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. Author manuscript; available in PMC: 2013 Nov 7.
Published in final edited form as: Cell Metab. 2012 Nov 7;16(5):579–587. doi: 10.1016/j.cmet.2012.10.003

Brainstem nutrient sensing in the nucleus of the solitary tract inhibits feeding

Clemence Blouet 1, Gary J Schwartz 1
PMCID: PMC3537851  NIHMSID: NIHMS415210  PMID: 23123165

SUMMARY

Direct detection of circulating nutrients by the central nervous system has been implicated in the regulation of energy balance, and the mediobasal hypothalamus is considered the primary sensing site mediating these effects. Neurons sensitive to energy-related signals have also been identified outside the hypothalamus, particularly within the caudomedial nucleus of the solitary tract (cmNTS) in brainstem, but the consequences of direct NTS nutrient detection on energy balance remain poorly characterized. Here we determined the behavioral and metabolic consequences of direct L-leucine detection by the cmNTS and investigated the intracellular signaling and neurochemical pathways implicated in cmNTS L-leucine sensing in rats. Our results support the distributed nature of central nutrient detection, evidence a role for the cmNTS S6K1 pathway in the regulation of meal size and body weight, and suggest that the cmNTS integrates direct cmNTS nutrient detection with gut-derived, descending forebrain, and adiposity signals of energy availability to regulate food intake.

Introduction

Within the central nervous system (CNS), specialized metabolic-sensing neurons modify their electrical and synaptic activity in response to signals related to body’s fuel status (Levin, 2006). Metabolic-sensing neurons are believed to play a critical role in the regulation of energy balance as a unique site of anatomical convergence of multiple signals of both acute and chronic energy availability, coupling detection and integration of these signals to coordinate effector functions that determine energy intake and utilization (Woods, 2009). However, the mechanisms underlying the integration of multiple fuel-related signals within the CNS, as well as the neuroanatomical distribution and organization of CNS metabolic-sensing circuits remain poorly characterized.

Metabolic-sensing neurons have been mainly localized in selective nuclei of the hypothalamus, particularly within the mediobasal hypothalamus (ventromedial and arcuate nuclei of the hypothalamus), historically considered as the cornerstone of CNS fuel detection (Jordan et al., 2011; Levin et al., 2011; Woods, 2009). Mediobasal hypothalamic neurons have been shown to detect adiposity and gut-derived signals of energy availability (Konner et al., 2009; Nogueiras et al., 2008), as well as representatives of all 3 classes of macronutrients (glucose, fatty acids and amino acids) (Blouet et al., 2009; Ibrahim et al., 2003; Jo et al., 2009; Obici et al., 2002; Wang et al., 2004). In response to these various signals, mediobasal hypothalamic metabolic-sensing neurons engage a complex set of neurochemical and neurophysiological responses to produce integrated commands to hindbrain neurons regulating behavioral and metabolic determinants of energy homeostasis.

Neurons sensitive to energy-related signals have also been identified outside the hypothalamus, challenging this hypothalamo-centric view of CNS fuel-sensing. Evidence supporting the physiological relevance of a more distributed nature of CNS fuel sensing (Grill, 2006) include the feeding and motivational responses to leptin, GLP-1 and ghrelin detection in the midbrain ventral tegmental area (Dickson et al., 2012; Hommel et al., 2006; Skibicka et al., 2011), or the feeding consequences of leptin, GLP-1 and ghrelin signaling in the nucleus of the solitary tract in the hindbrain (NTS) (Faulconbridge et al., 2003; Grill et al., 2002; Hayes et al., 2009; Hayes et al., 2011b; Huo et al., 2007). The hypothalamic framework of CNS fuel-sensing is further challenged by data supporting a role for the NTS as an important center of convergence and integration of multiple signals of energy availability. Initially described as a primary site receiving post-ingestive afferent information from the gastro-intestinal tract important in the negative feedback control of meal size (Schwartz et al., 2000), the NTS has recently been shown to integrate forebrain descending melanocortinergic input and leptin signals with gut-derived satiety signals to determine food intake (Blevins et al., 2009; Blevins et al., 2004; Sutton et al., 2005; Sutton et al., 2004; Zhao et al., 2012). Surprisingly, the functional consequences of direct nutrient detection by NTS neurons in the regulation of energy balance have received little attention, apart from work demonstrating the role of NTS glucose sensing in the feeding and glucoregulatory response to hypoglycemia (Ritter et al., 2000; Sanders et al., 2004) Furthermore, the potential NTS integration of direct NTS nutrient sensing with other signals of energy availability remain unexplored.

We previously found that L-leucine sensing by the mediobasal hypothalamus regulates energy balance, and implicated the activation of the amino-acid sensing p70 S6 kinase 1 (S6K1) signaling pathway in l-leucine detection (Blouet et al., 2009; Blouet et al., 2008). In the current work, in part based on its previously determined NTS nutrient sensing capabilities and its proximity to the circumventricular area postrema (AP), a region of specialized access to circulatory factors at the blood brain barrier interface, we hypothesized that CNS amino acid sensing may be localized to the caudal brainstem. Consequently, we evaluated the amino acid nutrient sensing capabilities of the rat NTS by examining the feeding and metabolic effects of direct NTS L-leucine administration. We characterized the neurochemical identities of NTS L-leucine sensing neurons, investigated the intracellular signaling mechanisms underlying NTS L-leucine sensing and directly tested the role of the NTS S6K1 pathway in the control of energy homeostasis. Last, we employed signaling, behavioral and physiological measurements to examine whether NTS neurons could integrate L-leucine stimulation with other signals of energy availability to determine food intake and body weight.

RESULTS

NTS L-leucine administration reduces food intake and body weight

To identify whether the caudomedial NTS (cmNTS) senses the local availability of amino acids in ways relevant for energy balance, we examined the behavioral and metabolic consequences of direct L-leucine administration into the cmNTS (spanning the rostrocaudal extent of the area postrema) in terms of food intake and body weight. We used a dose of L-leucine 2-fold lower than the dose previously used in MBH parenchymal injection studies, which was in the physiological range of the postprandial cerebrospinal L-leucine increase (Blouet et al., 2009). Bilateral NTS L-leucine administration (25 ng in 100 nl aCSF, per side) to 24 h fasted rats significantly reduced 24 h food intake (Figure 1A), and decreased 24 h body weight change (Fig. 1B) compared with aCSF vehicle injections. Meal pattern analyses revealed that NTS L-leucine administration rapidly produced a 40% decrease in the first meal size within the first 20 min following the injection (Fig. 1C), and reduced meal size was maintained throughout the 24h following the injection (Fig 1D). NTS L-leucine-also decreased meal number beginning 6 h after the injection (Figure 1E).

Figure 1.

Figure 1

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NTS L-leucine administration reduces food intake and body weight.

(A) Food intake, (B) 24h body weight change, (C) first meal size, (D) mean meal size and (E) cumulative meal number in rats following a bilateral injection of aCSF or L-leucine into the cmNTS. Data are means ± SEM (n=12). *: p<0.05, **: p<0.01.

To assess the specificity of the DVC L-leucine response with respect to other branched-chain amino acids, we examined the feeding inhibitory effect an equimolar dose of the branched-chain amino acid valine. NTS Valine administration failed to alter food intake and body weight change compared with aCSF control injections (Fig. S1).

NTS L-leucine sensing regulates food intake and body weight through the activation of the S6K1 pathway

Unlike valine, L-leucine is a strong activator of the mTORC1/p70 S6 kinase 1 signaling pathway (Proud, 2004). Therefore, we hypothesized that L-leucine administration into the NTS activates the amino-acid sensing mTORC1/p70 S6 kinase 1 signaling pathway, and that activation of this pathway is implicated in NTS L-leucine sensing and its effects on food intake and body weight. Consistent with this hypothesis, NTS administration of L-leucine at a dose that suppressed feeding significantly increased the expression of the activated phosphorylated form of p70 S6 kinase 1 (pS6K1, phosphorylated a threonine 789) in the DVC dorsal vagal complex (DVC; which includes NTS, dorsal motor nucleus of the vagus (DMV) and area postrema (AP)) within 30 minutes of injection (Figure 2A). NTS administration of L-leucine also increased the number of ribosomal protein S6 (pS6, phosphorylated at Ser240 and Ser244, a downstream effector of S6K1) immunoreactive neurons in the NTS (Figure 2B), compared to NTS aCSF administration. In contrast, NTS L-leucine administration did not alter the activity of the AMP-activated protein kinase (AMPK) in the DVC, as assessed by α2-AMPK threonine 172 phosphorylation (data not shown).

Figure 2.

Figure 2

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NTS L-leucine activates NTS S6K1 this signaling and this activation is required for NTS L-leucine’s anorexigenic effects.

(A) NTS S6K1 Thr389 phosphorylation (n=5) and (B) NTS pS6 immunoreactivity (n=4) in 24h fasted rats 30 min after a bilateral NTS aCSF or L-leucine injection. (C–E) Colocalization (right, orange) of DBH (C), TH (D) or POMC (E) (left, red) with pS6 (middle, green) in 24h fasted rats 30 min after a bilateral NTS L-leucine injection (n=4). (F) Food intake, first meal size and meal size following an icv injection of aCSF or SHU9119 combined with a bilateral injection of aCSF or L-leucine into the cmNTS. (G) Food intake, 24h body weight change, first meal size, mean meal size and cumulative meal number in rats following a bilateral injection of aCSF or L-leucine combined to a bilateral injection of DMSO or rapamycin into the cmNTS. Data are means ± SEM (n=12). *: p<0.05. scale bar: 100 μm.

We next characterized the neurochemical phenotype of NTS neurons that exhibit L-leucine-induced pS6 expression using immunofluorescence. Our attention was focused on the catecholaminergic neurons located in the NTS because they have been strongly implicated in the regulation of meal size (Rinaman, 2010; Zhang et al., 2010), and NTS POMC neurons, because we previously found that L-leucine directly depolarizes POMC neurons in the arcuate nucleus of the hypothalamus (Blouet et al., 2009), and NTS POMC neurons have been shown to contribute to the regulation of meal size (Babic et al., 2009; Fan et al., 2004). We found that NTS L-leucine administration induces pS6 expression in 36±3.61% of neurons expressing dopamine β hydroxylase (Figure 2C), 45±2.78% of neurons expressing tyrosine hydroxylase (Figure 2D), and 52±7.3% of neurons expressing POMC (Figure 2E).

To test whether activation of NTS POMC neurons may be involved in NTS leucine-induced anorexia, we assessed the effect of the melanocortin analog SHU9119, a MC3/4R antagonist. Coadministration of NTS leucine with a subthreshold dose of SHU9119 icv blocked NTS leucine-induced decreases in food intake, first meal size and meal size (Fig. 2F), indicating that NTS leucine’s effects on food intake require activation of MC3/4R.

To determine whether the stimulation of NTS S6K1 signaling is required for L-leucine–induced reduction of food intake, we combined NTS administration of the well-characterized mTORC1 inhibitor rapamycin, at a dose that did not affect food intake, with an NTS injection of L-leucine. Rapamycin completely blocked L-leucine–induced decreases in food intake, body weight, first meal size and meal size, but did not affect L-leucine-induced decreases in meal number (Figure 2G). These data suggest that activation of the NTS mTORC1/p70 S6 kinase 1 pathway following NTS L-leucine treatment induces both an acute and a sustained decrease in food intake that is mediated by a decrease in meal size, and a reduction in 24h body weight gain.

NTS S6K1 mediates the control of energy balance

Rapamycin’s ability to block NTS L-leucine-induced reductions in meal size indicate that the mTORC1/S6K1 pathway is an important effector of NTS L-leucine signaling in the regulation of energy balance. Accordingly, we asked whether NTS S6K1 activity is also sensitive to nutritional whole body energy status. Previous findings indicate that fasting and refeeding modify the expression of pS6K1 and pS6 exclusively in the arcuate nucleus of the hypothalamus in the rat forebrain, with no change in the activation of extra-arcuate hypothalamic sites, hippocampus or cortex (Cota et al., 2006). Using immunofluorescence, we found that the NTS is also a CNS site where the S6K1 signaling pathway is activated upon refeeding. Expression of pS6 was absent in the cmNTS of overnight fasted rats (Figure 3A), and was robustly induced during refeeding (Figure 3B).

Figure 3.

Figure 3

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Acute inhibition of NTS S6K1 signaling increases food intake.

(A–B) DVC pS6 immunoreactivity in 24h fasted (A) and 30 min refed rats (B). (C) First meal latency, (D) first meal size, (E) food intake, (F) mean meal size and (G) cumulative meal number in rats following a bilateral injection of DMSO or rapamycin into the cmNTS. Data are means ± SEM (n=12). *: p<0.05.

To directly assess the role of endogenous NTS S6K1 signaling in the regulation of energy balance, we next investigated the feeding effects of rapamycin. NTS bilateral administration of rapamycin (2.5 μg in 100 nl DMSO, per side) to pre-satiated rats (which were overnight fasted and refed for 1h) dramatically decreased first meal latency (figure 3C) and increased first meal size (Figure 3D) compared with DMSO vehicle injections. NTS rapamycin-induced hyperphagia was sustained through the 24h following the injection (Figure 3E) and was completely attributable to an increase in meal size (Figure 3F) with no change in meal number (Figure 3G).

We then bilaterally targeted the NTS with recombinant adenoviruses expressing a constitutively active mutant of S6K1 (CA S6K) or LACZ (control). We previously confirmed the functional validity of the CA S6K in GT1–7 hypothalamic cell lines (Blouet et al., 2008), and here we confirmed that expression of the constitutively active mutant of S6K1 induced a significant increase in DVC S6K1 protein expression (Figure 4A, S6K1/actin expression: 1.39 ± 0.06 vs. 2.37 ± 0.18 in LACZ vs. CA S6K1 infected NTS). Overactivation of S6K1 in the NTS induced a sustained reduction in food intake beginning on the first day following the viral injection, and persisting for the following week (figure 4B). This reduction in food intake was due to a reduction in meal size (figure 4C), with no change in meal number (figure 4D). Anorexia in S6K1 overexpressing rats lead to a modest but significant reduction in body weight gain starting 4 days after the NTS viral injection (Figure 4E), and a 25% reduction in fat mass (Figure 4F). Taken together, these data support an important role for NTS S6K signaling in the control of food intake and energy balance.

Figure 4.

Figure 4

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NTS S6K1 is a physiologically relevant nutrient sensor regulating the control of energy balance

(A) NTS S6K1 Thr389 phosphorylation in fasted rats bilaterally infected with the LACZ or CA S6K adenovirus in the NTS. (B) Food intake, (C) mean meal size, (D) cumulative meal number and (E) body weight in rats bilaterally infected with the LACZ or CA S6K adenoviruses in the NTS (n=6). (F) Fat mass and (G) fat free mass in rats 10 days following NTS bilateral infection with the LACZ or CA S6K adenovirus. Data are means ± SEM (n=6). *: p<0.05.

Integration of NTS L-leucine sensing with acute and chronic signals of energy availability in the control of meal size

NTS neurons have been shown to process gut-derived neural and humoral satiation signals following ingestion of food (Grill and Hayes, 2009; Hayes et al., 2009; Moran, 2006), adiposity signals conveying information about long-term energy storage such as leptin (Grill et al., 2002; Hayes et al., 2011b), and descending forebrain melanocortinergic signals (Wan et al., 2008), and accumulating evidence indicates that NTS neurons integrate these signals to determine meal size. Thus, we asked whether nutrient signals such as L-leucine also interacted with these various signals of acute and chronic energy availability.

We first investigated the interaction between L-leucine and the well-characterized gut-derived satiety signal cholecystokinin (CCK). We found that a subthreshold dose of ip CCK (0.5 μg/kg BW) enhanced the acute anorexigenic effect of NTS L-leucine (25 ng in 100 nl aCSF, per side), suggesting that CCK and L-leucine signals interact in the NTS to acutely limit food intake by limiting meal size (Figure 5A). Likewise, a subthreshold dose of NTS leptin (3 ng in 100nl/side) enhanced the acute anorexigenic effect of NTS L-Leucine (Figure 5B). Last, a subthreshold dose of NTS melanotan II (2 pmol in 100nl per side) enhanced the acute anorexigenic effect of NTS L-Leucine (Figure 5C). Together, these data indicated that NTS L-leucine sensing is integrated within the NTS with gut-derived, forebrain-descending and adiposity signals of energy availability to control meal size.

Figure 5.

Figure 5

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The NTS integrates ip CCK, NTS leptin and NTS MTII signals with L-leucine sensing to acutely decrease meal size.

First meal size following NTS bilateral administration of an anorexigenic dose of L-leucine combined with (A) a subthreshold dose of ip CCK, (B) a subthreshold dose of NTS leptin, or (C) a subthreshold dose of NTS MTII. Data are means ± SEM (n=12). Means sharing a common letter are not significantly different (p>0.05).

NTS L-leucine sensing activates the NTS ERK1/2 signaling pathway

NTS Erk1/2 signaling has been implicated in the regulation of meal size and proposed as a molecular integrator of various signals of energy availability in the NTS (Sutton et al., 2005; Sutton et al., 2004). Thus, we tested whether NTS L-leucine administration activates the Erk1/2 signaling pathway in the NTS. NTS administration of an anorexigenic dose of L-leucine rapidly and significantly increased the expression of the activated phosphorylated form of ERK1/2 (pERK1/2, phosphorylated at Threonine 202/Tyrosine204) in the DVC within 30 minutes (Figure 6A).

Figure 6.

Figure 6

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Activation of NTS Erk1/2 signaling is required for NTS L-leucine anorexigenic effect, and NTS Erk1/2 acts as a molecular integrator of ip CCK and NTS L-leucine.

(A) DVC Erk1/2 Thr202/Tyr204 phosphorylation in 24h fasted 30 min following an NTS administration of aCSF or L-leucine (n=5). (B) Food intake, (C) 24h body weight change, (D) first meal size, and (E) mean meal size in rats following a bilateral injection of aCSF or L-leucine combined to a bilateral injection of DMSO:saline (1:1) or U0126 into the cmNTS (n=12). (F) DVC Erk1/2 Thr202/Tyr204 phosphorylation in 24h fasted 30 min following an NTS administration of aCSF or L-leucine combined with an ip injection of saline or CCK. Data are means ± SEM (n=5). *: p<0.05.

To test whether activation of NTS ERK1/2 signaling pathway is required for NTS L-leucine anorexigenic effect, we co-administered NTS L-leucine with a subthreshold dose of NTS U0126 (25 ng in 100nl per side in DMSO:saline 50:50), a specific Erk1/2 inhibitor. Subthreshold NTS U0126 blocked the effect of NTS L-leucine on food intake, body weight gain, first meal size and meal size (Figure 6B–6E).

Last, we asked whether the ERK1/2 signaling pathway acts as a molecular integrator of L-leucine signals and gut-derived signals of acute energy availability. We coadministered an NTS anorexigenic dose of L-leucine with a subthreshold dose of ip CCK and assessed the activity of the DVC ERK1/2 signaling pathway. Intraperitoneal CCK and NTS L-leucine had an additive effect on DVC ERK1/2 phosphorylation (Figure 6F). In contrast, ip CCK, that did not modify S6K1 activity alone, did not further increase L-leucine-induced S6K1 activation (data not shown).

DISCUSSION

Mediobasal hypothalamic nutrient detection has been importantly implicated in the regulation of energy balance, but little is known about extra-hypothalamic nutrient-sensing sites and their contribution to the negative feedback control of food intake and body weight. Here we show that direct L-leucine detection by the cmNTS regulates food intake and body weight gain through the activation of the S6K1 signaling pathway, and we identify NTS catecholaminergic and POMC neurons as L-leucine- sensing cells. We show that bidirectional modulation of NTS S6K1 activity is sufficient to produce reciprocal alterations in food intake through specific changes in meal size, with significant consequences for body weight gain. We found that NTS L-leucine administration also activates the Erk1/2 signaling pathway in the NTS, and this activation is required for NTS L-leucine’s effects on food intake and body weight. Co- administration of subthreshold doses of ip CCK, NTS leptin or NTS melanotan II with NTS L-leucine all increase the acute anorexigenic effects of NTS L-leucine. Last, m subthreshold ip CCK and NTS L-leucine have additive effects on NTS Erk1/2 activation. Together, our results: 1) support the distributed nature of central nutrient detection, 2) evidence a role for the NTS S6K1 pathway in the regulation of meal size and body weight, and 3) suggest that NTS Erk1/2 signaling integrates direct NTS nutrient detection with forebrain-descending, gut-derived and adiposity signals of energy availability to regulate food intake.

This work demonstrates that extra-hypothalamic nutrient detection contributes to the negative feedback control of energy balance. NTS glucose sensing has been previously implicated in the glucoprivic feeding and homeostatic control of blood glucose (Balfour and Trapp, 2007; Ritter et al., 2000; Sanders et al., 2004), but the potential anorectic response to increased NTS glucose levels remains unexplored. Together with the recent findings establishing the role of direct NTS leptin, GLP-1 and ghrelin sensing in the homeostatic regulation of food intake (Faulconbridge et al., 2003; Grill et al., 2002; Hayes et al., 2009; Hayes et al., 2011b), these data support a role for the cmNTS as an important metabolic-sensing niche in the regulation of energy balance and a site of neuroanatomical convergence of all types of acute and chronic signals of energy availability.

Our data suggest that several mechanisms are engaged by NTS L-leucine to decrease food intake. First, both NTS L-leucine treatment and constitutive activation of NTS S6K1 activity reduce meal size. Such a behavioral response has been typically associated with the intake suppressive effects of GI-derived satiation signals such as CCK or gastric distention (Moran, 2006). Consequently, L-leucine detection could modulate pre-synaptic transmission from vagal terminals to NTS neurons or increase the sensitivity of NTS neurons to gut-derived glutamatergic inputs. These two mechanisms have been proposed to be implicated in the feeding-modulatory effect of hindbrain melanotan II, hindbrain leptin and hindbrain ghrelin (Cui et al., 2011; Emond et al., 1999; Huo et al., 2007; Schwartz and Moran, 2002; Sutton et al., 2005). Consistent with this, we found that ip CCK and NTS L-leucine synergistically decrease meal size and activate the NTS Erk1/2 pathway. In addition, NTS L-leucine administration increases pS6 immunoreactivity in NTS POMC neurons that modulate glutamatergic pre-synaptic activity of vagal terminals (Wan et al., 2008), and in NTS catecholaminergic neurons, required for CCK’s anorexigenic effects (Rinaman, 2003; Rinaman et al., 1993). In fact, NMDAR and AMPA/KA signaling activate S6K1 phosphorylation in cultured glial and neuronal cells (Gonzalez-Mejia et al., 2006; Lenz and Avruch, 2005) and this activation is implicated in long-term potentiation, supporting a potential role for S6K1 signaling in increasing sensitivity to depolarizing inputs. However, activation of feeding-regulatory mechanisms independent of gut-derived signals cannot be ruled out. For example, we found that NTS L-leucine decreases meal frequency, a behavioral response that is not characteristically triggered by peripheral detection of meal-related gut-derived signals (Smith, 1996). Anorexia mediated by a decrease in meal number has also been reported following hindbrain GLP1-receptor activation (Hayes et al., 2011a), supporting the idea that NTS feeding-modulatory actions are not restricted to the regulation of meal size but rather encompass multiple behavioral determinants of food intake.

Another important aspect of our work is the demonstration of a role for NTS S6K1, an evolutionary conserved nutrient-sensing enzyme, as a physiologically relevant nutrient sensor contributing to the NTS mechanisms regulating energy balance. We found that: 1) NTS S6K1 bidirectionally regulates food intake through a specific effect on meal size, and 2) NTS S6K1 activity is modulated by signals of energy availability (L-leucine and refeeding). Importantly, we found that acute inhibition of NTS mTOR/S6K1 signaling rapidly increases food intake in satiated rats, with a 5-fold decrease in the first meal latency and a 2-fold increase in the first meal size following NTS rapamycin administration, supporting a role for endogenous NTS S6K1 tone in both meal initiation and meal termination. This latter effect is sustained during the 24h of the test, suggesting that NTS S6K1 signaling regulates both acute and longer-term determinants of meal size. Previously shown to be an important MBH nutrient-sensor modulating the regulation of energy balance (Blouet et al., 2008), these results broaden the neuroanatomical basis for the contribution of the S6K1 pathway in central nutrient-sensing and the regulation of energy homeostasis. In contrast to NTS leucine, bidirectional manipulations of NTS mTORC1 pathway do not affect meal frequency, indicating that mechanisms independent from the activation of mTORC1 regulate mea size following leucine detection. These could include leucine catabolism to AcetylCoA, as has been previously shown in the mediobasal hypothalamus (Blouet et al., 2009). Interestingly, although NTS and MBH upregulation of S6K1 both led to a sustained reduction in food intake that is mediated by a reduction in meal size, these two manipulations did not produce similar consequences on body weight and body composition. MBH S6K1 upregulation produced sustained reductions in body weight gain, whereas overactivation of NTS S6K1 led to a modest and transient decrease in body weight. In contrast, MBH S6K1 upregulation did not affect body composition, whereas upregulation of S6K1 in the NTS decreased fat mass and increased fat free mass, these two effects compensating and minimizing overall body weight changes. Together, these results support the conclusion that MBH and NTS nutrient-sensing are both redundant and complementary.

The finding that the NTS integrates L-leucine sensing with the detection of gut-derived, adiposity and descending forebrain signals to regulate meal size extends previous evidence supporting NTS integrative capacity to include nutrient signals. Consistent with findings supporting a role for Erk1/2 signaling as a point of molecular convergence and integration of CCK, leptin and melanocortinergic signals in NTS neurons to reduce meal size (Campos et al., 2012; Hayes et al., 2011a; Sutton et al., 2005; Sutton et al., 2004), we found that NTS L-leucine detection also converges on the Erk1/2 signaling pathway. The mechanisms underlying L-leucine-induced Erk1/2 activation remain to be established and could implicate both an intracellular molecular interaction between Erk1/2 and S6K1 signaling, or an indirect activation of Erk1/2 in the NTS through modulations of melanocortinergic and glutamatergic tone following NTS L-leucine sensing, both of which have been shown to be coupled to Erk1/2 activation (Campos et al., 2012; Daniels et al., 2003).

mTOR and AMPK are both nutrient sensors importantly implicated in CNS fuel sensing and the regulation of energy balance (Hardie et al., 2012; Woods et al., 2008), but the cross-talk between these two pathways remains only partially characterized. While the current understanding is that AMPK is an upstream regulator of mTOR through TSC2 (Inoki et al., 2003), intra-cerebroventricular leucine was found to decrease hypothalamic α2-AMPK threonine 172 phosphorylation, suggesting that leucine-induced mTOR activation inhibits AMPK activity (Ropelle et al., 2008). Here, we found that NTS L-leucine administration did not modify α2-AMPK threonine 172 phosphorylation, consistent with the finding that constitutive activation of S6K1 in hypothalamic GT1-7 neurons does not affect α2-AMPK threonine 172 phosphorylation (Dagon et al., 2012). Recently, S6K1 has been described as a negative regulator of AMPK activity through novel phosphorylation sites (Dagon et al., 2012), but the recruitment of these sites following leucine-induced S6K1 activation remains to be tested.

The ability of icv SHU9119 to block both the acute and longer term anorexia induced by cmNTS leucine demonstrates a critical role NTS leucine-induced activation of NTS POMC neurons in these feeding effects. Whether NTS POMC neurons engage melanocortin receptors specifically within the NTS to suppress feeding remains to be determined.

Our results build on a growing challenge to a hypothalamic-centered view of central nutrient sensing and support a role for the NTS as a place of convergence and integration of multiple fuel-related signals important to the regulation of food intake. Our data also broaden the behavioral and metabolic contributions of NTS fuel-sensing to include the regulation of meal frequency, fat mass and fat-free mass. It remains to be established whether the anorectic effects NTS L-leucine require hindbrain ascending projections to the forebrain and/or local caudal brainstem effector circuitry. Identification of these circuits will be critical to understanding the behavioral and physiological relationships among forebrain and hindbrain nutrient-sensing capabilities.

Methods

Animals and reagents

Sprague Dawley rats (275–300 g, Charles River Laboratories) were housed in individual cages and maintained in a temperature-controlled room under a standard 12 h/12 h light/dark cycle with ad libitum access to water and standard chow, unless specifically indicated. Before any behavioral, signaling and immunostaining studies, animals were trained daily to be attached to the brain injection system for at least the 4 consecutive days preceding the injection. All experimental protocols were approved by the Institute for Animal Studies of the Albert Einstein College of Medicine.

CNS chronic cannula implantation

Surgical procedures were performed under ketamine/xylazine anesthesia. For 3rd ventricle cannula implantation, rats were implanted with a steel guide cannula (Plastics One) using the following coordinates relative to bregma: A/P: −2.5mm, D/V −8.5mm. Animals were stereotaxically implanted with a bilateral steel guide cannula (Plastics One) positioned 2mm above the caudomedial nucleus of the solitary tract (cannula holding bar in a 10° rostro-caudal angle, coordinates relative to occipital suture: A/P +1.5 mm, D/V −6.8 mm. +/− 0.75 lateral to midline). Beveled stainless steel injectors (30 gauge for the fourth ventricle and 33 gauge mounted onto a 26-gauge sleeve for the NTS) extending 2.0 mm from the tip of the guide cannulas were used for injections. Animals were allowed a 1 week recovery. Accurate cannula placement was confirmed by consumption of more than 1.5 g of chow within the 60 minutes following a parenchymal injection of 24μg of 5-thio-D-glucose (Sigma) in 100nl of artificial cerebrospinal fluid (aCSF; Harvard Apparatus) per side (Ritter et al., 2000).

Feeding behavior

Before all experiments, animals were adapted for 1 week to individual feeding chambers (Med Associates) with ad libitum access to a standard chow diet (Bioserv 45 mg precision pellets, F0165). Meal patterns were determined as previously described (Azzara et al., 2002).

NTS injection studies

Injections were performed in a crossover manner according the injection designs described below, and at least 4 days elapsed between each injection.

Injection design 1

Rats fasted overnight received a 2 min NTS injection (100 nl per side at 50 nl/min) of aCSF alone or together with 2.1mM L-leucine (i.e., 210 pmol per side, Sigma) or 2.1mM L-valine (Sigma) 1 h before the onset of the dark. Access to food was restored immediately after the injection, food intake was continuously recorded for the following 24 h, and body weight was measured before and 24 h after the injection. Using fluorescently-labeled leucine (Phoenix Pharmaceuticals, custom-made), we confirmed that in these conditions, injections targeted the cmNTS, spreading throughout the rostrocaudal extent of the area postrema, from approximately −13.5 to −14.0 mm posterior to bregma (Paxinos and Franklin, 2001) (Figure S2).

Injection design 2

Rats fasted overnight received a 6 min icv injection of 2 μl aCSF alone or with 0.1 nmol SHU9119 (Bachem) followed 2h later by a 2 min NTS injection of aCSF alone or together with 2.1mM L-leucine 1h before the onset of the dark. Access to food was restored immediately after the second injection and food intake was continuously recorded for the following 24 h.

Injection design 3

Rats fasted overnight received (1) a 2 min bilateral NTS injection of 100 nl DMSO (Sigma) alone or with 1.25μg rapamycin (EMD Chemicals) per side, (2) an ip injection of 100 μg/kg BW saline alone or with 0.5 μg/kg BW non-sulfated CCK octapeptide (Bachem), (3) a 2 min bilateral NTS injection of 100nl aCSF alone or with 3 ng rat recombinant leptin (R&D Systems) per side, (4) a 2 min bilateral NTS injection of 100nl aCSF alone or with 2 pmol melanotan 2 (Bachem) per side, or (5) a 2 min bilateral NTS injection of 100 nl DMSO:saline (50:50) alone or with 25 ng U0126 (Bachem) per side, followed by a bilateral injection of 100 nl aCSF alone or together with 2.1mM L-leucine 1 h before the onset of the dark. Access to food was restored immediately after the injections, food intake was continuously recorded for the following 24 h, and body weight was measured before and 24 h after the injection.

Injection design 4

Rats were trained to rapidly eat a chow meal after an overnight fast. On the day of the test, overnight fasted rats were refed for 1h and then received a 2 min NTS injection (100 nl per side at 50 nl/min) of DMSO alone or together with 2.5 μg rapamycin. Access to food was restored immediately after the injection, food intake was continuously recorded for the following 24 h, and body weight was measured before and 24 h after the injection.

Viral preparation and brain injection

Adenovectors expressing LACZ or a constitutively active mutant of p70 S6 kinase 1 were prepared as previously described (Blouet et al., 2008). Briefly, rat p70 S6 kinase 1 (shorter form of Rps6kb1) cDNA was subcloned using RT-PCR from rat liver. Mutations in S6K at F5A, T389E and RSPRR to ASPAA (AA410–414) were respectively introduced using PCR-based mutagenesis, with confirmation of whole sequences. Adenovirus for S6K mutants were generated using the Adeno-X expression system version 1 (BD Clontech), with a substitution of the promoter in the shuttle vector from CMV to CAG (Ono et al., 2003). LACZ adenovirus was prepared as previously described (Ono et al., 2003). Adenovirus were amplified in 293 cells and purified with an Adenopure kit (Puresyn Inc.). Adenoviral titers were measured by endpoint dilution assay with 1:3 serial dilutions on a 96-well plate of HEK293 cells.

Adenoviral injections and metabolic phenotyping

Using stereotaxic surgery performed under ketamine/xylazine anaesthesia, rats were bilaterally injected with adenovirus (2 .108 pfu in 200 nl/side over a 4 min period) expressing a constitutively active mutant of S6K (CA S6K), or LACZ in the NTS. Body weight and feeding behavior were monitored from 4 days before to 7–10 days after virus administration. Body composition was determined by magnetic resonance spectroscopy using an ECHO MRS instrument (Echo Medical Systems). Leptin sensitivity was measured in 24h fasted rats that received an ip injection of saline alone or with xx/kg BW rat recombinant leptin. 24h food intake and body weight gain following the ip leptin injection were adjusted to 24h food intake and body weight gain following the ip saline injection and the delta were used as indexes or leptin sensitivity. Successful adenovirus administration in the NTS was confirmed by immunoblot analysis with S6K antibody, as described below.

Biochemical analyses

Plasma insulin and leptin levels were measured using ELISA assays (ALPCO).

Tissue collection for immunostaining

Brains were perfused transcardially via a 23 gauge needle placed in the left ventricle with 200 ml of 0.1M heparinized PBS, pH 7.4, followed by 200 ml of 4% paraformaldehyde in PBS, and the fixed brains were cryoprotected in 30% sucrose. Coronal hypothalamic sections of 30 μm thickness were prepared on a freezing microtome.

Immunostaining

Free floating sections were incubated for 15 min in 1% hydrogen peroxide, washed 2 times in PBS, blocked 2h in 0.3% Triton X-100 and 5% NGS in PBS, before being incubated overnight at 4° C in phospho-S6 ribosomal protein (Ser235/236) rabbit antibody (1:50, Cell Signaling Technology), mouse anti-dopamine β hydroxylase (1:1000, Millipore), mouse anti tyrosine hydroxylase (1:100, Immunostar) or rabbit anti-proopiomelanocortin precursor (1:200, Phoenix Pharmaceuticals) in 0.3% Triton X-100 and 5% NGS. Sections were then washed and incubated for 2 h with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, floated onto superfrost Plus microscope slides (Fisher), and coverslipped with Vectashield (Vector). Specificity of the antibody against proopiomelanocortin precursor was confirmed using POMC-GFP mice (C57BL/6J-Tg(Pomc-EGFP)1Low/J from the Jackson laboratories) (Figure S3). Briefly, free floating sections were incubated in goat anti-GFP (1:1000, Abcam) and rabbit anti-proopiomelanocortin precursor (1:200, Phoenix Pharmaceuticals) and secondary detection was performed with Alexa Fluor 488 donkey anti-goat and Alexa Fluor 594 donkey anti-rabbit.

Image analysis

Images of tissue sections were digitized, and areas of interest were outlined based on cellular morphology. pS6-, TH-, DBH- and POMC-positive nuclei within the nucleus of cmNTS at the midlevel of the area postrema (7.3–7.4 mm caudal to bregma) were quantified with automated image analysis software (Image J), and were based on four animals/group.

Micropunch dissection

Brainstem sections spanning the rostrocaudal extent of the area postrema, from approximately −13.5 to −14.0 mm posterior to bregma (Paxinos and Franklin, 2001), were prepared from freshly extracted rat brain. The cerebellum was quickly removed with fine forceps. Sections were placed on a stainless steel plate cooled by liquid nitrogen and immersed in liquid nitrogen for 2 s. The spinal trigeminal nuclei forming the lateral borders of the section were excised and discarded. The remaining brainstem slice was then excised horizontally at the ventral aspect of the central canal, dissecting the dorsal vagal complex (DVC) from the more ventral hypoglossal nucleus and basal brainstem. The remaining DVC, including the area postrema, the NTS, and the dorsal motor vagal nucleus, was collected. All micropunches were snap frozen in liquid nitrogen.

Western blot analysis

Western blot analyses were performed as previously described (Blouet et al., 2008). Briefly brain micropunches were homogenized in 50mM Tris,1 mM EGTA, 1 mM EDTA, 50 mM sodium fluoride, 10 mM glycerophosphate, 20 mM sodium pyrophosphate, 2 mM orthovanadate, 2mM PMSF, 1% Triton, and Complete phosphatase inhibitor cocktail (Roche). Protein extracts were run on Criterion gels (Bio-Rad) and blotted onto nitrocellulose membranes. Immunoblots were incubated in phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, phospho-p70 S6 kinase (Thr389), phospho-S6 ribosomal protein (Ser235/236), p70 S6 kinase (Millipore), or β-actin (Santa Cruz Biotechnology) IgG, followed by incubation in fluorescent (Rockland Immunochemicals) or HRP-linked secondary antibodies. Proteins were detected using either the fluorescence-based Odyssey Infrared Imaging System (LI-COR Biosciences) or enhanced chemiluminescence (ECL Plus, GE Healthcare). We confirmed that the bands used to quantify the activation status of the mTOR/S6K1 pathway from phospho-p70 S6 kinase (Thr389), phospho-S6 ribosomal protein (Ser235/236) blots were rapamycin-sensitive (Figure S4).

Statistical analysis

All data, presented as means ± SEM, were analyzed using GraphPad Prism 5. For all statistical tests, an α risk of 5% was used. All kinetics were analyzed using a mixed model for repeated measurements. Multiple comparisons were tested with an ANOVA and adjusted with Tukey tests.

Supplementary Material

01

Highlights.

  • NTS L-leucine sensing regulates food intake and body weight gain

  • Activation of NTS p70 S6 kinase 1 is required for NTS L-leucine’s anorexigenic effect.

  • Activation of NTS Erk1/2 is required for NTS L-leucine’s anorexigenic effect.

  • NTS integrates L-leucine sensing with signals of acute and chronic fuel availability.

Acknowledgments

This work was supported by a grant to CB from National Institute of Health (1K99DK093724-01) and grants to GJS from the Ajinomoto Research Program (3-ARP), and the National Institutes of Health (DK026687 and DK020541).

Footnotes

The authors have no conflicts of interest to declare.

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