The Brain Renin-Angiotensin System Controls Divergent Efferent Mechanisms to Regulate Fluid and Energy Balance

The Brain Renin-Angiotensin System Controls DivergentEfferent Mechanisms to Regulate Fluid and Energy Balance

Justin L. Grobe1, Connie L. Grobe2, Terry G. Beltz2, Scott G. Westphal1, Donald A.Morgan1, Di Xu1, Willem J. de Lange1, Huiping Li1, Koji Sakai1, Daniel R. Thedens3, Lisa A.Cassis4, Kamal Rahmouni1, Allyn L. Mark1, Alan Kim Johnson2, and Curt D. Sigmund1

1 Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University ofIowa, Iowa City, IA 522422 Department of Psychology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa,Iowa City, IA 522423 Department of Radiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa,Iowa City, IA 522424 Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40536

SummaryThe renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role withinindividual tissues such as the brain. The brain RAS is thought to control blood pressure througheffects on fluid intake, vasopressin release and sympathetic nerve activity (SNA), and mayregulate metabolism through mechanisms which remain undefined. We used a double-transgenicmouse model that exhibits brain-specific RAS activity to examine mechanisms contributing tofluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenalsteroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptorblockade. They are also hyperphagic but lean because of a marked increase in body temperatureand metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalizedmetabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluidintake and energy expenditure.

IntroductionDespite a growing body of evidence supporting the existence of local, tissue-levelexpression and activity of the renin-angiotensin system (RAS), there persists a general lackof appreciation for the roles of these autocrine/paracrine systems in both normal physiologyand in pathophysiological states. Indeed, major roles for the RAS in the vasculature, heart,and kidney in the development and maintenance of hypertension and its sequelae have beenreported (Paul et al., 2006).

Corresponding Author: Curt D. Sigmund, Ph.D., Departments of Internal Medicine & Molecular Physiology & Biophysics, 3181MERF, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, Tel: (319) 335-7604, Fax: (319)353-5350, [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptCell Metab. Author manuscript; available in PMC 2011 November 3.

Published in final edited form as:Cell Metab. 2010 November 3; 12(5): 431–442. doi:10.1016/j.cmet.2010.09.011.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

The brain, like several peripheral organ systems, expresses all components of the RAS, andgenerates angiotensin peptides locally. There is evidence to support the hypothesis thatangiotensin peptides may be utilized as neurotransmitters, acting in certain cardiovascular-control substructures of the brain (reviewed in Grobe et al., 2008, Ferguson et al., 2001, andJohnson 1985). Through autocrine/paracrine signaling and acting as a neurotransmitter, thebrain RAS is thought to control blood pressure (Bickerton and Buckley, 1961), fluid balance(Booth, 1968, Daniels et al., 1969, and Epstein et al., 1970), learning, memory and anxiety(reviewed in Wright et al., 2008), and metabolic function (Porter et al., 2003, and Porter andPotratz, 2004). Abnormalities of the brain RAS leading to its over-activity have beenimplicated in several forms of genetic and experimental hypertension (Veerasingham andRaizada, 2003). Despite the recognition that the brain RAS is involved in these processes,the efferent mechanisms mediating these functions remain unclear.

To examine the functions of the brain RAS, we developed a double-transgenic mouse modelexpressing human renin controlled by the neuron-specific synapsin promoter (“sR”) and thehuman angiotensinogen gene controlled by its own promoter (“A”) (Sakai et al., 2007). Dueto the strict species-specificity of the renin-mediated cleavage of angiotensinogen, thehuman angiotensinogen protein is only cleaved into angiotensin I in the presence of humanrenin, so, despite a widespread expression of human angiotensinogen, RAS hyperactivityonly occurs in tissues where both transgenes are active. Double-transgenic “sRA” miceexhibit RAS hyperactivity only in the brain regions where angiotensinogen is normallyexpressed, and thus this model may phenocopy hypertension caused by increased brain RASactivity. Using this unique sRA animal model, we have identified divergent efferentsignaling mechanisms that mediate the marked hydromineral and metabolic consequences ofincreased brain RAS activity. The physiological relevance of our model was confirmed byshowing similar metabolic consequences in an independent experimental model of brainRAS-mediated hypertension.

ResultssRA Mice Exhibit Polydipsia and Polyuria

sRA mice exhibit robust polydipsia and polyuria (Figure 1A–B). Remarkably, they consumethe equivalent of their own body mass of fluids, per day, when offered tap water and 0.15 MNaCl in a two-bottle choice paradigm. To examine whether the elevated intake of fluids insRA mice was dependent upon the concentration of the NaCl solutions offered, weperformed a NaCl preference-aversion experiment by observing 24 hour intakes of waterand a range of hypotonic to hypertonic NaCl drink solutions (Figure 1B). sRA micedisplayed preference-aversion behavior that was indistinguishable from control mice, buttotal fluid intake remained greatly elevated in sRA mice across all solute concentrations.Total sodium intake from both food and drink solutions was similar between sRA andcontrol mice at hypotonic and hypertonic concentrations, but sodium intake was greatlyincreased when sRA mice were offered isotonic (0.15 M) NaCl. The elevation in sodiumintake when offered at an isotonic concentration may represent a taste-dependent or a post-ingestive effect, but does not fit the classical definition of an increased sodium appetitebecause the sRA mice did not consume excess sodium when presented at unpalatable,hypertonic concentrations. In addition, sRA mice consistently produced a large volume ofdilute urine across all solute concentrations. Urine sodium (UNaC) and potassium (UKC)concentrations, and urine osmolality were significantly suppressed in sRA mice (Figure 1B,Figure S1). Renal sympathetic nerve activity (RSNA) was markedly elevated (Figure 1C),perhaps as a compensatory mechanism to retain sodium in the face of an exaggerateddiuresis. Indeed, sRA mice exhibit an 8% increase in hematocrit suggesting that they may bedehydrated (Figure 1D). This is unlikely to be due to increased hematopoiesis, as renalerythropoietin expression is not elevated in sRA mice (P=0.42). Although serum osmolality

Grobe et al. Page 2

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

and serum potassium concentrations are normal in sRA mice, serum sodium concentrationsare significantly decreased (Figure 1E), thus suggesting sRA mice exhibit selective, chronichyponatremia. Interestingly, there is increased lethality associated with the sRA genotype, asonly 1-in-3 sRA pups survive until weaning, suggesting neonatal sRA mice may havedifficulty retaining sodium (Figure 1F).

Dehydration and brain angiotensin-II are known stimuli for the release of argininevasopressin (AVP) and the brain RAS is known to regulate AVP synthesis. Female sRAmice demonstrated a large elevation in plasma AVP at baseline (Figure 1G). Whereas malesRA mice did not exhibit increased AVP at baseline, a brief period (4 hours) of waterrestriction markedly increased plasma AVP in sRA but not control mice. We next testedwhether increased AVP is responsible for the elevated hydromineral turnover by treatingsRA mice with conivaptan, a non-peptide V1A/V2 receptor antagonist (Vaprisol, 10 mg/kg/day for 3 days). AVP receptor blockade suppressed sodium intake and preference for 0.15 MNaCl, but had no effect on total fluid intake or urine output (Figure 1H). Conivaptan alsohad no effect on serum electrolytes (not shown). This suggests that AVP has selective effectson intake behaviors in sRA mice.

Elevated Adrenal Steroids Cause Polydipsia and PolyuriaAdrenal hormones, particularly steroids, are suggested to play a role in food intake,metabolic expenditure, and fluid/electrolyte balance (Mastorakos et al., 2004). The adrenalglands are hypertrophied, and steroid levels are elevated in urine from sRA mice (Table 1).We performed bilateral complete adrenalectomy (ADX) to examine if adrenal steroids aremechanistically involved in the polydipsia exhibited by sRA mice. Four weeks aftercomplete removal of adrenal tissue, steroids were no longer detectable in urine. ADXcompletely normalized total fluid and sodium intake, and urine volume and osmolality(Figure 2A). ADX normalized urine potassium concentration, and whereas it had no effecton urine sodium concentration, it completely normalized daily sodium loss (Figure 2B). Wetreated a separate cohort of male mice with high doses of either the mineralocorticoidreceptor antagonist, spironolactone, and/or the progesterone/glucocorticoid receptorantagonist, mifepristone (RU-486) for four days to assess the importance of these signalingpathways. Spironolactone treatment, with or without co-treatment by mifepristone,significantly blunted total fluid and sodium intake, and sodium loss (Figure 2C), though themagnitude of the effect was small in comparison to full ADX, possibly due to dose orduration of treatment. In contrast, mifepristone treatment had no effects on these endpoints.These data support a role for adrenal steroids and mineralocorticoid receptor signaling in thehydromineral phenotypes of the sRA mouse, though other non-mineralocorticoid, adrenal-derived hormones (such as various catecholamines) may also be involved.

sRA Mice Exhibit Increased Energy Expenditure and ThermogenesisConcurrent with polydipsia and polyuria, adult sRA mice exhibit a greatly reduced bodymass regardless of sex (Figure 3A). This reduction is not observed until weaning (Figure3B). MRI revealed that adult sRA mice exhibit a profound reduction in both subcutaneousand visceral adiposity (Figure 3C). There was a 20% reduction in adipocyte cross-sectionalarea within interscapular brown, peri-genital white, and peri-renal white adipose pads(Figure 3D). Despite reduced fat mass at baseline (Table 1), sRA mice gain weight when feda high fat diet (Figure S2).

To identify the mechanism causing the lean phenotype, we examined both food intake andenergy expenditure. Over seven consecutive days in metabolism cages, sRA mice consumed7% less food than littermate controls. However, since the body masses of sRA mice werereduced by 25%, they exhibited hyperphagia and increased fecal output when the data were

Grobe et al. Page 3

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

normalized to body mass (Figure 3E, S3). Telemetry revealed an approximate 1°C elevationof core body temperature during multiple portions of the light-dark cycle (Figure 3F).Importantly, this was not correlated with any genotype-related differences in locomotoractivity (Figure 3G), thus providing evidence that their elevated energy expenditure is due toan elevation in activity-independent thermogenesis. Oxygen consumption atthermoneutrality was elevated by 16% in sRA mice aged 9–12 weeks, during both sleep andstationary wakefulness (Figure 3H). The increased metabolic rate in sRA mice was evidentin other cohorts ranging from 15 to 36 weeks of age (N>60 in both groups). There was nochange in preferred ambient temperature, suggesting a normal thermoneutral set-point(Figure S3). sRA mice did not exhibit signs of anxiety (Figure S3).

Unlike hydromineral phenotypes, bilateral ADX had no effect on body mass, metabolic rate,food intake or fecal output of sRA mice (Figure 4A, Table S1). Mineralocorticoid orglucocorticoid receptor antagonists similarly had no effect on these endpoints. sRA miceexhibited a normal body length, normal fasting blood glucose and plasma insulin levels, andnormal levels of triiodothyronine (T3) and thyroxine (T4) suggesting growth hormone andthyroid hormone signaling are not mechanistically involved in the increased metabolismobserved in sRA mice (Table 1).

Elevated Sympathetic Nervous Activity Increases Metabolic RateWe next measured the electrical activity of the sympathetic nerves (SNA) innervating theinterscapular brown adipose tissue (BAT) and the peri-genital white adipose tissue. sRAmice exhibited a remarkable elevation in BAT SNA (Figure 4B). Unlike controls, sRA micewere incapable of increasing BAT SNA more than 2–3% to stepwise decreases in coretemperature (Figure 4C). Similar trends in SNA were observed in peri-genital white adiposetissue (Figure S4).

We next sought to determine if elevated SNA mediated the elevation in basal metabolic rate.Acute delivery of the β-adrenergic receptor antagonist, propranolol, resulted in largereductions in metabolic rate (Figure 4D). sRA mice exhibited a robust dose-dependentresponse to propranolol, compared to controls (group × dose interaction P=0.017), indicatingincreased metabolic sensitivity to β-adrenergic blockade and dependency upon β-adrenergicsignaling. Interestingly, expression of uncoupling protein-1 (UCP-1) mRNA, the typicaleffector of β-adrenergic signaling, and surrogate marker for sympathetic tone, was notincreased in BAT from sRA mice (Figure 4E). The reason for this discrepancy was notimmediately obvious, as the expression of β-adrenergic receptors was normal. Interestingly,expression of endogenous mouse angiotensinogen, also a known sympathetic- and cAMP-sensitive gene, was significantly up-regulated in BAT. This prompted us to measure othercomponents of the RAS in BAT. There was no change in the expression of angiotensin(Ang) II type 1A (AT1A) or Ang-(1–7) (a.k.a. Mas) receptors. Mouse renin, AT1B and AngII type 2 (AT2) receptor mRNAs were not detected (Figure 4E).

Elevated Brain and Decreased Circulating RAS Increases Metabolic RateWhereas Ang II levels were unchanged in whole-brain homogenates (Control 35±7 vs sRA34±16 pg Ang II/mg protein), a significant elevation in Ang II was noted in the combinedanteroventral third ventricle (AV3V) and hypothalamic region of the brain (Control 5.2±0.7vs sRA 10.5±0.2 pg Ang II/mg protein, P=0.002) (Figure 5A). This highlights the regionallocalization of elevated Ang in sRA mice previously identified by immunohistochemistry(Sakai et al., 2007). In contrast, RAS peptides were significantly suppressed in thecirculation (Figure 5B), likely the result of suppressed renal renin gene expression (Figure5C). We hypothesize that this is the result of chronic hypertension in sRA mice (Figure 5D).We next chronically infused mice with Ang II to test whether RAS replacement/

Grobe et al. Page 4

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

supplementation would be sufficient to normalize the metabolic phenotype. Chronic infusionof a non-pressor dose of Ang II resulted in a decrease in metabolic rate back to the baselinelevel of control mice and a significant shift in respiratory quotient toward the increasedutilization of carbohydrates (Figure 5E). These data suggest that the circulating RAS plays amechanistic role in metabolic regulation.

Elevated Metabolic Rate in DOCA-salt Treated C57BL/6J MiceIn order to establish the physiological relevance of the sRA mouse model, we examinedmetabolic parameters in a well-established independent model of hypertension. Thedeoxycorticosterone acetate (DOCA)-salt model of hypertension is known to be dependentupon increased brain RAS activity and characterized by decreased circulating renin (Itaya etal., 1986). Consequently, we hypothesized that DOCA-salt mice should exhibit metabolicalterations similar to those of sRA mice. We treated adult non-transgenic C57BL/6J micewith subcutaneous DOCA and ad libitum 0.15 M NaCl (DOCA-salt treatment) for threeweeks. Consistent with our observations in sRA mice, DOCA salt-treated C57BL/6J miceexhibited a large increase in basal metabolic rate (Figure 6A). This was accompanied by amodest decrease in body mass, a trend toward decreased interscapular BAT mass, and asignificant reduction in peri-genital WAT mass (Figure 6B). DOCA-salt-treated mice, likesRA, exhibited decreased renal renin expression (Figure 6C). Similar effects of DOCA wereobserved in female mice (data not shown). These data demonstrate that sRA mice parallelthe phenotypes of the DOCA-salt model of hypertension (moderate hypertension, highmetabolic rate leading to weight loss and decreased adiposity), likely through both anelevation in brain RAS activity and a decrease in circulating RAS activity, therebyunderscoring the physiological relevance of the sRA mouse model.

DiscussionFrom the data presented here, we conclude that a diverse, but specific, set of efferentsignaling modalities are activated by brain RAS hyperactivity, and combinations of thesesignals converge to elicit the robust alterations in fluid and energy balance in the sRA mousemodel. A schematic representation of these pathways is illustrated in Figure 6D. Whileadrenal steroids are absolutely necessary for the maintenance of polydipsia and polyuria inthese animals, these hormones are dispensable for the increased energy expenditure of sRAmice. In contrast, the metabolic phenotypes of sRA mice are the result of elevatedsympathetic nerve activity and a suppression of the peripheral RAS. Further validation forthis mechanism comes from the observation that the metabolic phenotypes of sRA mice arereplicated in an independent model of neurogenic hypertension caused by an elevated brainand depressed circulating RAS.

Hydromineral regulation by the brain RASThe hydromineral phenotypes of sRA mice are the result of a combination of brain RASsignaling and peripheral steroids. We previously demonstrated the dependence of thepolydipsia in these animals upon both brain AT1 stimulation and hyperactivity of the RASwithin the subfornical organ (Sakai et al., 2007), and here we have demonstrated thenecessity of adrenal steroids in these behaviors. This synergy of adrenal steroids and brainAng has previously been documented in rats, as co-administration of peripheral steroids andbrain Ang II synergize to elicit robust drinking responses (Epstein 1982, Fluharty andEpstein 1983, Johnson et al., 2003, Krause and Sakai, 2007). This overlap of thirstmechanisms between mice and rats is important to note, as many investigators havepreviously documented that while peripheral Ang peptides are potent dipsogens in rats, andboth species exhibit robust responses to intra- and extracellular dehydration, mice are largelyinsensitive to peripheral Ang (Hoshishima et al., 1962, Kobayashi et al., 1979, Rowland,

Grobe et al. Page 5

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

1986, Rowland and Fregly, 1988). Presumably the synergy between brain angiotensinergicsignaling and adrenal steroids is mediated through mineralocorticoid regulation of brain AT1receptors (Thornton et al., 2007). Others have demonstrated binding sites for adrenalsteroids in brain regions involved in water and sodium intake (Birmingham et al., 1979,Gerlach and McEwen, 1972, McEwen et al., 1986); and interference with brainmineralocorticoid receptor activation (McEwen et al., 1986) or gene expression (Sakai et al.,2000) has inhibitory effects on hydromineral phenotypes in rats. Thus, the determinationhere that ADX, and more specifically mineralocorticoid receptor antagonism, attenuates thehydromineral phenotypes of the sRA mouse supports a similar mechanism in mice.Additional studies will nevertheless be required to directly test if the responses tomineralocorticoids are mediated within the brain.

The mechanism by which adrenal steroids are elevated in sRA mice is unclear. ACTH,vasopressin, interleukin-6 and tumor necrosis factor-α all participate in the regulation ofcorticosterone levels (Tanoue et al., 2004, and Judd et al., 2000), while circulating and localadrenal RAS activity, potassium, corticotropin, catecholamines, and prostaglandins allcontribute to the regulation of aldosterone levels (Willenberg et al., 2008). Vasopressin isknown to regulate steroid production through modulation of ACTH levels, but plasmaACTH levels were depressed in sRA mice (Table 1). We considered the possibility thatectopic expression of the hREN transgene may cause local RAS hyperactivity in the adrenalgland. We previously reported that hREN was not detected in the adrenal glands of sR mice(Morimoto et al., 2002). Nevertheless we examined adrenal expression of human and mouserenin by realtime RT-PCR, and determined that while the sR construct is expressed, its levelof expression is 1000-fold lower its expression in the brain, and 30-fold lower thanexpression of endogenous mouse renin in the adrenal gland (Table S2). These data rule outectopic expression of hREN in the adrenal gland as a cause for adrenal hypertrophy andelevated steroid levels in sRA mice. We also considered the possibility that there may bealtered circadian rhythms in the adrenal gland of sRA mice. Doi, et al. (2010) recentlyreported that expression of 3β-hydroxyl-steroid dehydrogenase type 6 (Hsd3b6) but notHsd3b1 is regulated by the circadian clock. Deficiency of the core clock componentsCryptochrome-1 and -2 resulted in aldosterone-dependent hypertension through markedlyincreased expression of Hsd3b6. Unlike their study, we found no evidence for alteredHsd3b6 or Hsd3b1 expression in adrenal gland (Table S2). This is consistent with other dataendpoints suggesting a retention of circadian rhythms in sRA mice (Figures 3F, 3G, 5D).Sympathetic stimulation of adrenal glands may also play a role in the elevated adrenalsteroid production. Preliminary studies utilizing the α-adrenergic antagonist, prazosin,suggest no effect on steroid levels, although it was previously reported that sympatheticregulation of adrenal steroid production is mediated through β-adrenergic (and possiblydopaminergic) stimulation of cAMP signaling (Pratt et al., 1985, Bugajski et al., 1991,Missale et al., 1990, Ehrhart-Bornstein et al., 1995), and thus catecholaminergic signalingmay represent the primary stimulus for the elevated steroid production in sRA mice.

Metabolic regulation by the brain RASThe metabolic phenotypes of sRA mice are dependent upon a combination of elevatedsympathetic nerve activity and suppression of the peripheral RAS, as blockade of β-adrenergic signaling or supplementation with subcutaneous angiotensin II both corrected theelevation in basal metabolic rate in sRA mice. The decrease in renal renin expression andcirculating Ang was at first puzzling given the remarkable increase in RSNA. Indeed, renalrenin production is regulated through many inputs, including endothelin-1, Ang II,mechanical stretch, inflammatory cytokines, α- and β-adrenergic stimulation, salt, and renalperfusion pressure (Pan and Gross, 2005). We hypothesize that the chronic hypertension inthis model is the primary cause for decreased renal renin production in the sRA mouse

Grobe et al. Page 6

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

model, just as it appears to be in the DOCA-salt model (Goodwin et al., 1969, Makrides etal., 1988).

Previous investigators have demonstrated that global knockout of the genes encoding renin(Takahashi et al., 2007), angiotensinogen (Massiera et al., 2001), angiotensin convertingenzyme (Jayasooriya et al., 2008), AT1A receptors (Kouyama et al., 2005), AT2 receptors(Yvan-Charvet et al., 2005), or the Mas receptor (Santos et al., 2008) result in leanphenotypes and/or resistance to weight gain. Thus, we originally hypothesized that sRAmice, with increased RAS activity in the brain, might exhibit weight gain when compared tocontrol littermates. Discovering that the sRA mice are lean and exhibit many of thecharacteristics of mouse models of global RAS knockout, we investigated the activity of thecirculating RAS. We determined that two indexes of circulating RAS activity (circulatingAng II and renal renin expression) were greatly suppressed. Consequently, from thestandpoint of the circulating RAS, our model phenocopies models of RAS deficiency orblockade. Importantly, we also documented an inverse correlation between circulating RASactivity and metabolic rate in the DOCA-salt model, a well documented experimental modelof neurogenic hypertension (Itaya et al., 1986). The determination that hyperactivity of thebrain RAS and hypoactivity of the peripheral RAS results in a negative energy balance isalso in agreement with previous findings from studies in rats utilizing brain Ang infusion(Porter et al., 2003, Porter and Potratz 2004), peripheral pharmacological inhibition of Angconverting enzyme (ACE) (Weisinger et al., 2008, Santos et al., 2008, Carter et al., 2004),and peripheral pharmacological antagonism of AT1 receptors (Zorad et al., 2006). Weightloss in humans in response to ACE inhibition (Beevers 1984) or AT1 receptor antagonism(Shimabukuro et al., 2007) has also been reported previously. Interestingly, doubletransgenic mice from our laboratory which exhibit RAS hyperactivity throughout the bodyincluding the brain, and therefore do not have a suppressed circulating RAS (Merrill et al.,1996), exhibit normal body masses (25–27 weeks of age; control 31.2±1.2g N=9; doubletransgenic 29.7±1.3g, N=5, P=0.46). It appears that regardless of mechanism (geneticdeletion, pharmacological inhibition or antagonism, feedback inhibition, or suppression dueto elevated brain RAS activity), suppression of the peripheral RAS elicits increasedmetabolic rate, altered adipose development, and generally a negative energy balance.

Conclusions, implications, and future directionsSignificant evidence supports the existence of a local RAS within the brain of rodents andhumans (reviewed in Grobe et al., 2008). Evidence also directly supports the function of thebrain RAS in the physiological and pathophysiological regulation of fluid balance andenergy metabolism, though the mechanisms mediating these phenotypes were largelyunknown. Here we have demonstrated robust hydromineral and metabolic consequences ofbrain RAS hyperactivity in mice, and identified the efferent signaling mechanisms involvedin the modulation of these endpoints. From these findings, we hypothesize selectiveparticipation by fore-/mid-brain versus hindbrain regions in the regulation of hydromineraland metabolic endpoints. Further, we hypothesize the involvement of altered brain RASsignaling in the hydromineral and metabolic consequences of various high- and low-reninforms of hypertension and the metabolic syndrome.

Experimental ProceduresAnimals

Double-transgenic sRA mice were generated as previously described (Sakai et al., 2007).C57BL/6J mice expressing human renin via the synapsin promoter (sR mice) were crossedwith mice expressing human angiotensinogen via its own promoter (A mice). A closelyrelated mouse model (sRAflox) was also used, in which the A strain is replaced by a human

Grobe et al. Page 7

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

angiotensinogen construct in which exon 2 of the gene is flanked by LoxP sequences (Aflox).Just as with A mice, Aflox mice are phenotypically normal, unless human renin is alsopresent. sRAflox mice have the same metabolic, blood pressure, and hydromineralphenotypes as sRA mice, and thus, sRA and sRAflox lines were used interchangeably. MRIimaging, histological examinations, high fat diet, radiotelemetric core temperature andactivity, quantitative RT-PCR from adipose and renal samples, plasma hormones weredetermined only with the sRA line. Sympathetic nerve recordings, Ang II replacement, andpropranolol treatment were performed in the sRAflox line. Baseline metabolic endpointswere examined using both lines. Single-transgenic (sR, A and Aflox) and non-transgeniclittermate mice are phenotypically normal for every endpoint examined, and have thus beencombined into a single control group for all studies. Both sexes of mice were utilized for allendpoints, with the exceptions that only males were used for steroid antagonist injections,sympathetic nerve activity recordings, and angiotensin II infusions. All animals were housedin shoebox-style forced-air cages, with ad libitum access to standard chow and water at alltimes, unless otherwise noted. Lighting was maintained on a standard 12:12 hr cycle, andhousing and experimental rooms were maintained between 23–25°C and 10–40% humidity.All procedures performed herein were approved by the University of Iowa’s InstitutionalAnimal Care and Use Committee.

MRIAdipose imaging by magnetic resonance was performed as previously described (Morgan etal., 2008, Rahmouni et al., 2008). Mice were anesthetized using a mixture of ketamine andxylazine (87.5 & 12.5 mg/kg, i.p.), and images were captured in the axial and coronal planeswith a Varian Unity/Inova 4.7 T small-bore MRI system (Varian, Palo Alto, CA), using aT1-weighted fast spin-echo sequence (TR/TE = 625/12 ms) with in-plane resolution of 0.13× 0.25 mm2 and slice thickness of 1 mm.

Metabolic CagesMice were acclimated to Nalgene (Rochester, NY) single-mouse sized metabolism cages forat least two days before any studies were performed. All animals received ad libitum accessto powdered chow (Harlan Teklad, NIH-31 modified 6% mouse/rat diet), water, and NaCldrink solutions (0.00 to 0.50 M, for two-bottle choice tests). Bottle positions were alternateddaily during two-bottle choice paradigms, to account for side biases.

Metabolic RateAnimals were placed into a water-jacketed two liter beaker (Ace Glass, Vineland, NJ)maintained at 30°C, and room air was drawn through the chamber at 300 mL/min (R2 flowcontrol, AEI). Air samples were dried by passage through two successive columns of CaSO4(Drierite, Arcos) dessicant, then analyzed for CO2 (CD-31, AEI) and O2 (S-3A/II, AEI)content. Data were recorded and analyzed using a PowerLab (ADInstruments) andassociated Chart software on a PC computer.

Telemetric Blood PressureMice were anesthetized with ketamine/xylazine, and a radiotelemetric blood pressure probe(DSI, Model TA11PA-C10) was implanted into the common carotid artery, as previously(Halabi et al., 2008). The telemeter was within the abdomen, and animals were allowed torecover for two days. Blood pressure and heart rate data were collected for 30 seconds every5 minutes and recorded using the Dataquest program (DSI) on a PC computer. Data werecollapsed within animal across two days of recording for statistical analyses.

Grobe et al. Page 8

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Telemetric Core TemperatureAnimals were anesthetized using ketamine/xylazine. A midline incision through theabdominal wall was made, and a radiotelemetric probe (Model TA10TA-F20, DSI) wasinserted. The abdominal wall and the overlying skin were separately sutured shut, andsingly-housed animals were allowed one week of recovery. Following recovery, the homecages of the animals were placed on top of receiver platforms (DSI). Core temperature andphysical activity data (30 seconds every 5 minutes) were recorded using the associatedDataquest program (DSI) on a PC computer. Individual time-points from five consecutivedays were collapsed within each animal for statistical analyses.

Hormone AssaysUrine collected during metabolism cage studies, serum (trunk blood collected into cleanmicrocentrifuge tubes allowed to stand 5 minutes before a 5 minute centrifugation at 5,000 ×g), and plasma (400 μL trunk blood collected into a microcentrifuge tube containing 50 μL0.5 M EDTA, gently mixed, then immediately centrifuged for 5 minutes at 5,000 × g) wereanalyzed for various hormones. Hormones were analyzed by selective ELISAs, as per themanufacturer’s instructions, including urine corticosterone and aldosterone (CaymanChemical), serum total triiodothyronine (T3) and thyroxine (T4) (Alpco), plasma leptin(Crystal Chem), plasma insulin (Alpco), and plasma ACTH (Bachem). Blood glucose levelswere determined using an Accu-Check meter (Roche).

Sympathetic Nerve ActivitySympathetic nerve recordings were performed as previously described (Morgan et al., 2008,Rahmouni et al., 2008). Mice were anesthetized using ketamine/xylazine, and instrumentedwith a colonic temperature probe. The left carotid artery and jugular veins were cannulated,and the brown adipose pad was exposed. A sympathetic nerve innervating the brownadipose was isolated and suspended on a 36-gauge platinum-iridium electrode and securedin place with silicone gel (World Precision Instruments). Electrodes were attached to a high-impedance probe (HIP-511, Grass Instruments), and the nerve signal was amplified (0.5–1.0×105 for RSNA, 5.5×106 for BAT, 6.0×105 for WAT) with a Grass P5 AC preamplifier,filtered at a 100- and 1000-Hz cutoff, and routed to a resetting voltage integrator (modelB600c, University of Iowa Bioengineering). Data were recorded and analyzed using aPowerLab unit and associated Chart software (ADInstruments) on a Macintosh computer.

Gene ExpressionTotal RNA was extracted using TriReagent (Molecular Research Center). RNA was thentreated with DNase-I (Fermentas), and cDNA generated by reverse transcriptase usingSuperscript III (Invitrogen). Realtime PCR was performed using primer/probe sets fromApplied Biosystems, and gene expression was determined by the Livak method (Livak andSchmittgen, 2001).

RAS Peptide AnalysesAfter isolation from whole blood as above, 150 μL of plasma was diluted with 15 μL ofmethanol and rapidly frozen at −80°C. Plasma samples were then analyzed by HPLC aspreviously described (Cassis et al., 2004, Daugherty et al., 2004). Tissues were snap frozenin liquid nitrogen, homogenized in 0.1 N HCl containing 0.5 mM o-phenanthroline and 0.1mM pepstatin. After centrifugation (20,000 × g, 20 min, 4°C), the supernatant was placed at−20°C overnight, and centrifugation was repeated the next day. The supernatant was diluted(1:1) with 0.1% orthophosphoric acid, and stored at 4°C for 6 hours before centrifugation afinal time at 20,000 × g (4°C). The supernatant was diluted with 0.02% orthophosphoricacid before applying to C18 minicolumns. Tissue extracts and plasma were then purified by

Grobe et al. Page 9

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

passage through a C18 column. Angiotensin peptides were then resolved using an HPLCsystem with ultraviolet detection (254 nm). The mobile phase at time 0 was 20% buffer A(0.05% trifluoroacetic acid), 80% buffer B (100% acetonitrile). Gradient elution wasachieved by increasing the concentration of buffer B to 40% over 30 minutes, followed by100% buffer B for 15 minutes to clean the column. With each set of plasma samples, a set ofangiotensin standards (Ang II, Ang-(2–8), Ang-(3–8), Ang-(4–8), Ang-(5–8); 5 μg/ml each;Sigma) was injected onto the system to define angiotensin peptide retention times. A bufferblank was injected and collected intermittently to check for carryover of peptides acrossindividual runs. Fractions (0.5 mL) from the HPLC were collected, evaporated to dryness,and reconstituted for Ang II RIA.

Bilateral Adrenalectomy (ADX)Adult sRA and littermate control mice, between 20–30 weeks of age, underwent bilateralADX. For two days preceding and one day following surgery, mice were injected daily with20 μg dexamethasone, i.p. Mice were anesthetized with ketamine/xylazine. The adrenalswere exposed through bilateral dorsal incisions, isolated by silk suture and removed bycautery, and the muscle wall and skin were closed separately with silk suture. Mice werethen housed singly, with ad libitum access to food, tap water, and 0.15 M NaCl for fourweeks. Four control and four sRA mice survived to the end of the experiment followingADX (44% and 36% survival after ADX, respectively).

DOCA-Salt ModelTen week old wildtype C57BL/6J mice were anesthetized with ketamine/xylazine and apellet of deoxycorticosterone acetate (DOCA, 50 mg × 21 days, Innovative Research ofAmerica) was implanted subcutaneously. Notably, animals were not uninephrectomized forthis study. Animals were allowed ad libitum access to chow, tap water, and 0.15 mM NaClfor four weeks.

Statistical AnalysesData were analyzed by 1- or 2-way ANOVA (with/without repeated measures), with P<0.05considered significant. Bonferroni post-hoc analyses were used when main effects reachedsignificance. Non-parametric analyses were utilized (Mann-Whitney, Friedman’s ANOVA)were utilized when data failed normality or equal variance tests.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThe authors would like to thank Victoria L. English, Mark S. Blumberg, Andrew J. Gall, Sara A. Romig-Martin,Ralph F. Johnson, Judith A. Herlein, Brian D. Fink, William I. Sivitz, Ella J. Born, Deborah R. Davis, and VickieL. Akers for assistance/input on this project. This work was supported through Institutional T32 Post-DoctoralFellowships funded by the NIH (JLG, CLG), a Post-Doctoral Fellowship in Physiological Genomics from theAmerican Physiological Society (JLG), a K99/R00 Pathway to Independence Award (JLG, HL098276), Pre-Doctoral (DX, 0910035G) and Post-Doctoral (HL, 0825813G) Fellowships from the American Heart Association, aPost-Doctoral Fellowship from the Japan Society for the Promotion of Science (KS), and through research supportfrom the National Institutes of Health (HL048058, HL061446, HL084207 to CDS; HL014388, DK066086, andMH080241 to AKJ; HL073085 to LAC; HL084207 to KR and ALM). We also gratefully acknowledge thegenerous research support of the Roy J. Carver Trust.

Grobe et al. Page 10

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

ReferencesBeevers DG. Enalapril in essential hypertension: a comparative study with propranolol. Enalapril in

hypertension study group (UK). Br J Clin Pharmacol. 1984; 18:51–56. [PubMed: 6331482]Bickerton RK, Buckley JP. Evidence for a central mechanism in angiotensin-induced hypertension.

Proc Soc Exp Biol Med. 1961; 106:834–837.Birmingham MK, Stumpf WE, Sar M. Nuclear localization of aldosterone in rat brain cells assessed by

autoradiography. Experientia. 1979; 35:1240–1241. [PubMed: 488294]Booth DA. Mechanism of action of norepinephrine in eliciting an eating response on injection into the

rat hypothalamus. J Pharmacol Exp Ther. 1968; 160:336–348. [PubMed: 4296696]Bugajski J, Turoń M, Gadek-Michalska A, Borycz JA. Catecholaminergic regulation of the

hypothalamic-pituitary-adrenocortical activity. J Physiol Pharmacol. 1991; 42:93–103. [PubMed:1681965]

Carter CS, Cesari M, Ambrosius WT, Hu N, Diz D, Oden S, Sonntag WE, Pahor M. Angiotensin-converting enzyme inhibition, body composition, and physical performance in aged rats. J GerontolA Biol Sci Med Sci. 2004; 59:416–423. [PubMed: 15123750]

Cassis LA, Huang J, Gong MC, Daugherty A. Role of metabolism and receptor responsiveness in theattenuated responses to Angiotensin II in mice compared to rats. Regul Pept. 2004; 117:107–116.[PubMed: 14700746]

Daniels AE, Ogden E, Vernikos-Danellis J. Effects of centrally administered angiotensin II in theunanesthetized rat. Physiologist. 1969; 12:205.

Daugherty A, Rateri DL, Lu H, Inagami T, Cassis LA. Hypercholesterolemia stimulates angiotensinpeptide synthesis and contributes to atherosclerosis through the AT1A receptor. Circulation. 2004;110:3849–3857. [PubMed: 15596561]

Doi M, Takahashi Y, Komatsu R, Yamazaki F, Yamada H, Haraguchi S, Emoto N, Okuno Y,Tsujimoto G, Kanematsu A, Ogawa O, Todo T, Tsutsui K, van der Horst GTJ, Okamura H. Salt-Salt-sensitive hypertension in circadian clock-deficient Cry-null mice involves dysregulatedadrenal Hsd3b6. Nat Med. 2010; 16:67–74. [PubMed: 20023637]

Epstein A. Mineralocorticoids and cerebral angiotensin may act together to produce sodium appetite.Peptides. 1982; 3:493–494. [PubMed: 7122274]

Epstein AN, Fitzsimons JT, Rolls BJ. Drinking induced by injection of angiotensin into the rain of therat. J Physiol. 1970; 210:457–474. [PubMed: 4322723]

Ehrhart-Bornstein M, Bornstein SR, González-Hernández J, Holst JJ, Waterman MR, Scherbaum WA.Sympathoadrenal regulation of adrenocortical steroidogenesis. Endocr Res. 1995; 21:13–24.[PubMed: 7588375]

Ferguson AV, Washburn DL, Latchford KJ. Hormonal and neurotransmitter roles for angiotensin inthe regulation of central autonomic function. Exp Biol Med (Maywood). 2001; 226:85–96.[PubMed: 11446443]

Fluharty SJ, Epstein AN. Sodium appetite elicited by intracerebroventricular infusion of angiotensin IIin the rat: II. Synergistic interaction with systemic mineralocorticoids. Behavioral Neuroscience.1983; 97:746–758. [PubMed: 6639747]

Gerlach J, McEwen BS. Rat brain binds adrenal steroid hormone: Radioautography of hippocampuswith corticosterone. Science. 1972; 175:1133–1136. [PubMed: 5060047]

Goodwin FJ, Knowlton AI, Laragh JH. Absence of renin suppression by deoxycorticosterone acetatein rats. Am J Physiol. 1969; 216:1476–1480. [PubMed: 5786734]

Grobe JL, Xu D, Sigmund CD. An intracellular renin-angiotensin system in neurons: fact hypothesis,or fantasy. Physiology. 2008; 23:187–193. [PubMed: 18697992]

Halabi CM, Beyer AM, de Lange WJ, Keen HL, Baumbach GL, Faraci FM, Sigmund CD. Interferencewith PPAR gamma function in smooth muscle causes vascular dysfunction and hypertension. CellMetab. 2008; 7:215–226. [PubMed: 18316027]

Hoshishima K, Yokoyama S, Seto K. Taste sensitivity in various strains of mice. Am J Physiol. 1962;202:1200–1204. [PubMed: 14449173]

Grobe et al. Page 11

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Itaya Y, Suzuki H, Matsukawa S, Kondo K, Saruta T. Central renin-angiotensin system and thepathogenesis of DOCA-salt hypertension in rats. Am J Physiol. 1986; 251:H261–H268. [PubMed:3526927]

Jayasooriya AP, Mathai ML, Walker LL, Begg DP, Denton DA, Cameron-Smith D, Egan GF,McKinley MJ, Rodger PD, Sinclair AJ, Wark JD, Weisinger HS, Jois M, Weisinger RS. Micelacking angiotensin converting enzyme have increased energy expenditure, with reduced fat massand improved glucose clearance. Proc Natl Acad Sci U S A. 2008; 105:6531–6536. [PubMed:18443281]

Johnson AK. The periventricular anteroventral third ventricle (AV3V): its relationship with thesubfornical organ and neural systems involved in maintaining body fluid homeostasis. Brain ResBull. 1985; 15:595–601. [PubMed: 3910170]

Johnson RF, Beltz TG, Thunhorst RL, Johnson AK. Investigations on the physiological controls ofwater and saline intake in C57BL/6 mice. Am J Physiol Regul Integr Comp Physiol. 2003;285:R394–R403. [PubMed: 12714354]

Judd AM, Call GB, Barney M, McIlmoil CJ, Balls AG, Adams A, Oliveira GK. Possible function ofIL-6 and TNF as intraadrenal factors in the regulation of adrenal steroid secretion. Ann N Y AcadSci. 2000; 917:628–637. [PubMed: 11268391]

Kobayashi H, Uemura H, Wada M, Takei Y. Ecological adaptation of angiotensin-induced thirstmechanism in tetrapods. Gen Comp Endocrinol. 1979; 38:93–104. [PubMed: 467974]

Kouyama R, Suganami T, Nishida J, Tanaka M, Toyoda T, Kiso M, Chiwata T, Miyamoto Y,Yoshimasa Y, Fukamizu A, Horiuchi M, Hirata Y, Ogawa Y. Attenuation of diet-induced weightgain and adiposity through increased energy expenditure in mice lacking angiotensin II type 1areceptor. Endocrinology. 2005; 146:3481–3489. [PubMed: 15878965]

Krause EG, Sakai RR. Richter and sodium appetite: From adrenalectomy to molecular biology.Appetite. 2007; 49:353–367. [PubMed: 17561308]

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCRand the 2-ΔΔCT method. Methods. 2001; 25:402–408. [PubMed: 11846609]

Makrides SC, Mulinari R, Zannis VI, Gavras H. Regulation of renin gene expression in hypertensiverats. Hypertension. 1988; 12:405–410. [PubMed: 3049341]

Massiera F, Seydoux J, Geloen A, Quignard-Boulange A, Turban S, Saint-Marc P, Fukamizu A,Negrel R, Ailhaud G, Teboul M. Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotoractivity. Endocrinology. 2001; 142:5220–5225. [PubMed: 11713218]

Mastorakos G, Zapanti E. The hypothalamic-pituitary-adrenal axis in the neuroendocrine regulation offood intake and obesity: the role of corticotropin releasing hormone. Nutr Neurosci. 2004; 7:271–280. [PubMed: 15682923]

McEwen BS, Lambdin LT, Rainbow TC, De Nicola AF. Aldosterone effects on salt appetite inadrenalectomized rats. Neuroendocrinology. 1986; 43:38–43. [PubMed: 3713988]

Merrill DC, Thompson MW, Carney CL, Granwehr BP, Schlager G, Robillard JE, Sigmund CD.Chronic hypertension and altered baroreflex responses in transgenic mice containing the humanrenin and human angiotensinogen genes. J Clin Invest. 1996; 97:1047–1055. [PubMed: 8613528]

Missale C, Lombardi C, Sigala S, Spano PF. Dopaminergic regulation of aldosterone secretion.Biochemical mechanisms and pharmacology. Am J Hypertens. 1990; 3:93S–95S. [PubMed:2166536]

Morgan DA, Thedens DR, Weiss R, Rahmouni K. Mechanisms mediating renal sympathetic activationto leptin in obesity. Am J Physiol Regul Integr Comp Physiol. 2008; 295:R1730–R1736.[PubMed: 18815209]

Morimoto S, Cassell MD, Sigmund CD. Glia- and neuron-specific expression of the renin-angiotensinsystem in brain alters blood pressure, water intake, and salt preference. J Biol Chem. 2002;277:33235–33241. [PubMed: 12080069]

Pan L, Gross KW. Transcriptional regulation of renin: an update. Hypertension. 2005; 45:3–8.[PubMed: 15545507]

Paul M, Poyan Mehr A, Kreutz R. Physiology of local renin-angiotensin systems. Physiol Rev. 2006;86:747–803. [PubMed: 16816138]

Grobe et al. Page 12

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Porter JP, Potratz KR. Effect of intracerebroventricular angiotensin II on body weight and food intakein adult rats. Am J Physiol Regul Integr Comp Physiol. 2004; 287:R422–R428. [PubMed:15117728]

Porter JP, Anderson JM, Robison RJ, Phillips AC. Effect of central angiotensin II on body weight gainin young rats. Brain Res. 2003; 959:20–28. [PubMed: 12480154]

Pratt JH, Turner DA, McAteer JA, Henry DP. Beta-adrenergic stimulation of aldosterone productionby rat adrenal capsular explants. Endocrinology. 1985; 117:1189–1194. [PubMed: 2410237]

Rahmouni K, Fath MA, Seo S, Thedens DR, Berry CJ, Weiss R, Nishimura DY, Sheffield VC. Leptinresistance contributes to obesity and hypertension in mouse models of Bardet-Biedl syndrome. JClin Invest. 2008; 118:1458–1467. [PubMed: 18317593]

Rowland NE. Comparative physiological psychology of feeding and salt appetite in rodents. Nutritionand Behavior. 1986; 3:27–42.

Rowland NE, Fregly MJ. Characteristics of thirst and sodium appetite in mice (Mus musculus). BehavNeurosci. 1988; 102:969–974. [PubMed: 3063282]

Sakai K, Agassandian K, Morimoto S, Sinnayah P, Cassell MD, Davisson RL, Sigmund CD. Localproduction of angiotensin II in the subfornical organ causes elevated drinking. J Clin Invest. 2007;117:1088–1095. [PubMed: 17404622]

Sakai RR, McEwen BS, Fluharty SJ, Ma LY. The amygdala: Site of genomic and nongenomic arousalof aldosterone-induced sodium intake. Kidney International. 2000; 57:1337–1345. [PubMed:10760064]

Santos EL, de Picoli Souza K, Guimarães PB, Reis FC, Silva SM, Costa-Neto CM, Luz J, PesqueroJB. Effect of angiotensin converting enzyme inhibitor enalapril on body weight and composition inyoung rats. Int Immunopharmacol. 2008; 8:247–253. [PubMed: 18182235]

Santos SH, Fernandes LR, Mario EG, Ferreira AV, Pôrto LC, Alvarez-Leite JI, Botion LM, Bader M,Alenina N, Santos RA. Mas deficiency in FVB/N mice produces marked changes in lipid andglycemic metabolism. Diabetes. 2008; 57:340–347. [PubMed: 18025412]

Shimabukuro M, Tanaka H, Shimabukuro T. Effects of telmisartan on fat distribution in individualswith the metabolic syndrome. J Hypertens. 2007; 25:841–848. [PubMed: 17351377]

Takahashi N, Li F, Hua K, Deng J, Wang CH, Bowers RR, Bartness TJ, Kim HS, Harp JB. Increasedenergy expenditure, dietary fat wasting, and resistance to diet-induced obesity in mice lackingrenin. Cell Metab. 2007; 6:506–512. [PubMed: 18054319]

Tanoue A, Ito S, Honda K, Oshikawa S, Kitagawa Y, Koshimizu TA, Mori T, Tsujimoto G. Thevasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity underboth stress and resting conditions. J Clin Invest. 2004; 113:302–309. [PubMed: 14722621]

Thornton SN, Omouessi ST, Falconetti C. Mineralocorticoid modulation of central angiotensin-induced neuronal activity, water intake and sodium appetite. Braz J Med Biol Res. 2007; 40:699–705. [PubMed: 17464433]

Veerasingham SJ, Raizada MK. Brain renin-angiotensin system dysfunction in hypertension: recentadvances and perspectives. Br J Pharmacol. 2003; 139:191–202. [PubMed: 12770924]

Weisinger HS, Begg DP, Egan GF, Jayasooriya AP, Lie F, Mathai ML, Sinclair AJ, Wark JD,Weisinger RS. Angiotensin converting enzyme inhibition from birth reduces body weight andbody fat in Sprague-Dawley rats. Physiol Behav. 2008; 93:820–825. [PubMed: 18191966]

Willenberg HS, Schinner S, Ansurudeen I. New mechanisms to control aldosterone synthesis. HormMetab Res. 2008; 40:435–441. [PubMed: 18493881]

Wright JW, Yamamoto BJ, Harding JW. Angiotensin receptor subtype mediated physiologies andbehaviors: new discoveries and clinical targets. Prog Neurobiol. 2008; 84:157–181. [PubMed:18160199]

Yvan-Charvet L, Even P, Bloch-Faure M, Guerre-Millo M, Moustaid-Moussa N, Ferre P, Quignard-Boulange A. Deletion of the angiotensin type 2 receptor (AT2R) reduces adipose cell size andprotects from diet-induced obesity and insulin resistance. Diabetes. 2005; 54:991–999. [PubMed:15793237]

Zorad S, Dou JT, Benicky J, Hutanu D, Tybitanclova K, Zhou J, Saavedra JM. Long-term angiotensinII AT1 receptor inhibition produces adipose tissue hypotrophy accompanied by increased

Grobe et al. Page 13

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

expression of adiponectin and PPARgamma. Eur J Pharmacol. 2006; 552:112–122. [PubMed:17064684]

Grobe et al. Page 14

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 1. Mice with brain renin-angiotensin system hyperactivity (sRA mice) exhibit potenthydromineral phenotypes(A) Ad libitum 24-hour intake volumes for tap water and 0.15 M NaCl solutions (ControlN=6, sRA N=3). (B) NaCl preference, total fluid intake, total sodium intake, urine volume,urine sodium concentration (UNaC), and urine osmolality from sRA and control littermatemice offered ad libitum access to varying concentrations of NaCl drink solution, distilledwater, and standard chow (Control N=6, sRA N=6). (C) Renal sympathetic nerve activity(RSNA) (Males only; Control N=7, sRA N=6). (D) Blood hematocrit (Control N=9, sRAN=8). (E) Serum osmolality (Control N=10, sRA N=6), potassium concentration, andsodium concentration (Control N=17, sRA N=15). (F) Analysis of pup survival to birth andto weaning. (G) Plasma arginine vasopressin (AVP) concentrations at baseline andfollowing a 4-hour water restriction (N=4 for all). (H) Total 24-hour sodium intake,preference for 0.15 M NaCl, fluid intake and urine volume after treatment with conivaptan(Saline N=5, Conivaptan N=6). * P<0.05 vs. control, or sRA+Saline. All data are mean ±SEM. See also Figure S1.

Grobe et al. Page 15

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 2. Adrenal hormones are necessary for the hydromineral phenotypes of sRA mice(A) Total 24-hour fluid intake, sodium intake, urine volume, and urine osmolality whenoffered ad libitum access to tap water and 0.15 M NaCl (Control N=7, Intact sRA N=5,ADX sRA N=4). (B) Urine potassium concentration (UKC), total daily potassium loss in theurine (UKV), urine sodium concentration (UNaC), and total daily sodium loss in the urine(UNaV). (C) Total 24-hour fluid intake, sodium intake, and total daily sodium loss (UNaV)following spironolactone (30 mg/kg/day) and/or mifepristone (50 mg/kg/day) treatment(Males only; Control N=15, Untreated sRA N=15, Spironolactone N=5, Mifepristone N=4,Combined N=6). * P<0.05 vs. control. † P<0.05 vs. intact/untreated sRA. All data are mean± SEM. See also Table S1.

Grobe et al. Page 16

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 3. sRA mice are small and lean, primarily due to a large increase in activity-independentenergy expenditure(A) Body masses of male and female mice from 9–22 weeks of age (Control males N=9,females N=6, sRA males N=6, females N=4). (B) Body masses of mice at day 17 ofgestation (Control N=15, sRA N=8), and 4-weeks (Control N=9, sRA N=4), and 8-weeks(Control N=17, sRA N=9) after birth. (C) MRI scans of adult male mice. (D) Hematoxylin& Eosin stained sections of interscapular brown adipose (BAT), and peri-genital and peri-renal white adipose (WAT) from adult mice. Scale bars = 100 microns. (E) Body masses,total 24-hour ad libitum intake of standard chow, and food intake data normalized to bodymass (Control N=12, sRA N=8). (F) Core body temperatures determined by telemetricprobes (N=6 for all). (G) Spontaneous physical activity determined by telemetric probes. (H)Metabolic rate, determined by indirect calorimetry (N=8 for all). * P<0.05 vs. control. Alldata are mean ± SEM. See also Figure S3.

Grobe et al. Page 17

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 4. Elevated sympathetic nerve activity contributes to the metabolic phenotypes of sRAmice(A) Body masses, metabolic rate, and food intake four weeks following bilateraladrenalectomy (ADX) (Control N=7, Intact sRA N=5, ADX sRA N=4). (B) Exampletracings from sympathetic nerves innervating interscapular brown adipose tissue (BAT), andquantification of BAT sympathetic nerve activity (SNA) by both frequency analysis andintegrated voltage (RVI) with core temperatures maintained at 37.5°C (Males only; N=6 forall). (C) BAT SNA RVI from mice with core temperatures decreased to 34°C (N=6 for all).(D) Metabolic rate, determined by indirect calorimetry, following graded doses ofpropranolol (Control N=9, sRA N=10). (E) Gene expression profile from interscapularbrown adipose tissue (BAT) (N=4 for all). * P<0.05 vs. control. All data are mean ± SEM.See also Figure S4.

Grobe et al. Page 18

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 5. Suppression of the peripheral RAS contributes to the metabolic phenotypes of sRAmice(A) Angiotensin II in whole-brain homogenate (Control N=5, sRA N=7), cortex (ControlN=4, sRA N=4), combined anteroventral third ventricle (AV3V) and hypothalamic regions(Control N=4, sRA N=3), and brain stem (Control N=4, sRA N=3). (B) Plasma RASpeptides (N=8 for all). (C) Renal murine renin mRNA expression (N=4 for all). (D) Meanarterial blood pressure determined by carotid artery cannula during anesthesia (Control N=7,sRA N=6), and in conscious, freely-moving mice by radiotelemetry during dark and lightphases (Control N=4, sRA N=4). (E) Metabolic rate and respiratory quotient followingangiotensin II treatment (100 ng/kg/min, s.c., for 8 weeks. Males only; Control saline, AngII N=4 each. sRA saline N=7, sRA Ang II N=3). * P<0.05 vs. control. † P≤0.05 vs. saline-treated sRA. All data are mean ± SEM.

Grobe et al. Page 19

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 6. DOCA-salt treated C57BL/6J mice exhibit similar phenotypes to sRA mice, and aworking modelAdult C57BL/6J male mice were treated for 3 weeks with DOCA-salt (N=3 for all). (A)Metabolic rate determined by indirect calorimetry. (B) Body masses, interscapular brownadipose, and peri-genital white adipose tissue masses. (C) Renal murine renin mRNAexpression. (* P<0.05 vs. sham. All data are mean ± SEM) (D) A working model for theefferent mechanisms mediating the elevated fluid turnover, hypertension, and elevatedmetabolic rate in sRA mice. Interventions supporting this model include adrenalectomy(ADX), spironolactone-mediated blockade of mineralocorticoid receptors (MR antagonism),intracerebroventricular blockade of angiotensin receptors (ARB, from Sakai et al., 2007),propranolol-mediated blockade of β-adrenergic signaling (β-AR antagonism), and chronicsubcutaneous angiotensin II supplementation (Ang II infusion).

Grobe et al. Page 20

Cell Metab. Author manuscript; available in PMC 2011 November 3.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Grobe et al. Page 21

Table 1

Tissue masses and endocrine measures in adult sRA mice.

Control sRA t-test

Anatomical Measures

Body Mass (g) 28.53 ± 0.96 (6) 23.94 ± 1.18 (7) P=0.013

Nose-Anus (cm) 9.20 ± 0.13 (6) 9.03 ± 0.16 (7) P=0.295

Organ Masses

Heart(mg) 112.0 ± 4.7 (6) 113.6 ± 10.3 (7) P=0.731

(mg/g) 12.15 ± 0.34 12.50 ± 0.91 P=0.742

Lungs(mg) 182.7 ± 9.2 (6) 136.8 ± 3.6 (7) P<0.001

(mg/g) 19.85 ± 0.93 15.17 ± 0.38 P<0.001

Liver(mg) 1285.5 ± 75.6 (6) 1053.4 ± 62.5 (7) P=0.051

(mg/g) 139.3 ± 6.4 116.4 ± 5.5 P=0.019

Kidney(mg) 170.2 ± 7.4 (6) 118.1 ± 8.2 (7) P<0.001

(mg/g) 18.47 ± 0.60 13.03 ± 0.74 P<0.001

Adrenal Gland(mg) 2.7 ± 0.3 (9) 3.8 ± 0.3 (10) P=0.032

(mg/kg) 83 ± 10 166 ± 15 P<0.001

Adipose Masses

Interscapular BAT(mg) 61.1 ± 5.2 (6) 48.0 ± 4.1 (7) P=0.073

(mg/g) 6.64 ± 0.57 5.28 ± 0.37 P=0.063

Peri-Genital WAT(mg) 284.0 ± 27.8 (6) 151.4 ± 21.2 (7) P=0.003

(mg/g) 30.73 ± 2.63 16.60 ± 2.03 P=0.001

Peri-Renal WAT(mg) 77.8 ± 16.7 (6) 48.5 ± 10.5 (7) P=0.154

(mg/g) 8.49 ± 1.87 5.41 ± 1.19 P=0.179

Mesenteric WAT(mg) 394.1 ± 25.4 (6) 304.9 ± 9.5 (7) P=0.002

(mg/g) 43.00 ± 3.20 33.75 ± 0.75 P=0.014

Endocrine Measures

Serum Total T3 ng/mL 12.29 ± 1.41 (9) 14.43 ± 3.47 (8) P=0.847

Serum Total T4 μg/dL 6.33 ± 1.39 (4) 5.11 ± 1.11 (4) P=0.520

Serum Leptin ng/mL 1.16 ± 0.69 (3) 1.39 ± 0.41 (3) P=0.788

Plasma Insulin (Fasted) ng/mL 0.218 ± 0.026 (8) 0.174 ± 0.011 (8) P=0.148

Blood Glucose (Fasted) mg/dL 123.6 ± 10.6 (11) 137.3 ± 9.8 (8) P=0.182

Plasma ACTH ng/mL 5.474 ± 2.235 (6) 1.423 ± 0.689 (5) P=0.052

Urinary Aldosteroneng/mL 23.04 ± 1.59 (8) 20.00 ± 1.82 (8) P=0.231

ng/day 30.63 ± 4.11 65.99 ± 9.20 P=0.003

Urinary Corticosteroneng/mL 1.15 ± 0.03 (8) 1.61 ± 0.10 (8) P<0.001

ng/day 1.63 ± 0.27 6.37 ± 1.37 P=0.004

See also Figure S2.

Cell Metab. Author manuscript; available in PMC 2011 November 3.