Evodiamine inhibits insulin-stimulated mTOR-S6K activation and IRS1 serine phosphorylation in adipocytes and improves glucose tolerance in obese/diabetic mice.

Evodiamine, an alkaloid extracted from the dried unripe fruit of the tree Evodia rutaecarpa Bentham (Rutaceae), reduces obesity and insulin resistance in obese/diabetic mice; however, the mechanism underlying the effect of evodiamine on insulin resistance is unknown. This study investigated the effect of evodiamine on signal transduction relating to insulin resistance using obese/diabetic KK-Ay mice and an in vitro adipocyte culture. There is a significant decrease in the mammalian target of rapamycin (mTOR) and ribosomal S6 protein kinase (S6K) signaling in white adipose tissue (WAT) in KK-Ay mice treated with evodiamine, in which glucose tolerance is improved.

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.

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.

Impaired basal thermal homeostasis in rats lacking capsaicin-sensitive peripheral small sensory neurons.

We studied the effects of selective loss of capsaicin-sensitive primary sensory neurons on thermosensation and thermoregulation in rats. Neonatal capsaicin treatment in rats caused a remarkable decrease in the number of small-diameter neurons in the dorsal root ganglion (DRG) compared with their number in the control rats. Gene expression analysis for various thermo-sensitive transient receptor potential (TRP) channels indicated marked reductions in the mRNA levels of TRPV1 (70%), TRPM8 (46%) and TRPA1 (64%), but not of TRPV2, in the DRG of capsaicin-treated rats compared with those in the control rats.

Evodiamine improves diet-induced obesity in a uncoupling protein-1-independent manner: involvement of antiadipogenic mechanism and extracellularly regulated kinase/mitogen-activated protein kinase signaling.

Evodiamine is an alkaloidal compound with antiobesity effects that have been thought to be due to uncoupling protein-1 (UCP1) thermogenesis similar to the effects of capsaicin, but the underlying mechanisms are poorly understood. To clarify the mechanisms, we first examined whether the antiobesity effect of evodiamine could be attributed to the involvement of UCP1. When UCP1-knockout mice were fed a high-fat diet with 0.

Potential contribution of vasoconstriction to suppression of heat loss and homeothermic regulation in UCP1-deficient mice.

To investigate the thermoregulatory mechanism in mice lacking uncoupling protein 1 (UCP1) from the viewpoint of heat loss, we measured oxygen consumptions (VO2), skin-surface temperatures (Tskin, an index of heat release), blood flows in the tails, and rectal temperatures (Trectal) of mice housed in an animal room under the standard thermal condition of approximately 23 degrees C. Compared with wild-type (Ucp1+/+) mice, adult UCP1-deficient (Ucp1-/-) mice tended to show a reduced VO2. Thermograhic analysis of the acute response of Ucp1-/- mice to a small change (a drop of 1-2 degrees C) in the ambient temperature revealed a sustained fall in the Tskin of Ucp1-/- mice; but this fall was only transient in Ucp1+/+ mice.

A lentiviral expression system demonstrates that L* protein of Theiler's murine encephalomyelitis virus (TMEV) has an anti-apoptotic effect in a macrophage cell line.

DA subgroup strains of TMEV persist in the CNS of infected mice and induce demyelination. The mechanism(s) of virus persistence and demyelination remains unknown. DA subgroup strains synthesize a 17-kDa protein, called L*, from an initiation site out-of-frame with the polyprotein.

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 degrees 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.

A lentiviral expression system demonstrates that L* protein of Theiler's murine encephalomyelitis virus (TMEV) is essential for virus growth in a murine macrophage-like cell line.

The DA subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein, which is translated out of frame with the polyprotein from an alternative AUG, 13 nucleotides downstream from the authentic polyprotein AUG. By a 'loss of function' experiment using a mutant virus, DAL*-1, in which the L* AUG is mutated to an ACG, L* protein is shown to play an important role in virus persistence, TMEV-induced demyelination, and virus growth in macrophages. In the present study, we established an L* protein-expressed macrophage-like cell line and confirmed the importance of L* protein in virus growth in this cell line.

Muscle type difference in the regulation of UCP3 under cold conditions.

We found opposite regulation of uncoupling protein 3 (UCP3) in slow-twitch soleus and fast-twitch gastrocnemius muscles of rats during cold exposure. Namely, the UCP3 mRNA level was downregulated in the soleus muscles, but upregulated in the gastrocnemius muscles after a 24-h cold exposure. In the analysis of UCP3 protein, we first succeeded in detecting UCP3 short-form as well as the long-form in vivo, which levels were decreased markedly in the cold-exposed soleus muscles.

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats.

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy.

Behavior of alginate gel beads containing chitosan salt prepared with water-soluble vitamins.

Alginate gel beads were prepared which contained weak acid salts of chitosan (Alg-CS) and water-soluble vitamins (e.g. ascorbic acid (AS)) and the behavior of the beads, uptake of bile acids was investigated in vitro.

Influence of dietary protein levels on beta-alanine aminotransferase expression and activity in rats.

beta-Alanine-oxoglutarate aminotransferase (beta-AlaAT I) and beta-alanine-pyruvate aminotransferase (beta-AlaAT II) catalyze the transamination reaction of omega-amino acids such as beta-alanine, beta-aminoisobutyrate, and gamma-aminobutyrate, amino acids that are not protein constituents. The influence of dietary protein levels on the expression and activities of these enzymes was investigated by using male rats. Both beta-AlaAT I and beta-AlaAT II activities in the liver were increased with the level of protein in the diet in accordance with changes in their mRNA levels.

The mature size of rat 4-aminobutyrate aminotransferase is different in liver and brain.

The amino acid sequence predicted from a rat liver cDNA library indicated that the precursor of beta-AlaAT I (4-aminobutyrate aminotransferase, beta-alanine-oxoglutarate aminotransferase) consists of a mature enzyme of 466 amino acid residues and a 34-amino acid terminal segment, with amino acids attributed to the leader peptide. However, the mass of beta-AlaAT I from rat brain was larger than that from rat liver and kidney, as assessed by Western-blot analysis, mass spectroscopy and N-terminal sequencing. The mature form of beta-AlaAT I from the brain had an ISQAAAK- peptide on the N-terminus of the liver mature beta-AlaAT I.

Cloning of rat dihydropyrimidine dehydrogenase and correlation of its mRNA increase in the rat liver with age.

Overlapping cDNAs encoding dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of pyrimidine degradation, were isolated from a rat liver cDNA library, and a 4,353 bp cDNA sequence with a 3,075 bp open reading frame encoding a polypeptide of 1,025 residues with a molecular mass of 111,458 Da was determined. Western blot analysis of COS-7 cells that were transfected with an RT-PCR-derived DPD cDNA showed that the product from the cDNA binds to an anti-serum for DPD. Northern blot analysis was performed by using a partial fragment of the cloned cDNA as a probe.

Inhibitory effect of ethanol administration on beta-alanine-2-oxoglutarate aminotransferase (GABA aminotransferase) in disulfiram-pretreated rats.

Ethanol in the presence of disulfiram (N,N,N',N'-tetraethylthiuram disulfide, an inhibitor of aldehyde dehydrogenase) inhibited liver beta-alanine-oxoglutarate aminotransferase (beta-AlaAT I) activity yet activated tyrosine aminotransferase (TAT) in weanling rats in vivo. The effect on beta-AlaAT I was followed by the inhibitory expression of beta-AlaAT I mRNA. The beta-AlaAT I activity was reduced with a pseudo-first-order profile with time, and the half-life was calculated to be 12.

Identity of D-3-aminoisobutyrate-pyruvate aminotransferase with alanine-glyoxylate aminotransferase 2.

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.

Inhibition of D-3-aminoisobutyrate-pyruvate aminotransferase by 5-fluorouracil and alpha-fluoro-beta-alanine.

Among pyrimidine derivatives, we found that 5-fluorouracil potently inhibited purified rat liver D-3-aminoisobutyrate-pyruvate aminotransferase, whereas 5-fluorouridine did so to a much lesser extent. 5-Fluorouracil acted as a competitive inhibitor against beta-alanine with a Ki of 56 microM, and was uncompetitive against pyruvic acid, with a Ki of 73 microM. alpha-Fluoro-beta-alanine, a metabolite of 5-fluorouracil, was also a competitive inhibitor with respect to beta-alanine with a Ki of 8.

Effect of dietary protein on pyrimidine-metabolizing enzymes in rats.

The effect of dietary protein on pyrimidine-metabolizing enzymes was studied in the rat. The activities of dihydropyrimidine dehydrogenase and beta-ureidopropionase in the livers of rats fed a protein-free diet were significantly decreased, while the activity of dihydropyrimidinase was unaffected. Protein deficiency (5%) also decreased the activity of beta-ureidopropionase.