Hypothalamic mTORC1 Signaling Controls Sympathetic Nerve Activity and Arterial Pressure and Mediates Leptin Effects

Cell Metabolism

Short Article Hypothalamic mTORC1 Signaling Controls Sympathetic Nerve Activity and Arterial Pressure and Mediates Leptin Effects Shannon M. Harlan,1,3 Deng-Fu Guo,1,3 Donald A. Morgan,1 Caroline Fernandes-Santos,2,4 and Kamal Rahmouni1,2,* 1Department

of Pharmacology of Internal Medicine University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA 3These authors contributed equally to this work 4Present address: Department of Basic Sciences, Universidade Federal Fluminense, Nova Friburgo, RJ 28625-650, Brazil *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cmet.2013.02.017 2Department

SUMMARY

The fundamental importance of the hypothalamus in the regulation of autonomic and cardiovascular functions is well established. However, the molecular processes involved are not well understood. Here, we show that the mammalian (or mechanistic) target of rapamycin (mTOR) signaling in the hypothalamus is tied to the activity of the sympathetic nervous system and to cardiovascular function. Modulation of mTOR complex 1 (mTORC1) signaling caused dramatic changes in sympathetic traffic, blood flow, and arterial pressure. Our data also demonstrate the importance of hypothalamic mTORC1 signaling in transducing the sympathetic and cardiovascular actions of leptin. Moreover, we show that the PI3K pathway links the leptin receptor to mTORC1 signaling and that changes in its activity impact sympathetic traffic and arterial pressure. These findings establish mTORC1 activity in the hypothalamus as a key determinant of sympathetic and cardiovascular regulation and suggest that dysregulated hypothalamic mTORC1 activity may influence the development of cardiovascular diseases. INTRODUCTION The evolutionarily conserved mammalian (or mechanistic) target of rapamycin (mTOR) protein belongs to the phosphatidylinositol-kinase-related kinase family (Laplante and Sabatini, 2012). mTOR is critically involved in the regulation of a number of cellular functions, including protein synthesis, cell growth and proliferation, ribosome and mitochondria biogenesis, cytoskeleton organization, and autophagy (Hay and Sonenberg, 2004; Mamane et al., 2006; Wullschleger et al., 2006). mTOR activity is modulated by various cues arising from growth factors, stress, nutrients, and energy status. Active mTOR interacts with several other proteins, leading to the formation of two functionally distinct large complexes (mTORC1 and mTORC2) that can be distinguished from each other based on their unique compositions and the downstream

pathways associated with each complex (Wullschleger et al., 2006; Laplante and Sabatini, 2012). For instance, activated mTORC1 causes the phosphorylation of S6 kinase (S6K) and its downstream effector, the ribosomal protein S6 (Laplante and Sabatini, 2012). In the central nervous system, the mTOR pathway contributes to the regulation of a wide range of processes including synaptic plasticity, neuronal excitability, learning, and memory (Jaworski and Sheng, 2006; Swiech et al., 2008). Dysregulated mTOR signaling has been implicated in a number of neurological disorders such as tuberous sclerosis and epilepsy, as well as neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease (Bove´ et al., 2011; Swiech et al., 2008). The proven beneficial effects of rapamycin and its analogs in cellular and animal models of various neurodegenerative diseases have led to significant efforts to pharmacologically target the mTOR pathway in neurodegeneration (Bove´ et al., 2011). Recently, increasing attention has been paid to mTOR signaling in the hypothalamus, a region that has a well-known role in homeostasis through coordination of the regulation of several physiological processes. For instance, although mTOR and S6K are widely expressed in the brain, modulation of their activity by changes in body energy status was found to be limited to specific neurons of the hypothalamus in rats (Cota et al., 2006). Altering mTORC1 signaling in the hypothalamus affected food intake and body weight (Cota et al., 2006; Blouet et al., 2008), peripheral insulin sensitivity (Ono et al., 2008), and reproductive function (Roa et al., 2009). Moreover, hypothalamic mTORC1 was found to be critically involved in mediating the anorectic and weight-reducing effects of leptin (Cota et al., 2006). The well-established role of the hypothalamus in autonomic and cardiovascular regulation (Dampney et al., 2005; Guyenet, 2006; Nakamura et al., 2009) led us to hypothesize that hypothalamic mTORC1 signaling is involved in the control of the sympathetic nervous system and cardiovascular function. RESULTS AND DISCUSSION Leucine Increases Hypothalamic mTORC1, Sympathetic Outflow, and Arterial Pressure To test our hypothesis, we took advantage of the unique ability of leucine, a branched amino acid, to activate mTORC1 signaling Cell Metabolism 17, 599–606, April 2, 2013 ª2013 Elsevier Inc. 599

Cell Metabolism Hypothalamic mTORC1 and Cardiovascular Regulation

Figure 1. Sympathetic and Hemodynamic Responses Evoked by Hypothalamic Action of Leucine (A) Representative western blots of p-S6K (Thr389) and total S6K in hypothalamic GT1-7 cells that were treated with 10 mM of leucine or valine (n = 5 each). (B and C) Effects of i.c.v. injection of leucine (5 mg) on p-S6K (Thr389, B) and p-S6 (Ser240/244, C) in the mediobasal hypothalamus of Sprague-Dawley rats (n = 3 each). AU, arbitrary units. (D–F) Renal SNA response to i.c.v. leucine and valine in rats (n = 13 each). Representative neurograms of renal SNA recording at baseline and 6 hr after i.c.v. leucine (5 mg, D), time course of renal SNA responses to i.c.v. vehicle (Veh) versus leucine (Leu) (5 mg, E), and averages of the last hour of renal SNA (F) after i.c.v. vehicle, leucine (1 or 5 mg), or valine (5 mg) are shown. (G and H) Arterial pressure response to i.c.v. leucine and valine in rats (n = 13 each). Time course of mean arterial pressure (MAP) response to i.c.v. vehicle versus leucine (5 mg, G) and averages of the last hour of MAP (H) following i.c.v. vehicle, leucine (1 or 5 mg), or valine (5 mg) are shown. (I) Effect of i.c.v. injection of 5 mg of leucine or valine on blood flow (conductance) recorded from various vascular beds in rats (averages of the last hour of recordings are displayed, n = 13–15). (J) Peak decrease in MAP in i.c.v. vehicle- and leucine (5 mg)-treated rats after a bolus i.v. injection of hexamethonium (1 mg/g body weight, n = 5–6). Data are presented as the mean ± SEM. *p < 0.05 versus vehicle; yp < 0.01 versus vehicle and valine.

(Avruch et al., 2009; Han et al., 2012). In hypothalamic GT1-7 cells, leucine stimulated S6K in a time-dependent manner (Figure 1A) as reported previously (Morrison et al., 2007; Cota et al., 2006; Blouet et al., 2008). We also performed in vivo experiments in rats to test the effect of leucine when administered intracerebroventricularly (i.c.v.) into the third cerebral ventricle, which is anatomically adjacent to the hypothalamus. As shown previously (Cota et al., 2006; Blouet et al., 2009), i.c.v. leucine (5 mg) activated hypothalamic mTORC1 signaling, as indicated by the robust and time-dependent increase in phosphorylated levels of S6K (p-S6K, Figure 1B) and S6 (p-S6, Figure 1C). Notably, the increase in mTORC1 signaling was restricted to the hypothalamus; no increase in p-S6K and p-S6 was detected in extrahypothalamic regions such as the hippocampus, cortex, or brainstem (Figures S1A and S1B available online, and data not shown). Thus, third-ventricle i.c.v. leucine activates the mTORC1 signaling pathway specifically in the hypothalamus. We then assessed the sympathetic and hemodynamic effects of hypothalamic mTORC1 activation with i.c.v. leucine. Sympathetic nerve activity (SNA) was recorded directly from the nerve 600 Cell Metabolism 17, 599–606, April 2, 2013 ª2013 Elsevier Inc.

fibers subserving the kidney and measured simultaneously with arterial pressure in anesthetized rats (Rahmouni and Morgan, 2007). We found that i.c.v. administration of leucine (1 and 5 mg) caused a significant, dose-dependent increase in renal SNA (Figures 1D–1F) and a parallel, dose-related rise in arterial pressure (Figures 1G and 1H). The pressor effect of i.c.v. leucine was reproduced using radiotelemetric measurement in freely moving, conscious rats (Figures S1C–S1F). Notably, the increase in arterial pressure was associated with a significant decrease in the blood flow recorded from various arteries— aortic, iliac, mesenteric, and renal—with variable intensity (Figure 1I). To investigate whether i.c.v.-leucine-induced arterial pressure elevation was dependent on the sympathetic outflow, we assessed the effect of ganglionic blockade with hexamethonium (1 mg/g body weight, intravenously [i.v.]). The magnitude of the arterial pressure decrease caused by hexamethonium was significantly greater in leucine-treated rats than in controls (Figure 1J), highlighting the importance of sympathetic transmission in mediating the pressor effect of i.c.v. leucine.

Cell Metabolism Hypothalamic mTORC1 and Cardiovascular Regulation

Injection (i.c.v.) of another branched-chain amino acid, valine, which does not activate mTORC1 signaling in the GT1-7 cell line (Figure 1A) or in mediobasal hypothalamic explants (Figures S1G), failed to alter renal SNA (Figure 1F), arterial pressure (Figure 1H), or regional blood flows (Figure 1I), indicating that the sympathetic and hemodynamic responses to leucine are specific. It is interesting to note that a previous study reported elevated circulating leucine in hypertensive subjects (Newgard et al., 2009). Moreover, plasma leucine levels correlated with cardiovascular events and even predicted their occurrence (Shah et al., 2010, 2012). These reports combined with our findings suggest circulating leucine as a major cardiovascular risk factor. We therefore investigated whether systemic administration of leucine will alter SNA and arterial pressure in rats. Infusion of leucine (1 mg/kg, i.v.) caused a slow but significant increase in renal SNA, with a 76% ± 10% increase in the sixth hour (Figure S1H). This was associated with a 13 ± 4 mmHg elevation in mean arterial pressure (Figure S1I). Notably, transection of the renal nerve distal to the recording site did not affect the sympathetic activation (84% ± 9% increase) caused by i.v. administration of leucine, demonstrating that leucine stimulated efferent rather than afferent sympathetic traffic, which is consistent with a centrally mediated effect of leucine. Rapamycin Inhibits the Sympathetic and Cardiovascular Effects of Leucine To confirm that the hypothalamic effects of leucine are mediated by mTORC1 signaling, we tested the renal SNA and arterial pressure responses to leucine in the presence of rapamycin. As expected, i.c.v. pretreatment with rapamycin (10 ng) blocked the increase in hypothalamic p-S6K and p-S6 caused by i.c.v. leucine (Figures 2A and 2B). In addition, i.c.v. pretreatment with rapamycin abolished the leucine-induced increase in both renal SNA (Figure 2C) and arterial pressure (Figure 2D), as well as the reduction in renal blood flow (Figure 2E). Together, these data demonstrate that hypothalamic mTORC1 plays an important role in controlling SNA and cardiovascular function. mTORC1 Mediates the Sympathetic and Cardiovascular Actions of Leptin The mTORC1 pathway has emerged as a critical signaling mechanism of the leptin receptor in the hypothalamus (Cota et al., 2006; Blouet et al., 2008). Given the compelling evidence supporting the notion that brain action of leptin promotes sympathoexcitation and arterial pressure increase (Hall et al., 2010; Harlan et al., 2011a), we hypothesized that mTORC1 activation is required for the sympathetic and hemodynamic responses induced by leptin. In rats, i.c.v. administration of leptin (10 mg) activated hypothalamic mTORC1 signaling (Figures 3A and 3B). Leptin (i.c.v.) caused no significant change in mTORC1 signaling in extrahypothalamic regions such as the hippocampus, cortex, and brainstem (data not shown). Pretreatment with rapamycin completely abolished leptin-evoked activation of mTORC1 signaling (Figures 3A and 3B). Moreover, i.c.v. pretreatment with rapamycin substantially reduced the leptininduced increase in renal SNA (Figure 3C) and arterial pressure (Figure 3D). We also tested the effects of rapamycin on the sympathetic and hemodynamic effects induced by another

Figure 2. The Sympathetic and Hemodynamic Actions of Hypothalamic Leucine Are Mediated by mTORC1 Signaling (A and B) Effects of mTORC1 inhibition with i.c.v. rapamycin (Rap; 10 ng) on the i.c.v. leucine (5 mg)-induced increase in p-S6K (Thr389, A) and p-S6 (Ser240/244, B) in rat mediobasal hypothalamic explants (n = 4 each). (C–E) Effects of i.c.v. pretreatment with rapamycin (10 ng) on the renal SNA (C), MAP (D), and renal blood flow (E) responses evoked by 5 mg of leucine i.c.v. (n = 10–15). Data are presented as the mean ± SEM. *p < 0.05 versus other groups.

stimulus, melanotan II (MTII), which activates the melanocortin 3 and 4 receptors that have been implicated as downstream mediators of the sympathetic and cardiovascular actions of leptin (Hall et al., 2010; Rahmouni et al., 2003b). However, pretreatment with rapamycin had no effect on i.c.v. MTII-induced increases in renal SNA or arterial pressure (Figures S2A and S2B), indicating that blockade of sympathetic and cardiovascular effects of leptin by rapamycin is upstream of the melanocortin system. The evidence implicating the hypothalamic arcuate nucleus (Arc) as the site of leptin’s action to increase sympathetic traffic and arterial pressure (Harlan et al., 2011a; Rahmouni and Morgan, 2007) prompted us to test whether blocking mTORC1 signaling in the Arc is sufficient to inhibit the sympathetic and hemodynamic effects of leptin. To do this, an adenoviral vector expressing a dominant-negative form of S6K (AdDNS6K) (Blouet et al., 2008) was delivered specifically to the Arc via bilateral stereotaxic microinjections. Adenovirus expressing GFP (AdGFP) was used as a control and coinjected with the AdDNS6K for facilitating the identification of the infected region (Figure 3E). The effectiveness of AdDNS6K in inhibiting S6K signaling was validated in the hypothalamic GT1-7 cell line and the Arc (Figures S2C and S2D and previous studies by Blouet et al., 2008 and Ono et al., 2008). We then examined the effect of selectively decreasing mTORC1 signaling in the Arc on the sympathetic response evoked by i.v. administration of leptin (0.75 mg/g). Interestingly, overexpression of DNS6K in the Arc decreased baseline renal Cell Metabolism 17, 599–606, April 2, 2013 ª2013 Elsevier Inc. 601

Cell Metabolism Hypothalamic mTORC1 and Cardiovascular Regulation

Figure 3. Critical Role of Hypothalamic mTORC1 Signaling in the Sympathoexcitatory and Pressor Effects of Leptin (A and B) Effects of pretreatment with i.c.v. rapamycin (10 ng) on the i.c.v. leptin (10 mg)-induced increase in hypothalamic p-S6K (Thr389, A) and p-S6 (Ser240/244, B) in rats (n = 4–5). (C and D) Renal SNA (C) and MAP (D) responses to i.c.v. leptin (10 mg) in the absence or presence of rapamycin (10 ng) administered i.c.v. in rats (n = 8 each). (E) GFP staining (counterstained with DAPI) in the Arc of a rat that received bilateral microinjection of AdGFP into this nucleus. Scale bar represents 100 mm. (F and G) Comparison of baseline renal SNA (F) and MAP (G) between rats that received AdDNS6K 1 week earlier (with AdGFP) or AdGFP alone (control group) hitting the Arc (n = 5–11). (H and I) Renal SNA response to i.v. infusion of leptin (0.75 mg/g body weight) in rats that received AdDNS6K or AdGFP hitting the Arc. (I) Renal SNA response to leptin was compared between rats that received AdDNS6K microinjections, which ‘‘hit’’ or ‘‘missed’’ the Arc (n = 5–11). Data are presented as the mean ± SEM. *p < 0.05 versus other group(s).

SNA and arterial pressure (Figures 3F and 3G). Notably, these effects are absent in the leptin receptor mutant Zucker rats (Figures S2E and S2F). We also found that overexpression of DNS6K in the Arc virtually eliminated the renal sympathoexcitation caused by i.v. leptin (Figures 3H and 3I). Administration of leptin i.v. caused a modest increase in arterial pressure (6 ± 6 mmHg), which was reversed (2 ± 6 mmHg) when Arc mTORC1 signaling was blocked with the DNS6K. Notably, the renal SNA response to leptin in rats that underwent AdDNS6K microinjection wherein the injections missed the Arc did not differ from control rats treated with AdGFP (Figure 3I). Moreover, bilateral microinjection of AdDNS6K into the ventromedial hypothalamus, which is adjacent to the Arc, failed to alter the renal SNA response evoked by leptin (Figure S2G). Together, these results implicate mTORC1 in the Arc as a key downstream pathway in the control of renal SNA and arterial pressure by leptin. 602 Cell Metabolism 17, 599–606, April 2, 2013 ª2013 Elsevier Inc.

PI3K Links the Leptin Receptor to mTORC1 Signaling To gain insight into the mechanisms mediating leptin-receptor activation of mTORC1 signaling, we investigated the role of the PI3K, which is a critical upstream element in the intracellular cascade regulating mTORC1 signaling (Hay and Sonenberg, 2004; Wang et al., 2006). Leptin stimulates hypothalamic PI3K through insulin receptor substrates (IRS) (Niswender et al., 2001; Rahmouni et al., 2009), and inhibition of PI3K signaling blocks the renal SNA response to leptin (Rahmouni et al., 2003a, 2009). Therefore, we postulated that PI3K signaling links the leptin receptor to mTORC1 in the control of renal SNA and arterial pressure. To test this, we first evaluated the effect of pharmacological blockade of PI3K (using LY29004 or wortmannin) on leptin-induced stimulation of mTORC1 signaling. Leptin activation of S6 was blocked by LY29004 and wortmannin in hypothalamic GT1-7 cells that were transfected with ObRb, the long-signaling form of the leptin receptor (Figures 4A and 4B). Knockdown of the catalytic p110a subunit of PI3K (Figure S3A) also inhibited the leptin activation of S6 in GT1-7 cells (Figure 4C). Moreover, pretreatment with LY29004 abolished the ability of i.c.v. leptin to increase hypothalamic p-S6K and p-S6 in mice (Figure 4D and data not shown). Second, we used two contrasting mouse models with either an abolished or an enhanced effect of leptin on PI3K: the leptin-resistant p110aD933A/WT mice, which carry a knockin

Cell Metabolism Hypothalamic mTORC1 and Cardiovascular Regulation

Figure 4. PI3K Mediates Leptin-Induced mTORC1 Activation, Sympathoexcitation, and Arterial Pressure Increase (A–C) Western blot analysis of the effect of PI3K blockade (A, LY294002 [LY]; B, wortmannin [Wort]; and C, knockdown of p110a subunit with siRNA) on the leptininduced increase in p-S6 (Ser240/244) in hypothalamic GT1-7 cells transfected with the leptin receptor (ObRb, n = 4 each). (D) Effect of PI3K inhibition (i.c.v. LY294002, 0.1 mg) on the i.c.v. leptin (2 mg)-induced increase in p-S6 (Ser240/244) in the mediobasal hypothalamus of C57BL/6J mice (n = 4 each). (E and F) Comparison of p-S6 (Ser240/244) levels in the Arc of wild-type (WT), PtenDObRb, and p110aD933A/WT mice that were treated with vehicle or leptin (60 mg, i.p.) (n = 3 each). (G–I) Renal SNA response (G, time course; H and I, average of last hour) to i.c.v. vehicle, leptin (2 mg), or MTII (100 pM) in WT, PtenDObRb, and p110aD933A/WT mice (n = 3–17). (J and K) Comparison of baseline radiotelemetric MAP (J, hourly averages over 24 hr; K, 24 hr averages) between WT, PtenDObRb, and p110aD933A/WT mice (n = 4–8). (L) MAP response to leptin (60 mg, i.p.) in WT, PtenDObRb, and p110aD933A/WT mice (n = 4–8). Data are presented as the mean ± SEM. *p < 0.05 versus vehicle controls; yp < 0.05 versus WT mice.

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Cell Metabolism Hypothalamic mTORC1 and Cardiovascular Regulation

mutation that abrogates p110a kinase activity (Foukas et al., 2006), and the leptin-sensitive PtenDObRb mice, which have the lipid phosphatase Pten (phosphatase and tensin homolog) ablated in cells expressing the ObRb, leading to an enhanced PI3K pathway specifically in leptin-sensitive neurons (Plum et al., 2007). To avoid differences in body weight as a variable, we used mutant mice with body weights matching those of the littermate controls (Figures S3B and S3C). Notably, the PtenDObRb mice had elevated p-S6 staining in the Arc, at baseline and in response to leptin, as detected by immunohistochemistry (Figure 4E and 4F). Conversely, the p110aD933A/WT mice had reduced p-S6 immunoreactivity in the Arc, both at baseline and in response to leptin (Figures 4E and 4F). In line with a recent report (Dagon et al., 2012), these data implicate PI3K as a mediator underlying leptin receptor activation of mTORC1 signaling. It should be noted, however, that both Pten and p110a are major regulators of mTORC1 activity. Thus, the altered baseline mTORC1 activity in the Arc of PtenDObRb mice and p110aD933A/WT mice may be expected. Additional experiments are needed to determine the contribution of leptin to the changes in baseline mTORC1 activity in the Arc of the two mouse models. PI3K Activity Is Crucial for the Sympathetic and Cardiovascular Effects of Leptin Lastly, we assessed the sympathetic and cardiovascular ramifications of the contrasting change in the activity of the PI3KmTORC1 axis. Relative to control mice, the PtenDObRb mice had a strikingly enhanced renal SNA response to i.c.v. leptin, whereas the p110aD933A/WT mice displayed a blunted response (Figures 4G and 4H). In contrast, the MTII-induced renal SNA increase was similar in PtenDObRb, p110aD933A/WT, and control mice (Figure 4I), arguing for the specificity of the altered renal SNA responses to leptin. The lack of arterial pressure response to i.c.v. leptin in anesthetized mice (Harlan et al., 2011b) prompted us to use radiotelemetry to assess the hemodynamic consequences of the bidirectional change in PI3K-mTORC1 activity. Interestingly, baseline arterial pressure was elevated in PtenDObRb mice compared to controls, whereas the p110aD933A/WT mice had a normal baseline arterial pressure (Figures 4J and 4K). However, alteration of PI3K signaling in mice did not alter heart rate (Figures S3D and S3E). Finally, we examined the effect of leptin on arterial pressure in the two mouse models. The leptin-induced arterial pressure increase was enhanced in PtenDObRb mice but blunted in p110aD933A/WT mice (Figure 4L). In PtenDObRb mice, leptin also attenuated the arterial pressure decrease caused by fasting (Figure S3F). Together, these findings demonstrate the importance of the PI3K pathway for sympathetic and hemodynamic regulation by leptin. This study identifies a role for hypothalamic mTORC1 in the regulation of sympathetic nerve traffic and arterial pressure. Our data also indicate the importance of PI3K-mTORC1 signaling in mediating the sympathetic and hemodynamic effects of leptin. Our findings broaden the role of hypothalamic mTORC1 signaling, which has previously been implicated in the control of other physiological processes such as feeding (Blouet et al., 2008; Cota et al., 2006), glucose metabolism (Ono et al., 2008), and reproductive function (Roa et al., 2009). 604 Cell Metabolism 17, 599–606, April 2, 2013 ª2013 Elsevier Inc.

The ability of mTORC1 to sense and integrate various signals, combined with its prominent role in the control of a plethora of cellular processes ranging from gene transcription to neuronal excitability, raises the possibility that mTORC1 may serve as a ‘‘molecular hub’’ that allows hypothalamic neurons to integrate various inputs including hormones and nutrients for the production of coordinated responses that maintain homeostasis, with potential pathophysiological consequences. Indeed, hyperactive hypothalamic mTORC1 signaling has recently been implicated in age-related obesity (Yang et al., 2012). Moreover, inhibition of mTORC1 signaling (via rapamycin treatment or deletion of the s6k1 gene) extends lifespan in mice (Harrison et al., 2009; Selman et al., 2009). It will be interesting to examine whether hypothalamic mTORC1 activation accounts for the sympathetic overdrive, hypertension, and deteriorating cardiovascular health that are commonly associated with aging (Seals and Dinenno, 2004; North and Sinclair, 2012). EXPERIMENTAL PROCEDURES Animals All animal protocols were approved by the University of Iowa Animal Research Committee. Details about the strains of rats and mice used are provided in the Supplemental Information. Hypothalamic mTORC1 Signaling Animals were fasted for 3 hr before i.c.v. injections. Animals were killed at the indicated time points via CO2 asphyxiation. The mediobasal hypothalamus was quickly removed from each animal. The total proteins were extracted using lysis buffer and processed for western blotting. Sympathetic Nerve Recording Renal SNA was assessed using multifiber recording. In brief, anesthesia was induced with intraperitoneal (i.p.) injection of a ketamine (91 mg/kg) and xylazine (9.1 mg/kg) cocktail and sustained with i.v. administration of a-chloralose (6 mg/kg/hr). The renal nerve was identified and mounted on bipolar platinumiridium electrodes, and the ensemble was fixed with silicone gel. Background noise was excluded in the assessment of renal SNA by correcting for postmortem activity. Regional Blood Flows Rats were anesthetized i.p. with the ketamine/xylazine cocktail. After careful dissection and separation of each artery, individual Doppler ultrasound probes were placed around the left renal artery (0.8 mm in diameter), superior mesenteric artery (1.0 mm), left iliac artery (1.0 mm), and lower abdominal aorta (1.3 mm). Once a clear Doppler echo signal was achieved, each probe was securely tied into place, and wires were tunneled subcutaneously out of the nape of the animal’s neck. Data Analysis Data are expressed as means ± SEM and analyzed using one- or two-way ANOVA with or without repeated measure. When analysis of ANOVA reached significance, appropriate post hoc tests were used. A value of p < 0.05 was considered statistically significant. SUPPLEMENTAL INFORMATION Supplemental Information includes three figures and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi. org/10.1016/j.cmet.2013.02.017. ACKNOWLEDGMENTS We are grateful to Pamela Mellon for the GT1-7 cell line, Gary Schwartz for providing the AdDNS6K (and the University of Iowa Gene Transfer Vector

Cell Metabolism Hypothalamic mTORC1 and Cardiovascular Regulation

Core for its amplification), Jens Bru¨ning for the PtenDObRb mice, and Bart Vanhaesebroeck for the p110aD933A/WT mice. We thank Martin Cassell for assistance with the neuroanatomical aspects of the studies and Allyn Mark and Paul Casella for their helpful comments on the manuscript. This study was supported by grants from National Institutes of Health (HL084207 and HL014388), the American Diabetes Association (1-11-BS-127) and the University of Iowa Fraternal Order of Eagles Diabetes Center. S.M.H. was supported by a Postdoctoral Fellowship Award from the American Heart Association (12POST9410009). Received: October 4, 2012 Revised: January 15, 2013 Accepted: February 25, 2013 Published: March 28, 2013 REFERENCES Avruch, J., Long, X., Ortiz-Vega, S., Rapley, J., Papageorgiou, A., and Dai, N. (2009). Amino acid regulation of TOR complex 1. Am. J. Physiol. Endocrinol. Metab. 296, E592–E602. Blouet, C., Ono, H., and Schwartz, G.J. (2008). Mediobasal hypothalamic p70 S6 kinase 1 modulates the control of energy homeostasis. Cell Metab. 8, 459–467. Blouet, C., Jo, Y.H., Li, X., and Schwartz, G.J. (2009). Mediobasal hypothalamic leucine sensing regulates food intake through activation of a hypothalamusbrainstem circuit. J. Neurosci. 29, 8302–8311. Bove´, J., Martı´nez-Vicente, M., and Vila, M. (2011). Fighting neurodegeneration with rapamycin: mechanistic insights. Nat. Rev. Neurosci. 12, 437–452.

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