Modulatory effects of estrous cycle on ingestive behaviors and energy balance in young adult C57BL/6J mice maintained on a phytoestrogen-free diet.

The estrous cycle is known to modify food, fluid, and electrolyte intake behaviors and energy homeostasis in various species, in part through fluctuations in estrogen levels. Simultaneously, commonly commercially available rodent dietary formulations greatly vary in soy protein content, and thereby the delivery of biologically active phytoestrogens. To explore the interactions among the estrous cycle, sodium, fluid, and caloric seeking behaviors, and energy homeostasis, young adult C57BL/6J female mice were maintained on a soy protein-free 2920x diet and provided water, or a choice between water and 0.15 mol/L NaCl drink solution. Comprehensive metabolic phenotyping was performed using a multiplexed Promethion (Sable Systems International) system, and estrous stages were determined via daily vaginal cytology. When provided food and water, estrous cycling had no major modulatory effects on intake behaviors or energy balance. When provided a saline solution drink choice, significant modulatory effects of the transition from diestrus to proestrus were observed upon fluid intake patterning, locomotion, and total energy expenditure. Access to saline increased total daily sodium consumption and aspects of energy expenditure, but these effects were not modified by the estrous stage. Collectively, these results indicate that when supplied a phytoestrogen-free diet, the estrous cycle has minor modulatory effects on ingestive behaviors and energy balance in C57BL/6J mice that are sensitive to sodium supply.NEW & NOTEWORTHY When provided a phytoestrogen-free diet, the estrous cycle had very little effect on food and water intake, physical activity, or energy expenditure in C57BL/6J mice. In contrast, when provided an NaCl drink in addition to food and water, the estrous cycle was associated with changes in intake behaviors and energy expenditure. These findings highlight the complex interactions among estrous cycling, dietary formulation, and nutrient presentation upon ingestive behaviors and energy homeostasis in mice.

Early-life sodium deprivation programs long-term changes in ingestive behaviors and energy expenditure in C57BL/6J mice.

Postnatal growth failure remains a significant problem for infants born prematurely, despite aggressive efforts to improve perinatal nutrition. Though often dysregulated in early life when children are born preterm, sodium (Na) homeostasis is vital to achieve optimal growth. We hypothesize that insufficient Na supply in this critical period contributes to growth restriction and programmed risks for cardiometabolic disease in later adulthood. Thus, we sought to ascertain the effects of prolonged versus early-life Na depletion on weight gain, body composition, food and water intake behaviors, and energy expenditure in C57BL/6J mice. In one study, mice were provided a low (0.04%)- or normal/high (0.30%)-Na diet between 3 and 18 wk of age. Na-restricted mice demonstrated delayed growth and elevated basal metabolic rate. In a second study, mice were provided 0.04% or 0.30% Na diet between 3 and 6 wk of age and then returned to standard (0.15%)-Na diet through the end of the study. Na-restricted mice exhibited growth delays that quickly caught up on return to standard diet. Between 6 and 18 wk of age, previously restricted mice exhibited sustained, programmed changes in feeding behaviors, reductions in total food intake, and increases in water intake and aerobic energy expenditure while maintaining normal body composition. Although having no effect in control mice, administration of the ganglionic blocker hexamethonium abolished the programmed increase in basal metabolic rate in previously restricted mice. Together these data indicate that early-life Na restriction can cause programmed changes in ingestive behaviors, autonomic function, and energy expenditure that persist well into adulthood.

Role of the Peroxisome Proliferator Activated Receptors in Hypertension.

Nuclear receptors represent a large family of ligand-activated transcription factors which sense the physiological environment and make long-term adaptations by mediating changes in gene expression. In this review, we will first discuss the fundamental mechanisms by which nuclear receptors mediate their transcriptional responses. We will focus on the PPAR (peroxisome proliferator-activated receptor) family of adopted orphan receptors paying special attention to PPARγ, the isoform with the most compelling evidence as an important regulator of arterial blood pressure. We will review genetic data showing that rare mutations in PPARγ cause severe hypertension and clinical trial data which show that PPARγ activators have beneficial effects on blood pressure. We will detail the tissue- and cell-specific molecular mechanisms by which PPARs in the brain, kidney, vasculature, and immune system modulate blood pressure and related phenotypes, such as endothelial function. Finally, we will discuss the role of placental PPARs in preeclampsia, a life threatening form of hypertension during pregnancy. We will close with a viewpoint on future research directions and implications for developing novel therapies.

Melanocortin MC4R receptor is required for energy expenditure but not blood pressure effects of angiotensin II within the mouse brain.

The brain renin-angiotensin system (RAS) is implicated in control of blood pressure (BP), fluid intake, and energy expenditure (EE). Angiotensin II (ANG II) within the arcuate nucleus of the hypothalamus contributes to control of resting metabolic rate (RMR) and thereby EE through its actions on Agouti-related peptide (AgRP) neurons, which also contribute to EE control by leptin. First, we determined that although leptin stimulates EE in control littermates, mice with transgenic activation of the brain RAS (sRA) exhibit increased EE and leptin has no additive effect to exaggerate EE in these mice. These findings led us to hypothesize that leptin and ANG II in the brain stimulate EE through a shared mechanism. Because AgRP signaling to the melanocortin MC4R receptor contributes to the metabolic effects of leptin, we performed a series of studies examining RMR, fluid intake, and BP responses to ANG II in mice rendered deficient for expression of MC4R via a transcriptional block (Mc4r-TB). These mice were resistant to stimulation of RMR in response to activation of the endogenous brain RAS via chronic deoxycorticosterone acetate (DOCA)-salt treatment, whereas fluid and electrolyte effects remained intact. These mice were also resistant to stimulation of RMR via acute intracerebroventricular (ICV) injection of ANG II, whereas BP responses to ICV ANG II remained intact. Collectively, these data demonstrate that the effects of ANG II within the brain to control RMR and EE are dependent on MC4R signaling, whereas fluid homeostasis and BP responses are independent of MC4R signaling.

Cardiometabolic effects of DOCA-salt in male C57BL/6J mice are variably dependent on sodium and nonsodium components of diet.

Hypertension characterized by low circulating renin activity accounts for roughly 25%-30% of primary hypertension in humans and can be modeled experimentally via deoxycorticosterone acetate (DOCA)-salt treatment. In this model, phenotypes develop in progressive phases, although the timelines and relative contributions of various mechanisms to phenotype development can be distinct between laboratories. To explore interactions among environmental influences such as diet formulation and dietary sodium (Na) content on phenotype development in the DOCA-salt paradigm, we examined an array of cardiometabolic endpoints in young adult male C57BL/6J mice during sham or DOCA-salt treatments when mice were maintained on several common, commercially available laboratory rodent "chow" diets including PicoLab 5L0D (0.39% Na), Envigo 7913 (0.31% Na), Envigo 2920x (0.15% Na), or a customized version of Envigo 2920x (0.4% Na). Energy balance (weight gain, food intake, digestive efficiency, and energy efficiency), fluid and electrolyte homeostasis (fluid intake, Na intake, fecal Na content, hydration, and fluid compartmentalization), renal functions (urine production rate, glomerular filtration rate, urine Na excretion, renal expression of renin, vasopressin receptors, aquaporin-2 and relationships among markers of vasopressin release, aquaporin-2 shedding, and urine osmolality), and blood pressure, all exhibited changes that were subject to interactions between diet and DOCA-salt. Interestingly, some of these phenotypes, including blood pressure and hydration, were dependent on nonsodium dietary components, as Na-matched diets resulted in distinct phenotype development. These findings provide a broad and robust illustration of an environment × treatment interaction that impacts the use and interpretation of a common rodent model of low-renin hypertension.

Chronic intracerebroventricular infusion of angiotensin II causes dose- and sex-dependent effects on intake behaviors and energy homeostasis in C57BL/6J mice.

The renin-angiotensin system (RAS) within the brain is implicated in the control of fluid and electrolyte balance, autonomic functions, blood pressure, and energy expenditure. Mouse models are increasingly used to explore these mechanisms; however, sex and dose dependencies of effects elicited by chronic intracerebroventricular (ICV) angiotensin II (ANG II) infusion have not been carefully established in this species. To examine the interactions among sex, body mass, and ICV ANG II on ingestive behaviors and energy balance, young adult C57BL/6J mice of both sexes were studied in a multiplexed metabolic phenotyping system (Promethion) during chronic infusion of ANG II (0, 5, 20, or 50 ng/h). At these infusion rates, ANG II caused accelerating dose-dependent increases in drinking and total energy expenditure in male mice, but female mice exhibited a complex biphasic response with maximum responses at 5 ng/h. Body mass differences did not account for sex-dependent differences in drinking behavior or total energy expenditure. In contrast, resting metabolic rate was similarly increased by ICV ANG II in a dose-dependent manner in both sexes after correction for body mass. We conclude that chronic ICV ANG II stimulates water intake, resting, and total energy expenditure in male C57BL/6J mice following straightforward accelerating dose-dependent kinetics, but female C57BL/6J mice exhibit complex biphasic responses to ICV ANG II. Furthermore, control of resting metabolic rate by ANG II is dissociable from mechanisms controlling fluid intake and total energy expenditure. Future studies of the sex dependency of ANG II within the brain of mice must be designed to carefully consider the biphasic responses that occur in females.

The Renin-Angiotensin System in the Central Nervous System and Its Role in Blood Pressure Regulation.

PURPOSE OF THE REVIEW: The main goal of this article is to discuss how the development of state-of-the-art technology has made it possible to address fundamental questions related to how the renin-angiotensin system (RAS) operates within the brain from the neurophysiological and molecular perspective. RECENT FINDINGS: The existence of the brain RAS remains surprisingly controversial. New sensitive in situ hybridization techniques and novel transgenic animals expressing reporter genes have provided pivotal information of the expression of RAS genes within the brain. We discuss studies using genetically engineered animals combined with targeted viral microinjections to study molecular mechanisms implicated in the regulation of the brain RAS. We also discuss novel drugs targeting the brain RAS that have shown promising results in clinical studies and trials. Over the last 50 years, several new physiological roles of the brain RAS have been identified. In the coming years, efforts to incorporate cutting-edge technologies such as optogenetics, chemogenetics, and single-cell RNA sequencing will lead to dramatic advances in our full understanding of how the brain RAS operates at molecular and neurophysiological levels.

Renal release of N-acetyl-seryl-aspartyl-lysyl-proline is part of an antifibrotic peptidergic system in the kidney.

The antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is released from thymosin-ß4 (Tß4) by the meprin-α and prolyl oligopeptidase (POP) enzymes and is hydrolyzed by angiotensin-converting enzyme (ACE). Ac-SDKP is present in urine; however, it is not clear whether de novo tubular release occurs or if glomerular filtration is the main source. We hypothesized that Ac-SDKP is released into the lumen of the nephrons and that it exerts an antifibrotic effect. We determined the presence of Tß4, meprin-α, and POP in the kidneys of Sprague-Dawley rats. The stop-flow technique was used to evaluate Ac-SDKP formation in different nephron segments. Finally, we decreased Ac-SDKP formation by inhibiting the POP enzyme and evaluated the long-term effect in renal fibrosis. The Tß4 precursor and the releasing enzymes meprin-α and POP were expressed in the kidneys. POP enzyme activity was almost double that in the renal medulla compared with the renal cortex. With the use of the stop-flow technique, we detected the highest Ac-SDKP concentrations in the distal nephron. The infusion of a POP inhibitor into the kidney decreased the amount of Ac-SDKP in distal nephron segments and in the proximal nephron to a minor extent. An ACE inhibitor increased the Ac-SDKP content in all nephron segments, but the increase was highest in the distal portion. The chronic infusion of a POP inhibitor increased kidney medullary fibrosis, which was prevented by Ac-SDKP. We conclude that Ac-SDKP is released by the nephron and is part of an important antifibrotic system in the kidney.

The anti-inflammatory peptide Ac-SDKP is released from thymosin-ß4 by renal meprin-α and prolyl oligopeptidase.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-ß4 (Tß4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and Tß4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, Tß4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-α metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, Tß4 is hydrolyzed by meprin-α. In vitro, we found that the incubation of Tß4 with both meprin-α and POP released Ac-SDKP, whereas no Ac-SDKP was released when Tß4 was incubated with either meprin-α or POP alone. Incubation of Tß4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-α inhibitor actinonin. In addition, kidneys from meprin-α knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-α KO mice failed to release Ac-SDKP from Tß4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 ± 0.2 nmol/l; captopril, 15.1 ± 0.7 nmol/l; captopril + actinonin, 6.1 ± 0.3 nmol/l; P < 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from Tß4 is mediated by successive hydrolysis involving meprin-α and POP.

Ac-SDKP suppresses TNF-α-induced ICAM-1 expression in endothelial cells via inhibition of IκB kinase and NF-κB activation.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that, in angiotensin II-induced hypertension, Ac-SDKP decreased the activation of nuclear transcription factor NF-κB, whereas, in experimental autoimmune myocarditis and hypertension animal models, it also reduced the expression of endothelial leukocyte adhesion molecule ICAM-1. However, the mechanisms by which Ac-SDKP downregulated ICAM-1 expression are still unclear. TNF-α is a proinflammatory cytokine that induces ICAM-1 expression in various cell types via TNF receptor 1 and activation of the classical NF-κB pathway. We hypothesized that in endothelial cells Ac-SDKP suppresses TNF-α-induced ICAM-1 expression by decreasing IKK phosphorylation that as a consequence leads to a decrease of IκB phosphorylation and NF-κB activation. To test this hypothesis, human coronary artery endothelial cells were treated with Ac-SDKP and then stimulated with TNF-α. We found that TNF-α-induced ICAM-1 expression was significantly decreased by Ac-SDKP in a dose-dependent manner. Ac-SDKP also decreased TNF-α-induced NF-κB translocation from cytosol to nucleus, as assessed by electrophoretic mobility shift assay, which correlated with a decrease in IκB phosphorylation. In addition, we found that Ac-SDKP decreased TNF-α-induced IKK phosphorylation and IKK-ß expression. However, Ac-SDKP had no effect on TNF-α-induced phosphorylation of p38 MAP kinase or ERK. Thus we conclude that Ac-SDKP inhibition of TNF-α activation of canonical, i.e., IKK-ß-dependent, NF-κB pathway and subsequent decrease in ICAM-1 expression is achieved via inhibition of IKK-ß.

Cardiac-deleterious role of galectin-3 in chronic angiotensin II-induced hypertension.

Galectin-3 (Gal-3), a member of the ß-galactoside lectin family, has an important role in immune regulation. In hypertensive rats and heart failure patients, Gal-3 is considered a marker for an unfavorable prognosis. Nevertheless, the role and mechanism of Gal-3 action in hypertension-induced target organ damage are unknown. We hypothesized that, in angiotensin II (ANG II)-induced hypertension, genetic deletion of Gal-3 prevents left ventricular (LV) adverse remodeling and LV dysfunction by reducing the innate immune responses and myocardial fibrosis. To induce hypertension, male C57BL/6J and Gal-3 knockout (KO) mice were infused with ANG II (3 µg·min-1·kg-1 sc) for 8 wk. We assessed: 1) systolic blood pressure by plethysmography, 2) LV function and remodeling by echocardiography, 3) myocardial fibrosis by histology, 4) cardiac CD68+ macrophage infiltration by histology, 5) ICAM-1 and VCAM-1 expression by Western blotting, 6) plasma cytokines, including interleukin-6 (IL-6), by enzyme-linked immunosorbent assay, and 7) regulatory T (Treg) cells by flow cytometry as detected by their combined expression of CD4, CD25, and FOXP3. Systolic blood pressure and cardiac hypertrophy increased similarly in both mouse strains when infused with ANG II. However, hypertensive C57BL/6J mice suffered impaired ejection and shortening fractions. In these mice, the extent of myocardial fibrosis and macrophage infiltration was greater in histological sections, and cardiac ICAM-1, as well as plasma IL-6, expression was higher as assessed by Western blotting. However, all these parameters were blunted in Gal-3 KO mice. Hypertensive Gal-3 KO mice also had a higher number of splenic Treg lymphocytes. In conclusion, in ANG II-induced hypertension, genetic deletion of Gal-3 prevented LV dysfunction without affecting blood pressure or LV hypertrophy. This study indicates that the ANG II effects are, in part, mediated or triggered by Gal-3 together with the related intercellular signaling (ICAM-1 and IL-6), leading to cardiac inflammation and fibrosis.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline: mechanisms of renal protection in mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease characterized by the development of auto antibodies against a variety of self-antigens and deposition of immune complexes that lead to inflammation, fibrosis, and end-organ damage. Up to 60% of lupus patients develop nephritis and renal dysfunction leading to kidney failure. N-acetyl-seryl-aspartyl-lysyl-proline, i.e., Ac-SDKP, is a natural tetrapeptide that in hypertension prevents inflammation and fibrosis in heart, kidney, and vasculature. In experimental autoimmune myocarditis, Ac-SDKP prevents cardiac dysfunction by decreasing innate and adaptive immunity. It has also been reported that Ac-SDKP ameliorates lupus nephritis in mice. We hypothesize that Ac-SDKP prevents lupus nephritis in mice by decreasing complement C5-9, proinflammatory cytokines, and immune cell infiltration. Lupus mice treated with Ac-SDKP for 20 wk had significantly lower renal levels of macrophage and T cell infiltration and proinflammatory chemokine/cytokines. In addition, our data demonstrate for the first time that in lupus mouse Ac-SDKP prevented the increase in complement C5-9, RANTES, MCP-5, and ICAM-1 kidney expression and it prevented the decline of glomerular filtration rate. Ac-SDKP-treated lupus mice had a significant improvement in renal function and lower levels of glomerular damage. Ac-SDKP had no effect on the production of autoantibodies. The protective Ac-SDKP effect is most likely achieved by targeting the expression of proinflammatory chemokines/cytokines, ICAM-1, and immune cell infiltration in the kidney, either directly or via C5-9 proinflammatory arm of complement system.

N-acetyl-seryl-aspartyl-lysyl-proline reduces cardiac collagen cross-linking and inflammation in angiotensin II-induced hypertensive rats.

We have reported previously that Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) reduces fibrosis and inflammation (in macrophages and mast cells). However, it is not known whether Ac-SDKP decreases collagen cross-linking and lymphocyte infiltration; lymphocytes modulate both collagen cross-linking and ECM (extracellular matrix) formation in hypertension. Thus we hypothesized that (i) in AngII (angiotensin II)-induced hypertension, Ac-SDKP prevents increases in cross-linked and total collagen by down-regulating LOX (lysyl oxidase), the enzyme responsible for cross-linking, and (ii) these effects are associated with decreased pro-fibrotic cytokine TGFß (transforming growth factor ß) and the pro-inflammatory transcription factor NF-κB (nuclear factor κB) and CD4+/CD8+ lymphocyte infiltration. We induced hypertension in rats by infusing AngII either alone or combined with Ac-SDKP for 3 weeks. Whereas Ac-SDKP failed to lower BP (blood pressure) or LV (left ventricular) hypertrophy, it did prevent AngII-induced increases in (i) cross-linked and total collagen, (ii) LOX mRNA expression and LOXL1 (LOX-like 1) protein, (iii) TGFß expression, (iv) nuclear translocation of NF-κB, (v) CD4+/CD8+ lymphocyte infiltration, and (vi) CD68+ macrophages infiltration. In addition, we found a positive correlation between CD4+ infiltration and LOXL1 expression. In conclusion, the effect of Ac-SDKP on collagen cross-linking and total collagen may be due to reduced TGFß1, LOXL1, and lymphocyte and macrophage infiltration, and its effect on inflammation could be due to lower NF-κB.

Insights Into the Role of Angiotensin-II AT1 Receptor-Dependent ß-Arrestin Signaling in Cardiovascular Disease.

ß-arrestins are a family of intracellular signaling proteins that play a key role in regulating the activity of G protein-coupled receptors. The angiotensin-II type 1 receptor is an important G protein-coupled receptor involved in the regulation of cardiovascular function and has been implicated in the progression of cardiovascular diseases. In addition to canonical G protein signaling, G protein-coupled receptors including the angiotensin-II type 1 receptor can signal via ß-arrestin. Dysregulation of ß-arrestin signaling has been linked to several cardiovascular diseases including hypertension, atherosclerosis, and heart failure. Understanding the role of ß-arrestins in these conditions is critical to provide new therapeutic targets for the treatment of cardiovascular disease. In this review, we will discuss the beneficial and maladaptive physiological outcomes of angiotensin-II type 1 receptor-dependent ß-arrestin activation in different cardiovascular diseases.

Genetic Deletion of ß-Arrestin 2 From the Subfornical Organ and Other Periventricular Nuclei in the Brain Alters Fluid Homeostasis and Blood Pressure.

BACKGROUND: ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the AT1R [angiotensin type 1 receptor])-mediated AT1R in the subfornical organ. Recently, we demonstrated that ARRB2 (ß-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical AT1R signaling. METHODS: Male and female Arrb2FLOX mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (Arrb2ICV-Cre). Arrb2FLOX mice receiving ICV-AAV-GFP were used as control (Arrb2ICV-Control). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl. RESULTS: Arrb2ICV-Cre mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, Arrb2ICV-Cre mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, Arrb2ICV-Cre mice exhibited elevated saline intake. CONCLUSIONS: Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical AT1R responses to both exogenous and endogenous ANG. Stimulation of the AT1R/ARRB axis in the brain may represent a novel strategy to treat hypertension.

Cell-specific transcriptome changes in the hypothalamic arcuate nucleus in a mouse deoxycorticosterone acetate-salt model of hypertension.

A common preclinical model of hypertension characterized by low circulating renin is the "deoxycorticosterone acetate (DOCA)-salt" model, which influences blood pressure and metabolism through mechanisms involving the angiotensin II type 1 receptor (AT1R) in the brain. More specifically, AT1R within Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) has been implicated in selected effects of DOCA-salt. In addition, microglia have been implicated in the cerebrovascular effects of DOCA-salt and angiotensin II. To characterize DOCA-salt effects upon the transcriptomes of individual cell types within the ARC, we used single-nucleus RNA sequencing (snRNAseq) to examine this region from male C57BL/6J mice that underwent sham or DOCA-salt treatment. Thirty-two unique primary cell type clusters were identified. Sub-clustering of neuropeptide-related clusters resulted in identification of three distinct AgRP subclusters. DOCA-salt treatment caused subtype-specific changes in gene expression patterns associated with AT1R and G protein signaling, neurotransmitter uptake, synapse functions, and hormone secretion. In addition, two primary cell type clusters were identified as resting versus activated microglia, and multiple distinct subtypes of activated microglia were suggested by sub-cluster analysis. While DOCA-salt had no overall effect on total microglial density within the ARC, DOCA-salt appeared to cause a redistribution of the relative abundance of activated microglia subtypes. These data provide novel insights into cell-specific molecular changes occurring within the ARC during DOCA-salt treatment, and prompt increased investigation of the physiological and pathophysiological significance of distinct subtypes of neuronal and glial cell types.

Genetic Ablation of Prorenin Receptor in the Rostral Ventrolateral Medulla Influences Blood Pressure and Hydromineral Balance in Deoxycorticosterone-Salt Hypertension.

Non-enzymatic activation of renin via its interaction with prorenin receptor (PRR) has been proposed as a key mechanism of local renin-angiotensin system (RAS) activation. The presence of renin and angiotensinogen has been reported in the rostral ventrolateral medulla (RVLM). Overactivation of bulbospinal neurons in the RVLM is linked to hypertension (HTN). Previous studies have shown that the brain RAS plays a role in the pathogenesis of the deoxycorticosterone (DOCA)-salt HTN model. Thus, we hypothesized that PRR in the RVLM is involved in the local activation of the RAS, facilitating the development of DOCA-salt HTN. Selective PRR ablation targeting the RVLM (PRRRVLM-Null mice) resulted in an unexpected sex-dependent and biphasic phenotype in DOCA-salt HTN. That is, PRRRVLM-Null females (but not males) exhibited a significant delay in achieving maximal pressor responses during the initial stage of DOCA-salt HTN. Female PRRRVLM-Null subsequently showed exacerbated DOCA-salt-induced pressor responses during the "maintenance" phase with a maximal peak at 13 d on DOCA-salt. This exacerbated response was associated with an increased sympathetic drive to the resistance arterioles and the kidney, exacerbated fluid and sodium intake and output in response to DOCA-salt, and induced mobilization of fluids from the intracellular to extracellular space concomitant with elevated vasopressin. Ablation of PRR suppressed genes involved in RAS activation and catecholamine synthesis in the RVLM but also induced expression of genes involved in inflammatory responses. This study illustrates complex and sex-dependent roles of PRR in the neural control of BP and hydromineral balance through autonomic and neuroendocrine systems. Graphical abstract.

Cardiometabolic Effects of DOCA-Salt in Mice Depend on Ambient Temperature.

BACKGROUND: Mice prefer warmer environments than humans. For this reason, behavioral and physiological thermoregulatory responses are engaged by mice in response to a standard room temperature of 22 to 24 °C. Autonomic mechanisms mediating thermoregulatory responses overlap with mechanisms activated in hypertension, and, therefore, we hypothesized that housing at thermoneutral temperatures (TNs; 30 °C) would modify the cardiometabolic effects of deoxycorticosterone acetate (DOCA)-salt in mice. METHODS: The effects of DOCA-salt treatment upon ingestive behaviors, energy expenditure, blood pressure, heart rate (HR), and core temperature were assessed in C57BL/6J mice housed at room temperature or TN. RESULTS: Housing at TN reduced food intake, energy expenditure, blood pressure, and HR and attenuated HR responses to acute autonomic blockade by chlorisondamine. At room temperature, DOCA-salt caused expected increases in fluid intake, sodium retention in osmotically inactive pools, blood pressure, core temperature, and also caused expected decreases in fat-free mass, total body water, and HR. At TN, the effects of DOCA-salt upon fluid intake, fat gains, hydration, and core temperature were exaggerated, but effects on energy expenditure and HR were blunted. Effects of DOCA-salt upon blood pressure were similar for 3 weeks and exaggerated by TN housing in the fourth week. CONCLUSIONS: Ambient temperature robustly influences behavioral and physiological functions in mice, including metabolic and cardiovascular phenotype development in response to DOCA-salt treatment. Studying cardiometabolic responses of mice at optimal ambient temperatures promises to improve the translational relevance of rodent models.

Angiotensin AT1A receptor signal switching in Agouti-related peptide neurons mediates metabolic rate adaptation during obesity.

Resting metabolic rate (RMR) adaptation occurs during obesity and is hypothesized to contribute to failed weight management. Angiotensin II (Ang-II) type 1 (AT1A) receptors in Agouti-related peptide (AgRP) neurons contribute to the integrative control of RMR, and deletion of AT1A from AgRP neurons causes RMR adaptation. Extracellular patch-clamp recordings identify distinct cellular responses of individual AgRP neurons from lean mice to Ang-II: no response, inhibition via AT1A and Gαi, or stimulation via Ang-II type 2 (AT2) receptors and Gαq. Following diet-induced obesity, a subset of Ang-II/AT1A-inhibited AgRP neurons undergo a spontaneous G-protein "signal switch," whereby AT1A stop inhibiting the cell via Gαi and instead begin stimulating the cell via Gαq. DREADD-mediated activation of Gαi, but not Gαq, in AT1A-expressing AgRP cells stimulates RMR in lean and obese mice. Thus, loss of AT1A-Gαi coupling within the AT1A-expressing AgRP neuron subtype represents a molecular mechanism contributing to RMR adaptation.