Hitoshi Yamashita

Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity.

The mRNA levels for the mitochondrial uncoupling protein (UCP1) in fat tissues of A/J and C57BL/6J inbred strains of mice varied in a regional-specific manner after stimulation of adrenergic signaling by cold exposure or treatment with a beta3-adrenergic agonist. While the differences between strains were minimal in interscapular brown fat, large differences occurred in white fat tissues, particularly in retroperitoneal fat. Among the AXB recombinant inbred strains, the Ucp1 mRNA levels varied up to 130-fold. This large induction at the mRNA level was accompanied by a corresponding increase in brown adipocytes as revealed by immunohistology with anti-UCP1 antibodies. A high capacity to induce brown fat in areas of traditional white fat had no impact on the ability to gain weight in response to high fat and sucrose diets, but did correlate with the loss of weight in response to treatment with a beta3-adrenergic agonist (CL 316,243). This genetic variation in mice provides an experimental approach to identify genes controlling the induction of brown adipocytes in white fat tissues.

Forkhead transcription factor FOXO1 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation

A forkhead-type transcription factor, DAF-16, is located in the most downstream part of the insulin signalling pathway via PI3K (phosphoinositide 3-kinase). It is essential for the extension of life-span and is also involved in dauer formation induced by food deprivation in Caenorhabditis elegans. In the present study, we addressed whether or not FOXO members AFX, FKHR (forkhead homologue in rhabdomyosarcoma) and FKHRL1 (FKHR-like protein 1), mammalian counterparts of DAF-16, are involved in starvation stress. We found a remarkable selective induction of FKHR and FKHRL1 transcripts in skeletal muscle of mice during starvation. The induction of FKHR gene expression was observed at 6 h after food deprivation, peaked at 12 h, and returned to the basal level by 24 h of refeeding. The induction was also found in skeletal muscle of mice with glucocorticoid treatment. Moreover, we found that the levels of PDK4 (pyruvate dehydrogenase kinase 4) gene expression were up-regulated through the direct binding of FKHR to the promoter region of the gene in C2C12 cells. These results suggest that FKHR has an important role in the regulation of energy metabolism, at least in part, through the up-regulation of PDK4 gene expression in skeletal muscle during starvation.

UCP1 deficiency increases susceptibility to diet-induced obesity with age.

Loss of nonshivering thermogenesis in mice by inactivation of the mitochondrial uncoupling protein gene (Ucp1−/– mice) causes increased sensitivity to cold and unexpected resistance to diet‐induced obesity at a young age. To clarify the role of UCP1 in body weight regulation throughout life and influence of UCP1 deficiency on longevity, we longitudinally analyzed the phenotypes of Ucp1−/– mice maintained in a room at 23 °C. There was no difference in body weight and lifespan between genotypes under the standard chow diet condition, whereas the mutant mice developed obesity with age under the high‐fat (HF) diet condition. Compared with Ucp1+/+ mice, Ucp1−/– mice showed increased expression of genes related to thermogenesis and fatty acid metabolism, such as β3‐adrenergic receptor, in adipose tissues of the 3‐month‐old mutants; however, the augmented expression was reduced in Ucp1+/+ mice in 11‐month‐old Ucp1−/– mice fed the HF diet. Likewise, the increased levels of UCP3 and cAMP‐dependent protein kinase in the brown adipose tissue of Ucp1−/– mice given the standard diet were decreased significantly in that of Ucp1−/– mice fed the HF diet, which animals showed impaired norepinephrine‐induced lipolysis in their adipose tissues. These results suggest profound attenuation of β‐adrenergic responsiveness and fatty acid utilization in Ucp1−/– mice fed the HF diet, bringing them to late‐onset obesity. Our findings provide evidence that UCP1 is neither essential for body weight regulation nor for longevity under conditions of standard diet and normal housing temperature, but deficiency increases susceptibility to obesity with age in combination with HF diet.

Evodiamine inhibits adipogenesis via the EGFR–PKCα–ERK signaling pathway

The molecular mechanism of the anti‐adipogenic effect of evodiamine (which has several capsaicin‐like pharmacological actions) was investigated. The evodiamine effect was not blocked by the specific TRPV1 antagonist capsazepine in 3T3‐L1 preadipocytes, whereas its effect was greatly curtailed by inhibitors of protein kinase C (PKC) and epidermal growth factor receptor (EGFR). Signal analyses showed that evodiamine stimulated the phosphorylation of EGFR, PKCα, and ERK, all of which were reduced by an EGFR inhibitor. Silencing experiments of EGFR mRNA supported the involvement of these signaling molecules in the inhibitory effect of evodiamine. An unidentified mechanism whereby evodiamine inhibits adipogenesis via the EGFR–PKCα–ERK signaling pathway was revealed.

TRPV4 deficiency increases skeletal muscle metabolic capacity and resistance against diet-induced obesity

Transient receptor potential channel V4 (TRPV4) functions as a nonselective cation channel in various cells and plays physiological roles in osmotic and thermal sensation. However, the function of TRPV4 in energy metabolism is unknown. Here, we report that TRPV4 deficiency results in increased muscle oxidative capacity and resistance to diet-induced obesity in mice. Although no difference in body weight was observed between wild-type and Trpv4(-/-) mice when fed a standard chow diet, obesity phenotypes induced by a high-fat diet were significantly improved in Trpv4(-/-) mice, without any change in food intake. Quantitative analysis of mRNA revealed the constitutive upregulation of many genes, including those for transcription factors such as peroxisome proliferator-activated receptor α and for metabolic enzymes such as phosphoenolpyruvate carboxykinase. These upregulated genes were especially prominent in oxidative skeletal muscle, in which the activity of Ca(2+)-dependent phosphatase calcineurin was elevated, suggesting that other Ca(2+) channels function in the skeletal muscle of Trpv4(-/-) mice. Indeed, gene expressions for TRPC3 and TRPC6 increased in the muscles of Trpv4(-/-) mice compared with those of wild-type mice. The number of oxidative type I fiber also increased in the mutant muscles following myogenin gene induction. These results strongly suggested that inactivation of Trpv4 induces compensatory increases in TRPC3 and TRPC6 production, and elevation of calcineurin activity, affecting energy metabolism through increased expression of genes involved in fuel oxidation in skeletal muscle and thereby contributing to increased energy expenditure and protection from diet-induced obesity in mice.

Proteose peptone enhances production of tissue-type plasminogen activator from human diploid fibroblasts

Proteose peptone markedly enhanced the production of human tissue-type plasminogen activator (t-PA) from confluently cultured cell of human diploid fibroblasts. The cells continued synthesizing and secreting high levels of t-PA under periodic replacement of medium containing proteose peptone for more than one month. The highly increased activity correlated with equally increased levels of t-PA antigen and concomitantly increased levels of t-PA specific mRNA.

Difference in induction of uncoupling protein genes in adipose tissues between young and old rats during cold exposure.

Induction of uncoupling protein (UCP) genes in adipose tissues from young and old rats exposed to cold was compared. UCP1 mRNA expression in brown adipose tissue (BAT) was enhanced in both young and old rats after cold exposure, but the expression was downregulated at 72 h after the exposure only in the young rats. The UCP2 gene was induced notably in BAT of young rats instead of the downregulation of the UCP1 gene, whereas the induction in old rats was almost blunted. The pattern of UCP3 expression was similar to that of UCP1 expression in each group. The effect of cold exposure on the expression of UCP2 genes was also observed in white adipose tissue from the young rats. These results indicate a change in induction of UCP genes in adipose tissues with aging.

Characterization of the differential expression of uncoupling protein 2 and ROS production in differentiated mouse macrophage-cells (Mm1) and the progenitor cells (M1).

The expression status of mitochondrial uncoupling protein 2 (UCP2) was investigated in undifferentiated mouse myeloid leukemia (M1) and its differentiated macrophage-like cells (Mm1). Mm1 cells have a high ability of phagocytosis along with significantly high levels of reactive oxygen species (ROS) production, UCP2 protein and manganese superoxide dismutase (Mn-SOD), in contrast to undifferentiated leukemia cells (M1). Mm1 cells expressed 10-fold more UCP2 protein compared with undifferentiated M1 cells, although the UCP2 mRNA levels in both cell types were similar. The higher expression of UCP2 in the Mm1 cells suggests a regulatory role of UCP2 in the ROS production. Furthermore, the transfection of UCP2-GFP-expression vector in Mm1 cells dissipated the mitochondrial membrane potential and reduced ROS production, which was shown by their direct visualization using MitoTracker Red CM-H2Xros. The macrophage gp91phox protein, a membrane catalytic component of the NADPH oxidase complex, was at a similar level in both of UCP2-GFP expressed and non-expressed Mm1 cells. These results suggest that the UCP2 protein of the undifferentiated cell is regulated at a quite low level and the higher UCP2 protein of the differentiated macrophages involves with the regulation of ROS production.

Chronic Exposure to Low Frequency Noise at Moderate Levels Causes Impaired Balance in Mice

We are routinely exposed to low frequency noise (LFN; below 0.5 kHz) at moderate levels of 60–70 dB sound pressure level (SPL) generated from various sources in occupational and daily environments. LFN has been reported to affect balance in humans. However, there is limited information about the influence of chronic exposure to LFN at moderate levels for balance. In this study, we investigated whether chronic exposure to LFN at a moderate level of 70 dB SPL affects the vestibule, which is one of the organs responsible for balance in mice. Wild-type ICR mice were exposed for 1 month to LFN (0.1 kHz) and high frequency noise (HFN; 16 kHz) at 70 dB SPL at a distance of approximately 10–20 cm. Behavior analyses including rotarod, beam-crossing and footprint analyses showed impairments of balance in LFN-exposed mice but not in non-exposed mice or HFN-exposed mice. Immunohistochemical analysis showed a decreased number of vestibular hair cells and increased levels of oxidative stress in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. Thus, the results of this study indicate the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance.

Induction of fatty acid-binding protein 3 in brown adipose tissue correlates with increased demand for adaptive thermogenesis in rodents

We investigated the contribution of fatty acid-binding protein 3 (FABP3) to adaptive thermogenesis in brown adipose tissue (BAT) in rodents. The expression of FABP3 mRNA in BAT was regulated discriminatively in response to alteration of the ambient temperature, which regulation was similar and reciprocal to the regulation of uncoupling protein 1 (UCP1) and leptin, respectively. FABP3 expression in the BAT was significantly higher in the UCP1-knockout (KO) mice than in the wild-type ones, and these KO mice showed a higher clearance rate of free fatty acid from the plasma. In addition, FABP3 expression in the BAT was increased greatly with the development of diet-induced obesity in mice. These results indicate that the induction of FABP3 in BAT correlates with an increased demand for adaptive thermogenesis in rodents. FABP3 appears to be essential for accelerating fatty acid flux and its oxidation through UCP1 activity for non-shivering thermogenesis in BAT.