Molecular mechanisms for sweet-suppressing effect of gymnemic acids.

Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 µg/ml) inhibited the [Ca(2+)]i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3.

Chronic restraint stress in rats suppresses sweet and umami taste responses and lingual expression of T1R3 mRNA.

Effects of chronic restraint stress on the taste responses to five basic taste qualities were investigated electrophysiologically in the rat chorda tympani. In addition, the mRNA expression for T1R3, the common G-protein-coupled receptor (GPCR) for sweet and umami tastes, was studied quantitatively by RT-PCR after such stress. Rats were restrained in a small cylindrical restrainer made of steel wire for 8h daily for 14 successive days. The integrated responses to sweet and umami tastes, as recorded from the chorda tympani, were significantly suppressed after such stress, but the other three basic taste responses were unaffected. Expression of T1R3 mRNA in the fungiform papillae, as estimated by RT-PCR, was slightly reduced by the stress, and a quantitative real time RT-PCR study revealed a significant suppression of T1R3 mRNA expression in the stress group. These results suggest that the observed stress-induced changes in taste sensation could be caused by a peripheral disorder of the transduction mechanism in taste-receptor cells, involving in particular a stress-induced inhibition of T1R3 expression.

Systemic administration of polymyxin B induces hypothermia in rats via an inhibitory effect on metabolic rate.

Polymyxin B, a cyclic cationic polypeptide antibiotic, binds to the lipid A of bacterial endotoxin (lipopolysaccharide; LPS) to inhibit LPS-induced fever. On the basis of a casual observation, we hypothesised that in rats (unlike in rabbits and goats), intravenous (i.v.) polymyxin B would decrease resting body temperature. A single i.v. injection of polymyxin B (10, 100 or 1000 microg/kg) induced a rapid, marked drop in body temperature in a dose-related manner, with no change in physical activity. However, the highest dose (1000 microg/kg) seemed to impair heat-loss mechanisms and/or functions controlling the animal's day-night cycle [because the day-time body temperature remained elevated for two days after the injection (versus the pre-injection level)]. By contrast, rats given 100 or 10 microg/kg of the drug showed a normal day-night cycle after recovery from the initial hypothermic effect of the drug. Therefore, we used the middle dose of polymyxin B (100 microg/kg) in the subsequent experiments. In these experiments, significant decreases in metabolic rate and heat-loss responses were observed immediately after an i.v. injection of polymyxin B (100 microg/kg). By contrast, intracerebroventricular injection of polymyxin B (3 microg) had no effect on resting body temperature. These results suggest that the observed decrease in metabolic rate is responsible for the polymyxin-B-induced hypothermia. Further, rats may react with a reduction in heat-loss responses so as to prevent the body temperature decreasing too far in response to polymyxin B. Thus, polymyxin B modulates or interferes with the peripheral mechanisms underlying body temperature regulation in rats.

Induction of salivary kallikreins by the diet containing a sweet-suppressive peptide, gurmarin, in the rat.

Gymnema sylvestre (gymnema) contains gurmarin that selectively inhibits responses to sweet substances in rodents. The present study investigated possible interaction between gurmarin and the submandibular saliva in rats fed diet containing gymnema. Electrophoretic analyses demonstrated that relative amounts of two proteins in the saliva clearly increased in rats fed the gymnema diet. However, rats previously given section of the bilateral glossopharyngeal nerve showed no such salivary protein induction. Analyses of amino acid sequence indicate that two proteins are rat kallikrein 2 (rK2) and rat kallikrein 9 (rK9). rK2 and rK9, a family of serine proteases, have a striking resemblance of cleavage site in the protein substrates. Interestingly, gurmarin possesses comparable residues with those rK2 and rK9 prefer. The kallikreins significantly inhibited immunoreaction between gurmarin and antigurmarin antiserum. These results suggest that rK2 and rK9 increased by chemosensory information for the gymnema diet via the glossopharyngeal nerve might cleave gurmarin or at least cause specific binding with it.

Effect of natriuretic peptide receptor antagonist on lipopolysaccharide-induced fever in rats: is natriuretic peptide an endogenous antipyretic?

We investigated whether natriuretic peptide (NP) acts as an endogenous antipyretic inside and/or outside the blood-brain barrier in rats made febrile by systemic administration of bacterial endotoxin (lipopolysaccharide; LPS). Intravenous (i.v.) injection of LPS induced a triphasic fever, the second phase of which was significantly enhanced by an i.v. injection of the NP receptor (A-type and B-type) antagonist HS-142-1, a glucose-caproic acid polymer. In contrast, the same antagonist (i.v.) had no effect on the fever induced by i.v. injection of interleukin (IL)-1beta. An i.v. administration of HS-142-1 enhanced the LPS (i.v.)-induced IL-1beta response in the rat spleen. An i.v. treatment with atrial NP (ANP) significantly attenuated the second phase of the LPS-induced fever. On the other hand, i.c.v. injection of the above-mentioned NP receptor antagonist resulted in an augmentation of the third phase of the fever induced by i.v. administration of LPS, the same phase that was attenuated by ANP given i.c.v. When given intracerebro-ventricularly (i.c.v.), the antagonist had no effect on the fever induced by i.v. IL-1beta. Finally, the fever induced by i.c.v. injection of LPS was not affected even by an i.c.v. administration of the antagonist. These results suggest that the production of pyrogenic cytokines (such as IL-1beta) that follows i.v. LPS injection may be inhibited by NP acting outside the blood-brain barrier, leading to an inhibition of the fever. In contrast, inside the blood-brain barrier NP may inhibit cytokine-independent mechanisms present within the rat brain that mediate LPS (i.v.)-induced fever.

Effects of central injection of angiotensin-converting-enzyme inhibitor and angiotensin type 1 receptor antagonist on the brain NF-kappaB and AP-1 activities of rats given LPS.

Angiotensin II (ANG II) activation of the angiotensin type 1 (AT1) receptor facilitates the production of brain interleukin-1beta (IL-1beta) and contributes to the induction of the fever following the intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). The purpose of the present study was to investigate whether proinflammatory transcription factors [nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1)] contribute to the ANG II-dependent production of cytokines within the brain. Interestingly, we found that a single i.c.v. injection of LPS had no effect on NF-kappaB and AP-1 activities in the hypothalamus, hippocampus, and cerebellum at either 1 or 3 h post-injection (except for a decrease in hypothalamic AP-1 activity at 1 h). Furthermore, both an angiotensin-converting-enzyme (ACE) inhibitor and an AT1 receptor antagonist enhanced (rather than reduced) the NF-kappaB and AP-1 activities in the hippocampus and/or cerebellum of rats given LPS. In contrast, an i.c.v. injection of ANG II increased the NF-kappaB activity in the hypothalamus. These results suggest that while "endogenous" ANG II exerts (via AT1 receptors) inhibitory effects on the activation of transcription factors in the brain of rats given LPS, a large dose of exogenous ANG II produces effects opposite to those induced by the presumably small amount of endogenous ANG II released locally by LPS. Our results seem not to support the idea that NF-kappaB and AP-1 play key roles in the ANG II-induced enhancement of the production of proinflammatory cytokines that is induced by LPS in the rat's brain.

Interaction of gymnemic acid with cyclodextrins analyzed by isothermal titration calorimetry, NMR and dynamic light scattering.

The physiological phenomenon that the antisweet taste effect of gymnemic acid (GA) is diminished by application of gamma-cyclodextrin (gamma-CD) to the mouth was evaluated at the molecular level using isothermal titration calorimetry, NMR and dynamic light scattering. These analyses showed that GA specifically binds to gamma-CD. Thermodynamic analysis using isothermal titration calorimetry revealed that the association constant of GA and gamma-CD is 10(5)-10(6) m(-1) with favorable enthalpy and entropy changes. The heat capacity change was negative and large, despite the change in accessible surface area upon binding being small. These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of gamma-CD. In addition, NMR measurements showed that in solution the spectra of GA are broad and sharpened by the addition of gamma-CD, indicating that unbound GA is in a water-soluble aggregate that is dispersed when it forms a complex with gamma-CD. Dynamic light scattering showed that the average diameter of unbound GA is > 30 nm and that of GA and gamma-CD complex is 2.2 nm, similar to unbound gamma-CD, supporting the aggregate property of GA and the inclusion complexation of GA by gamma-CD.

Gymnemic acids inhibit rabbit glyceraldehyde-3-phosphate dehydrogenase and induce a smearing of its electrophoretic band and dephosphorylation.

Gymnemic acids (GA) inhibited rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Binding of GA to GAPDH was observed by surface plasmon resonance measurement. Incubation of GAPDH with GA induced a smearing of the GAPDH band in SDS-PAGE. The GA-induced smearing was diminished by prior incubation of GA with gamma-cyclodextrin or by GA treatment with NAD. GA treatment did not affect the electrophoretic mobility of glucose-6-phosphate isomerase and dehydrogenase. GA treatment diminished the GAPDH band detected by an antibody to phosphoserine, but did not affect the phosphoserine bands of glucose-6-phosphate isomerase and dehydrogenase. These results indicated that GA specifically induced dephosphorylation of GAPDH.

Systemic administration of lipopolysaccharide upregulates angiotensin II expression in rat renal tubules: immunohistochemical and ELISA studies.

We investigated whether angiotensin II (AII) peptide is induced in the rat kidney under endotoxemic conditions. Immunohistochemistry revealed strong AII-like immunoreactivity in the renal tubules of rats given high-dose lipopolysaccharide (LPS; 1000 microg/kg) intraperitoneally (i.p.). AII-like immunoreactivity in renal tubules was slight at 1h after the LPS injection, but marked at 3 h. There were few signals in the kidney in saline-injected control rats. When injected at 0.1, 10, or 1000 microg/kg i.p., LPS-induced a dose-related increase in AII-like immunoreactivity in renal tubules that was unaffected by treatment with the prostaglandin-synthesis blocker indomethacin. ELISA measurement of the AII concentration in the whole kidney supported the above findings. These results suggest that systemically administered LPS induces AII peptide expression in renal tubules by a prostaglandin-independent mechanism.

Angiotensin II: its effects on fever and hypothermia in systemic inflammation.

Angiotensin II (ANG II), a bioactive peptide that plays important roles in blood-pressure and body-fluid regulation, has recently been reported to be involved in normal thermoregulation and fever. In the case of thermoregulation, ANG II lowers body temperature when administered centrally or systemically (i.e. "exogenous" ANG II acts as a hypothermia-inducing agent). In contrast, "endogenous" ANG II is involved both in heat-loss responses in a hot environment and in thermogenesis in the cold. It therefore seems likely that endogenous ANG II is involved in maintaining body temperature at the set-point. In the case of fever, it has been reported that endogenous brain ANG II and its type 1 receptor mediate or modulate the fever induced by "restraint stress". At the final step in "pyrogen-induced" fever, brain ANG II facilitates the fever induced by prostaglandin E2 (PGE2) through its action on the type 2 receptor, whereas at its first step the lipopolysaccharide (LPS, 2 microg/kg, i.v.)-induced production of pyrogenic cytokines [such as interleukin-1 (IL-1)] involves an action of endogenous ANG II through its type 1 receptor. On the other hand, it is well known that a very high dose of LPS (50-5000 microg/kg) injected systemically induces hypothermia in rodents. This hypothermia is presumably initiated by tumor necrosis factor (TNF). Since ANG II contributes to the LPS-induced production of cytokines such as IL-1beta, as described above, it is possible that the generation of TNF by LPS involves an action of ANG II, too, and that this TNF production leads to the LPS-induced hypothermia. Together, these findings suggest that ANG II and its receptors make a number of contributions to normal thermoregulation, to fever, and to the hypothermia in systemic inflammation.

Endogenous cryogens existing in the blood of a hypothermic patient.

We treated a young female patient who suffered from severe hypothermia. The etiology of the hypothermia was not identified, though various medical examinations were performed. Intraperitoneal (i.p.) injections of the patient's serum produced a significant and profound hypothermia in rats that was not associated with circulatory shock. The patient's serum was separated into 4 fractions by the use of various types of centrifugal filtration units according to molecular weights (MW), i.e., below 10 kDa, 10-30 kDa, 30-100 kDa, and beyond 100 kDa. The first two fractions never induced hypothermia in rats, but the i.p. injections of the fraction with MW from 30 to 100 kDa consistently produced hypothermia. The fraction with MW beyond 100 kDa caused marked hypothermia in one out of 3 rats tested. The results suggest that the patient was excessively producing endogenous cryogenic substances of which MW may be greater than 30 kDa.

The effect of central injection of angiotensin-converting enzyme inhibitor and the angiotensin type 1 receptor antagonist on the induction by lipopolysaccharide of fever and brain interleukin-1beta response in rats.

We recently reported an involvement of peripheral angiotensin II (ANG II) in the development of both the fever and the peripheral interleukin (IL)-1beta production induced in rats by a systemic injection of lipopolysaccharide (LPS). The present study was performed to investigate whether brain ANG II contributes to the fever and IL-1beta production in the rat brain induced by i.c.v. injection of LPS. LPS (0.2 and 2 microg i.c.v.) induced dose-related fevers and increases in the brain (hypothalamus, hippocampus, and cerebellum) concentrations of IL-1beta. These effects were significantly inhibited by i.c.v. administration of either an angiotensin-converting-enzyme (ACE) inhibitor or an angiotensin type 1 (AT(1)) receptor antagonist. By contrast, the ACE inhibitor had no effect on the IL-1beta (i.c.v.)-induced fever, whereas the AT(1) receptor antagonist enhanced (rather than reduced) it. The AT(1) receptor antagonist had no effect on the brain levels of prostaglandin E(2) in rats given an i.c.v. injection of IL-1beta. These results suggest that in rats, brain ANG II and AT(1) receptors are involved in the LPS-induced production of brain IL-1beta, thus contributing to the fever induced by the presence of LPS within the brain.

6-Formylpterin protects retinal neurons from transient ischemia-reperfusion injury in rats: a morphological and immunohistochemical study.

Neuroprotective effects of 6-formylpterin (6FP) on transient retinal ischemia-reperfusion injury were evaluated in rats by means of counting the number of retinal ganglion cells, measuring the thicknesses of the inner plexiform and inner nuclear layers, and by immunohistochemical detection of apoptotic cells in the retina. Sixty-one Sprague-Dawley rats (12 weeks, male, 295-330 g) were subjected to transient retinal ischemia-reperfusion by elevated intra-ocular pressure (80 mmHg for 60 min). Intraperitoneal injection of 6FP (3.8 mg/kg) was performed before or after ischemia. The retina was histologically better preserved in rats with 6FP treatment than without 6FP treatment. 6FP showed more strong neuroprotective effects when it was administered before ischemia. The number of single-stranded DNA-positive cells in the retina also decreased remarkably in rats with 6FP treatment, especially when administered before ischemia. These results suggest that 6FP protects retinal neurons from transient ischemia-reperfusion injury, at least in part by inhibiting apoptotic cell death.

Differential gurmarin suppression of sweet taste responses in rat solitary nucleus neurons.

We examined the effect of the sweet transduction blocker gurmarin on taste responses recorded from neurons in the rat solitary nucleus (NST) to determine how gurmarin sensitivity is distributed across neuronal type. Initially, responses evoked by washing the anterior tongue and palate with 0.5 M sucrose, 0.1 M NaCl, 0.01 M HCl, and 0.01 M quinine-HCl were recorded from 35 neurons. For some cells, responses to a sucrose concentration series (0.01-1.0 M) or an array of sweet-tasting compounds were also measured. Gurmarin (10 microg/ml, 2-4 ml) was then applied to the tongue and palate. Stimuli were reapplied after 10-15 min. Neurons were segregated into groups based on similarities among their initial response profiles using hierarchical cluster analysis (HCA). Results indicated that sucrose responses recorded from neurons representative of each HCA-defined class were suppressed by gurmarin. However, a disproportionate percentage of cells in each group displayed sucrose responses that were substantially attenuated after gurmarin treatment. Postgurmarin sucrose responses recorded from neurons that composed 57% of class S, 40% of class N, and 33% of class H were suppressed by >or=50% relative to control. On average, attenuation was statistically significant only in class S and N neurons. Although the magnitude of gurmarin-induced response suppression did not differ across sucrose concentration, responses to different sweet-tasting compounds were differentially affected. Responses to NaCl, HCl, or quinine were not suppressed by gurmarin. Results suggest that information from gurmarin-sensitive and -insensitive receptor processes converges onto single NST neurons.

ANG II is involved in the LPS-induced production of proinflammatory cytokines in dehydrated rats.

We have previously reported results that led us to speculate that ANG II is involved in the LPS-induced production of proinflammatory cytokines, especially under dehydrated conditions. To test this possibility, in this study we examined the effects of an angiotensin-converting enzyme (ACE) inhibitor and an antagonist of the type-1 ANG II receptor (AT(1) receptor) on the LPS-induced production of the proinflammatory cytokines IL-1 and IL-6 in dehydrated rats. A single intravenous injection of LPS induced a marked increase in the expression of IL-1beta mRNA in the liver, an effect that was significantly attenuated by pretreatment with the ACE inhibitor. Furthermore, the ACE inhibitor reduced the LPS-induced increase in the hepatic concentration of IL-1beta protein. When the AT(1)-receptor antagonist was given intravenously before the LPS, the increase in the hepatic concentration of IL-1beta was significantly reduced. Finally, the ACE inhibitor reduced the LPS-induced increase in the plasma concentration of IL-6. These results represent the first in vivo evidence that ANG II and its AT(1) receptor play important roles in the production of proinflammatory cytokines that is induced by LPS under dehydrated conditions.