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.

Diet in Food Insecurity: A Mediator of Metabolic Health?

Objective: Food insecurity (FI) is associated with poor metabolic health. It is assumed that energy intake and diet quality underlie this association. We tested the hypothesis that dietary factors (quantity and quality) mediate the association of FI with excess weight, waist circumference and glycemic control [glycohemoglobin (A1C)]. Methods: A mediation analysis was performed on data from the National Health And Nutrition Examination Survey using FI as an independent variable; body mass index (BMI), waist circumference, and A1C as metabolic outcome variables and total energy intake, macronutrients, and diet quality measured by the Healthy Eating Index-2015 (HEI-2015) as potential mediators. Results: Despite a greater prevalence of obesity in participants experiencing FI, daily reported energy intake was similar in food-secure and -insecure subjects. In adjusted analyses of the overall cohort, none of the examined dietary factors mediated associations between FI and metabolic outcomes. In race-stratified analyses, total sugar consumption was a partial mediator of BMI in non-Hispanic Whites, while diet quality measures (HEI-2015 total score and added sugar subscore) were partial mediators of waist circumference and BMI, respectively, for those in the "other" ethnic group. Conclusion: Dietary factors are not the main factors underlying the association of FI with metabolic health. Future studies should investigate whether other social determinants of health commonly present in the context of FI play a role in this association.

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.

Differences in Fluid, Electrolyte, and Energy Balance in C57BL/6J Mice (Mus musculus) in Metabolic Caging at Thermoneutral or Standard Room Temperatures.

The Guide for the Care and Use of Laboratory Animals recommends mice be pair or group housed and provided with nesting materials. These provisions support social interactions and are also critical for thermoregulatory behaviors such as huddling and burrowing. However, studies of fluid and electrolyte balance and digestive function may involve use of metabolic caging (MC) systems in which mice are housed individually on wire-mesh floors that permit quantitative collection of urine and feces. MC housing prevents mice from performing their typical huddling and burrowing behaviors. Housing in MC can cause weight loss and behavioral changes in rodents. Here, we tested the hypothesis that MC housing of mice at standard room temperature (SRT, 22 to 23 °C) exposes them to cold stress, which causes metabolic changes in the mice as compared with standard housing. We hypothesized that performing MC studies at a thermoneutral temperature (TNT, 30 °C) would minimize these changes. Fluid, electrolyte, and energy balance and body composition were assessed in male and female C57BL/6J mice housed at SRT or TNT in MC, static microisolation cages, or a multiplexed metabolic phenotyping system designed to mimic static microisolation cages (Promethion, Sable Systems International). In brief, as compared with MC housing at SRT, MC housing at TNT was associated with lower food intake and energy expenditure, absence of weight loss, and lower urine and fecal corticosterone levels. These results indicate that housing in MC at SRT causes cold stress that can be mitigated if MC studies are performed at TNT.

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.

Krüppel-like factor 4 in transcriptional control of the three unique isoforms of Agouti-related peptide in mice.

Agouti-related peptide (AgRP/Agrp) within the hypothalamic arcuate nucleus (ARC) contributes to the control of energy balance, and dysregulated Agrp may contribute to metabolic adaptation during prolonged obesity. In mice, three isoforms of Agrp are encoded via distinct first exons. Agrp-A (ENSMUST00000005849.11) contributed 95% of total Agrp in mouse ARC, whereas Agrp-B (ENSMUST00000194654.2) dominated in placenta (73%). Conditional deletion of Klf4 from Agrp-expressing cells (Klf4Agrp-KO mice) reduced Agrp mRNA and increased energy expenditure but had no effects on food intake or the relative abundance of Agrp isoforms in the ARC. Chronic high-fat diet feeding masked these effects of Klf4 deletion, highlighting the context-dependent contribution of KLF4 to Agrp control. In the GT1-7 mouse hypothalamic cell culture model, which expresses all three isoforms of Agrp (including Agrp-C, ENSMUST00000194091.6), inhibition of extracellular signal-regulated kinase (ERK) simultaneously increased KLF4 binding to the Agrp promoter and stimulated Agrp expression. In addition, siRNA-mediated knockdown of Klf4 reduced expression of Agrp. We conclude that the expression of individual isoforms of Agrp in the mouse is dependent upon cell type and that KLF4 directly promotes the transcription of Agrp via a mechanism that is superseded during obesity.NEW & NOTEWORTHY In mice, three distinct isoforms of Agouti-related peptide are encoded via distinct first exons. In the arcuate nucleus of the hypothalamus, Krüppel-like factor 4 stimulates transcription of the dominant isoform in lean mice, but this mechanism is altered during diet-induced obesity.

Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

The Power of the Heterogeneous Stock Rat Founder Strains in Modeling Metabolic Disease.

Metabolic diseases are a host of complex conditions, including obesity, diabetes mellitus, and metabolic syndrome. Endocrine control systems (eg, adrenals, thyroid, gonads) are causally linked to metabolic health outcomes. N/NIH Heterogeneous Stock (HS) rats are a genetically heterogeneous outbred population developed for genetic studies of complex traits. Genetic mapping studies in adult HS rats identified loci associated with cardiometabolic risks, such as glucose intolerance, insulin resistance, and increased body mass index. This study determined underappreciated metabolic health traits and the associated endocrine glands within available substrains of the HS rat founders. We hypothesize that the genetic diversity of the HS rat founder strains causes a range of endocrine health conditions contributing to the diversity of cardiometabolic disease risks. ACI/EurMcwi, BN/NHsdMcwi, BUF/MnaMcwi, F344/StmMcwi, M520/NRrrcMcwi, and WKY/NCrl rats of both sexes were studied from birth until 13 weeks of age. Birth weight was recorded, body weight was measured weekly, metabolic characteristics were assessed, and blood and tissues were collected. Our data show wide variation in endocrine traits and metabolic health states in ACI, BN, BUF, F344, M520, and WKY rat strains. This is the first report to compare birth weight, resting metabolic rate, endocrine gland weight, hypothalamic-pituitary-thyroid axis hormones, and brown adipose tissue weight in these rat strains. Importantly, this work unveils new potential for the HS rat population to model early life adversity and adrenal and thyroid pathophysiology. The HS population likely inherited risk alleles for these strain-specific traits, making the HS rat a powerful model to investigate interventions on endocrine and metabolic health.

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.

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.

Sodium and Growth in Preterm Infants: A Review.

Aim: This article is intended to review the relationship between sodium homeostasis and growth, outline reasons why preterm infants may become sodium deficient, and share data from our group and others regarding the potential benefits of dietary sodium supplementation. Background: Despite tremendous efforts over the past 20 years to optimize neonatal nutrition, postnatal growth failure in preterm infants remains a significant problem. Compelling associations have been identified between in-hospital growth failure and cardiometabolic and neurodevelopmental disorders, heightening the need to further identify the optimal nutritional needs of preterm infants. Results: The impact of sodium deficiency may have on somatic growth is poorly studied and reported upon within the human literature. In contrast, animal studies dating back almost 100 years highlight the nutritional importance of dietary sodium. Sodium homeostasis during early postnatal life is understudied and underappreciated by neonatologists. Conclusion: Insufficient sodium intake during early life is likely a critical yet underappreciated contributor to growth failure. Total body sodium depletion may be an important risk factor driving complications of premature birth. Clinical significance: Increased awareness of sodium homeostasis in preterm infants may improve outcomes in this population. Sodium intake recommendations are provided based on the interpretation of currently available literature.

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.

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.

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 background in the rat affects endocrine and metabolic outcomes of bisphenol F exposure.

Environmental bisphenol compounds like bisphenol F (BPF) are endocrine-disrupting chemicals (EDCs) affecting adipose and classical endocrine systems. Genetic factors that influence EDC exposure outcomes are poorly understood and are unaccounted variables that may contribute to the large range of reported outcomes in the human population. We previously demonstrated that BPF exposure increased body growth and adiposity in male N/NIH heterogeneous stock (HS) rats, a genetically heterogeneous outbred population. We hypothesize that the founder strains of the HS rat exhibit EDC effects that were strain- and sex-dependent. Weanling littermate pairs of male and female ACI, BN, BUF, F344, M520, and WKY rats randomly received either vehicle (0.1% EtOH) or 1.125 mg BPF/l in 0.1% EtOH for 10 weeks in drinking water. Body weight and fluid intake were measured weekly, metabolic parameters were assessed, and blood and tissues were collected. BPF increased thyroid weight in ACI males, thymus and kidney weight in BUF females, adrenal weight in WKY males, and possibly increased pituitary weight in BN males. BUF females also developed a disruption in activity and metabolic rate with BPF exposure. These sex- and strain-specific exposure outcomes illustrate that HS rat founders possess diverse bisphenol-exposure risk alleles and suggest that BPF exposure may intensify inherent organ system dysfunction existing in the HS rat founders. We propose that the HS rat will be an invaluable model for dissecting gene EDC interactions on health.

Appraising the Preclinical Evidence of the Role of the Renin-Angiotensin-Aldosterone System in Antenatal Programming of Maternal and Offspring Cardiovascular Health Across the Life Course: Moving the Field Forward: A Scientific Statement From the American Heart Association.

There is increasing interest in the long-term cardiovascular health of women with complicated pregnancies and their affected offspring. Emerging antenatal risk factors such as preeclampsia appear to increase the risk of hypertension and cardiovascular disease across the life course in both the offspring and women after pregnancy. However, the antenatal programming mechanisms responsible are complex and incompletely understood, with roots in alterations in the development, structure, and function of the kidney, heart, vasculature, and brain. The renin-angiotensin-aldosterone system is a major regulator of maternal-fetal health through the placental interface, as well as kidney and cardiovascular tissue development and function. Renin-angiotensin-aldosterone system dysregulation plays a critical role in the development of pregnancy complications such as preeclampsia and programming of long-term adverse cardiovascular health in both the mother and the offspring. An improved understanding of antenatal renin-angiotensin-aldosterone system programming is crucial to identify at-risk individuals and to facilitate development of novel therapies to prevent and treat disease across the life course. Given the inherent complexities of the renin-angiotensin-aldosterone system, it is imperative that preclinical and translational research studies adhere to best practices to accurately and rigorously measure components of the renin-angiotensin-aldosterone system. This comprehensive synthesis of preclinical and translational scientific evidence of the mechanistic role of the renin-angiotensin-aldosterone system in antenatal programming of hypertension and cardiovascular disease will help (1) to ensure that future research uses best research practices, (2) to identify pressing needs, and (3) to guide future investigations to maximize potential outcomes. This will facilitate more rapid and efficient translation to clinical care and improve health outcomes.

ARRB2 (ß-Arrestin-2) Deficiency Alters Fluid Homeostasis and Blood Pressure Regulation.

BACKGROUND: GPCRs (G protein-coupled receptors) are implicated in blood pressure (BP) and fluid intake regulation. There is a developing concept that these effects are mediated by both canonical G protein signaling and noncanonical ß-arrestin mediated signaling, but the contributions of each remain largely unexplored. Here, we hypothesized that ß-arrestin contributes to fluid homeostasis and blood pressure (BP) regulation in deoxycorticosterone acetate (DOCA) salt hypertension, a prototypical model of salt-sensitive hypertension. METHODS: Global ß-arrestin1 (Arrb1) and ß-arrestin2 (Arrb2) knockout mice were employed to evaluate drinking behavior, and BP was evaluated in Arrb2-knockout mice. Age- and sex-matched C57BL/6 mice served as controls. We measured intake of water and different sodium chloride solutions and BP employing a 2-bottle choice paradigm with and without DOCA. RESULTS: Without DOCA (baseline), Arrb2-knockout mice exhibited a significant elevation in saline intake with no change in water intake. With DOCA treatment, Arrb2-knockout mice exhibited a significant increase in both saline and water intake. Although Arrb2-knockout mice exhibited hypernatremia at baseline conditions, we did not find significant changes in total body sodium stores or sodium palatability. In a separate cohort, BP was measured via telemetry in Arrb2-knockout and C57BL/6 mice with and without DOCA. Arrb2-knockout did not exhibit significant differences in BP before DOCA treatment when provided water alone, or when provided a choice of water and saline. However, Arrb2-knockout exhibited an increased pressor response to DOCA-salt. CONCLUSIONS: These findings suggest that in salt-sensitive hypertension, ARRB2, but not ARRB1 (ß-arrestin 1), might counterbalance the canonical signaling of GPCRs.

Elucidating the role of Rgs2 expression in the PVN for metabolic homeostasis in mice.

OBJECTIVE: RGS2 is a GTPase activating protein that modulates GPCR-Gα signaling and mice lacking RGS2 globally exhibit metabolic alterations. While RGS2 is known to be broadly expressed throughout the body including the brain, the relative contribution of brain RGS2 to metabolic homeostasis remains unknown. The purpose of this study was to characterize RGS2 expression in the paraventricular nucleus of hypothalamus (PVN) and test its role in metabolic homeostasis. METHODS: We used a combination of RNAscope in situ hybridization (ISH), immunohistochemistry, and bioinformatic analyses to characterize the pattern of Rgs2 expression in the PVN. We then created mice lacking Rgs2 either prenatally or postnatally in the PVN and evaluated their metabolic consequences. RESULTS: RNAscope ISH analysis revealed a broad but regionally enriched Rgs2 mRNA expression throughout the mouse brain, with the highest expression being observed in the PVN along with several other brain regions, such as the arcuate nucleus of hypothalamus and the dorsal raphe nucleus. Within the PVN, we found that Rgs2 is specifically enriched in CRH+ endocrine neurons and is further increased by calorie restriction. Functionally, although Sim1-Cre-mediated prenatal deletion of Rgs2 in PVN neurons had no major effects on metabolic homeostasis, AAV-mediated adult deletion of Rgs2 in the PVN led to significantly increased food intake, body weight (both fat and fat-free masses), body length, and blood glucose levels in both male and female mice. Strikingly, we found that prolonged postnatal loss of Rgs2 leads to neuronal cell death in the PVN, while rapid body weight gain in the early phase of viral-mediated PVN Rgs2 deletion is independent of PVN neuronal loss. CONCLUSIONS: Our results provide the first evidence to show that PVN Rgs2 expression is not only sensitive to metabolic challenge but also critically required for PVN endocrine neurons to function and maintain metabolic homeostasis.

Cardiometabolic Consequences of Deleting the Regulator of G protein Signaling-2 (Rgs2) From Cells Expressing Agouti-Related Peptide or the ANG (Angiotensin) II Type 1A Receptor in Mice.

BACKGROUND: RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in the RGS2 gene in humans have been linked to hypertension, preeclampsia, and anxiety disorders. Mice deficient for Rgs2 (Rgs2Null) exhibit hypertension, anxiety, and altered adipose development and function. METHODS: To study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for the Rgs2 gene (Rgs2Flox) was developed. These mice were bred with mice expressing Cre-recombinase via the Agouti-related peptide locus (Agrp-Cre) to cause deletion of Rgs2 from all cells expressing Agrp (Rgs2Agrp-KO), or a novel transgenic mouse expressing Cre-recombinase via the ANG (angiotensin) type 1A receptor (Agtr1a/ AT1A) promoter encoded in a bacterial artificial chromosome (BAC-AT1A-Cre) to delete Rgs2 in all Agtr1a-expressing cells (Rgs2AT1A-KO). RESULTS: Whereas Rgs2Flox, Rgs2Agrp-KO, and BAC-AT1A-Cre mice exhibited normal growth and survival, Rgs2AT1A-KO exhibited pre-weaning lethality. Relative to littermates, Rgs2Agrp-KO exhibited reduced fat gains when maintained on a high fat diet, associated with increased energy expenditure. Similarly, surviving adult Rgs2AT1A-KO mice also exhibited increased energy expenditure. Surprisingly, given the hypertensive phenotype previously reported for Rgs2Null mice and evidence supporting a role for RGS2 in terminating AT1A signaling in various cell types, Rgs2AT1A-KO mice exhibited normal blood pressure, ingestive behaviors, and renal functions, both before and after chronic infusion of ANG (490 ng/kg/min, sc). CONCLUSIONS: These results demonstrate the development of a novel mouse with conditional expression of Rgs2 and illustrate the role of Rgs2 within selected cell types for cardiometabolic control.

Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons.

Acute regulation of CO2 and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO2/pH sensitive cells - so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO2 sensitive or insensitive based on differences in embryonic origin, suggesting these functionally distinct subpopulations may have unique transcriptional profiles. Here, we used Patch-to-Seq to determine if the CO2 responses in brainstem 5-HT neurons could be correlated to unique transcriptional profiles and/or unique molecular markers and pathways. First, firing rate changes with hypercapnic acidosis were measured in fluorescently labeled 5-HT neurons in acute brainstem slices from transgenic, Dahl SS (SSMcwi) rats expressing T2/ePet-eGFP transgene in Pet-1 expressing (serotonin) neurons (SS ePet1-eGFP rats). Subsequently, the transcriptomic and pathway profiles of CO2 sensitive and insensitive 5-HT neurons were determined and compared by single cell RNA (scRNAseq) and bioinformatic analyses. Low baseline firing rates were a distinguishing feature of CO2 sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO2 sensitive and insensitive 5-HT neurons. Pathway analyses yielded novel predicted upstream regulators, including the transcription factor Egr2 and Leptin. Additional bioinformatic analyses identified 6 candidate gene markers of CO2 sensitive 5-HT neurons, and 2 selected candidate genes (CD46 and Iba57) were both expressed in 5-HT neurons determined via in situ mRNA hybridization. Together, these data provide novel insights into the transcriptional control of cellular chemoreception and provide unbiased candidate gene markers of CO2 sensitive 5-HT neurons. Methodologically, these data highlight the utility of the patch-to-seq technique in enabling the linkage of gene expression to specific functions, like CO2 chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.