Calcium taste preferences: genetic analysis and genome screen of C57BL/6J x PWK/PhJ hybrid mice.

To characterize the genetic basis of voluntary calcium consumption, we tested C57BL/6J mice (B6; with low avidity for calcium), PWK/PhJ mice (PWK; with high avidity for calcium) and their F(1) and F(2) hybrids. All mice received a series of 96-h two-bottle preference tests with a choice between water and the following: 50 mm CaCl(2), 50 mm calcium lactate, 50 mm MgCl(2), 100 mm KCl, 100 mm NH(4)Cl, 100 mm NaCl, 5 mm citric acid, 30 microm quinine hydrochloride and 2 mm saccharin. Most frequency distributions of the parental and F(1) but not F(2) groups were normally distributed, and there were few sex differences. Reciprocal cross analysis showed that B6 x PWK F(1) mice had a non-specific elevation of fluid intake relative to PWK x B6 F(1) mice. In the F(2) mice, trait correlations were clustered among the divalent salts and the monovalent chlorides. A genome screen involving 116 markers showed 30 quantitative trait loci (QTLs), of which six involved consumption of calcium chloride or lactate. The results show pleiotropic controls of calcium and magnesium consumption that are distinct from those controlling consumption of monovalent chlorides or exemplars of the primary taste qualities.

Licking for taste solutions by potassium-deprived rats: specificity and mechanisms.

There has been little work on the specificity and mechanisms underlying the appetite of potassium (K(+)) deprived rats, and there are conflicting results. To investigate the contribution of oral factors to changes in intake induced by K(+) deficiency, we conducted two experiments using 20-s "brief access" tests. In Experiment 1, K(+)-deprived rats licked less for water than did replete rats. After adjusting for this difference, K(+)-deprived rats exhibited increased licking for 100 mM CaCl(2), 100 mM MgCl(2), and 100 mM FeCl(2) compared with K(+)-replete rats. In Experiment 2, which used larger rats, the K(+)-deprived and replete groups licked equally for water, 500 mM Na.Gluconate, 350 mM KCl, 500 mM KHCO(3), and 1 mM quinine.HCl, but the K(+)-deprived rats licked more for 500 mM KCl, 500 mM CsCl, and 500 mM NaCl than did the replete rats. Licking was unaffected by addition to NaCl of 200 muM amiloride, an epithelial Na(+) channel (ENaC) blocker, or 100 muM ruthenium red, a vanilloid receptor 1 (VR-1) antagonist, or by addition to KCl of 50 muM 4-aminopyridine, a K(+) channel blocker. These findings suggest that K(+)-deprivation produces a non-specific appetite that is guided by oral factors. We found no evidence that this response was mediated by ENaC, VR-1, or K(+) channels in taste receptor cells.

Polymorphisms in the taste receptor gene (Tas1r3) region are associated with saccharin preference in 30 mouse strains.

The results of recent studies suggest that the mouse Sac (saccharin preference) locus is identical to the Tas1r3 (taste receptor) gene. The goal of this study was to identify Tas1r3 sequence variants associated with saccharin preference in a large number of inbred mouse strains. Initially, we sequenced approximately 6.7 kb of the Tas1r3 gene and its flanking regions from six inbred mouse strains with high and low saccharin preference, including the strains in which the Sac alleles were described originally (C57BL/6J, Sac(b); DBA/2J, Sac(d)). Of the 89 sequence variants detected among these six strains, eight polymorphic sites were significantly associated with preferences for 1.6 mm saccharin. Next, each of these eight variant sites were genotyped in 24 additional mouse strains. Analysis of the genotype-phenotype associations in all 30 strains showed the strongest association with saccharin preference at three sites: nucleotide (nt) -791 (3 bp insertion/deletion), nt +135 (Ser45Ser), and nt +179 (Ile60Thr). We measured Tas1r3 gene expression, transcript size, and T1R3 immunoreactivity in the taste tissue of two inbred mouse strains with different Tas1r3 haplotypes and saccharin preferences. The results of these experiments suggest that the polymorphisms associated with saccharin preference do not act by blocking gene expression, changing alternative splicing, or interfering with protein translation in taste tissue. The amino acid substitution (Ile60Thr) may influence the ability of the protein to form dimers or bind sweeteners. Here, we present data for future studies directed to experimentally confirm the function of these polymorphisms and highlight some of the difficulties of identifying specific DNA sequence variants that underlie quantitative trait loci.

Influence of the number of alcohol and water bottles on murine alcohol intake.

BACKGROUND: Most work to understand the controls of alcohol intake by animals involves the two-bottle choice method. Recent experiments involving other nutrients suggest that intakes are profoundly influenced by the number of nutrient choices available. Here, we extended these observations by measuring the alcohol consumption of mice and rats given multiple choices of water and alcohol. METHODS: Four experiments were conducted. In experiments 1 and 2, male C57BL6/J (B6) mice, 129X1/SvJ mice, or Sprague-Dawley rats received a series of six 72- or 48-hr tests in which the number of bottles of 10% alcohol and water was manipulated. One test involved the typical two-bottle choice. In the other five, the rodents always had six bottles with one, two, three, four, or five containing 10% alcohol and the rest containing water. In experiment 3, separate groups of B6 mice received for 16 days (a) the typical two-bottle test, (b) five alcohol bottles and one water bottle, (c) three alcohol bottles and three water bottles, or (d) one alcohol bottle and five water bottles. In experiment 4, groups of B6 mice received either a two-bottle test or five alcohol bottles and one water bottle for 24 days. RESULTS: In all experiments, the volume of alcohol consumed was strongly and positively related to the number of alcohol bottles available and inversely related to the number of water bottles available. The effect of alcohol availability on alcohol intake persisted for at least 24 days. CONCLUSIONS: Alcohol intake is strongly influenced by availability. The results point to a simple method of manipulating murine alcohol intake over a wide range. They provide an animal model that might be useful for understanding the influence of alcohol availability on human alcohol consumption.

Role of vagal afferent innervation in feeding and brain Fos expression produced by metabolic inhibitors.

Hepatic vagal afferent fibers have been implicated in the feeding responses initiated by administration of 2,5-anhydro-D-mannitol (2,5-AM; an inhibitor of hepatic metabolism) and methyl palmoxirate (MP; an inhibitor of fat metabolism). 2,5-AM and MP also increase brain Fos expression, an indicator of neural activity, which suggests that Fos expression can reveal the central neural pathways involved in the stimulation of feeding by these agents. To more closely test the hypothesis that brain Fos expression is related to the effects of 2,5-AM and MP on feeding, the vagus was lesioned by application of capsaicin, which destroys afferent fibers, directly to the cervical vagi. Perivagal capsaicin treatment blocked 2,5-AM-induced eating and attenuated MP-induced eating. Although perivagal capsaicin treatment attenuated MP-induced Fos expression, capsaicin treatment did not affect brain Fos expression produced by 2,5-AM. It is concluded that (1) brain Fos expression is not always related to the effects of 2,5-AM on feeding, (2) capsaicin-sensitive hepatic vagal afferent fibers carry the signal that stimulates feeding following 2,5-AM treatment, and (3) MP-induced feeding and brain Fos expression is mediated in part by capsaicin-sensitive fibers.

NaCl consumption is attenuated in female KCNE1 null mutant mice.

The role of potassium channels in the regulation of NaCl intake has not been investigated previously. One potassium channel, KCNQ1, and its regulator, KCNE1, are expressed in salivary glands and kidneys, and KCNE1 null mutant mice are deficient in KCNQ1 potassium currents. To understand the role of the KCNQ1/KCNE1 channel complex in NaCl taste and intake, we compared the NaCl consumption of KCNE1 +/+ (129/Sv), KCNE1 +/-, and KCNE1 -/- mice using two-bottle intake tests and lick rate tests. Although KCNE1 +/+ and KCNE1 +/- mice exhibited consumption patterns for 75-150 mM NaCl solutions considered typical for 129/Sv mice, the KCNE1 -/- null mutant 129/Sv mice were indifferent to or rejected them. This effect was observed in female mice only, required prior exposure to NaCl solutions, and the extent of rejection was greater after prior exposure to 150 mM NaCl solution than 75 mM NaCl solution. No differences were observed in the avidity for KCl solutions or in lick rates of naive mice for 150 or 300 mM NaCl solutions. These results demonstrate that a single potassium channel gene can influence voluntary NaCl intake. We speculate that disruption of the KCNE1 gene impairs sodium metabolism in female mice drinking high levels of 150 mM NaCl, which causes malaise that becomes associated with NaCl taste, and as a consequence, reduced preference for NaCl.

Calcium: taste, intake, and appetite.

This review summarizes research on sensory and behavioral aspects of calcium homeostasis. These are fragmented fields, with essentially independent lines of research involving gustatory electrophysiology in amphibians, ethological studies in wild birds, nutritional studies in poultry, and experimental behavioral studies focused primarily on characterizing the specificity of the appetite in rats. Recently, investigators have begun to examine potential physiological mechanisms underlying calcium intake and appetite. These include changes in the taste perception of calcium, signals related to blood calcium concentrations, and actions of the primary hormones of calcium homeostasis: parathyroid hormone, calcitonin, and 1,25-dihydroxyvitamin D. Other influences on calcium intake include reproductive and adrenal hormones and learning. The possibility that a calcium appetite exists in humans is discussed. The broad range of observations documenting the existence of a behavioral limb of calcium homeostasis provides a strong foundation for future genetic and physiological analyses of this behavior.

Influence of oral and gastric NaCl preloads on NaCl intake and gastric emptying of sodium-deficient rats.

Evidence is mixed as to whether oral metering contributes to the satiation of NaCl intake. To examine this in detail, we measured NaCl intake of sodium-deficient rats given preloads of NaCl that were sham ingested, normally ingested, or intubated into the stomach. Intake of 500 mM NaCl was reduced by prior ingestion, but not by sham ingestion, of an NaCl preload. NaCl intubation reduced NaCl intake if the test began 15 min, but not 60 min, after the preload. Gastric emptying of NaCl was initially more rapid after intubated than after ingested NaCl. Plasma aldosterone concentrations dropped more rapidly after ingested than after intubated NaCl and also dropped after sham ingestion of NaCl, raising the possibility of a cephalic-phase influence on aldosterone levels. These findings suggest that oral factors do not directly control the amount of NaCl consumed by sodium-deprived rats. Differences between the physiological effects of voluntary ingestion and intubation may be responsible for the results of several early studies purported as evidence for oral metering of sodium consumption.

Sweetener preference of C57BL/6ByJ and 129P3/J mice.

Previous studies have shown large differences in taste responses to several sweeteners between mice of the C57BL/6ByJ (B6) and 129P3/J (129) inbred strains. The goal of this study was to compare behavioral responses of B6 and 129 mice to a wider variety of sweeteners. Seventeen sweeteners were tested using two-bottle preference tests with water. Three main patterns of strain differences were evident. First, sucrose, maltose, saccharin, acesulfame-K, sucralose and SC-45647 were preferred by both strains, but the B6 mice had lower preference thresholds and higher solution intakes. Second, the amino acids D-phenylalanine, D-tryptophan, L-proline and glycine were highly preferred by B6 mice, but not by 129 mice. Third, glycyrrhizic acid, neohesperidin dihydrochalcone, thaumatin and cyclamate did not evoke strong preferences in either strain. Aspartame was neutral to all 129 and some B6 mice, but other B6 mice strongly preferred it. Thus, compared with the 129 mice the B6 mice had higher preferences for sugars, sweet tasting amino acids and several but not all non-caloric sweeteners. Glycyrrhizic acid, neohesperidin, thaumatin and cyclamate are not palatable to B6 or 129 mice.

Positional cloning of the mouse saccharin preference (Sac) locus.

Differences in sweetener intake among inbred strains of mice are partially determined by allelic variation of the saccharin preference (Sac) locus. Genetic and physical mapping limited a critical genomic interval containing Sac to a 194 kb DNA fragment. Sequencing and annotation of this region identified a gene (Tas1r3) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial backcrossing of the 194 kb chromosomal fragment containing the Tas1r3 allele from the high-sweetener-preferring C57BL/6ByJ strain onto the genetic background of the low-sweetener-preferring 129P3/J strain rescued its low-sweetener-preference phenotype. Polymorphisms of Tas1r3 that are likely to have functional significance were identified using analysis of genomic sequences and sweetener-preference phenotypes of genealogically distant mouse strains. Tas1r3 has two common haplotypes, consisting of six single nucleotide polymorphisms: one haplotype was found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor responds to sweeteners.

Calcium deprivation alters gustatory-evoked activity in the rat nucleus of the solitary tract.

Calcium-deprived rats develop a compensatory appetite for substances that contain calcium. To investigate the role of gustatory factors in calcium appetite, we recorded the extracellular activity of single neurons in the nucleus of the solitary tract of calcium-deprived and replete rats. The activity evoked by a broad array of taste stimuli was examined in 51 neurons from replete rats and 47 neurons from calcium-deprived rats. There were no differences between the groups in the responses of all neurons combined. However, neurons with sugar-oriented response profiles gave significantly larger responses to 3, 10, and 100 mM CaCl(2) in the calcium-deprived group than did corresponding cells in the replete group. This difference in taste-evoked responding may underlie an increase in the palatability of CaCl(2) and, in turn, contribute to the expression of calcium appetite.

Nutrient preference and diet-induced adiposity in C57BL/6ByJ and 129P3/J mice.

Purified carbohydrates and fats are usually palatable to humans and other animals, and their consumption often induces weight gain and accumulation of fat. In this study, we examined consumption of complex carbohydrates (cornstarch and Polycose) and fats (soybean oil and margarine) in mice from two inbred strains, C57BL/6ByJ and 129P3/J. At lower concentrations of liquid nutrients tested using two-bottle tests, when the amounts consumed had negligible energy content, the C57BL/6ByJ mice had higher acceptance of Polycose and soybean oil. This was probably due to strain differences in chemosensory perception of Polycose and oil. At higher concentrations, the mice consumed a substantial part of their daily energy from the macronutrient sources, however, there were no or only small strain differences in nutrient consumption. These small differences were probably due to strain variation in body size. The two strains also did not differ in chow intake. Despite similar energy intakes, access to the nutrients resulted in greater body weight (BW) gain in the C57BL/6ByJ mice than in the 129P3/J mice. The diet-induced weight gain was examined in detail in groups of 2-month-old C57BL/6ByJ and 129P3/J mice given ether chow, or chow and margarine to eat. Access to margarine did not increase total energy consumption of either strain. It increased BW and adiposity of the C57BL/6ByJ mice, but only after they reached the age of approximately 3 months. There were no differences in BW and adiposity between control and margarine-exposed 129P3/J mice. The results suggest that diet-induced adiposity in the B6 mice depends on age and does not depend on hyperphagia.

High-resolution genetic mapping of the saccharin preference locus (Sac) and the putative sweet taste receptor (T1R1) gene (Gpr70) to mouse distal Chromosome 4.

The Sac (saccharin preference) locus affecting mouse behavioral and neural responsiveness to sweeteners has been mapped to distal Chr 4. A putative sweet taste receptor, T1R1, has been recently cloned, and the gene encoding it, Gpr70, has also been mapped to mouse distal Chr 4. To assess Gpr70 as a candidate gene for Sac, we compared the Gpr70 sequences of C57BL/6ByJ and 129P3/J mouse strains with different alleles of Sac. Using Gpr70 sequence variation between the C57BL/6ByJ and 129P3/J strains, we conducted a high-resolution analysis of the chromosomal localization of the Gpr70 and Sac loci in the F2 hybrids and 129.B6-Sac partially congenic mice originating from these two strains. The Gpr70 gene maps proximal to Sac, which demonstrates that they are different loci.

Calcium-deprived rats sham-drink CaCl2 and NaCl.

Calcium-deprived rats are often thought to increase their calcium intake as a result of learning, but recent studies indicate that there is also an unlearned component to the appetite. They also ingest large amounts of some non-calcium minerals, including sodium. We examined the contribution of post-ingestive feedback to drinking using calcium-deprived and replete rats that could sham-drink CaCl2 and NaCl. Rats fitted with gastric cannulae in order to allow ingested fluids to drain freely drank 0.3 M NaCl in six 1-h sessions with their cannulae open (sham), followed by two sessions with their cannulae closed. Their intake of 0.03 M CaCl2 was then measured in a similar series of tests (six with cannula open followed by two with it closed). Ingestion of both NaCl and CaCl2 was significantly greater in calcium-deprived than in replete subjects under both open and closed conditions. These differences reached significance within 15 min after the onset of drinking during the first test with NaCl, and within 5 min in subsequent tests. The differences in CaCl2 intake generally reached significance within 5 min, including during the first test. Because there was minimal opportunity for post-ingestive NaCl or CaCl2 to mediate learning, the results provide additional support that the appetite for CaCl2 and NaCl in calcium-deprived rats can be driven solely by orosensory factors.

Intake of umami-tasting solutions by mice: a genetic analysis.

In two-bottle preference tests with water and solutions of monosodium glutamate (MSG) and inosine-5'-monophosphate (IMP), mice from the C57BL/6ByJ inbred strain consumed more and had higher preferences for these solutions compared with mice from the 129/J strain. The C57BL/6ByJ mice consumed 300 mmol/L MSG in large amounts, which were comparable to intakes of highly preferred solutions of sweeteners. The strain differences in voluntary consumption of 300 mmol/L MSG depended at least in part on postingestive effects because prior experience with MSG influenced the expression of the strain difference in MSG acceptance. The strain difference in MSG acceptance was in the opposite direction to the strain difference in NaCl acceptance and was not affected by previous consumption of saccharin. Although the C57BL/6ByJ mice had higher avidity for both MSG and sweeteners than did the 129/J mice, there was no correlation between preferences for these solutions in the second hybrid generation (F(2)) derived from these two strains. Thus, the strain differences in MSG acceptance are not related to the strain differences in salty or sweet taste responsiveness and most likely represent specific umami taste responsiveness. High acceptance of MSG solutions by the C57BL/6ByJ mice was inherited as a recessive trait in the F(2) generation. Further genetic linkage analyses using the F(2) hybrids are being conducted to map chromosomal locations of genes determining the strain difference in MSG acceptance.

Fatty acid oxidation affects food intake by altering hepatic energy status.

Inhibition of fatty acid oxidation stimulates feeding behavior in rats. To determine whether a decrease in hepatic fatty acid oxidation triggers this behavioral response, we compared the effects of different doses of methyl palmoxirate (MP), an inhibitor of fatty acid oxidation, on food intake with those on in vivo and in vitro liver and muscle metabolism. Administration of 1 mg/kg MP selectively decreased hepatic fatty acid oxidation but did not stimulate food intake. In contrast, feeding behavior increased in rats given 5 or 10 mg/kg MP, which inhibited hepatic fatty acid oxidation to the same extent as did the low dose but in addition suppressed fatty acid oxidation in muscle and produced a marked depletion of liver glycogen. Dose-related increases in food intake tracked dose-related reductions in liver ATP content, ATP-to-ADP ratio, and phosphorylation potential. The findings suggest that a decrease in hepatic fatty acid oxidation can stimulate feeding behavior by reducing hepatic energy production.

Modification of behavioral and neural taste responses to NaCl in C57BL/6 mice: effects of NaCl exposure and DOCA treatment.

To investigate the possible role of peripheral gustatory responsiveness to changes in NaCl acceptance, we studied NaCl consumption and the chorda tympani nerve responses to lingual application of NaCl in C57BL/6ByJ mice. The mice were treated with 300 mM NaCl (given to drink in 96-h two-bottle tests with water) or with injections of deoxycorticosterone acetate (DOCA; 33 mg/kg daily). Naive mice were neutral to 75 mM NaCl, but mice previously exposed to 300 mM NaCl avoided 75 mM NaCl. The NaCl-exposed (300 mM for 4 days and 75 mM for 2 days) mice had enhanced amiloride-sensitive components of the chorda tympani responses to 10-30 mM NaCl applied at room temperature (24 degrees C). DOCA injections increased acceptance of 300 mM NaCl, but did not change the chorda tympani responses to 100-1000 mM NaCl. However, the DOCA-treated mice had enhanced amiloride-sensitive components of the chorda tympani responses to cold (12 degrees C) 10-30 mM NaCl. These data suggest that peripheral gustatory responsiveness possibly contributes to the NaCl aversion induced by exposure to concentrated NaCl, but not to the DOCA-induced increase of NaCl acceptance.

Consumption of electrolytes and quinine by mouse strains with different blood pressures.

Daily fluid intakes were measured using two-bottle tests in female mice of inbred strains with high (BPH/2), normal (BPN/3) or low (BPL/1) blood pressure. The mice were offered a choice between water and different concentrations of NaCl (37.5-600 mM), KCl (1-400 mM), CaCl2 (1-100 mM) and quinine hydrochloride (0.003-1.0 mM). Compared with the normotensive strain, the hypertensive mice had higher water and total fluid intakes, and lower intakes of NaCl, KCl (only 200 mM) and quinine; the hypotensive mice had higher intakes of KCl (only 10-50 mM) and lower intakes of CaCl2 and quinine. These data suggest that fluid and salt intake are not linearly related to blood pressure, but are independently determined in these strains. Certain concentrations of the salts were preferred relative to water, which depended on mouse genotype: the BPN/3 and BPL/1 mice strongly preferred 37.5-150 mM NaCl, the BPL/1 mice preferred 10-100 mM KCl, and the BPN/3 mice preferred 1-10 mM CaCl2.

Calcium-deprived rats avoid sweet compounds.

To characterize the link between calcium status and sweet solution intake, rats fed a diet containing 25 mmol Ca2+/kg (Ca-25, low calcium) or 150 mmol Ca2+/kg (Ca-150, control) were given 48-h two-bottle tests with a choice between water and various concentrations of a nutrient (sucrose, Polycose, ethanol and/or corn oil). Rats fed the Ca-25 diet had significantly lower sucrose intakes and preferences over the entire range tested (10-320 g/L) even though the same (Experiment 1), or identically treated (Experiment 2a) rats had normal Polycose and ethanol intakes and normal (Experiment 1, 2b) or significantly greater (Experiment 2a) corn oil intakes. In additional tests, rats fed the Ca-25 diet had significantly lower intakes relative to rats fed the Ca-150 diet of other sweeteners (30 mmol/L D-phenylalanine, 1 mmol/L saccharin and 0.3 mmol/L aspartame), significantly higher intakes of 0.5 mg/L capsaicin and 300 mmol/L monosodium glutamate, and normal intakes of 10 g/L or 80 g/L safflower oil and 10 g/L peanut oil. In a three-cup macronutrient selection experiment (Experiment 3), calcium-deprived rats ate significantly less of a high sucrose carbohydrate source and significantly more of a protein source than did controls. These results suggest that calcium deficiency reduces the rat's liking for sweetness, irrespective of the type or form of sweetener, and that this is not due to a general reduction in energy intake.