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. In addition, reduction of insulin receptor substrate 1 (IRS1) serine phosphorylation, an indicator of insulin resistance, was detected in their WAT, suggesting suppression of the negative feedback loop from S6K to IRS1. As well as the stimulation of IRS1 and Akt serine phosphorylation, insulin-stimulated phosphorylation of mTOR and S6K is time-dependent in 3T3-L1 adipocytes, whereas evodiamine does not affect their phosphorylation except for an inhibitory effect on mTOR phosphorylation. Moreover, evodiamine inhibits the insulin-stimulated phosphorylation of mTOR and S6K, leading to down-regulation of IRS1 serine phosphorylation in the adipocytes. Evodiamine also stimulates phosphorylation of AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, which may cause down-regulation of mTOR signaling in adipocytes. A similar effect on AMPK, mTOR and IRS1 phosphorylation was found in adipocytes treated with rosiglitazone. These results suggest evodiamine improves glucose tolerance and prevents the progress of insulin resistance associated with obese/diabetic states, at least in part, through inhibition of mTOR-S6K signaling and IRS1 serine phosphorylation in adipocytes.

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.

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.

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. In addition to the heat and cold insensitivity, capsaicin-treated rats showed lower rectal core temperature, higher skin temperature and decreased sensitivity to ambient temperature alteration under normal housing at room temperature, suggesting impaired thermosensation and change in thermoregulation in the rats. Uncoupling protein 1 (UCP1) expression and the thermogenic ability in brown adipose tissues were attenuated in the capsaicin-treated rats. These results indicate a critical role of capsaicin-sensitive sensory neurons in both heat and cool sensation and hence in basal thermal homeostasis, which is balanced by heat release and production including UCP1 thermogenesis, following sensation of the ambient temperature.

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 0.03% evodiamine (wt/wt) for 2 months, the increases in body weight, adiposity, and the serum levels of leptin and insulin were reduced in a manner indistinguishable from control mice fed a high-fat diet with evodiamine, suggesting that evodiamine triggered a UCP1-independent mechanism to prevent diet-induced obesity. By using preadipocyte cultures, we found that evodiamine, but not capsaicin, increased phosphorylation of ERK/MAPK, reduced the expression of transcription factors such as peroxisome proliferator-activated receptor-gamma, and strongly inhibited adipocyte differentiation. Evodiamine treatment also reduced insulin-stimulated phosphorylation of Akt, a crucial regulator of adipocyte differentiation; and the reduction of phosphorylated-Akt and augmentation of phosphorylated ERK were reversed by blockade of the MAPK kinase/MAPK signaling pathway, restoring adipogenesis in the cultures. The changes in ERK and Akt phosphorylation levels were also observed in white adipose tissues of UCP1-knockout mice fed the evodiamine diet. These findings suggest that evodiamine has a potential to prevent the development of diet-induced obesity in part by inhibiting adipocyte differentiation through ERK activation and its negative cross talk with the insulin signaling pathway.

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. Analysis of tail blood flow under anesthesia clearly showed a stronger vasoconstrictor response in Ucp1-/- mice than in Ucp1+/+ mice. Administration of a vasodilator, evodiamine, transiently increased Tskin in Ucp1+/+ and Ucp1-/- mice similarly; whereas the induction of vasodilation caused a greater and more prolonged reduction in Trectal in Ucp1-/- mice than in Ucp1+/+ mice. These results indicate that Ucp1-/- mice highly, or at least partly, rely on vasoconstriction for heat conservation to compensate for their UCP1 deficiency and to maintain homeothermy under the condition of normal housing temperature.

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. The previous study using a mutant virus, DAL*-1 (in which the L* AUG is substituted by an ACG) showed that L* has an anti-apoptotic effect in a macrophage cell line, P388D1. Therefore, we established P388D1 cells that continuatively express L*, in order to confirm its role in TMEV-induced apoptosis. The anti-apoptotic activity of L* may be important in TMEV pathogenesis.

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. However, the levels of UCP3 and cytochrome oxidase subunit IV were well maintained in the cold-exposed gastrocnemius muscles with a rise in the total mitochondrial protein level, suggesting an increase of total oxidative ability. The fast-twitch muscle rather than the slow-twitch one may play an important role in adaptive responses, including thermogenesis under acute cold exposure.

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. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.