Brain Renin-angiotensin System Activity Research Articles

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.

Studies of salt and stress sensitivity on arterial pressure in renin-b deficient mice.

Excessive sodium intake is known to increase the risk for hypertension, heart disease, and stroke. Individuals who are more susceptible to the effects of high salt are at higher risk for cardiovascular diseases even independent of their blood pressure status. Local activation of the renin-angiotensin system (RAS) in the brain, among other mechanisms, has been hypothesized to play a key role in contributing to salt balance. We have previously shown that deletion of the alternative renin isoform termed renin-b disinhibits the classical renin-a encoding preprorenin in the brain resulting in elevated brain RAS activity. Thus, we hypothesized that renin-b deficiency results in higher susceptibility to salt-induced elevation in blood pressure. Telemetry implanted Ren-bNull and wildtype littermate mice were first offered a low salt diet for a week and subsequently a high salt diet for another week. A high salt diet induced a mild blood pressure elevation in both Ren-bNull and wildtype mice, but mice lacking renin-b did not exhibit an exaggerated pressor response. When renin-b deficient mice were exposed to a high salt diet for a longer duration (4 weeks), there was a trend for increased myocardial enlargement in Ren-bNull mice when compared with control mice, but this did not reach statistical significance. Multiple studies have also demonstrated the association of environmental stress with hypertension. Activation of the RAS in the rostral ventrolateral medulla and the hypothalamus is required for stress-induced hypertension. Thus, we next questioned whether the lack of renin-b would result in exacerbated response to an acute restraint-stress. Wildtype and Ren-bNull mice equally exhibited elevated blood pressure in response to restraint-stress, which was similar in mice fed either a low or high salt diet. These studies suggest that mechanisms unrelated to salt and acute stress alter the cardiovascular phenotype in mice lacking renin-b.

BRAIN-PENETRATING AMINOPEPTIDASE A INHIBITORS FOR NEW TREATMENT OF HYPERTENSION AND HEART FAILURE

Objective: Brain renin-angiotensin system (RAS) hyperactivity is involved in the pathophysiology of hypertension and heart failure (HF). Angiotensin III/(AngIII), one of the main effector peptides of the brain RAS, exerts tonic stimulatory control over blood pressure/(BP) in hypertensive rats and plays a pivotal role in sympathetic hyperactivity and left ventricular/(LV) dysfunction in rats post-myocardial infarction/(MI). Thus, aminopeptidase A/(APA), the enzyme generating brain AngIII represents a potential therapeutic target for the treatment of these pathologies. Our aim was to evaluate the mode of action of firibastat, a brain-penetrating prodrug of the APA inhibitor, EC33, to reduce BP in hypertensive rats and prevent LV dysfunction in mice post-MI. Design and method: Firibastat and MLN4760, an angiotensin-converting enzyme2/(ACE2) inhibitor were administered by central route in hypertensive DOCA-salt rats and mean arterial BP (MABP) was recorded following insertion of a catheter into the femoral artery. Brain APA and ACE2 activities were measured 30 min post-treatment. Two days post-MI induced by the left anterior descending artery ligation, mice were randomized to receive during 4–8 weeks oral treatment with vehicle, firibastat (150 mg/kg) or the ACE inhibitor, enalapril (1 mg/kg). Cardiac function was evaluated by echocardiography Results: 1-Firibastat, when given centrally in hypertensive rats, is immediately cleaved by brain reductases, generating EC33 which inhibits brain APA activity, blocks brain AngIII formation and decreases BP. At the same time, firibastat treatment increases the activity of brain ACE2, majoring the conversion of AngII to Ang1-7, which by acting on the Mas-receptor, participate to the firibastat-induced BP decrease. 2-Chronic firibastat treatment in mice post-MI normalizes brain APA activity (normalizing brain RAS activity) and prevents sympathetic hyperactivity. It reduced LV end-diastolic pressure, LV end-systolic diameter and volume, and increased LV ejection fraction. It decreased the expression of HF and fibrosis biomarkers. Firibastat treatment prevents cardiac dysfunction and attenuates cardiac hypertrophy and fibrosis. Conclusions: Following these data, firibastat was selected for clinical development. Phase IIa/IIb clinical trials provided pharmacological proof-of-principle of firibastat efficacy for decreasing BP in hypertensive patients. The ongoing phase II study/QUORUM will determine if firibastat treatment may constitute a new therapeutic approach for preventing HF post-MI.

Increased Susceptibility of Mice Lacking Renin-b to Angiotensin II-Induced Organ Damage.

Several cardiac and renal diseases are attributed to a dysregulation of the renin-angiotensin system. Renin, the rate-limiting enzyme of the renin-angiotensin system, has 2 isoforms. The classical renin isoform (renin-a) encoding preprorenin is mainly confined to the juxtaglomerular cells and released into the circulation upon stimulation. Alternatively, renin-b is predicted to remain intracellular and is expressed in the brain, heart, and adrenal gland. In the brain, ablation of renin-b (Ren-bNull mice) results in increased brain renin-angiotensin system activity. However, the consequences of renin-b ablation in tissues outside the brain remain unknown. Therefore, we hypothesized that renin-b protects from hypertensive cardiac and renal end-organ damage in mice. Ren-bNull mice exhibited normal blood pressure at baseline. Thus, we induced hypertension by using a slow pressor dose of Ang II (angiotensin II). Ang II increased blood pressure in both wild type and Ren-bNull to the same degree. Although the blood pressure between Ren-bNull and wild-type mice was elevated equally, 4-week infusion of Ang II resulted in exacerbated cardiac remodeling in Ren-bNull mice compared with wild type. Ren-bNull mice also exhibited a modest increase in renal glomerular matrix deposition, elevated plasma aldosterone, and a modestly enhanced dipsogenic response to Ang II. Interestingly, ablation of renin-b strongly suppressed plasma renin, but renal cortical renin mRNA was preserved. Altogether, these data indicate that renin-b might play a protective role in the heart, and thus renin-b could be a potential target to treat hypertensive heart disease.

Susceptibility of Mice Lacking Renin‐b to Chronic Angiotensin II Infusion

In addition to the classical endocrine renin angiotensin system (RAS), local activity of the RAS has been detected in many tissues including the brain. The brain renin angiotensin system is known for its importance in blood pressure regulation and water homeostasis. The brain specific isoform of renin, termed renin‐b, has been proposed as a negative regulator of the brain renin‐angiotensin system (RAS) since the selective ablation of ren‐b (Ren‐b KO) in mice results in brain RAS disinhibition leading to blood pressure (BP) elevation. New cohorts of Ren‐b KO mice, however, do not exhibit elevated BP and the retrospective analysis of five previous cohorts indicated that Ren‐b KO model exhibits a high degree of variability in BP, which may be attributed to the susceptibility to different stressors. Importantly, Ren‐b KO (including recent cohorts) exhibit suppressed plasma renin and angiotensin II (Ang II) implying that suppression of the endocrine RAS might compensate for the elevation of the brain RAS activity in those Ren‐b KO mice exhibiting normal BP. Therefore, we hypothesized that Ren‐b KO are sensitized to exogenous systemic infusion of Ang II. Wildtype (WT) or Ren‐b KO were infused with either vehicle or Ang II (400 ng/kg/min; sc.) via osmotic minipumps for 4 weeks. Ang II significantly increased BP in both WT and Ren‐b KO to the same degree (WT+veh: 103±4 vs WT+Ang: 120±4 mmHg; p = 0.01 (n=10–12) and KO+veh: 104±3 vs KO+Ang: 118±5 mmHg; p = 0.03 (n=10–14)). Although the BP between Ren‐b KO and WT mice was elevated equally, Ren‐b KO exhibited enhanced Ang II‐induced dipsogenic response (WT+Ang: 3.7±0.3 vs KO+Ang: 4.6±0.4 ml/day; p = 0. 05; n=10–12) and exhibited a trend toward increase in levels of urinary aldosterone (WT+Ang: 14.6±1.4 vs KO+Ang: 17.8±1.7 mmHg) and copeptin, a marker of arginine vasopressin (WT+Ang: 11.5±2.6 vs KO+Ang: 22.2±5.2 mmHg). BP, drinking, aldosterone, and copeptin data in Ang II‐treated Ren‐b KO exhibits a bi‐modal distribution indicating that some mice are, whereas others, are not sensitized to Ang II. Ang II‐treated Ren‐b KO exhibited elevated heart weight (WT+Ang: 4.8±0.2 vs KO±Ang: 5.2±0.1 mg/g of body weight; p = 0.03; n=10–12) concomitant with an increased cardiac collagen type I alpha‐2 mRNA expression (p = 0.03; n=7–9) and trend toward increase in tumor necrosis factor‐α. Ang II induced an exaggerated suppression of plasma renin in Ren‐b KO compared to WT (WT+Ang: 14.8±2.1 vs KO+Ang: 7.2±2.6 ng/ml; p = 0. 03; n=7–9). However, there was no difference in the renin mRNA and protein levels of renin in the renal cortex. We used in situ hybridization technique to localize renin expression in the renin cortex. We did not observe a shift from glomerular to tubular renin expression in Ren‐b KO. We conclude Ren‐b KO exhibits higher sensitivity to Ang II‐induced aldosterone secretion and drinking responses as well as elevated susceptibility to Ang II‐evoked cardiac remodeling. The mechanism causing suppressed plasma renin in Ren‐b KO remains unclear. Studies of a conditional Ren‐b KO model will be used to elucidate the physiological consequences of downregulating renin‐b in specific neuroanatomical regions.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

Assessing the Effects of Leptin and Angiotensin II Upon Expression of Agouti‐Related Peptide in the GT1‐7 Cell Model

Leptin acts at its receptor (Lepr) within the arcuate nucleus of the hypothalamus (ARC) to reduce feeding behavior and increase resting metabolic rate (RMR) in part through the inhibition of Agouti‐related peptide (Agrp) signaling. Previously we demonstrated that a unique subset of Agrp neurons express the angiotensin II (ANG) type 1a receptor (Agtr1a). Genetic deletion of Agtr1a from Agrp neurons of mice was sufficient to disinhibit expression of Agrp within the ARC, and to attenuate adipose sympathetic nerve activity responses to leptin. In contrast, RMR responses to brain renin‐angiotensin system (RAS) stimulation are intact in the dysfunctional Lepr db/db mouse model, and acute leptin administration does not additively exaggerate RMR activation in a transgenic mouse model of increased brain RAS activity. New unpublished data also indicate that ANG signaling within the ARC stimulates RMR through suppression of Agrp expression and Agrp neuron electrical activity, and thereby disinhibition of melanocortin Mc4r receptor signaling. To further dissect the interaction of Lepr and Agtr1a within Agrp neurons, here we examined the effects of leptin and ANG stimulation of the “GT1‐7” immortalized cell culture model of mouse Agrp‐expressing cells. We confirmed that cultured GT1‐7 cells express critical markers of the Agtr1a‐positive Agrp neuron subtype, including Agrp, Agtr1a, and Neuropeptide‐Y (Npy), but not pro‐opiomelanocortin (Pomc). Dose‐ and time‐course experiments were performed to identify mid‐range doses and time courses for Agrp gene expression responses to acute application of leptin and ANG. Subsequently, Agrp expression responses to low doses of leptin (100 nM) and ANG (100 nM), which would allow detection of any additive or synergistic effect, were assessed after 1 and 2–2.5 hours to evaluate the hypothesis that leptin and ANG signaling are non‐additive for the suppression of Agrp expression. After 1 hour, ANG (0.66 (0.49–0.91), fold of vehicle (± 1 sem), n=0.5) and leptin (0.61 (0.44–0.84), n=6) had similar effects on Agrp mRNA expression compared to vehicle (1.00 (0.86–1.17), n=7), but co‐administration of both peptides had no synergistic effect (0.96 (0.78–1.18), n=5). Likewise after 2–2.5 hours, compared to vehicle (1.00 (0.78–1.28), n=6), ANG (0.56 (0.34–0.94), n=6) and leptin (0.69 (0.57–0.85), n=6) had similar effects that were not additive (0.81 (0.67–0.98), n=6). These findings support the preliminary conclusion that the effects of leptin and ANG upon Agrp expression are not additive in the GT1‐7 cell culture model of Agrp neurons. These findings support our working model in which leptin action at the ARC results in increased Agtr1a signaling to suppress Agrp, ultimately to disinhibit Mc4r signaling and thus increase resting energy expenditure.Support or Funding InformationHL134850, HL084207

Orally Active Aminopeptidase A Inhibitor Prodrugs: Current State and Future Directions.

To review the data supporting the use of aminopeptidase A (APA) inhibitor prodrugs as centrally acting antihypertensive agents. Brain renin-angiotensin system (RAS) hyperactivity has been implicated in the development and maintenance of hypertension. Angiotensin III, generated by APA, one of the main effector peptides of the brain RAS, exerts a tonic stimulatory control over blood pressure in hypertensive rats. This identified brain APA as a potential therapeutic target for the treatment of hypertension, leading to the development of RB150/firibastat, an orally active prodrug of the specific and selective APA inhibitor, EC33. When given orally, RB150/firibastat crosses the gastrointestinal and blood-brain barriers, enters the brain, and generates two active molecules of EC33 which inhibit brain APA activity, blocking brain angiotensin III formation, and decrease blood pressure for several hours in hypertensive rats. Orally active APA inhibitor prodrugs, by blocking brain RAS activity, represent promising novel strategy for treating hypertension.

Abstract P204: Induction of Endoplasmic Reticulum Stress Upregulates Renin Expression in Immortalized Mouse Hypothalamic N43/5 Cells

Dysregulation of the brain renin-angiotensin system (RAS) has been implicated in many forms of hypertension, including resistant hypertension. However, the mechanism by which the brain RAS is activated is unknown. We have recently published on the activation of brain RAS following disinhibition of renin-a expression after deletion of the brain-specific renin-b isoform. Endoplasmic reticulum (ER) stress, which is an accumulation of unfolded proteins in the ER, has been linked to hypertension as central angiotensin II administration induced ER stress markers. Here, we hypothesize that ER stress induces brain RAS activation by upregulating renin expression in the brain. We first tested if ER stress could induce renin expression in vitro in a mouse neuroblastoma hypothalamic cell line, N43/5 cells. After treating cells with ER stressor thapsigargin, a SERCA pump inhibitor, (100nM) for 8 hours we observed transcriptional upregulation of ER stress marker Bip (28.9±5.0 fold increase vs vehicle, p<0.05, n=6). This same treatment also induced an increase in renin expression (13.8±5.8 fold increase vs vehicle, p<0.05, n=6). We have previously shown that deletion of renin-b in the brain causes an increase in renin-a expression specifically in the rostral ventrolateral medulla (RVLM). This data suggests that ER stress may induce RAS activation through a similar renin-a disinhibitory mechanism. Under basal conditions, the RVLM of renin-b knockout mice showed no difference in gene expression of ER stress marker Bip (0.7±0.5 fold increase vs control, p=0.67, n=3-4) or Chop (0.7±0.35 fold increase vs control, p=0.64, n=3-4). This suggests that ER stress may be upstream of renin induction. To test this, we treated C57BL/6 mice with tunicamycin (40mg/mL, 2uL) via intracerebellar ventricle cannula. We detected a positive endogenous renin transcript signal via in situ hybridization bilaterally in the medulla in the vicinity of the RVLM (possibly the nucleus ambiguous) from vehicle and tunicamycin treated mice. We are currently testing if ER stress quantitatively increases renin expression using qPCR. These data extend previous studies showing that RAS activation in the brain causes ER stress by suggesting that ER stress may also induce the brain RAS.

Selective Deletion of Renin-b in the Brain Alters Drinking and Metabolism.

The brain-specific isoform of renin (Ren-b) has been proposed as a negative regulator of the brain renin-angiotensin system (RAS). We analyzed mice with a selective deletion of Ren-b which preserved expression of the classical renin (Ren-a) isoform. We reported that Ren-bNull mice exhibited central RAS activation and hypertension through increased expression of Ren-a, but the dipsogenic and metabolic effects in Ren-bNull mice are unknown. Fluid intake was similar in control and Ren-bNull mice at baseline and both exhibited an equivalent dipsogenic response to deoxycorticosterone acetate-salt. Dehydration promoted increased water intake in Ren-bNull mice, particularly after deoxycorticosterone acetate-salt. Ren-bNull and control mice exhibited similar body weight when fed a chow diet. However, when fed a high-fat diet, male Ren-bNull mice gained significantly less weight than control mice, an effect blunted in females. This difference was not because of changes in food intake, energy absorption, or physical activity. Ren-bNull mice exhibited increased resting metabolic rate concomitant with increased uncoupled protein 1 expression and sympathetic nerve activity to the interscapular brown adipose tissue, suggesting increased thermogenesis. Ren-bNull mice were modestly intolerant to glucose and had normal insulin sensitivity. Another mouse model with markedly enhanced brain RAS activity (sRA mice) exhibited pronounced insulin sensitivity concomitant with increased brown adipose tissue glucose uptake. Altogether, these data support the hypothesis that the brain RAS regulates energy homeostasis by controlling resting metabolic rate, and that Ren-b deficiency increases brain RAS activity. Thus, the relative level of expression of Ren-b and Ren-a may control activity of the brain RAS.

Telmisartan attenuates diabetes induced depression in rats

Role of brain renin angiotensin system (RAS) is well understood and various clinical studies have proposed neuroprotective effects of ARB's. It is also assumed that diabetic depression is associated with activation of brain RAS, HPA axis dysregulation and brain inflammatory events. Therefore, the present study was designed to investigate the antidepressant effect of low dose telmisartan (TMS) in diabetes induced depression (DID) in rats. Diabetes was induced by injecting streptozotocin. After 21days of treatment the rats were subjected to forced swim test (FST). The rats, with increased immobility time, were considered depressed and were treated with vehicle or TMS (0.05mg/kg, po) or metformin (200mg/kg, po) or fluoxetine (20mg/kg, po). A separate group was also maintained to study the combination of metformin and TMS. At the end of 21days of treatments, FST, open field test (OFT) and elevated plus maze (EPM) paradigm were performed. Blood was drawn to estimate serum cortisol, nitric oxide (NO), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Persistent hyperglycemia resulted in depression and anxiety in rats as observed by increased immobility, reduced latency for immobility, reduced open arm entries and time spent. The depressed rats showed a significant rise in serum cortisol, NO, IL-6 and IL-1β (p<0.001). TMS antagonized depression and anxiety. It also significantly attenuated serum cortisol, NO, IL-6 and IL-1β (p<0.001). Low dose TMS and its combination with metformin normalizes depressive mood, reduces pro-inflammatory mediators and ameliorates the HPA axis function; thereby providing beneficial effects in DID.

Selective Deletion of the Brain-Specific Isoform of Renin Causes Neurogenic Hypertension.

The renin-angiotensin system (RAS) in the brain is a critical determinant of blood pressure, but the mechanisms regulating RAS activity in the brain remain unclear. Expression of brain renin (renin-b) occurs from an alternative promoter-first exon. The predicted translation product is a nonsecreted enzymatically active renin whose function is unknown. We generated a unique mouse model by selectively ablating the brain-specific isoform of renin (renin-b) while preserving the expression and function of the classical isoform expressed in the kidney (renin-a). Preservation of renal renin was confirmed by measurements of renin gene expression and immunohistochemistry. Surprisingly, renin-b-deficient mice exhibited hypertension, increased sympathetic nerve activity to the kidney and heart, and impaired baroreflex sensitivity. Whereas these mice displayed decreased circulating RAS activity, there was a paradoxical increase in brain RAS activity. Physiologically, renin-b-deficient mice exhibited an exaggerated depressor response to intracerebroventricular administration of losartan, captopril, or aliskiren. At the molecular level, renin-b-deficient mice exhibited increased expression of angiotensin-II type 1 receptor in the paraventricular nucleus, which correlated with an increased renal sympathetic nerve response to leptin, which was dependent on angiotensin-II type 1 receptor activity. Interestingly, despite an ablation of renin-b expression, expression of renin-a was significantly increased in rostral ventrolateral medulla. These data support a new paradigm for the genetic control of RAS activity in the brain by a coordinated regulation of the renin isoforms, with expression of renin-b tonically inhibiting expression of renin-a under baseline conditions. Impairment of this control mechanism causes neurogenic hypertension.

Abstract P278: On the Origin of Brain Renin

A renin-angiotensin system (RAS) in the brain is believed to contribute to blood pressure regulation. Given the presence of the blood-brain barrier, brain RAS activity most likely depends on synthesis of (pro)renin in the brain. In support of this concept, an intracellular, non-secreted form of renin has been described in the brain, depending on an alternative transcript of the renin gene. To what degree this intracellular truncated prorenin truly displays angiotensin (Ang) I-generating activity (AGA) is unknown. In the present study, we set out to quantify brain (pro)renin, both before and after buffer perfusion of the brain, in normal mice, renin knockout (KO) mice, DOCA-salt-treated mice (which have been reported to display brain RAS activation), and angiotensin II-infused mice. Brain nuclei were homogenized and incubated with excess angiotensinogen to detect AGA, both before and after acid activation of prorenin, with or without the renin inhibitor aliskiren to correct for non-renin-mediated AGA. Renin-dependent (i.e., aliskiren-inhibitable) AGA was readily detectable in brain nuclei, the highest AGA being present in brainstem (&gt;thalamus ≈cerebellum ≈striatum ≈midbrain &gt;hippocampus ≈cortex). Brain AGA increased non-significantly after prorenin activation, suggesting that brain prorenin levels are low or absent. Buffer perfusion reduced AGA in all brain areas by &gt;60%. Plasma renin (expressed per mL plasma) was 40-800 x higher than brain renin (expressed per g tissue). Plasma prorenin levels resembled plasma renin levels. AGA was undetectable in plasma and brain of renin KO mice, both before and after prorenin activation. DOCA-salt and Ang II suppressed plasma renin, and parallel decreases were observed for brain renin. In conclusion, brain renin levels (per g tissue) correspond with the amount of renin present in 1-20 μL blood plasma. Brain renin disappears after buffer perfusion, and varies in association with plasma renin. This indicates that renin detected in brain nuclei represents plasma renin and/or locally activated plasma prorenin. DOCA-salt exposure does not selectively increase brain renin expression.

Abstract P061: Selective Deletion of Intracellular Renin in the Brain Causes Hypertension and Elevated Sympathetic Nervous System Activity

Renin expression is regulated by two distinct promoter-1st exon combinations that target renin either for secretion (exon 1a for secreted renin) or cytoplasmic retention (exon 1b for intracellular renin, icREN). We developed icREN knockout (KO) mice by selectively deleting exon 1b. icREN KO mice are essentially brain-specific knockouts of icREN because icREN is predominantly expressed in the brain. Notably, systolic blood pressure measured by telemetry was increased in icREN KO mice (130±2 mmHg, n=8 vs 122±2 mmHg in controls, n=7, P&lt;0.01). The low- to high-frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability, a parameter of sympathetic nerve activity (SNA), was increased in icREN KO mice (KO: 1.24±0.21, n=7 vs control: 0.70±0.11, n=7, P&lt;0.05). Body weight (BW) was normal in icREN KO mice compared to controls, but the BW gain and fat accumulation induced by high fat diet (HFD) were attenuated in male icREN KO mice (BW at 16 wks of HFD- KO: 36.8±1.2 g, n=8 vs control: 41.9±1.4 g, n=9; relative fat mass at 14 wks of HFD- KO: 27.7±1.7%, n=8 vs control: 34.4±2.3%, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in icREN KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53), whereas food consumption and absorbed calories were not different. We previously reported that the brain renin-angiotensin system facilitates renal SNA (RSNA) response to acute intracerebroventricular (ICV) injection of leptin. Interestingly, the RSNA response to ICV leptin was greater in icREN KO mice (KO: 214±40 % baseline, n=5 vs control: 114±18 % baseline, n=10, P&lt;0.01). AT1a receptor mRNA was upregulated in the paraventricular nucleus of icREN KO mice (P&lt;0.05). Chronic ICV injection of losartan not only abolished the elevated blood pressure in icREN KO mice, but reduced it to below baseline in controls (systolic blood pressure, 111±3 mmHg in KO, n=5; 124±4 mmHg in controls, n=6). These data suggest that icREN deletion increases the activity of brain renin-angiotensin system and elevates blood pressure and metabolic rate through sympathetic activation. We conclude that this novel icREN isoform contributes to cardiovascular and metabolic control possibly as part of an inhibitory neural circuit.

Activation of central PPAR-γ attenuates angiotensin II-induced hypertension.

Inflammation and renin-angiotensin system activity in the brain contribute to hypertension through effects on fluid intake, vasopressin release, and sympathetic nerve activity. We recently reported that activation of brain peroxisome proliferator-activated receptor (PPAR)-γ in heart failure rats reduced inflammation and renin-angiotensin system activity in the hypothalamic paraventricular nucleus and ameliorated the peripheral manifestations of heart failure. We hypothesized that the activation of brain PPAR-γ might have beneficial effects in angiotensin II-induced hypertension. Sprague-Dawley rats received a 2-week subcutaneous infusion of angiotensin II (120 ng/kg per minute) combined with a continuous intracerebroventricular infusion of vehicle, the PPAR-γ agonist pioglitazone (3 nmol/h) or the PPAR-γ antagonist GW9662 (7 nmol/h). Angiotensin II+vehicle rats had increased mean blood pressure, increased sympathetic drive as indicated by the mean blood pressure response to ganglionic blockade, and increased water consumption. PPAR-γ mRNA in subfornical organ and hypothalamic paraventricular nucleus was unchanged, but PPAR-γ DNA-binding activity was reduced. mRNA for interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2, and angiotensin II type 1 receptor was augmented in both nuclei, and hypothalamic paraventricular nucleus neuronal activity was increased. The plasma vasopressin response to a 6-hour water restriction also increased. These responses to angiotensin II were exacerbated by GW9662 and ameliorated by pioglitazone, which increased PPAR-γ mRNA and PPAR-γ DNA-binding activity in subfornical organ and hypothalamic paraventricular nucleus. Pioglitazone and GW9662 had no effects on control rats. The results suggest that activating brain PPAR-γ to reduce central inflammation and brain renin-angiotensin system activity may be a useful adjunct in the treatment of angiotensin II-dependent hypertension.

Brain ACE2 overexpression stimulates food intake and promotes glucose intolerance in mice (1126.15)

The brain Renin‐Angiotensin System (RAS) plays an important role in the regulation of metabolic function. While ACE2 has been reported to modulate brain RAS activity, its role in the central regulation of metabolism is unknown. To determine the role of central ACE2 in metabolism, Body weight and intake behaviors, assessed using metabolic cages, were measured in 24 wk old non‐transgenic (NT, n=8) and transgenic mice overexpressing ACE2 in the brain (SA, n=8). Fasting blood glucose was measured after a 14 h overnight fasting. Glucose tolerance test was performed by measuring blood glucose at 15, 30, 60 and 120 min after IP injection of glucose (2 g/kg). Plasma insulin level was measured by ELISA. Interestingly, body weight was significantly higher in SA than NT mice (39±1.5 vs. 31±0.8 g, P&lt;0.05). In addition, SA mice exhibited increased daily food intake (0.12±0.01 vs. 0.09±0.01 g/g body weight), water intake (0.14±0.01 vs. 0.08±0.01 ml/g body weight), fasting blood glucose (147.8±13 vs.106.5±7 mg/dl), impaired glucose tolerance (AUC: 51.6±2 vs.41.5±1.7) and increased plasma insulin level (5.5±1.2 vs.1.7±0.3 ng/ml), compared to NT mice (P&lt;0.05). These data suggest that ACE2 is involved in the central regulation of energy and glucose homeostasis. Brain ACE2 over‐expression leads to increased body weight, fasting glycemia and glucose intolerance, possibly through stimulating energy intake and insulin resistance.Grant Funding Source: Supported by NIH (NIGMS)

Randomised, Double-Blind, Placebo-Controlled, Dose-Escalating Phase I Study of QGC001, a Centrally Acting Aminopeptidase A Inhibitor Prodrug

Inhibition of brain aminopeptidase A (APA), which converts angiotensin II into angiotensin III, has emerged as a novel antihypertensive treatment, as demonstrated in several experimental animal models. QGC001 (originally named RB150) is a prodrug of the specific and selective APA inhibitor EC33, and as such it is the prototype of a new class of centrally acting antihypertensive agents. Given by the oral route in hypertensive rats, it enters the brain and generates EC33, which blocks the brain renin-angiotensin system activity and normalises blood pressure. The aim of the present study was to evaluate the safety, pharmacokinetics and pharmacodynamic effects of QGC001 in humans. Fifty-six healthy male volunteers were randomly assigned to receive in double-blind and fasted conditions single oral doses of 10, 50, 125, 250, 500, 750, 1,000 and 1,250 mg of QGC001 (n = 6/dose) or placebo (n = 2/dose). We measured plasma and urine concentrations of both QGC001 and EC33 by liquid chromatography-tandem mass spectrometry, plasma renin concentrations (PRC), plasma and free urine aldosterone (PAldo and UAldo), plasma copeptine (PCop), and plasma and urine cortisol (PCort and UCort) concentrations, and supine systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) at various time points. All doses of QGC001 were clinically and biologically well-tolerated. Peak plasma concentrations (Cmax) of QGC001 and EC33 increased linearly with the dose, with a median time to reach Cmax (tmax) of 1.5 h for QGC001 and 3.0 h for EC33. The median plasma elimination half-life of QGC001 was 1.6 h consistently throughout doses. Urinary excretion of QGC001 and EC33 was below 2% of the administered dose. When compared with placebo, QGC001 did not significantly change PRC, PAldo, UAldo, PCop, PCort or UCort. No significant change was observed for supine HR, SBP and DBP in any treatment group. Single oral administration of QGC001 up to 1,250 mg in healthy volunteers was well-tolerated. Following oral administration, QGC001 is absorbed via the gastrointestinal tract and converted partially into its active metabolite EC33 in plasma. As in animal experiments, in normotensive subjects QGC001 had no effect on the systemic renin-angiotensin-aldosterone parameters and on PCop concentrations, a marker of vasopressin release. In normotensive subjects, a single dose of QCG001 had no effect on SBP, DBP or HR. These data support further evaluation of multiple oral doses of QGC001 in human volunteers and its clinical efficacy in hypertensive patients.

Abstract 414: Adipose Angiotensin AT2 Receptors Modulate Thermogenesis Though Interactions with Epidermal Growth Factor Signaling

The brain renin-angiotensin system (RAS) stimulates resting metabolic rate (RMR) in part through a mechanism involving suppression of the circulating RAS. This effect appears to be mediated through the loss of tonic adipose angiotensin AT2 receptor (AT2R) activation specifically within inguinal fat. Mice with hyperactivity of the brain RAS (“sRA” mice, expressing human renin via the synapsin promoter and human angiotensinogen via its own promoter) and littermate controls were chronically infused with vehicle or the AT2R agonist, CGP-42112a (CGP, 90 ng/hr, 8 wk, sc). To identify altered signaling pathways in sRA mice and their response to CGP treatment, total RNA was isolated from inguinal adipose tissue and transcript abundance was quantitated by RNA-Seq (50 bp paired reads sequenced with the Illumina HiSeq 2000). There was a significant (P&lt;0.001) change in expression of 123 genes in sRA mice (n=3) compared to littermate controls (n=3), which were generally reversed by CGP treatment (n=4). By manual inspection, we observed that 51 of these genes are associated with epidermal growth factor (EGF) signaling. Consistent with this, Gene Set Enrichment Analysis of the RNA-Seq data demonstrated that genes in the EGF receptor signaling pathway were, as a group, statistically upregulated (P=0.02) in sRA and suppressed (P=0.02) by CGP treatment. Therefore, to further explore the impact of increased EGF signaling on RMR and to isolate a potential modulatory effect of AT2R activation on this response, wildtype C57Bl/6J male mice (9 wk) were infused with EGF (833 ng/hr, 2 wk, sc) with or without co-infusion of CGP. This brief EGF infusion caused a possible increase in RMR (vehicle n=20, 0.173 ± 0.009; EGF n=20, 0.192 ± 0.012 kcal/hr, P=0.13), which was significantly reduced with co-infusion of CGP (90 ng/hr, 2 wk, sc) (EGF+CGP n=17, 0.165 ± 0.005, P&lt;0.05 vs EGF alone; CGP alone n=9, 0.156 ± 0.010). Together these data implicate elevated EGF signaling in the elevated RMR of mice with elevated brain RAS activity. Further, these data suggest a suppressive effect of AT2R activation upon RMR specifically in the context of elevated EGF signaling. Studies utilizing primary inguinal adipose cultures are underway to investigate the molecular basis of the EGF-AT2R interaction.

Direct calorimetry exposes inadequacies of respirometry in the measurement of resting metabolic rate

Resting metabolic rate (RMR) in animals is frequently estimated by respirometry, but the dependence of this method upon untested or untestable assumptions (such as negligible anaerobic metabolism, simultaneously measured or negligible protein oxidation, and no substrate inter‐conversion) render respirometry inappropriate or inadequate for examining novel genetically‐manipulated animal models. A Seebeck‐style direct calorimeter apparatus was constructed to allow simultaneous measurement of total heat dissipation and respirometry gasses from unrestrained mice. Double‐transgenic “sRA” mice exhibit elevated brain renin‐angiotensin system activity by expression of human renin via the synapsin promoter and human angiotensinogen via its own promoter. Respirometry underestimated the RMR in sRA mice by 16% (n=6, male, 11.0±0.6 wks, 20.97±0.68 g; 0.889±0.036 vs 1.057±0.034 kcal/100g lean/hr, P=0.008), and by 14% in control littermates and C57Bl/6J mice (n=10, male, 11.6±0.5 wks, 27.02±0.60 g; 0.712±0.025 vs 0.828±0.018 kcal/100g lean/hr, P=0.005). While respirometry faithfully detected the 26% increase in sRA RMR vs control (P&lt;0.001 by both methods), the magnitude of RMR estimation error by respirometry was similar (~15%). Reanalysis of various other genetically‐manipulated models by direct calorimetry may therefore uncover previously unappreciated physiological regulators of RMR.

Hypertension in mice with transgenic activation of the brain renin-angiotensin system is vasopressin dependent

An indispensable role for the brain renin-angiotensin system (RAS) has been documented in most experimental animal models of hypertension. To identify the specific efferent pathway activated by the brain RAS that mediates hypertension, we examined the hypothesis that elevated arginine vasopressin (AVP) release is necessary for hypertension in a double-transgenic model of brain-specific RAS hyperactivity (the "sRA" mouse model). sRA mice experience elevated brain RAS activity due to human angiotensinogen expression plus neuron-specific human renin expression. Total daily loss of the 4-kDa AVP prosegment (copeptin) into urine was grossly elevated (≥8-fold). Immunohistochemical staining for AVP was increased in the supraoptic nucleus of sRA mice (~2-fold), but no quantitative difference in the paraventricular nucleus was observed. Chronic subcutaneous infusion of a nonselective AVP receptor antagonist conivaptan (YM-087, Vaprisol, 22 ng/h) or the V(2)-selective antagonist tolvaptan (OPC-41061, 22 ng/h) resulted in normalization of the baseline (~15 mmHg) hypertension in sRA mice. Abdominal aortas and second-order mesenteric arteries displayed AVP-specific desensitization, with minor or no changes in responses to phenylephrine and endothelin-1. Mesenteric arteries exhibited substantial reductions in V(1A) receptor mRNA, but no significant changes in V(2) receptor expression in kidney were observed. Chronic tolvaptan infusion also normalized the (5 mmol/l) hyponatremia of sRA mice. Together, these data support a major role for vasopressin in the hypertension of mice with brain-specific hyperactivity of the RAS and suggest a primary role of V(2) receptors.

Abstract 84: Angiotensin II Type 1a Receptor in the Subfornical Organ is Essential for Blood Pressure Regulation and Hydromineral Effects in Mouse Models of Elevated Brain Renin-Angiotensin System Activity.

Elevated brain renin-angiotensin system (RAS) activity is necessary to increase blood pressure in many animal models of hypertension. We tested the hypothesis that AT1A receptors (AT1AR) within the subfornical organ (SFO) are required for the phenotypes that result from an increased brain RAS. We examined the effect of SFO-targeted Cre-recombinase mediated ablation of AT1A in mice treated with DOCA-salt (deoxycorticosterone acetate, 50 mg s.c. + ad lib 0.15 M NaCl). Mice homozygous for a conditional allele of the endogenous AT1A gene (AT1ARflox) were administered an adenovirus encoding Cre-recombinase and eGFP (AdCre), or eGFP alone (AdGFP) into the lateral cerebral ventricle, then treated for 3 weeks with DOCA-salt. AdCre reduced DOCA-salt hypertension (AdGFP baseline: 108±3 mmHg; AdGFP pre-DOCA: 104±3; AdGFP post-DOCA: 136±6 vs AdCre baseline: 116±2; Adcre pre-DOCA: 109±3, Adre post-DOCA: 118±5; P≤0.01), polydipsia (AdGFP+DOCA: 20.6±2.1 mL/day; AdCre+DOCA: 11.6±1.1, P&lt;0.05), and sodium intake (AdGFP+DOCA: 2.6±0.3 mEq/day; AdCre+DOCA: 1.8±0.2, P&lt;0.05). AdCre reduced AT1AR mRNA in the SFO (0.4±0.3 fold of AdGFP), without significant effect in the paraventricular or arcuate nuclei, or cortex; this was paralleled by SFO-specific AT1AR genomic DNA recombination. AdCre also caused SFO-specific recombination in ROSA-TdTomato reporter mice. Complementing the DOCA-salt model, we also examined the effect of AT1AR ablation in the SFO of double-transgenic sRA mice. sRA mice exhibit life-long brain-specific angiotensin overproduction via expression of human angiotensinogen via its own promoter and neuron-specific expression of human renin via the synapsin promoter. In sRA mice bred onto the AT1A conditional genetic background, AdCre significantly attenuated the polydipsia (AdGFP: -0.2±2; AdCre: -9.7±2.6 mL/day) and sodium intake (AdGFP: +0.2±0.7; AdCre: -1.3±0.4 mEq/day). Blood pressure measures are in progress. Together, these data highlight the involvement of SFO AT1A receptors in blood pressure in DOCA-salt model and additionally in hydromineral balance in two different models of increased brain RAS activity.