Leptin as a modulator of sweet taste sensitivities in mice

Leptin acts as a potent inhibitory factor against obesity by regulating energy expenditure, food intake, and adiposity. The obese diabetic db/db mouse, which has defects in leptin receptor, displays enhanced neural responses and elevated behavioral preference to sweet stimuli. Here, we show the effects of leptin on the peripheral taste system. An administration of leptin into lean mice suppressed responses of peripheral taste nerves (chorda tympani and glossopharyngeal) to sweet substances (sucrose and saccharin) without affecting responses to sour, salty, and bitter substances. Whole-cell patch-clamp recordings of activities of taste receptor cells isolated from circumvallate papillae (innervated by the glossopharyngeal nerve) demonstrated that leptin activated outward K+ currents, which resulted in hyperpolarization of taste cells. The db/db mouse with impaired leptin receptors showed no such leptin suppression. Taste tissue (circumvallate papilla) of lean mice expressed leptin-receptor mRNA and some of the taste cells exhibited immunoreactivities to antibodies of the leptin receptor. Taken together, these observations suggest that the taste organ is a peripheral target for leptin, and that leptin may be a sweet-sensing modulator (suppressor) that may take part in regulation of food intake. Defects in this leptin suppression system in db/db mice may lead to their enhanced peripheral neural responses and enhanced behavioral preferences for sweet substances.

Dominant loss of responsiveness to sweet and bitter compounds caused by a single mutation in α-gustducin

Biochemical and genetic studies have implicated α-gustducin as a key component in the transduction of both bitter or sweet taste. Yet, α-gustducin-null mice are not completely unresponsive to bitter or sweet compounds. To gain insights into how gustducin mediates responses to bitter and sweet compounds, and to elicit the nature of the gustducin-independent pathways, we generated a dominant-negative form of α-gustducin and expressed it as a transgene from the α-gustducin promoter in both wild-type and α-gustducin-null mice. A single mutation, G352P, introduced into the C-terminal region of α-gustducin critical for receptor interaction rendered the mutant protein unresponsive to activation by taste receptor, but left its other functions intact. In control experiments, expression of wild-type α-gustducin as a transgene in α-gustducin-null mice fully restored responsiveness to bitter and sweet compounds, formally proving that the targeted deletion of the α-gustducin gene caused the taste deficits of the null mice. In contrast, transgenic expression of the G352P mutant did not restore responsiveness of the null mice to either bitter or sweet compounds. Furthermore, in the wild-type background, the mutant transgene inhibited endogenous α-gustducin's interactions with taste receptors, i.e., it acted as a dominant-negative. That the mutant transgene further diminished the residual bitter and sweet taste responsiveness of the α-gustducin-null mice suggests that other guanine nucleotide-binding regulatory proteins expressed in the α-gustducin lineage of taste cells mediate these responses.

(+)-Germacrene A Biosynthesis : The Committed Step in the Biosynthesis of Bitter Sesquiterpene Lactones in Chicory

The leaves and especially the roots of chicory (Cichorium intybus L.) contain high concentrations of bitter sesquiterpene lactones such as the guianolides lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and germacranolides are present in smaller amounts. Their postulated biosynthesis through the mevalonate-farnesyl diphosphate-germacradiene pathway has now been confirmed by the isolation of a (+)-germacrene A synthase from chicory roots. This sesquiterpene cyclase was purified 200-fold using a combination of anion-exchange and dye-ligand chromatography. It has a Km value of 6.6 μm, an estimated molecular mass of 54 kD, and a (broad) pH optimum around 6.7. Germacrene A, the enzymatic product, proved to be much more stable than reported in literature. Its heat-induced Cope rearrangement into (−)-β-elemene was utilized to determine its absolute configuration on an enantioselective gas chromatography column. To our knowledge, until now in sesquiterpene biosynthesis, germacrene A has only been reported as an (postulated) enzyme-bound intermediate, which, instead of being released, is subjected to additional cyclization(s) by the same enzyme that generated it from farnesyl diphosphate. However, in chicory germacrene A is released from the sesquiterpene cyclase. Apparently, subsequent oxidations and/or glucosylation of the germacrane skeleton, together with a germacrene cyclase, determine whether guaiane- or eudesmane-type sesquiterpene lactones are produced.

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Deficits in memory and motor performance in synaptotagmin IV mutant mice

Synaptotagmin (Syt) IV is a synaptic vesicle protein. Syt IV expression is induced in the rat hippocampus after systemic kainic acid treatment. To examine the functional role of this protein in vivo, we derived Syt IV null [Syt IV(−/−)] mutant mice. Studies with the rotorod revealed that the Syt IV mutants have impaired motor coordination, a result consistent with constitutive Syt IV expression in the cerebellum. Because Syt IV is thought to modulate synaptic function, we also have examined Syt IV mutant mice in learning and memory tests. Our studies show that the Syt IV mutation disrupts contextual fear conditioning, a learning task sensitive to hippocampal and amygdala lesions. In contrast, cued fear conditioning is normal in the Syt IV mutants, suggesting that this mutation did not disrupt amygdala function. Conditioned taste aversion, which also depends on the amygdala, is normal in the Syt IV mutants. Consistent with the idea that the Syt IV mutation preferentially affects hippocampal function, Syt IV mutant mice also display impaired social transmission of food preference. These studies demonstrate that Syt IV is critical for brain function and suggest that the Syt IV mutation affects hippocampal-dependent learning and memory, as well as motor coordination.

Reversible inactivation of the nucleus basalis magnocellularis induces disruption of cortical acetylcholine release and acquisition, but not retrieval, of aversive memories

The basal forebrain complex, which includes the nucleus basalis magnocellularis (NBM), provides widespread cholinergic and γ-aminobutyric acid-containing projections throughout the brain, including the insular and pyriform cortices. A number of studies have implicated the cholinergic neurons in the mediation of learning and memory processes. However, the role of basal forebrain activity in information retrieval mechanisms is less known. The aim of the present study is to evaluate the effects of reversible inactivation of the NBM by tetrodotoxin (TTX, a voltage-sensitive sodium channel blocker) during the acquisition and retrieval of conditioned taste aversion (CTA) and to measure acetylcholine (ACh) release during TTX inactivation in the insular cortex, by means of the microdialysis technique in free-moving rats. Bilateral infusion of TTX in the NBM was performed 30 min before the presentation of gustative stimuli, in either the CTA acquisition trial or retrieval trial. At the same time, levels of extracellular ACh release were measured in the insular cortex. The behavioral results showed significant impairment in CTA acquisition when the TTX was infused in the NBM, whereas retrieval was not affected when the treatment was given during the test trial. Biochemical results showed that TTX infusion into the NBM produced a marked decrease in cortical ACh release as compared with the controls during consumption of saccharin in the acquisition trial. Depleted ACh levels were found during the test trial in all groups except in the group that received TTX during acquisition. These results suggest a cholinergic-dependent process during acquisition, but not during memory retrieval, and that NBM-mediated cholinergic cortical release may play an important role in early stages of learning, but not during recall of aversive memories.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a “Deg” mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

Genetic Manipulation of Alcohol Dehydrogenase Levels in Ripening Tomato Fruit Affects the Balance of Some Flavor Aldehydes and Alcohols1

Tomato (Lycopersicon esculentum) plants were transformed with gene constructs containing a tomato alcohol dehydrogenase (ADH) cDNA (ADH 2) coupled in a sense orientation with either the constitutive cauliflower mosaic virus 35S promoter or the fruit-specific tomato polygalacturonase promoter. Ripening fruit from plants transformed with the constitutively expressed transgene(s) had a range of ADH activities; some plants had no detectable activity, whereas others had significantly higher ADH activity, up to twice that of controls. Transformed plants with fruit-specific expression of the transgene(s) also displayed a range of enhanced ADH activities in the ripening fruit, but no suppression was observed. Modified ADH levels in the ripening fruit influenced the balance between some of the aldehydes and the corresponding alcohols associated with flavor production. Hexanol and Z-3-hexenol levels were increased in fruit with increased ADH activity and reduced in fruit with low ADH activity. Concentrations of the respective aldehydes were generally unaltered. The phenotypes of modified fruit ADH activity and volatile abundance were transmitted to second-generation plants in accordance with the patterns of inheritance of the transgenes. In a preliminary taste trial, fruit with elevated ADH activity and higher levels of alcohols were identified as having a more intense “ripe fruit” flavor.

Identification of a Drosophila G protein alpha subunit (dGq alpha-3) expressed in chemosensory cells and central neurons.

We have identified another Drosophila GTP-binding protein (G protein) alpha subunit, dGq alpha-3. Transcripts encoding dGq alpha-3 are derived from alternative splicing of the dGq alpha locus previously shown to encode two visual-system-specific transcripts [Lee, Y.-J., Dobbs, M.B., Verardi, M.L. & Hyde, D.R. (1990) Neuron 5, 889-898]. Immunolocalization studies using dGq alpha-3 isoform-specific antibodies and LacZ fusion genes show that dGq alpha-3 is expressed in chemosensory cells of the olfactory and taste structures, including a subset of olfactory and gustatory neurons, and in cells of the central nervous system, including neurons in the lamina ganglionaris. These data are consistent with a variety of roles for dGq alpha-3, including mediating a subset of olfactory and gustatory responses in Drosophila, and supports the idea that some chemosensory responses use G protein-coupled receptors and the second messenger inositol 1,4,5-trisphosphate.