A high salt diet inhibits obesity and delays puberty in the female rat.

BACKGROUND/OBJECTIVES: Processed foods are considered major contributors to the worldwide obesity epidemic. In addition to high sugar and fat contents, processed foods contain large amounts of salt. Owing to the correlations with rising adiposity, salt has recently been proposed to be obesogenic. This study investigated three hypotheses: (i) high salt contributes to weight gain and adiposity in juvenile female rats, (ii) puberty onset would be altered because salt is known to affect neuronal systems involved in activating the reproductive system, and (iii) enhanced adiposity will act synergistically with salt to drive early puberty onset. DESIGN: Female weanling rats (post-natal day 21, n=105) were fed a low fat/low salt diet, low fat/high salt diet, high fat/low salt diet or a high salt/high fat diet for 24 days. Metabolic measures, including weight gain, food intake, fecal output, activity and temperature were recorded in subsets of animals. RESULTS: Body weight, retroperitoneal and perirenal fat pad weight, and adipocyte size were all lower in animals fed high fat/high salt compared with animals fed high fat alone. Leptin levels were reduced in high fat/high salt fed animals compared with high fat/low salt-fed animals. Daily calorie intake was higher initially but declined with adjusted food intake and was not different among groups after 5 days. Osmolality and corticosterone were not different among groups. Fecal analysis showed excess fat excretion and a decreased digestive efficiency in animals fed high fat/low salt but not in animals fed high fat/high salt. Although respiratory exchange ratio was reduced by high dietary fat or salt, aerobic-resting metabolic rate was not affected by the diet. High salt delayed puberty onset, regardless of dietary fat content. CONCLUSIONS: Salt delays puberty and prevents the obesogenic effect of a high fat diet. The reduced weight gain evident in high salt-fed animals is not due to differences in food intake or digestive efficiency.

Characterization of nuclear neurokinin 3 receptor expression in rat brain.

Ligand-induced translocation of the G-protein-coupled receptor, neurokinin 3 (NK3-R), to the nucleus of hypothalamic neurons was reported using antibodies (ABs) raised against the C-terminal region of NK3-R. The current work was undertaken to substantiate the ability of NK3-R to enter the nucleus and identify which portion of the NK3-R molecule enters the nucleus. ABs directed at epitopes in the N-terminal and second extracellular loop of the rat NK3-R molecule were used to evaluate western blots of whole tissue homogenates and nuclear fractions from multiple brain areas. Specificity of the protein bands recognized by these ABs was demonstrated using Chinese hamster ovary (CHO) cells transfected with rat or human NK3-R. Both ABs prominently recognized a diffuse protein band of ∼56-65 kDa (56 kDa=predicted size) and distinct ∼70-kDa and 95-kDa proteins in homogenates of multiple brain areas. The ∼95-kDa protein recognized by the extracellular loop AB was enriched in nuclear fractions. Recognition of these proteins by ABs directed at different regions of the NK3-R supports their identification as NK3-R. The size differences reflect variable glycosylation and possibly linkage to different cytosolic and nuclear proteins. Recognition of protein bands by both ABs in nuclear fractions is consistent with the full-length NK3-R entering the nucleus. Hypotension increased the density of the ∼95-kDa band in nuclear fractions from the supraoptic nucleus indicating activity-induced nuclear translocation. Since NK3-R is widely distributed in the CNS, the presence of NK3-R in nuclei from multiple brain regions suggests that it may broadly influence CNS gene expression in a ligand-dependent manner.

Expression of the nuclear transport protein importin ß-1 and its association with the neurokinin 3 receptor in the rat hypothalamus following acute hyperosmotic challenge.

The tachykinin NK3 receptor (NK3R) is a G-protein coupled receptor that is activated, internalized, and trafficked to the nuclei of magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVN) in response to acute hyperosmolarity. The lack of information on the nuclear import pathway raises concerns about the physiological role of nuclear NK3R. NK3R contains a nuclear localizing sequence (NLS) and this raises the possibility that importins are involved in transport of NK3R through the nuclear pore complex. The following experiments utilized: (1) co-immunoprecipitation to determine if NK3R is associated with importin ß-1 following activation in response to acute hyperosmolarity in vivo, and (2) immuno-neutralization of importin ß-1 in vitro to determine if nuclear transport of NK3R was blocked. Rats were given an i.v. injection of hypertonic saline (2 M) and 10 min after the infusion, the PVN was removed and homogenized. Importin ß-1 co-immunoprecipitated with the NK3R following treatment with 2 M NaCl, but not following isotonic saline treatment. Immuno-neutralization of importin ß-1 decreased the transport of NK3R into the nuclei in a time dependent fashion. The results indicate that in response to acute hyperosmotic challenge, NK3R associates with importin ß-1 which enables the nuclear transport of NK3R. This is the first in vivo study linking importin ß-1 and the nuclear transport of a G protein coupled receptor, the NK3R, in brain.

Trafficking of tachykinin neurokinin 3 receptor to nuclei of neurons in the paraventricular nucleus of the hypothalamus following osmotic challenge.

Tachykinin neurokinin 3 receptor (NK3R) is a G-protein (GTP binding protein) -coupled receptor that is heavily expressed by magnocellular neurons of the paraventricular nucleus of the hypothalamus (PVN). Osmotic challenge is reported to activate NK3R expressed by magnocellular neurons and cause the NK3R to be internalized to the cytoplasm and perhaps the cell nucleus. In this study we show using immuno-electron microscopy that isolated nuclei from neurons in the PVN of osmotic challenged animals (rats) show a robust labeling for the NK3R. NK3R immunoreactivity was detected by Western blot in isolated nuclei of PVN neurons following the 2 M NaCl injection. No nuclear NK3R immunoreactivity was detected in control animals. NK3R antibody specificity was confirmed by small interfering (SI) RNA technology. This study establishes that the NK3R is trafficked to the nucleus of PVN neurons following a peripheral osmotic challenge.

Distribution of Fos-like immunoreactivity within the rat brain following intraventricular injection of the selective NK(3) receptor agonist senktide.

Neurokinin B (NKB) is one member of an evolutionarily conserved family of neuropeptides, the tachykinins. Preferential binding of NKB to endogenous NK(3) receptors affects a variety of biological and physiological processes, including endocrine secretions, sensory transmission, and fluid and electrolyte homeostasis. In light of its widespread biological actions, immunohistochemical detection of the c-Fos protein product was used to study the distribution of neuronal activation in the rat brain caused by intraventricular (icv) injections of the selective NK(3) receptor agonist (succinyl-[Asp(6), N-Me-Phe(8)] substance P [6-11]), senktide. Quantitative analysis revealed that treatment with isotonic saline or 200 ng senktide resulted in the differential expression of Fos-like immunoreactivity (FLI) throughout the brain. Senktide induced the highest number of FLI neurons in the lateral septum, bed nucleus of the stria terminalis, amygdala, paraventricular nucleus of the hypothalamus, median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus, periaqueductal gray, and medial nucleus of the solitary tract compared to isotonic saline controls. Additional regions that contained elevated FLI following icv injection of senktide, relative to saline injection, included the cerebral cortex, lateral hypothalamic nucleus, suprachiasmatic nucleus, ventral tegmental area, substantia nigra, inferior colliculus, locus coeruleus, zona incerta, and arcuate nucleus. Our data indicate that activation of NK(3) receptors induces the expression of FLI within circumscribed regions of the rat brain. This pattern of neuronal activation overlaps with nuclei known to regulate homeostatic processes, such as endocrine secretion, cardiovascular function, salt intake, and nociception.

Intraventricular injection of tachykinin NK3 receptor agonists suppresses the ingestion of NaCl-associated tastes.

The present experiments evaluated whether a salty taste was required for injections of a neurokinin-3 (NK3) receptor agonist (senktide) to suppress intake or whether senktide would reduce the intake of tastes that are predictive of NaCl. During training, different groups of rats were given access to 1% almond + water, 1% almond + 0.3 M NaCl, or 1% almond + 0.1 M sucrose. On the test day, rats were administered intraventricular injections of either saline or 200 ng senktide and then given access to 1% almond + water. Senktide had no effect on the intake of the water-associated or sucrose-associated almond. In contrast, senktide significantly reduced the intake of NaCl-associated almond. Senktide had no effect on almond intake by water-deprived rats. These results show that activation of NK3 receptors reduces the intake of NaCl and of a neutral taste that is predictive of sodium but not of calories.

Differential effects of intraventricular injections of tachykinin NK1 and NK3 receptor agonists on normal and sham drinking of NaCl by sodium-deficient rats.

The effects of lateral ventricular injections of succinyl-[Asp6, N-Me-Phe8]-substance P (SENK; 25, 100, 200 ng), a tachykinin NK3 receptor agonist, and [Sar9, Met(O2)11]-substance P (Sar Met; 100, 200 ng), an NK1 receptor agonist, on normal (gastric fistula closed) and sham drinking (gastric fistula open) of hypertonic NaCl by sodium-deficient rats were compared. Intraventricular injections of Sar Met had no effect on NaCl intake in either condition. Injections of 100 ng and 200 ng SENK caused an equal suppression of NaCl intake in the 2 fistula conditions. The latency to drink was not affected, but the initial lick rate was significantly lower and decayed more rapidly after 100 ng SENK than after saline or 25 ng SENK. The results show that (a) the tachykinin subtypes are not equally involved in the control of need-induced salt intake; (b) negative feedback from the stomach and distal gastrointestinal tract is not required for intraventricular injections of SENK to suppress sodium appetite; (c) the activation of NK3 receptors decreases the oral excitatory influence of hypertonic NaCl in sodium-deficient rats.

Brain tachykinins and the regulation of salt intake.

The regulation of salt intake is achieved through the coordination of behavioral and physiological responses. Brain neuropeptides, such as the tachykinins, play an important role in orchestrating both of these responses. Intraventricular injections of NK3 receptor agonists, such as senktide, are potent in suppressing salt intake. Experimental results show that intraventricular injections of senktide that suppress salt intake have no effect on the ingestion of other tastes, such as sucrose. The means by which senktide suppresses salt intake was investigated in a series of experiments. Taste reactivity and lick rate analyses suggest that the activation of NK3 receptors reduces salt intake by modulating the oral-stimulating property of salt taste.

Lateral ventricular injections of the NK3 agonist senktide affect salt taste-elicited responses.

Central injections of the selective tachykinin NK3 receptor agonist senktide (SENK) suppresses salt appetite. Also, following SENK, intraoral infusions of hypertonic NaCl elicit fewer ingestive taste reactivity responses and more aversive responses than following intraventricular injections of isotonic saline. This pattern of taste reactivity results suggest that SENK affects the oral stimulating properties of salt taste. Before accepting this interpretation, however, alternative explanations need to be examined. The following experiments evaluated whether the effects of intraventricular SENK injection on taste reactivity could be due to: 1) a general oral motor impairment that reduces ingestive responding to tastes (Experiment 1) or; 2) SENK having aversive consequences (Experiment 2). In Experiment 1, the effects of intraventricular injections of SENK (200 ng) on taste reactivity responses to 0.5 M NaCl and 0.1 M sucrose were measured in sodium deficient rats. Intraoral infusions of 0.5 M NaCl elicited fewer ingestive taste reactivity responses following SENK than injections of isotonic saline in sodium deficient rats. Sucrose continued to elicit the same high number of ingestive taste reactivity responses following intraventricular injections of isotonic saline and SENK. Thus, SENK did not cause a general decrease in ingestive responding. A conditioned taste aversion test was employed in Experiment 2 to determine if SENK had aversive consequences. Rats were given 30 min access to alanine (0.3 M) and were then administered either lithium chloride (LiCl) or intraventricular injections of SENK (200 ng) on three consecutive days. Rats avoided alanine that was paired with LiCl, but those rats that had alanine paired with SENK showed no avoidance of the taste even after three pairings. These results replicate findings that intraventricular injections of the NK3 agonist SENK decreases the palatability of NaCl (as measured by taste reactivity) and suggest that its effect on NaCl-elicited taste reactivity is not due to the treatment causing a motor impairment or malaise.

Bombesin receptor antagonists block the effects of exogenous bombesin but not of nutrients on food intake.

The endogenous, meal-contingent release of bombesin (BN)-like peptides is thought to contribute to the termination of a meal. In the following experiments the potency of BN receptor antagonists to attenuate the ability of nutrients to suppress food intake was tested. First, the effectiveness of BN receptor subtype antagonists was verified by testing their ability to block the effects of exogenous BN on food intake. Rats were administered intraperitoneal (i.p.) injections of either saline or 0.1 mg/kg [D-Phe12,Leu14]BN (binds both GRP and NMB receptors), [D-Phe6]BN(6-13) ethyl amide (binds GRP > NMB), and cyclo-SS-octa (BIM-23042; binds NMB > GRP). Five minutes later rats were administered 8 micrograms/kg BN (i.p.) and milk intake was measured. Injections of [D-Phe12,Leu14]BN and [D-Phe6]BN(6-13) ethyl amide reliably attenuated the ability of BN to suppress milk intake whereas BIM-23042 was ineffective. The results show that the antagonists were behaviorally effective and that exogenous BN may exert its effects on food intake primarily through the GRP receptor subtype. Next, the antagonists were administered either 5 min prior to or 5 min after an intragastric nutrient load or no load in both overnight-deprived and nondeprived rats, and milk intake was then measured. Stomach loads reduced intake and this effect was not attenuated by BN receptor antagonists. Finally, rats were allowed to prefeed and the milk was then removed. Rats were then administered a BN receptor antagonist (0.1 and 1.0 mg/kg) or saline either immediately after the prefeed, 10 min later, or 20 min later. Milk diet was then returned and intake was measured. Peripheral injections of the BN receptor antagonist had no effect compared to saline on milk intake. Collectively, the results indicate that the blockade of peripheral Bn peptide receptors is not sufficient to attenuate the safety signals generated by stomach loads or prefeeding.

Mammalian bombesin-like peptides suppress sham drinking of salt by sodium-deficient rats.

Bombesin (BN) and its mammalian relatives, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been implicated in the control of food intake, and more recently systemic injections of BN have been shown to suppress need-free and sodium deficiency-induced salt (NaCl) intake. Postoral mechanisms that are activated by the ingestion of salt contribute to the termination of salt intake. The hypothesis that BN-like peptides potentiate postoral salt ingestion-contingent feedback and thereby accelerate the termination of salt intake was evaluated by measuring the effects of peripheral injections of BN (4, 6, 16 micrograms/kg), GRP (4, 8, 16 micrograms/kg), and NMB (4, 8, 16 micrograms/kg) on sham (gastric fistula open) and normal (gastric fistula closed) drinking of NaCl (0.5 M) by sodium-deficient male rats. Sodium deficiency was induced by injections of deoxy-corticosterone acetate and furosemide. Injections of 4 micrograms/kg BN produced a transient suppression in the sham drinking of salt and a more sustained reduction of salt intake when postoral factors were present in the normal drinking condition. In contrast, injections of 6 and 16 micrograms/kg BN produced the same pronounced suppression in salt intake in both the closed and open fistula test conditions. Only 16 micrograms/kg GRP was behaviorally effective, and this des: reliably suppressed both normal and sham drinking of salt. On an equimolar basis, BN was more potent than GRP. Injections of NMB had no effect on salt intake under any dose or fistula condition. The order of BN-like peptide potency in suppressing sham and normal drinking of salt by sodium-deficient rats (BN > GRP > NMB) is similar to the peptides' effects on feeding. When postoral feedback is minimized, the combination of BN or GRP and pregastric stimuli mimic the salt intake by rats in the fistula closed, normal drinking condition.

Dehydration anorexia in decerebrate rats.

Hypertonic saline (HS) administered intraperitoneally reduced the intake of sucrose solution infused intraorally in tube-fed decerebrate rats, as it did in control animals. Similarly, either intraperitoneal or intravenous HS markedly decreased the intake of laboratory chow by neurologically intact control rats. These observations complement recent findings that lesions of putative osmoreceptors in the ventral diencephalon, which eliminate thirst and blunt pituitary secretion of vasopressin and oxytocin in response to HS in rats, have no apparent effect on the HS-induced inhibition of food intake. Taken together they support previous studies indicating an important role for the caudal brainstem in the central control of food intake and suggest that such brainstem control may also include the inhibition of food intake induced by acute hyperosmolality.

Applications of taste reactivity to the study of the neural-hormonal controls of ingestive behavior.

Taste plays a central role in guiding ingestive behavior and the encoding of taste is affected by manipulations that influence ingestive behavior. In this article, the use of the taste reactivity test to provide a behavioral assessment of how changes in the oral reinforcing properties of a taste may initiate or sustain ingestive behaviors in several contexts are discussed. The affects of the animal's sex, sodium deficiency, exogenous bombesin administration, and the role of central gustatory lesions in mediating taste reactivity responses are discussed. Findings indicate that an enhancement of ingestive taste reactivity responses correlate with an increased preference and intake of taste stimuli for some, but not all situations. Such situations include the bombesin-like peptides that reduce sucrose and sodium chloride intake without influencing taste reactivity responses. Conversely, female rats, compared to males, show an elevated intake and preference for a range of NaCl concentrations and a greater number of ingestive taste reactivity responses to some, but not all of the preferred concentrations. Such mismatches of taste reactivity and intake measures shift attention to the contribution of nongustatory factors (trigeminal, visceral) in the control of intake.

Tachykinin NK3 receptor agonist blocks sodium deficiency-induced shift in taste reactivity.

Intracerebroventricular (i.c.v.) injections of tachykinin NK3 receptor agonists suppress NaCl intake by sodium deficient rats. The brief exposure, taste reactivity test was used to examine the effect of tachykinins on the immediate, oral reinforcing properties of NaCl (0.5 M) in sodium replete and sodium deficient male rats. In sodium replete rats, intraoral infusions of NaCl elicited a mixed response comprised of a similar number of ingestive and aversive responses following i.c.v. injections of saline and succinyl-[Asp6,N-Me-Phe8]substance P, (6-11), (SENK), a NK3 receptor agonist. When sodium deficient, saline injected rats showed a reliable increase in ingestive and a decrease in aversive taste reactivity responses. Lateral i.c.v. injections of SENK blocked the shift in taste reactivity responses by sodium deficient rats, indicating that concentrated NaCl retained its aversive taste property.

Bombesin suppresses need-free and need-induced salt intake in rats.

The researchers performed experiments to evaluate whether the effects of bombesin are selective for the satiation of ingestive behaviors related to energy balance or if ingestive behaviors associated with sodium balance are also suppressed by bombesin. Injections of 4 and 8 micrograms/kg bombesin reliably reduced need-free and sodium deficiency-induced NaCl intake in male rats. The effects of bombesin on the sodium-deficiency-induced change in taste reactivity was assessed. Injections of 4 micrograms/kg and 8 micrograms/kg bombesin had no effect on the sodium deficiency-induced shift in taste reactivity. These data indicate that bombesin suppresses NaCl intake and that bombesin does not appear to interact with gustatory sensibility in exerting its behavior-controlling action.

Lordosis response components can be elicited in decerebrate rats by combined flank and cervix stimulation.

Previous research has indicated that chronically decerebrate adult female rats are incapable of exhibiting lordosis in response to lumbosacral somatic stimuli, suggesting a dependency of this response on forebrain influence. Since vaginocervical stimulation facilitates lordosis to lumbosacral stimuli in rats, the present study investigated the possibility that chronically decerebrate rats could show lordosis in response to flank palpation if vaginocervical pressure was applied concurrently. Six of nine ovariectomized, decerebrate rats exhibited a coordinated display of lordosis-like responses to flank palpation if this stimulus was accompanied by vaginocervical stimulation, but not in response to flank and perineal pressure alone. Estradiol-progesterone treatment increased the magnitude of the response in five of six animals. As in intact rats, cervix pressure in the decerebrates also produced immobilization and blocked responses to nociceptive stimuli. Thus, the caudal neuraxis can mediate expression of essential components of the lordosis response in the absence of forebrain influence.

Fourth ventricular injection of selective bombesin receptor antagonists facilitates feeding in rats.

Exogenous bombesin (BN)-like peptides exert their effects on feeding through caudal brain stem BN-like peptides and their receptors. To determine if endogenous BN-like peptides are involved in the control of feeding and exert their actions on caudal brain stem neurons, nondeprived rats were administered fourth ventricular injections of either saline or BN receptor antagonists in the following doses: 5 and 25 ng [D-Phe12,Leu14]BN or 5 ng [D-Phe6]BN(6-13)methyl ester. Milk intake was measured every 5 min and each animal's behavior (eating, grooming, exploring, resting) was monitored every 1 min for 60 min. Compared with saline injections, fourth ventricular injections of 25 ng [D-Phe12,Leu14]BN and 5 ng [D-Phe6]BN(6-13)methyl ester reliably facilitated milk intake. The enhanced milk intake was evident within 5 min after the injection and persisted throughout the 1-h intake test. The behavioral sequence following fourth ventricular injections of saline and the antagonists that facilitated feeding were similar. Injections of [D-Phe12,Leu14]BN and [D-Phe6]BN(6-13)methyl ester did not produce a generalized arousal. The results indicate that food-contingent, endogenous BN-like peptides contribute to the termination of feeding and exert their behavior-controlling actions on BN-like peptide receptors in the vicinity of the fourth ventricle.

Sex differences in salt preference and taste reactivity in rats.

Previous studies have shown that female rats consume significantly more sodium chloride (NaCl) than do age-matched males. The gustatory contribution to this sex difference was examined in the following experiments. In Experiment 1, female rats demonstrated a higher two-bottle preference for NaCl ranging from 0.03 M to 1.0 M than did age-matched males. Next, to determine if the animal's sex modified gustatory sensitivity for NaCl, taste reactivity responses elicited by intraoral infusions (0.8 ml) of NaCl (0.03 M, 0.15 M, 0.3 M, and 1.0 M) were measured in age-matched male and female Sprague-Dawley rats. Intraoral infusions of NaCl were administered in ascending concentration order on successive days. During the intraoral infusion, the animal's oral motor taste reactivity responses were videotaped and subsequently analyzed to determine the number of ingestive and aversive responses. Intraoral infusions of 0.15 M and 0.3 M NaCl elicited reliably more ingestive responses and 1.0 M NaCl more aversive responses in females than in males. Because differences in taste reactivity were not found for all those concentrations for which female rats showed a higher preference than did males, changes in gustatory sensitivity contributes to, but does not appear to fully account for the female rats' preference for NaCl.