SR-A Mice Research Articles

Organic dust-induced lung injury and repair: Bi-directional regulation by TNFα and IL-10

Exposure to organic dust increases chronic airway inflammatory disorders. Effective treatment strategies are lacking. It has been reported that hog barn dust extracts (HDE) induce TNFα through protein kinase C (PKC) activation and that lung inflammation is enhanced in scavenger receptor A (SRA/CD204) knockout (KO) mice following HDE. Because interleukin (IL)-10 production can limit excessive inflammation, it was hypothesized here that HDE-induced IL-10 would require CD204 to effect inflammatory responses. C57BL/6 wild-type (WT), SRA KO, and IL-10 KO mice were intranasally challenged daily for 8 days with HDE and subsequently rested for 3 days with/without recombinant IL-10 (rIL-10) treatment. Primary peritoneal macrophages (PM) and murine alveolar macrophages (MH-S cells) were treated in vitro with HDE, SRA ligand (fucoidan), rIL-10, and/or PKC isoform inhibitors. HDE induced in vivo lung IL-10 in WT, but not SRA KO mice, and similar trends were demonstrated in isolated PM from same treated mice. Lung lymphocyte aggregates and neutrophils were elevated in in vivo HDE-treated SRA and IL-10 KO mice after a 3-d recovery, and treatment during recovery with rIL-10 abrogated these responses. In vitro rIL-10 treatment reduced HDE-stimulated TNFα release in MH-S and WT PM. In SRA KO macrophages, there was reduced IL-10 and PKC zeta (ζ) activity and increased TNFα following in vitro HDE stimulation. Similarly, blocking SRA (24 hr fucoidan pre-treatment) resulted in enhanced HDE-stimulated macrophage TNFα and decreased IL-10 and PKCζ activation. PKCζ inhibitors blocked HDE-stimulated IL-10, but not TNFα. Collectively, HDE stimulates IL-10 by an SRA- and PKCζ-dependent mechanism to regulate TNFα. Enhancing resolution of dust-mediated lung inflammation through targeting IL-10 and/or SRA may represent new approaches to therapeutic interventions.

All that is silver is not toxic: silver ion and particle kinetics reveals the role of silver ion aging and dosimetry on the toxicity of silver nanoparticles

BackgroundWhen suspended in cell culture medium, nano-objects composed of soluble metals such as silver can dissolve resulting in ion formation, altered particle properties (e.g. mass, morphology, etc.), and modulated cellular dose. Cultured cells are exposed not just to nanoparticles but to a complex, dynamic mixture of altered nanoparticles, unbound ions, and ion-ligand complexes. Here, three different cell types (RAW 264.7 macrophages and bone marrow derived macrophages from wild-type C57BL/6 J mice and Scavenger Receptor A deficient (SR-A(−/−)) mice) were exposed to 20 and 110 nm silver nanoparticles, and RAW 264.7 cells were exposed to freshly mixed silver ions, aged silver ions (ions incubated in cell culture medium), and ions formed from nanoparticle dissolution. The In Vitro Sedimentation, Diffusion, Dissolution, and Dosimetry Model (ISD3) was used to predict dose metrics for each exposure scenario.ResultsSilver nanoparticles, freshly mixed ions, and ions from nanoparticle dissolution were toxic, while aged ions were not toxic. Macrophages from SR-A(−/−) mice did not take up 20 nm silver nanoparticles as well as wild-types but demonstrated no differences in silver levels after exposure to 110 nm nanoparticles. Dose response modeling with ISD3 predicted dose metrics suggest that amount of ions in cells and area under the curve (AUC) of ion amount in cells are the most predictive of cell viability after nanoparticle and combined nanoparticle/dissolution-formed-ions exposures, respectively.ConclusionsResults of this study suggest that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to nanoparticles. Applying computational modeling (ISD3) to better understand dose metrics for soluble nanoparticles allows for better interpretation of in vitro hazard assessments.

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.

Evidence for intraventricular secretion of angiotensinogen and angiotensin by the subfornical organ using transgenic mice.

Direct intracerebroventricular injection of angiotensin II (ANG II) causes increases in blood pressure and salt and water intake, presumably mimicking an effect mediated by an endogenous mechanism. The subfornical organ (SFO) is a potential source of cerebrospinal fluid (CSF), ANG I, and ANG II, and thus we hypothesized that the SFO has a secretory function. Endogenous levels of angiotensinogen (AGT) and renin are very low in the brain. We therefore examined the immunohistochemical localization of angiotensin peptides and AGT in the SFO, and AGT in the CSF in two transgenic models that overexpress either human AGT (A+ mice), or both human AGT (hAGT) and human renin (SRA mice) in the brain. Measurements were made at baseline and following volumetric depletion of CSF. Ultrastructural analysis with immunoelectron microscopy revealed that superficially located ANG I/ANG II and AGT immunoreactive cells in the SFO were vacuolated and opened directly into the ventricle. Withdrawal of CSF produced an increase in AGT in the CSF that was accompanied by a large decline in AGT immunoreactivity within SFO cells. Our data provide support for the hypothesis that the SFO is a secretory organ that releases AGT and possibly ANG I/ANG II into the ventricle at least under conditions when genes that control the renin-angiotensin system are overexpressed in mice.

Suppression of Resting Metabolism by the Angiotensin AT2 Receptor

Activation of the brain renin-angiotensin system (RAS) stimulates energy expenditure through increasing of the resting metabolic rate (RMR), and this effect requires simultaneous suppression of the circulating and/or adipose RAS. To identify the mechanism by which the peripheral RAS opposes RMR control by the brain RAS, we examined mice with transgenic activation of the brain RAS (sRA mice). sRA mice exhibit increased RMR through increased energy flux in the inguinal adipose tissue, and this effect is attenuated by angiotensin II type 2 receptor (AT2) activation. AT2 activation in inguinal adipocytes opposes norepinephrine-induced uncoupling protein-1 (UCP1) production and aspects of cellular respiration, but not lipolysis. AT2 activation also opposes inguinal adipocyte function and differentiation responses to epidermal growth factor (EGF). These results highlight a major, multifaceted role for AT2 within inguinal adipocytes in the control of RMR. The AT2 receptor may therefore contribute to body fat distribution and adipose depot-specific effects upon cardio-metabolic health.

MTORC1 Signaling Contributes to Drinking But Not Blood Pressure Responses to Brain Angiotensin II.

Mechanistic target of rapamycin complex 1 (mTORC1) is a molecular node that couples extracellular cues to a wide range of cellular events controlling various physiological processes. Here, we identified mTORC1 signaling as a critical mediator of angiotensin II (Ang II) action in the brain. In neuronal GT1-7 cells, we show that Ang II stimulates neuronal mTORC1 signaling in an Ang II type 1 receptor-dependent manner. In mice, a single intracerebroventricular (ICV) injection or chronic sc infusion of Ang II activated mTORC1 signaling in the subfornical organ, a critical brain region in cardiovascular control and fluid balance. Moreover, transgenic sRA mice with brain-specific overproduction of Ang II displayed increased mTORC1 signaling in the subfornical organ. To test the functional role of brain mTORC1 in mediating the action of Ang II, we examined the consequence of mTORC1 inhibition with rapamycin on Ang II-induced increase in water intake and arterial pressure. ICV pretreatment with rapamycin blocked ICV Ang II-mediated increases in the frequency, duration, and amount of water intake but did not interfere with the pressor response evoked by Ang II. In addition, ICV delivery of rapamycin significantly reduced polydipsia, but not hypertension, of sRA mice. These results demonstrate that mTORC1 is a novel downstream pathway of Ang II type 1 receptor signaling in the brain and selectively mediates the effect of Ang II on drinking behavior.

Abstract P072: Adipocyte AT 2 Receptor Suppresses UCP1 Transcription and Thereby Resting Metabolic Rate

The renin-angiotensin system (RAS) contributes to energy balance through opposing actions in the brain and periphery. We hypothesize that tissue- and receptor-specific RAS modulation may represent a novel therapeutic approach to obesity. Transgenic “sRA” mice exhibit brain-specific RAS hyperactivity through expression of human renin in neurons (synapsin promoter) and human angiotensinogen via its own promoter. Previously we documented that sRA mice exhibit a suppressed circulating RAS, and an elevated resting metabolic rate (RMR) that is sensitive to replacement of circulating angiotensin II or the AT 2 receptor (AT 2 R) agonist, CGP-42112a (CGP, 100 ng/kg/min, s.c.). sRA mice exhibit a robust and specific elevation in uncoupling protein-1 (UCP1) expression and glucose uptake in inguinal white adipose tissue (iWAT). Chronic infusion of the AT 2 R agonist, CGP-42112a (CGP) into sRA mice increased weight gain and normalized RMR and inguinal fat expression of UCP1, but had no effect on food intake or digestive efficiency. RMR suppression by CGP was not mediated through attenuation of adipose sympathetic nervous activity (SNA, measured via multifiber nerve recordings), leading to the hypothesis that AT 2 R action on the adipocyte suppresses adipose sensitivity to SNA. Cultured primary mouse adipocytes were isolated from the iWAT, and norepinephrine (NE, 1 nM) treatment induced UCP1 mRNA (597 -fold of vehicle, P<0.05), however this UCP1 induction was attenuated with CGP co-treatment (10 nM, 0.2 -fold of NE alone, P<0.05). In human adipocytes, activation of AT 2 R also prevented the NE-induced increase in UCP1 mRNA. NE additionally significantly increased lipolysis by adipocytes (measured via free glycerol in the media; P<0.05), but AT 2 R activation had no effect (vehicle= 15.9 ug/mL; NE= 70.8 ug/mL; NE + CGP=64.7 ug/mL). These data support a suppressive action of AT 2 R upon RMR that is mediated specifically through the suppression of UCP1 expression, but not its activation by lipolysis, within subcutaneous adipocytes. Ongoing studies are examining the transcriptional regulation of UCP1 by adipose AT 2 R activation.

Abstract P003: Hyperactive Brain Renin Angiotensin System Induces Polydipsia Through mTORC1 Pathway

Mechanistic target of rapamycin complex 1 (mTORC1) is a molecular hub for signaling pathways mediating a wide range of cellular events involved in the regulation of various physiological and pathophysiological processes. We previously demonstrated that Angiotensin II (Ang II) activates mTORC1 and its downstream effector ribosomal protein S6 kinase in neurons in vitro . Here, we investigated the role of brain mTORC1 in hypertension and polydipsia induced by Ang II. In wild-type mice, acute stimulation of angiotensin type 1 receptor signaling by intracerebroventricular (ICV) injection of Ang II (1 μg, 30 min) activated mTORC1 signaling in the subfornical organ (SFO), a critical brain region in cardiovascular control and fluid balance, as indicated by the significant increase in the number of phosphorylated S6-positive cells (32±2 vs 13±3 in vehicle group). Similar upregulation of the mTORC1 pathway in the SFO was also found in the mice treated subcutaneously with Ang II (1000 ng/kg/min) using an osmotic minipump for 1 week (27±3 vs 11±2 in vehicle group). To verify functional roles of the Ang II activation of mTORC1 in the SFO, we utilized hypertensive and polydipsic transgenic mice (sRA) that have a hyperactive brain renin-angiotensin system, resulting in SFO-overproduction of Ang II. Interestingly, sRA mice exhibited substantially elevated phospho-S6 immunoreactivity only in the SFO (64±6 vs 36±8 in controls) but not in other cardiovascular regulatory regions including the paraventricular nucleus. ICV delivery of mTORC1 blocker, rapamycin (10 ng/day for 7 days) significantly (p<0.05) reduced daily water intake (-4.5±0.7 mL) compared to vehicle-treated sRA mice (-0.7±0.6 mL). In contrast, systolic blood pressure remained unchanged with rapamycin treatment (123±1 vs 125±4 mmHg in pre-treatment) and was consistently higher than the control group (110±4 mmHg). These results suggest that mTORC1 activity in the SFO is a critical determinant of the polydipsia evoked by Ang II.

Direct pro-inflammatory effects of prorenin on microglia.

Neuroinflammation has been implicated in hypertension, and microglia have been proposed to play an important role in the progression of this disease. Here, we have studied whether microglia are activated within cardiovascular regulatory area(s) of the brain during hypertension, especially in high blood pressure that is associated with chronic activation of the renin-angiotensin-system. In addition, we determined whether prorenin, an essential component of the renin-angiotensin-system, exerts direct pro-inflammatory effects on these microglia. Our data indicate that two rodent models which display neurogenic hypertension and over activation of the renin-angiotensin-system in the brain (sRA mice and spontaneously hypertensive rats) exhibit microglial activation, and increased levels of pro-inflammatory cytokines, in the paraventricular nucleus of the hypothalamus, an area crucial for regulation of sympathetic outflow. Further, the renin-angiotensin-system component prorenin elicits direct activation of hypothalamic microglia in culture and induction of pro-inflammatory mechanisms in these cells, effects that involve prorenin receptor-induced NFκB activation. In addition, the prorenin-elicited increases in cytokine expression were fully abolished by microglial inhibitor minocycline, and were potentiated by pre-treatment of cells with angiotensin II. Taken together with our previous data which indicate that pro-inflammatory processes in the paraventricular nucleus are involved in the hypertensive action of renin-angiotensin-system, the novel discovery that prorenin exerts direct stimulatory effects on microglial activation and pro-inflammatory cytokine production provides support for the idea that renin-angiotensin-system -induced neurogenic hypertension is not restricted to actions of angiotensin II alone.

Abstract 015: Regulation of Resting Metabolism by the Angiotensin AT 2 Receptor

The renin-angiotensin system (RAS) positively correlates with obesity, and contributes to energy homeostasis through opposing actions in the brain and adipose. We hypothesize that site- and receptor-specific modulation may represent a novel therapeutic target for obesity. Transgenic “sRA” mice exhibit brain-specific RAS hyperactivity through expression of human renin in neurons (synapsin promoter) and human angiotensinogen via its own promoter. Previously we documented that sRA mice exhibit a suppressed circulating RAS, and an elevated resting metabolic rate (RMR) that is sensitive to replacement of circulating angiotensin II or the AT 2 receptor (AT 2 R) agonist, CGP-42112a (CGP, 100 ng/kg/min, s.c.). sRA mice consume more food than littermate controls (con n=7, 12.98±0.65 vs sRA n=8, 15.41±0.70 g/d, P<0.05), but because of a major suppression of digestive efficiency (con 77.6±2.3 vs sRA 59.3±4.7 % consumed, P<0.05), sRA mice absorb a normal number of calories (con 10.04±0.50 vs sRA 9.07±0.76 kcal/d). Chronic CGP had no effect on total daily caloric absorption (con+CGP n=7, 9.83±0.95 vs sRA+CGP n=5, 9.32±0.80 kcal/d); however CGP appears to disproportionately increase weight gain in sRA mice (vehicle +0.33±0.49 vs CGP +1.54±0.47 g/8 wks, P=0.27) compared to control mice (vehicle +2.12±1.00 vs CGP +2.47±0.73 g/8 wks, P=0.73), consistent with a suppression of energy expenditure by CGP. Given the increased RMR and core temperature in sRA mice, we next examined the expression of uncoupling protein-1 (UCP1) content of thermogenic adipose tissues by Western blot (all n=3, vs perilipin). Interscapular brown adipose tissue UCP1 was unchanged in sRA mice (94±6%), and weakly suppressed by CGP treatment (84±4% of sRA). Inguinal adipose UCP1 was increased in sRA mice (198±13%), and this was suppressed by CGP treatment (72±9% of sRA). UCP1 mRNA levels paralleled protein in both fat types. Cultured adipocytes from 4 day old AT 2 -deficient mice exhibited increased UCP1 protein vs littermate controls (190%). Further, CGP (10 nM) reduced UCP1 in control adipocytes (by 30%). These data support a suppressive action of AT 2 R upon RMR most likely through UCP1. Inguinal adipose AT 2 R may therefore contribute to obesity through suppression of RMR.

Abstract 083: Role of Endoplasmic Reticulum Stress in the Subfornical Organ (SFO) in Fluid Balance and Metabolic Effects of Brain Renin-Angiotensin System (RAS) Activation

Endoplasmic reticulum (ER) stress has been identified as an important contributor to neurological diseases and implicated in mediating hypothalamic inflammation and the hypertensive effects of angiotensin II (AngII). We examined the role of ER stress in the metabolic and fluid balance effects of brain AngII in two mouse models: 1) sRA transgenic mice (expressing human renin in neurons and human angiotensinogen in glia and neurons), and 2) DOCA-salt treated C57BL/6J mice. Both DOCA-salt and sRA mice exhibit hyperactivity of the brain RAS, suppression of circulating RAS, hypertension, polydipsia, and an elevated resting metabolic rate (RMR). CCAAT-enhancer-binding protein homologous protein (CHOP), a marker of chronic ER stress, was examined by immunocytochemistry in the brain of both models. CHOP immunoreactivity was evident in the SFO of sRA and DOCA-salt mice but was absent in control and CHOP-/- mice. We infused the ER stress-reducing chemical chaperone tauroursodeoxycholic acid (TUDCA, 5.28 ug/day, or aCSF vehicle) to assess if ER stress is mechanistically related to the hypertension, polydipsia, and elevated RMR observed in both models. In initial studies, ICV TUDCA significantly attenuated the polydipsia (aCSF 20.7±0.9 vs TUDCA 10.8±1.0 mL/day, n=6,2) and RMR (aCSF, 3.38±0.07 vs TUDCA 3.16±0.06 mL O2/100g/min, P<0.05 n=13,11) in the DOCA-salt model. ICV TUDCA had similar effects on the polydipsia in the sRA model (51±10% of aCSF control, P<0.05 n=3,4). In the DOCA-salt model, daily ICV injections of TUDCA (10 days, 5ug/ul) markedly reduced drinking, but polydipsia returned one day after the injections were terminated (n=14,12). Daily ICV injection of another ER stress reducer 4-phenylbutyrate (5ug/ul) also reduced drinking (P<0.05 n=5,4). To assess the functional role of CHOP, we measured RMR and water intake in CHOP-/- mice. Interestingly, CHOP-/- mice exhibited increased baseline RMR (CHOP-/- 0.161±0.010 vs C57 0.140±0.005 kcal/hr, P<0.05 n=10,9). The increase in water intake in response to DOCA-salt was blunted (32.7±0.5 vs 22.8±1.1 ml/day, P<0.05, n=4,5) in CHOP-/- mice. Together these data mechanistically implicate ER stress in the fluid and metabolic responses to increased brain RAS activity and suggest CHOP may play a functional role.

Activity of protein kinase C-α within the subfornical organ is necessary for fluid intake in response to brain angiotensin.

Angiotensin-II production in the subfornical organ acting through angiotensin-II type-1 receptors is necessary for polydipsia, resulting from elevated renin-angiotensin system activity. Protein kinase C and mitogen-activated protein kinase pathways have been shown to mediate effects of angiotensin-II in the brain. We investigated mechanisms that mediate brain angiotensin-II-induced polydipsia. We used double-transgenic sRA mice, consisting of human renin controlled by the neuron-specific synapsin promoter crossed with human angiotensinogen controlled by its endogenous promoter, which results in brain-specific overexpression of angiotensin-II, particularly in the subfornical organ. We also used the deoxycorticosterone acetate-salt model of hypertension, which exhibits polydipsia. Inhibition of protein kinase C, but not extracellular signal-regulated kinases, protein kinase A, or vasopressin V₁A and V₂ receptors, corrected the elevated water intake of sRA mice. Using an isoform selective inhibitor and an adenovirus expressing dominant negative protein kinase C-α revealed that protein kinase C-α in the subfornical organ was necessary to mediate elevated fluid and sodium intake in sRA mice. Inhibition of protein kinase C activity also attenuated polydipsia in the deoxycorticosterone acetate-salt model. We provide evidence that inducing protein kinase C activity centrally is sufficient to induce water intake in water-replete wild-type mice, and that cell surface localization of protein kinase C-α can be induced in cultured cells from the subfornical organ. These experimental findings demonstrate a role for central protein kinase C activity in fluid balance, and further mechanistically demonstrate the importance of protein kinase C-α signaling in the subfornical organ in fluid intake stimulated by angiotensin-II in the brain.

Essential role of mTORC1 in the hypertension induced by overactivation of the brain renin‐angiotensin system (1067.2)

Angiotensin II (Ang II) is a major effector peptide of the renin‐angiotensin system (RAS). Ang II‐induced activation of angiotensin type 1 receptor (AT1R) signaling in cardiovascular‐related brain nuclei such as the subfornical organ (SFO) is thought to be important for the development of hypertension. However, the molecular machinery underlying the central pressor actions of Ang II is poorly defined. We previously demonstrated the involvement of neuronal mammalian target of rapamycin complex 1 (mTORC1) in blood pressure (BP) regulation. Interestingly, using in vitro systems we also found that mTORC1 is a downstream element of neuronal AT1R signaling. To assess the relevance of mTORC1 for Ang II action, we first examined mTORC1 activity in the cardiovascular brain regions in hypertensive transgenic mice with brain‐specific hyperactive RAS (sRA mice). Strikingly, the number of phospho‐S6 positive cells (used as a readout of mTORC1 activity) was significantly higher in the SFO of sRA mice (64±6) relative to controls (36±8, p<0.05, n=5). Moreover, central administration of an adenovirus encoding a dominant negative S6 kinase (an mTORC1 substrate) lowered systolic BP in sRA (from 148±8 to 130±8 mmHg, p<0.05, n=3) to comparable levels as control mice receiving GFP adenovirus (from 125±7 to 126±1 mmHg, n=3). These results indicate that neuronal mTORC1 activation is a critical event in brain RAS‐induced BP elevation.

Abstract 173: Modulation of the Metabolic Effects of Leptin by the Brain Renin-Angiotensin System

Genetic deletion or delivery of antagonists of the renin-angiotensin system (RAS) directly to the brain abolishes both renal and adipose sympathetic nervous activity (SNA) responses to leptin, suggesting a cross-talk between these central cardiovascular / metabolic control systems. To further explore this mechanism of cross-talk, we examined the sensitivity of metabolic responses in mice with transgenic hyperactivity of the brain RAS (“sRA” mice) to acute leptin treatment. sRA mice, previously shown to exhibit hypertension, polydipsia, and elevated SNA and resting metabolic rate, exhibit brain-specific RAS hyperactivity through neuronal expression of human renin via the synapsin promoter and expression of human angiotensinogen via its own promoter. When housed at standard room temperature (23°C), twice-daily leptin injections (1 mg/kg, i.p., 3 hrs into light phase and 2 hrs preceding dark phase of a 12:12-hr cycle) caused significant and similar reductions in body mass (control -0.76±0.13, n=5 male; sRA -0.65±0.09 g/d, n=5 male, P=0.46) and food intake (-1.43±0.69 vs -1.99±0.27 kcal/d, P=0.63) in littermate control and sRA mice. When housed at thermoneutrality (30°C) in an OxyMax (Columbus Instruments) CLAMS apparatus, leptin injections had minimal effects on total daily food intake in control mice (from 7.4±0.6 at baseline to 8.1±0.5 kcal/d with leptin, n=4 male + 4 female, P=0.29), but reduced food intake in sRA mice (from 8.3±0.7 to 6.0±0.4 kcal/d, n=4 male + 4 female, P=0.003). sRA mice were more sensitive to leptin’s suppressive effect on physical activity (control from 998±148 to 655±90, P=0.10; sRA from 1232±304 to 588±41 counts/d, P=0.005). Unexpectedly, leptin had a suppressive effect upon metabolic rate in sRA mice (control from 16.4±1.3 to 16.8±0.9, P=0.44; sRA from 20.2±1.1 to 17.5±0.9 kcal/kg lean/hr, P<0.001). Together, these data support a cross-talk between the brain RAS and leptin signaling in the control of metabolic function. These data suggest a complex and context-dependent mechanism of interaction between leptin and brain angiotensin, with gross alterations in leptin-induced food, physical activity, and metabolic rate responses when tested in a thermoneutral environment.

Abstract 219: Adipose Angiotensin AT 2 Receptors Inhibit Resting Metabolism

The brain renin-angiotensin system (RAS) modulates blood pressure and resting metabolic rate (RMR). Double-transgenic “sRA” mice exhibit brain-specific elevations in RAS activity, as a human renin transgene is expressed via the neuron-specific synapsin promoter and a human angiotensinogen transgene is expressed via its own promoter. sRA mice have a significantly lower body weight due primarily to increased RMR, which is sustained through chronically increased sympathetic nerve activity (SNA) and a hypertension-mediated suppression of the circulating RAS. Here we identified the anatomical site of increased thermogenesis, and the components of the adipose/circulating RAS that suppress RMR in the sRA model. Expression of uncoupling protein 1 (UCP1) was elevated specifically within the inguinal fat of sRA mice, with no changes in UCP1 in interscapular or epididymal fat or skeletal muscle, and no change in UCP3 in skeletal muscle or any fat pad. UCP1 mRNA in differentiated 3T3L1 adipocytes was increased following treatment with the AT 2 R antagonist PD-123,319 and suppressed with the AT 2 R agonist CGP-42112a (CGP), but was unaffected by the AT 1 R antagonist losartan. Infusion of CGP (50 ng/kg/min, 8 wk, sc) into adult sRA mice normalized body mass (control n=5, 25.0±1.8; sRA n=5, 17.7±0.6*; sRA+CGP n=7, 22.7±2.0** g), epididymal fat (control 317±100, sRA 127±16*, sRA+CGP 247±45** mg/g), and RMR (control 2.8±0.2, sRA 3.6±0.2*, sRA+CGP 3.1±0.2** mL O 2 /100g/min). Food intake was unchanged across groups (control 10.5±0.5, sRA 9.8±0.6, sRA+CGP 11.2±0.9 kcal/d). CGP treatment did not suppress SNA to inguinal fat in sRA mice (control 9.7±1.0, sRA 14.7±1.3*, sRA+CGP 18.9±3.3* spikes/sec). sRA mice exhibit generally normal glucose tolerance but grossly increased insulin sensitivity (0.5 U/kg; control nadir 72±8, sRA nadir 27±5 mg/dL), and ongoing studies are designed to determine whether adipose insulin signaling is modulated by AT 2 R activation. (Throughout, * P<0.05 vs control, ** P<0.05 vs sRA.) We conclude that hyperactivity of the brain RAS stimulates RMR (and possibly insulin sensitivity) specifically within the subcutaneous inguinal fat pad, and identifies AT 2 R signaling as an important regulator of thermogenic activity of adipose tissue.

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<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<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.

Abstract 176: Role of Endoplasmic Reticulum Stress in the SFO in Fluid Balance and Metabolic Effects of Brain Renin-Angiotensin System Activation

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been identified as important contributors to neurological diseases and have been implicated in mediating hypothalamic inflammation and the hypertensive response to angiotensin II. We examined the role of ER stress and the UPR in the metabolic and fluid balance effects of brain angiotensin in two mouse models: 1) “sRA” transgenic mice (expressing human renin in neurons via the synapsin promoter crossed with human angiotensinogen via its own promoter), and 2) DOCA-salt treated C57BL/6J mice. Both DOCA-salt and sRA mice exhibit hyperactivity of the brain RAS, suppression of circulating RAS, hypertension, polydipsia, and an elevated resting metabolic rate. We examined the accumulation of UPR biomarker CCAAT-enhancer-binding protein homologous protein (CHOP) by immunocytochemistry in the brain of both models. CHOP is considered a marker of chronic ER stress. Increased CHOP immunoreactivity was evident in the subfornical organ (SFO) of sRA mice but was absent in non-transgenic (NT) and CHOP-/- mice. There was also increased CHOP immunoreactivity in the SFO of DOCA-salt mice compared with untreated controls. Next, we infused the ER stress-reducing chemical chaperone tauroursodeoxycholic acid (TUDCA, 5.28 ug/day, or aCSF vehicle) to assess if ER stress is mechanistically related to the hypertension, polydipsia, and elevated resting metabolic rate observed in both models. ICV TUDCA (3-5 day pretreatment then continuously for 3 wks) significantly attenuated the polydipsia (aCSF 20.7±0.9 vs TUDCA 10.8±1.0 mL/day, P<0.05) and metabolic rate (aCSF, 3.38±0.07 vs TUDCA 3.16±0.06 mL O2/100g/min, P<0.05) in the DOCA-salt model. ICV TUDCA (3 wks) had similar effects on the polydipsia in the sRA model (51±10% of aCSF control, P<0.05). DOCA-salt caused (P<0.05) increases in 24 hr mean arterial pressure (MAP) that were unaffected by ICV TUDCA (aCSF baseline 114±3 to 128±4 mmHg with DOCA-salt; TUDCA 117±7 to 129±4). Heart rate responses to DOCA-salt were attenuated (P<0.05) with ICV TUDCA (aCSF baseline 528±10 to 448±15 BPM with DOCA-salt; TUDCA 529±6 to 486±11). Together these data mechanistically implicate ER stress in the fluid and metabolic responses to increased brain RAS activity.

Glycemic control by the brain renin‐angiotensin system: Role for peripheral AT2 receptors

The effect of hyperactivity of the brain renin‐angiotensin system (RAS) on metabolism was explored using double‐transgenic “sRA” mice, which express human renin via the synapsin promoter and human angiotensinogen via its own promoter. sRA mice exhibit hypertension, polydipsia, and elevated resting metabolic rate (RMR). Compared to control littermates (n=9), body mass was lower in sRA (n=9) mice (25.11±1.26 vs 18.95±0.87 g, P<0.05), and i.p. glucose tolerance (ipGTT, 2 g/kg total body) was improved (genotype x time P=0.03). Lean mass by NMR was reduced in sRA (16.54±0.79 vs 12.62±0.63, P<0.05), but proportional lean mass was normal (66.1±.52 vs 66.69±1.48 % total). Lean ipGTT (2g/kg lean body, P=0.29) was normal, and tolerance to a bolus dose of glucose was reduced (28 mg/mouse, P<0.001). sRA mice were more sensitive to insulin, as i.p. insulin (0.5 U/kg total body) caused dramatic drops in blood glucose (control nadir 72±8 vs sRA nadir 27±5 mg/dL and occasionally death, genotype x time P<0.02). As the RMR increase in sRA is mediated through suppression of adipose AT2 receptor activation, we tested the effects of chronic infusion of CGP‐42112a (50 ng/kg/min) on ipGTT. Chronic CGP‐42112a significantly reduced ipGTT (2 g/kg total body, AUC +23%, P=0.008) in both control and sRA mice. Together, these data suggest that the brain RAS may improve glycemic control through tonic suppression of peripheral AT2 activation.

Regulation of adipose thermogenesis by Epidermal Growth Factor and angiotensin AT2 receptor activation

Transgenic “sRA” mice with hyperactivity of the brain renin‐angiotensin system (RAS) express human renin via the synapsin promoter and human angiotensinogen via its own promoter. sRA mice exhibit increased resting metabolic rate (RMR) due to reduced activation of adipose angiotensin AT2 receptors. To identify downstream pathways, RNA‐Seq was performed using inguinal adipose from sRA and littermate controls, with or without infusion of the AT2 agonist CGP‐42112a (“CGP,” 50 ng/kg/min, s.c.). In sRA vs controls, there were significant expression changes in 123 genes, which were mostly normalized by CGP. 51 of these genes are associated with epidermal growth factor (EGF). To determine the sufficiency of EGF to increase RMR, male C57Bl/6J mice were infused with EGF (0.8 ug/hr, 2 weeks, n=8) or saline (n=9), resulting in increased RMR (+38±12 cal/hr vs control, P=0.006), and a trend toward reduced inguinal (176±21 vs 140±17 mg P=0.20) and increased brown (54±6 vs 68±4 mg, P=0.08) adipose. Co‐infusion of CGP (n=5) caused trends toward ameliorated RMR (+15±22 cal/hr vs control, P=0.31 vs control, P=0.13 vs EGF) and inguinal adipose (187±21 mg, P=0.15 vs EGF). Ongoing studies are aimed at optimizing doses of EGF and CGP in C57Bl/6J mice, and at quantifying EGF and evaluating the effects of chronic EGF blockade in sRA mice. Together these data support a role for EGF, and modulation of EGF signaling by AT2 receptors, in RMR control.

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<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.