Kiyohito Nakashima

Leptin and sweet taste.

Leptin, the product of the obese (ob) gene, is a hormone primarily produced in adipose cells, and also at smaller amounts in some other peripheral organs. It regulates food intake, energy expenditure, and body weight. Leptin is thought to promote weight loss, at least in rodents, by suppressing appetite and stimulating metabolism. Mutant mice that lack either leptin or functional leptin receptors, such as ob/ob and db/db mice, are hyperphagic, massively obese, and diabetic. Central hypothalamic targets are mainly responsible for the effects of leptin on food intake and weight loss. However, there are also direct effects on peripheral tissues. Recently, the taste organ was found to be one of the peripheral targets for leptin. The hormone specifically inhibits sweet taste responses in lean mice and not in db/db mice. Thus leptin appears to act as a modulator of sweet taste, provided a functional leptin receptor is expressed by the taste cells. This chapter reviews the genetics and molecular biology of leptin and its receptors, the receptor mechanisms for sweet taste, the modulating action of leptin on taste receptor cells, and the consequences for the regulation of food intake.

Transduction for sweet taste of saccharin may involve both inositol 1,4,5-trisphosphate and cAMP pathways in the fungiform taste buds in C57BL mice.

The transduction pathways for sweet and bitter tastes were investigated with assays of inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine monophosphate (cAMP) levels in mouse fungiform taste buds. Recordings of taste responses were also made in the chorda tympani nerve. Stimulation of the tongue with saccharin elicited a significant increase in IP3 levels in the fungiform papilla only at 20 mM but in cAMP levels at 3 and 20 mM, without affecting those of the nonsensory epithelial tissue. Formation of both IP3 and cAMP induced by 20 mM saccharin was suppressed by pretreatment of the tongue with pronase, a proteolytic enzyme which specifically inhibits sweet responses. Quinine and denatonium elicited both significant increases in IP3 levels at a concentration of 20 mM and slight decreases in cAMP levels at concentrations of 1–20 mM in the fungiform papilla. Recording of the chorda tympani nerve showed good responses by saccharin, quinine, and denatonium at concentrations of 1 mM and higher. These results suggest that the fungiform taste cells in C57BL mice have pronase-sensitive receptors for saccharin, coupled to both the IP3 and the cAMP pathways; the former participates only at high concentration, while the latter acts from low to high concentrations. The results also do not rule out the possibility that a phosphodiesterase-mediated cAMP decrease may be involved in bitter transduction for quinine and denatonium.

Increase in Inositol 1,4,5-Trisphosphate Levels of the Fungiform Papilla in Response to Saccharin and Bitter Substances in Mice

Inositol 1,4,5-trisphosphate (IP3) levels of the mouse fungiform papilla and nonsensory epithelial tissue in response to various taste stimuli were measured by use of a competitive protein binding assay. Stimulation of the tongue with 20 mM saccharin induced a significant increase in IP3 levels of the fungiform papillae without affecting those of the nonsensory epithelial tissue, whereas stimulations with 20 mM quinine and denatonium raised IP3 levels in not only the fungiform papilla but slightly also nonsensory epithelial tissue. No such increases in IP3 levels were observed to lower concentrations of saccharin, denatonium and quinine, and other basic taste stimuli, such as 0.5 M sucrose, 0.1 M NaCl and 10 mM HCl. Pretreatment of the tongue with pronase, a proteolytic enzyme which inhibits sweet responses, completely eliminated the increase in IP3 levels induced by saccharin, but not those induced by quinine or denatonium. These results suggest that an IP3 pathway in taste cells of the mouse fungiform taste bud is involved in the transduction not only for denatonium and quinine, but also for 20 mM saccharin. The increase of IP3 induced by saccharin, but not by denatonium and quinine, may occur through pronase-sensitive membrane components.

Enhanced responses of the chorda tympani nerve to nonsugar sweeteners in the diabetic db/db mouse.

Genetically diabetic db/db mice show greater neural and behavioral responses to sugars than lean control mice. The present study examined chorda tympani responses of db/db mice to nonsugar sweeteners and their inhibition by a sweet response inhibitor, gurmarin. The results showed that responses to sucrose, saccharin, glycine,l-alanine, andd-tryptophan, but not tod-phenylalanine, were ∼1.5 times greater in db/db mice than in control mice. Treatment of the tongue with gurmarin suppressed responses to these sweeteners in db/dband control mice, but the extent of suppression was considerably smaller in db/db mice. The magnitudes of gurmarin-sensitive components of the response to sweeteners in db/db mice were not significantly different from those in control mice, whereas the magnitudes of gurmarin-insensitive components in db/db mice were about twice as large as those in control mice. These results suggest that the enhancement of chorda tympani responses in db/db mice to sucrose and other nonsugar sweeteners may occur through gurmarin-insensitive membrane components.Genetically diabetic db/db mice show greater neural and behavioral responses to sugars than lean control mice. The present study examined chorda tympani responses of db/db mice to nonsugar sweeteners and their inhibition by a sweet response inhibitor, gurmarin. The results showed that responses to sucrose, saccharin, glycine, L-alanine, and D-tryptophan, but not to D-phenylalanine, were approximately 1.5 times greater in db/db mice than in control mice. Treatment of the tongue with gurmarin suppressed responses to these sweeteners in db/db and control mice, but the extent of suppression was considerably smaller in db/db mice. The magnitudes of gurmarin-sensitive components of the response to sweeteners in db/db mice were not significantly different from those in control mice, whereas the magnitudes of gurmarin-insensitive components in db/db mice were about twice as large as those in control mice. These results suggest that the enhancement of chorda tympani responses in db/db mice to sucrose and other nonsugar sweeteners may occur through gurmarin-insensitive membrane components.

Behavioral responses to glutamate receptor agonists and antagonists implicate the involvement of brain-expressed mGluR4 and mGluR1 in taste transduction for umami in mice

Recent molecular studies have identified many candidate receptors for umami, typically the taste of monosodium glutamate (MSG). The candidate receptors, including taste-mGluR4, T1R1+T1R3, and truncated mGluR1, respond to MSG in the millimolar concentration range. Expression of brain-expressed mGluR4 and mGluR1 with much higher sensitivities to glutamate has also been reported in taste papillae. To test the involvement of brain-expressed mGluRs in umami taste, we tested glutamate agonists and antagonists at concentration ranges relevant to both types of the receptors using a combination of a detection threshold and conditioned taste aversion (CTA) methods in mice. The detection threshold experiment showed that mice could detect the group III mGluR agonist L(+)-2-amino-4-phosphonobutyrate (L-AP4) taste thresholds at 0.0009-0.0019 mM. Mice conditioned using CTA methods to avoid either MSG or MPG showed aversive responses to MSG with and without amiloride or to MPG, respectively, at concentrations of 0.0001 mM and above. A CTA to L-AP4 or MSG showed comparable concentration-response ranges for L-AP4 and MSG. The Group III mGluR antagonist, (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), and the mGluR1 antagonist, 1-aminoindan-1,5-dicarboxylic acid (AIDA), suppressed aversive responses to glutamate agonists at concentrations between 0.0001 and 100mM in the CTA experiments. Our results suggest the possibility that brain-expressed mGluR4 and mGluR1 may contribute to umami taste in mice.

Multiple receptor systems for umami taste in mice.

Recent molecular studies proposed that the T1r1/T1r3 heterodimer, mGluR1 and mGluR4 might function as umami taste receptors in mice. However, the roles of each of these receptors in umami taste are not yet clear. In this paper, we summarize recent data for T1r3, mGluR1, and mGluR4 as umami taste receptors and discuss receptor systems responsible for umami detection in mice.

Salivary cystatins influence ingestion of capsaicin-containing diets in the rat.

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.

Ion channels and second messengers involved in transduction and modulation of sweet taste in mouse taste cells

Leptin, a hormone released from the adipose tissue, inhibits food intake and increases energy expenditure. We have found a novel function of leptin as a modulator of sweet taste sensitivity in mice. In lean normal mice, the gustatory nerve responses to sweet stimuli were selectively suppressed depending on plasma leptin level after an intraperitoneal injection of recombinant leptin. Patch-clamp studies using isolated taste cells of lean mice showed that extracellular leptin enhanced K+ currents of sweet-responsive taste cells, which led to membrane hyperpolarization and a reduction of sweetener-induced depolarization. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization analyses demonstrated specific expression of mRNA of the long-form functional leptin receptor (Ob-Rb) in taste tissue and cells of lean mice. The genetically diabetic db/db mice, which have defects in Ob-Rb, demonstrated neither a suppression of gustatory neural responses to sweeteners nor an increment of whole-cell K+ conductance of taste cells even with high doses of leptin. These results suggest that Ob-Rb is specifically expressed in sweet-responsive taste cells of lean mice and that leptin suppresses sweetener-induced depolarization via activation of K+ channels, leading to a decrease in impulses of sweet-best fibers. The enhanced sweet responses of db/db mice may result from the lack of inhibitory modulation by leptin.

Taste may not affect salt appetite in zinc deficient rats

Novel peptidergic neurons, metastin neurons, are thought to facilitate sexual maturation and ovulation by stimulating gonadotropin releasing hormone (GnRH) neurons, but their physiological functions are still largely unknown. Here we focused on the relationship between the metastin and hypothalamic/extrahypothalamic GnRH neurons. Taking advantage of well-developed teleost GnRH systems, we used medaka, which is a useful teleost for the application of various molecular genetic tools. First, we cloned and sequenced the metastin gene (Kiss1) in medaka for the first time in non-mammalian species. Then, Kiss1 expression in the brain was analyzed by in situ hybridization. We found two hypothalamic nuclei with Kiss1 expression and discovered prominent sexual difference in cell number (male female) in one of the two nuclei. On the other hand, we found no circadian variation in expression of Kiss1 by real-time PCR, although medaka shows daily ovulatory cyclicity.

Effects of the combined treatment of cadmium chloride and X-rays on 8-day mouse embryos in vitro

SummaryThe combined action of cadmium and X-rays was examined on 8-day mouse embryos using a whole embryo culture system. B6C3F1 mouse embryos were explanted onto the headfold stage and cultured for 40 hrs. To examine the dose-effect relationships of the single treatments, the embryos were exposed to 1.2−2.2 μM cadmium or to 0.82−2.64 Gy of X-rays at 1 hr. after the explantation. In combined treatments, 1.58 μM of cadmium was applied at 1 hr. and X-rays of 0.82 or 1.24 Gy was applied at 1.5 hr. The end-points measured were the yolk sac diameter, crown-rump and head lengths, somite number, protein contents of yolk sac and embryo, and formation of the neural tube defect.Cadmium-induced neural tube defect increased as a function of dose in a highly curvilinear manner. The remaining end-points affected by the single treatments were also indicative to be curvilinear. The combined action of cadmium and X-rays was found to exert an additive effect on the inhibition of embryonic growth and a synergistic effect on the teratogenicity. The form of the dose-effect curves of the single agents was suggested to be responsible for teratogenic synergism. Other possibilities of interaction remain to be resolved.