Endogenous central amygdala mu-opioid receptor signaling promotes sodium appetite in mice.

Due to the importance of dietary sodium and its paucity within many inland environments, terrestrial animals have evolved an instinctive sodium appetite that is commensurate with sodium deficiency. Despite a well-established role for central opioid signaling in sodium appetite, the endogenous influence of specific opioid receptor subtypes within distinct brain regions remains to be elucidated. Using selective pharmacological antagonists of opioid receptor subtypes, we reveal that endogenous mu-opioid receptor (MOR) signaling strongly drives sodium appetite in sodium-depleted mice, whereas a role for kappa (KOR) and delta (DOR) opioid receptor signaling was not detected, at least in sodium-depleted mice. Fos immunohistochemistry revealed discrete regions of the mouse brain displaying an increased number of activated neurons during sodium gratification: the rostral portion of the nucleus of the solitary tract (rNTS), the lateral parabrachial nucleus (LPB), and the central amygdala (CeA). The CeA was subsequently targeted with bilateral infusions of the MOR antagonist naloxonazine, which significantly reduced sodium appetite in mice. The CeA is therefore identified as a key node in the circuit that contributes to sodium appetite. Moreover, endogenous opioids, acting via MOR, within the CeA promote this form of appetitive behavior.

Involvement of central relaxin-3 signalling in sodium (salt) appetite.

NEW FINDINGS: What is the central question of this study? Sodium appetite is controlled by conserved neuronal transmitter-receptor systems. Here, we tested the contribution made by relaxin family peptide 3 receptor (RXFP3), the cognate G-protein-coupled receptor for the neuropeptide relaxin-3. What is the main finding and its importance? Intracerebroventricular infusion of an RXFP3 antagonist reduced in a dose-dependent manner the volume of 0.3 m NaCl consumed by sodium-depleted C57Bl/6J (wild-type) mice. This effect was absent in sodium-depleted Rxfp3 knockout mice, and RXFP3 antagonist infusion did not alter water consumption in wild-type mice subjected to multiple thirst tests, indicating both the pharmacological and the physiological specificity of observed effects. Our findings identify endogenous relaxin-3-RXFP3 signalling as a modulator of sodium appetite. Overconsumption of highly salted foods is common in Western diets and contributes significantly to metabolic disorders such as hypertension, renal dysfunction and diabetes. Sodium appetite, or the desire of terrestrial animals to seek and consume sodium-containing salts, is a behaviour mediated by a set of evolutionarily conserved neuronal systems. In these studies, we tested whether this instinctive behavioural drive is influenced by the G-protein-coupled relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the neuropeptide relaxin-3, because relaxin-3-RXFP3 signalling can modulate arousal, motivation and ingestive behaviours. Intracerebroventricular (i.c.v.) infusion of the selective RXFP3 antagonist, R3(B1-22)R, reduced in a dose-dependent manner the volume of 0.3 m NaCl solution consumed when offered to sodium-depleted C57Bl/6J wild-type mice, relative to vehicle-treated control animals. Notably, i.c.v. R3(B1-22)R infusion did not alter 0.3 m NaCl consumption relative to vehicle in sodium-depleted Rxfp3 knockout mice, confirming the pharmacological specificity of this effect. Furthermore, i.c.v. R3(B1-22)R did not alter the volume of water consumed by wild-type mice in three tests where water drinking was the normal physiological response, suggesting that the ability of R3(B1-22)R to reduce activated salt appetite is specific and not due to a generalized reduction in drinking behaviour. These findings identify, for the first time, that endogenous relaxin-3-RXFP3 signalling is a powerful mediator of salt appetite in mice and further elucidate the functional role of the relaxin-3-RXFP3 system in the integrative control of motivated behaviours.

Relation of addiction genes to hypothalamic gene changes subserving genesis and gratification of a classic instinct, sodium appetite.

Sodium appetite is an instinct that involves avid specific intention. It is elicited by sodium deficiency, stress-evoked adrenocorticotropic hormone (ACTH), and reproduction. Genome-wide microarrays in sodium-deficient mice or after ACTH infusion showed up-regulation of hypothalamic genes, including dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa (DARPP-32), dopamine receptors-1 and -2, α-2C- adrenoceptor, and striatally enriched protein tyrosine phosphatase (STEP). Both DARPP-32 and neural plasticity regulator activity-regulated cytoskeleton associated protein (ARC) were up-regulated in lateral hypothalamic orexinergic neurons by sodium deficiency. Administration of dopamine D1 (SCH23390) and D2 receptor (raclopride) antagonists reduced gratification of sodium appetite triggered by sodium deficiency. SCH23390 was specific, having no effect on osmotic-induced water drinking, whereas raclopride also reduced water intake. D1 receptor KO mice had normal sodium appetite, indicating compensatory regulation. Appetite was insensitive to SCH23390, confirming the absence of off-target effects. Bilateral microinjection of SCH23390 (100 nM in 200 nL) into rats' lateral hypothalamus greatly reduced sodium appetite. Gene set enrichment analysis in hypothalami of mice with sodium appetite showed significant enrichment of gene sets previously linked to addiction (opiates and cocaine). This finding of concerted gene regulation was attenuated on gratification with perplexingly rapid kinetics of only 10 min, anteceding significant absorption of salt from the gut. Salt appetite and hedonic liking of salt taste have evolved over >100 million y (e.g., being present in Metatheria). Drugs causing pleasure and addiction are comparatively recent and likely reflect usurping of evolutionary ancient systems with high survival value by the gratification of contemporary hedonic indulgences. Our findings outline a molecular logic for instinctive behavior encoded by the brain with possible important translational-medical implications.

Mice lacking angiotensin-converting enzyme have increased energy expenditure, with reduced fat mass and improved glucose clearance.

In addition to its role in the storage of fat, adipose tissue acts as an endocrine organ, and it contains a functional renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) plays a key role in the RAS by converting angiotensin I to the bioactive peptide angiotensin II (Ang II). In the present study, the effect of targeting the RAS in body energy homeostasis and glucose tolerance was determined in homozygous mice in which the gene for ACE had been deleted (ACE(-/-)) and compared with wild-type littermates. Compared with wild-type littermates, ACE(-/-) mice had lower body weight and a lower proportion of body fat, especially in the abdomen. ACE(-/-) mice had greater fed-state total energy expenditure (TEE) and resting energy expenditure (REE) than wild-type littermates. There were pronounced increases in gene expression of enzymes related to lipolysis and fatty acid oxidation (lipoprotein lipase, carnitine palmitoyl transferase, long-chain acetyl CoA dehydrogenase) in the liver of ACE(-/-) mice and also lower plasma leptin. In contrast, no differences were detected in daily food intake, activity, fed-state plasma lipids, or proportion of fat excreted in fecal matter. In conclusion, the reduction in ACE activity is associated with a decreased accumulation of body fat, especially in abdominal fat depots. The decreased body fat in ACE(-/-) mice is independent of food intake and appears to be due to a high energy expenditure related to increased metabolism of fatty acids in the liver, with the additional effect of increased glucose tolerance.

Changes in angiotensin type 1 receptor binding and angiotensin-induced pressor responses in the rostral ventrolateral medulla of angiotensinogen knockout mice.

ANG II, the main circulating effector hormone of the renin-angiotensin system, is produced by enzymatic cleavage of angiotensinogen. The present study aimed to examine whether targeted deletion of the angiotensinogen gene (Agt) altered brain ANG II receptor density or responsiveness to ANG II. In vitro autoradiography was used to examine the distribution and density of angiotensin type 1 (AT(1)) and type 2 receptors. In most brain regions, the distribution and density of angiotensin receptors were similar in brains of Agt knockout mice (Agt(-/-)) and wild-type mice. In Agt(-/-) mice, a small increase in AT(1) receptor binding was observed in the rostral ventrolateral medulla (RVLM), a region that plays a critical role in blood pressure regulation. To examine whether Agt(-/-) mice showed altered responses to ANG II, blood pressure responses to intravenous injection (0.01-0.1 microg/kg) or RVLM microinjection (50 pmol in 50 nl) of ANG II were recorded in anesthetized Agt(-/-) and wild-type mice. Intravenous injections of phenylephrine (4 microg/kg and 2 microg/kg) were also made in both groups. The magnitude of the pressor response to intravenous injections of ANG II or phenylephrine was not different between Agt(-/-) and wild-type mice. Microinjection of ANG II into the RVLM induced a pressor response, which was significantly smaller in Agt(-/-) compared with wild-type mice (+10 + or - 1 vs. +23 + or - 4 mmHg, respectively, P = 0.004). Microinjection of glutamate into the RVLM (100 pmol in 10 nl) produced a robust pressor response, which was not different between Agt(-/-) and wild-type mice. A diminished response to ANG II microinjection in the RVLM of Agt(-/-) mice, despite an increased density of AT(1) receptors suggests that signal transduction pathways may be altered in RVLM neurons of Agt(-/-) mice, resulting in attenuated cellular excitation.

Change in salt intake affects blood pressure of chimpanzees: implications for human populations.

BACKGROUND: Addition of up to 15.0 g/d salt to the diet of chimpanzees caused large rises in blood pressure, which reversed when the added salt was removed. Effects of more modest alterations to sodium intakes in chimpanzees, akin to current efforts to lower sodium intakes in the human population, are unknown. METHODS AND RESULTS: Sodium intakes were altered among 17 chimpanzees in Franceville, Gabon, and 110 chimpanzees in Bastrop, Tex. In Gabon, chimpanzees had a biscuit diet of constant nutrient composition except that the sodium content was changed episodically over 3 years from 75 to 35 to 120 mmol/d. In Bastrop, animals were divided into 2 groups; 1 group continued on the standard diet of 250 mmol/d sodium for 2 years, and sodium intake was halved for the other group. Lower sodium intake was associated with lower systolic, diastolic, and mean arterial blood pressures in Gabon (2-tailed P<0.001, unadjusted and adjusted for age, sex, and baseline weight) and Bastrop (P<0.01, unadjusted; P=0.08 to 0.10, adjusted), with no threshold down to 35 mmol/d sodium. For systolic pressure, estimates were -12.7 mm Hg (95% confidence interval, -16.9 to -8.5, adjusted) per 100 mmol/d lower sodium in Gabon and -10.9 mm Hg (95% confidence interval, -18.9 to -2.9, unadjusted) and -5.7 mm Hg (95% confidence interval, -12.2 to 0.7, adjusted) for sodium intake lower by 122 mmol/d in Bastrop. Baseline systolic pressures higher by 10 mm Hg were associated with larger falls in systolic pressure by 4.3/2.9 mm Hg in Gabon/Bastrop per 100 mmol/d lower sodium. CONCLUSIONS: These findings from an essentially single-variable experiment in the species closest to Homo sapiens with high intakes of calcium and potassium support intensified public health efforts to lower sodium intake in the human population.

Angiotensinogen and angiotensin-converting enzyme gene copy number and angiotensin and bradykinin peptide levels in mice.

OBJECTIVE: To test the hypothesis that changes in gene expression that may accompany angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) gene polymorphism cause alteration in angiotensin and bradykinin peptide levels. DESIGN: Mice with one or two genes for AGT and ACE allow assessment of the effects of modest alteration in AGT and ACE gene expression on angiotensin and bradykinin peptide levels. METHODS: Angiotensin and bradykinin peptides were measured in the blood, kidney, heart, lung, adrenal, brain, and aorta of mice that were either wild-type (+/+), heterozygous (+/-) or null (-/-) for either the AGT or ACE gene. RESULTS: Angiotensin I and angiotensin II were not detectable in blood or tissues of AGT -/- mice, which had increased bradykinin levels in kidney and lung. ACE -/- mice had markedly reduced angiotensin II levels and increased bradykinin levels in blood and tissues. However, despite reduced AGT and ACE gene expression, angiotensin and bradykinin peptide levels in AGT and ACE +/- mice were no different from the levels in wild-type mice. CONCLUSION: Although the AGT and ACE genes are fundamental determinants of angiotensin and bradykinin peptide levels, compensatory mechanisms attenuate the effect of modest change in AGT and ACE gene expression on the levels of these peptides. Identification of these compensatory mechanisms may provide new candidate genes for investigation in humans.

The relaxin gene-knockout mouse: a model of progressive fibrosis.

Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene-knockout (RLX-KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX-KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild-type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX-KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX-KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age-related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX-KO mice and was associated with organ dysfunction. From 6-9 months of age and onwards, all organs of RLX-KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P < .05) compared with wild-type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX-KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.

Angiotensin-converting enzyme (ACE) interacts with dopaminergic mechanisms in the brain to modulate prepulse inhibition in mice.

A renin-angiotensin system, separate to that in the periphery, has been found in the brain. Angiotensin-converting enzyme (ACE) is crucial in the synthesis of angiotensin II, breakdown of bradykinin and the hydrolysis of several other neuropeptides such as enkephalin, substance P, dynorphin and neurotensin. Changes in the levels of ACE have been found in brains of schizophrenia patients, suggesting an involvement of ACE in the illness which awaits further investigation. Prepulse inhibition (PPI) has been suggested to be an operational measure of sensorimotor gating and is disrupted in patients with schizophrenia. We found that ACE knockout mice have increased startle responses but no differences in baseline PPI compared to wildtype controls. Treatment with the dopamine receptor agonist, apomorphine, or the dopamine-releasing drug, amphetamine, produced significant disruption of PPI in control mice but not in ACE knockout mice. Pretreatment with the ACE inhibitor, captopril, which itself did not affect PPI, caused a reduction in the effect of apomorphine on PPI, similar to that seen in the ACE knockout mice. These data suggest an important role of ACE substrates in modulating dopaminergic mechanisms involved in PPI. Further studies are needed to ascertain if angiotensin or other neuropeptides are involved in these interactions and to investigate the neurochemical mechanism behind these effects.

Osmoregulatory fluid intake but not hypovolemic thirst is intact in mice lacking angiotensin.

Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.