Yasuhide Kontani

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.

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.

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 ∼23°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°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.

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.

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.

Structures of campanin and rubrocampanin, two novel acylated anthocyanins with p-hydroxybenzoic acid from the flowers of bellflower, Campanula medium L.

The structures of campanin 1 and rubrocampanin 2, major antocyanins from flowers of the purple and pink cultivars of Campanula medium L., have been identified as 3-O-[(6-O-α-L-rhamnosyl)-β-D-glucopyranosyl]-7-O-[6-O-(4-{6-O-[4-(6-O-p-hydroxybenzoyl-β-D- glucopyranosyloxy)benzoyl]-β-D-glucopyranosyloxy}benzoyl)-β-D-glucopyranosides] of delphinidin, and pelargonidin, respectively, by means of chromatography, FAB-MS, and NMR including negative NOE difference (DIFNOE) spectroscopy. The colour stabilities of both the p-hydroxybenzoylanthocyanins 1 and 2 were examined and found to be relatively unstable compared with that of platyconin, 3-O-[6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-7-O-{6-O-[(E)-4-O-{6-O-[(E)-4-O-(β-D-glucopyranosyl)-caffeoyl]-β-D-glucopyranosyl}caffeoyl]-β-D-glucopyranosyl}delphinidin, in neutral aqueous solution.