included full-length papers reporting RCTs that examined the clinical effects of acupuncture on patients in Japan published in or before 2019. We assessed the risk of bias (RoB), sample size, control setting, negative trial reporting, informed consent, ethics approval, trial registration, and adverse event reporting. RESULTS: A total of 99 articles reporting 108 eligible RCTs were identified. The number of RCTs published in each decade was 1, 6, 9, 5, 40, and 47 in the 1960s, 1970s, 1980s, 1990s, 2000s, and 2010s, respectively. Quality assessment using the Cochrane RoB tool revealed that "sequence generation" improved in and after 1990 (73%-80% of RCTs were rated as "low") and "blinding of outcome assessors" slightly improved in and after the 2000s (40%-50% judged as "low"). However, "high" or "unclear" remained the dominant grades in other domains. Clinical trial registration and adverse events were reported only in 9% and 28% of the included RCTs even in the 2010s, respectively. A different acupuncture method or different point selection (e.g., deep vs. shallow insertion) was the most dominant control setting before 1990, while sham (or "placebo") needling and/or sham acupoints became the most dominant in the 2000s. The proportion of RCTs with positive results was 80% in the 2000s and 69% in the 2010s. CONCLUSIONS: The quality of RCTs on acupuncture conducted in Japan did not appear to have improved over the decades except for "sequence generation." While the culture of submitting negative trial reports was prevalent in the Japanese acupuncture research milieu as late as the 1990s, the overall quality of the relevant trials needs to be further improved.

Relationship between health literacy and attitudes toward acupuncture: A web-based cross-sectional survey with a panel of Japanese residents.

The relationship between health literacy of Japanese people, their attitudes toward acupuncture, and their behavior in choosing this therapy is currently unclear. Therefore, for this study, we conducted a web-based survey to address this unknown relationship. A questionnaire comprising four categories (health status, health literacy, previous acupuncture experience, and attitudes toward acupuncture) was administered to 1,600 Japanese participants. For this study, we performed cross-tabulation and path analysis to examine the relationship between each questionnaire item. The mean score of participants' health literacy was 3.41 (SD = 0.74), and older, educated, female participants tended to have higher health literacy. The respondents perceived acupuncture to be effective for chronic low back pain, tension-type headache, and knee pain due to osteoarthritis (40.0%, 38.7%, and 21.8%, respectively). Contrastingly, acupuncture was perceived as far less effective for postoperative nausea/vomiting and prostatitis symptoms (8.3% and 8.7%, respectively). Of the total study respondents, 34.4% reported that they would try acupuncture only if recommended by clinical practice guidelines, and 35.6% agreed that acupuncture is safe. The path analysis showed that attitudes toward acupuncture were significantly influenced by the participants' health literacy, number of information sources, and previous acupuncture experience. However, it was also found that experience with acupuncture was not directly associated with health literacy. Although the Japanese population with higher health literacy is more likely to perceive acupuncture positively, they do not necessarily have sufficient relevant knowledge of the clinical evidence. Therefore, their decision to receive acupuncture may be more dependent on personal narratives rather than clinical evidence. Thus, future challenges lie in individual education of the population on how to choose a reliable health information source, and organizational efforts to provide more reliable health information.

Fatty acid-binding protein 3 stimulates glucose uptake by facilitating AS160 phosphorylation in mouse muscle cells.

We studied the relationship between fatty acid-binding protein 3 (FABP3) and obesity in vivo and the effects of FABP3 on signal transduction for glucose uptake in skeletal muscle cells in vitro. In obese mice, the level of FABP3 protein in gastrocnemius muscles increased significantly with an increase in body weight and metabolic phenotypes, suggesting a close relationship between FABP3 expression in the muscle and the development of obesity and/or insulin resistance in mice. In experiments using C2C12 myotubes infected with adenoviruses encoding human FABP3, induction stimulated glucose uptake without insulin stimulation in parallel with increases in the phosphorylation of AMP-activated protein kinase (AMPK) and AS160. Insulin enhanced glucose uptake in an additive fashion with increased phosphorylation of Akt and AS160 in FABP3-induced myotubes compared to control cells. This increased glucose uptake in FABP3-induced myotubes with insulin stimulation was found even in the presence of palmitate, in which a significantly higher Akt phosphorylation was detected compared to controls. These results suggest that FABP3 stimulates glucose uptake by facilitating AMPK-dependent AS160 phosphorylation in skeletal muscle. FABP3 may also contribute to AS160 phosphorylation by maintaining insulin-dependent Akt activation in the cells under a lipotoxic condition.

Mimosine-induced apoptosis in C6 glioma cells requires the release of mitochondria-derived reactive oxygen species and p38, JNK activation.

Growth-inhibitory effects of mimosine, a plant amino acid, on rat C6 glioma cells were analyzed. Mimosine markedly inhibited proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent manner. Mimosine-mediated apoptosis was accompanied by promoting reactive oxygen species (ROS) generation in mitochondria, and by decreased mitochondrial membrane potential (Δψ), and release of cytochrome c from mitochondria, followed by caspase 3 activation. Furthermore, mimosine increased the phosphorylation level of c-Jun-N-terminal protein kinase and p38, which was the downstream effect of ROS accumulation. Mimosine was confirmed to show profound effects on apoptosis of C6 glioma cells by ROS-regulated mitochondria pathway, and these results bear on the hypothesized potential for mimosine as promising agents in the treatment of malignant gliomas.

Traditional medicine in china, Korea, and Japan: a brief introduction and comparison.

Background and Purpose. Traditional medicine (TM) has been widely used in China (including the Taiwan region), Korea, and Japan. The purposes of this paper are to summarize the basic data on TM systems in these three countries and to compare them in terms of overall policy, education, and insurance. Methods. Government websites, national statistics, and authoritative papers from each country were fully searched. Further data were gathered by TM experts from each country. Results. China and Korea showed similar patterns in TM systems, whereas Japan showed different patterns. In China and Korea, TM was practiced in a dual system with conventional medicine (CM), and TM education was 6-year training programs on average for TM doctors, and acupuncture, moxibustion, and cupping were completely insured. Whereas, CM was dominant in Japan, and TM was practiced by each health care worker who has received different TM education respectively, and main TM therapies were partially insured. Conclusions. TM was developed similarly or somewhat differently based on differences in cultural background and national policies in East Asia. We cautiously propose that this study could contribute to the development of TM and also be used for reference in complementary and alternative medicine systems.

Quality assessment of Japanese clinical practice guidelines including recommendations for acupuncture.

Background: The quality of clinical practice guidelines (CPGs) should be extensively evaluated. This study aimed to evaluate Japanese CPGs that include recommendations for acupuncture. Methods: In a literature search, CPGs including recommendations for acupuncture published in Japan until October 2021 were sought. We assessed (1) whether the CPGs were developed in accordance with the Grading Recommendations Assessment, Development and Evaluation (GRADE) system, (2) the quality of the CPGs using the Appraisal of Guidelines for Research and Evaluation (AGREE) II, and (3) whether the strength of the recommendations for acupuncture was consistent with each CPG's predefined procedure. Results: Seventeen CPGs including 23 recommendations in total were identified and assessed. (1) Three CPGs were in accordance with the GRADE system. (2) The mean score of overall assessment using AGREE II was 4.5 on a 7-point Likert scale. The mean domain scores were 77% for domain 1 (scope and purpose), 54% for domain 2 (stakeholder involvement), 48% for domain 3 (rigor of development), 78% for domain 4 (clarity of presentation), 20% for domain 5 (applicability), and 51% for domain 6 (editorial independence). (3) The strength of the recommendations for acupuncture in two CPGs was judged to be underestimated. Some of the CPGs contained elementary problems that were not considered in AGREE II. Conclusion: The methodological quality of Japanese CPGs including recommendations for acupuncture was not necessarily high. Since technical issues exist in each field of therapy, the respective experts should be involved in developing and reviewing CPGs to disseminate accurate health information.

CREG1 administration stimulates BAT thermogenesis and improves diet-induced obesity in mice.

Brown and beige adipocytes, which express thermogenic uncoupling protein-1 (UCP1), stimulate glucose and lipid metabolism, improving obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes adipose tissue browning and inhibits diet-induced obesity (DIO) in mice. In this study, we investigated the effects of CREG1 administration on DIO inhibition and adipose browning. Subcutaneous administration of recombinant CREG1 protein to C57BL/6 mice stimulated UCP1 expression in interscapular brown adipose tissue (IBAT) and improved DIO, glucose tolerance and fatty liver compared with those in phosphate-buffered saline-treated mice. Injection of Creg1-expressing adenovirus into inguinal white adipose tissue (IWAT) significantly increased browning and mRNA expression of beige adipocyte marker genes compared with that in mice injected with control virus. The effect of Creg1 induction on beige adipocyte differentiation was supported in primary culture using preadipocytes isolated from IWAT of Creg1-transgenic mice compared with that of wild-type mice. Our results indicate a therapeutic effect of CREG1 on obesity and its associated pathology and a potential of CREG1 to stimulate brown/beige adipocyte formation.

Sustained high body temperature exacerbates cognitive function and Alzheimer's disease-related pathologies.

Global warming is a serious public health threat to people worldwide. High body temperature is one of the important risk factors for Alzheimer's disease (AD), and the body temperature of AD patients has been found to be significantly higher than that of elderly control subjects. However, the effects of high body temperature on cognitive function and AD pathologies have not been completely elucidated. We report here that Tg2576 mice housed at a high ambient temperature of 30 °C for 13 months showed an increase in the body temperature, which is accompanied by memory impairment and an enhancement of amyloid-ß peptides (Aß) generation through the upregulation of ß-site APP cleaving enzyme 1 (BACE1) level and decrease in the level of an Aß-degrading enzyme, neprilysin (NEP) in the brain, compared with those of Tg2576 mice at 23 °C. High body temperature also increased the levels of heat shock proteins (HSPs), stress-stimulated kinases such as JNK, and total tau, leading to the enhancement of tau phosphorylation at 30 °C. Taken together, our findings suggest that high body temperature exacerbates cognitive function and AD pathologies, which provides a mechanistic insight for its prevention.

Genetic organization and mode of action of a novel bacteriocin, bacteriocin 51: determinant of VanA-type vancomycin-resistant Enterococcus faecium.

Bacteriocin 51 (Bac 51) is encoded on the mobile plasmid pHY (6,037 bp), which was isolated from vancomycin-resistant Enterococcus faecium VRE38. Bacteriocin 51 is active against E. faecium, E. hirae, and E. durans. Sequence analysis of pHY showed that it encodes nine open reading frames (ORFs) from ORF1 to ORF9 (in that order). Genetic analysis suggested that ORF1 and ORF2, which were designated bacA and bacB, respectively, are the bacteriocin and immunity genes. bacA encodes a 144-amino-acid protein. The deduced BacA protein has a typical signal sequence at its amino terminus, and a potential signal peptidase-processing site corresponding to the V-E-A sequence is located between the 37th and 39th amino acids. The predicted mature BacA protein consists of 105 amino acids. A potential promoter sequence was identified upstream of the start codon. bacB encodes a 55-amino-acid protein. No obvious promoter or terminator sequence was identified between bacA and bacB. Northern blot analysis of bacA and bacB with a bacA RNA probe produced a transcript of approximately 700 nucleotides, which corresponded to the combined nucleotide sizes of bacA and bacB, indicating that transcription was initiated from the promoter upstream of bacA, continued through bacB, and was terminated at the terminator downstream of bacB. The transcription start site was determined to be the T nucleotide located 6 nucleotides downstream from the -10 promoter sequence. These results indicate that bacA and bacB constitute an operon and that bacA is the bacteriocin structural gene while bacB is the immunity gene. The purified C-terminally His tagged BacA protein of Bac 51 showed bacteriostatic activity against the indicator strain. The purified C-terminally His tagged BacA protein of Bac 32 (whose mature BacA protein has 54 amino acids) and the culture filtrates of the Bac 31- and Bac 43-producing E. faecalis strain FA2-2 showed bactericidal activity. Bac 31 and Bac 43 are pore-forming bacteriocins, unlike the newly characterized bacteriocin Bac 51.

Taxonomic Reassessment of the Izumo Lineage of Hynobius utsunomiyaorum: Description of a New Species from Chugoku, Japan.

Here, we describe a new species of the genus Hynobius from Chugoku, Japan. In populations from central to eastern Shimane Prefecture, the Izumo Lineage of Hynobius utsunomiyaorum was clearly distinguished from the true Hynobius utsunomiyaorum based on morphological and molecular evidence. Thus, we describe the former lineage as a new species, Hynobius kunibiki sp. nov. Morphological comparisons revealed that H. utsunomiyaorum lacks a distinct yellow line on the ventral side of its tail, whereas the new species possesses this yellow line; most H. utsunomiyaorum individuals have distinct white spots on the lateral sides of their body and lack a fifth toe, whereas the new species largely lacks these spots, and all examined individuals had a fifth toe. The two species also differed significantly by several other morphological characteristics. The lentic species Hynobius setoi is morphologically similar to H. kunibiki sp. nov., but they differ significantly by various morphological characteristics. Despite their partial morphological similarity, these two species differed substantially in terms of their genetics. Finally, we show, in a phylogenetic tree including all Japanese Hynobius species, that the subgenus Hynobius can be divided into four genetic clades. Overall, this information will help develop conservation management strategies and policies for these species.

Lack of UCP1 stimulates fatty liver but mediates UCP1-independent action of beige fat to improve hyperlipidemia in Apoe knockout mice.

Brown adipose tissue (BAT) plays a critical role in lipid metabolism and may protect from hyperlipidemia; however, its beneficial effect appears to depend on the ambient temperature of the environment. In this study, we investigated the effects of uncoupling protein 1 (UCP1) deficiency on lipid metabolism, including the pathophysiology of hyperlipidemia, in apolipoprotein E knockout (APOE-KO) mice at a normal (23 °C) and thermoneutral (30 °C) temperature. Unexpectedly, UCP1 deficiency caused improvements in hyperlipidemia, atherosclerosis, and glucose metabolism, regardless of an increase in hepatic lipid deposition, in Ucp1/Apoe double-knockout (DKO) mice fed a high-fat diet at 23 °C, with BAT hyperplasia and robust browning of inguinal white adipose tissue (IWAT) observed. Proteomics and gene expression analyses revealed significant increases in many proteins involved in energy metabolism and strong upregulation of brown/beige adipocyte-related genes and fatty acid metabolism-related genes in browned IWAT, suggesting an induction of beige fat formation and stimulation of lipid metabolism in DKO mice at 23 °C. Conversely, mRNA levels of fatty acid oxidation-related genes decreased in the liver of DKO mice. The favorable phenotypic changes were lost at 30 °C, with BAT whitening and disappearance of IWAT browning, while fatty liver further deteriorated in DKO mice compared with that in APOE-KO mice. Finally, longevity analysis revealed a significant lifespan extension of DKO mice compared with that of APOE-KO mice at 23 °C. Irrespective of the fundamental role of UCP1 thermogenesis, our results highlight the importance of beige fat for the improvement of hyperlipidemia and longevity under the atherogenic status at normal room temperature.

Dietary Supplementation with Dunaliella Tertiolecta Prevents Whitening of Brown Fat and Controls Diet-Induced Obesity at Thermoneutrality in Mice.

We investigated the effect of evodiamine-containing microalga Dunaliella tertiolecta (DT) on the prevention of diet-induced obesity in a thermoneutral C57BL/6J male (30 °C). It attenuates the activity of brown adipose tissue (BAT), which accelerates diet-induced obesity. Nine-week-old mice were fed a high-fat diet supplemented with 10 g (Low group) or 25 g (High group) DT powder per kg food for 12 weeks. Compared to control mice without DT supplementation, body weight gain was significantly reduced in the High group with no difference in food intake. Tissue analyses indicated maintenance of multilocular morphology in BAT and reduced fat deposition in liver in DT-supplemented mice. Molecular analysis showed a significant decrease in mammalian target of rapamycin-ribosomal S6 protein kinase signaling pathway in white adipose tissue and upregulation in mRNA expression of brown fat-associated genes including fibroblast growth factor-21 (Fgf21) and uncoupling protein 1 (Ucp1) in BAT in the High group compared to the control. In the experiments using C3H10T1/2 adipocytes, DT extract upregulated mRNA expression of brown fat-associated genes in dose-dependent and time-dependent manners, accompanied by a significant increase in secreted FGF21 levels. Our data show the ability of DT as a nutraceutical to prevent brown fat attenuation and diet-induced obesity in vivo.