Leptin potently reduces food intake by acting on leptin receptors (LepRb) in the brain.  The search for which of the many LepRb-expressing brain nuclei contribute to leptin’s food intake-suppressive effects began with attention focused on the arcuate hypothalamic nucleus (Arc), as its LepRb neurons express several neuropeptides associated with food intake control.  After many experiments, however, it became clear that Arc LepRb do not mediate leptin's effects on food intake.  An understanding of the neural basis of the food intake-suppressive effects of leptin required investigating the roles of extra-arcuate LepRb .  We focused first on evaluating the function of LepRb expressing NTS neurons.
 
Our laboratory performed a series of experiments whose results strongly support the hypothesis that NTS LepRb signaling is critical to the normal control of food intake. 

NTS leptin receptor signaling is critical to food intake control

Figure 1. (A) Leptin receptors (indicated by pSTAT3) are expressed in the medial subnucleus (m) of the NTS (Huo et al, 2007). (B) Microinjection of leptin into the mNTS results in a reduction in cumulative chow intake (Kanoski et al. 2013).

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Leptin receptors expressed in the NTS are found uniquely in the mNTS and are most concentrated at the level of the area postrema (Figure 1A). We investigated the effects of both increased and decreased mNTS LepRb signaling.  LepRb activation (via microinjection) in the mNTS significantly reduces food intake (Figure 1B) and 24h change in body weight.  Furthermore,  our data show that this food intake inhibition triggered by increased NTS LepRb signaling results from the amplification of the food intake-inhibitory actions of post-prandial satiation signals (e.g., gastric distention, intestinal nutrient stimulation, and the GI hormones) that are transmitted by vagal afferents. 
 
It was well known that medial NTS (mNTS) neurons are activated by inputs from vagal afferent terminals that convey GI satiation signals to the brain.  An anatomical substrate for the amplification of GI satiation signal effects on feeding was revealed using immunohistochemistry (IHC). 

 

Figure 2. (A) A population of LepRb and GI signal responsive neurons was revealed by double IHC.  mNTS LepRb expressing neurons shown using leptin-induced pSTAT3 (green immunofluorescence).  GI satiation signal responsive neurons shown with gastric distension-induced cFos (red immunofluorescence). Yellow fluorescence indicates neurons that express both pSTAT3 and cFos (Huo et al., 2007) (B) Leptin amplifies the intake-inhibitory effect of gastric distension (Huo et al., 2007).

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Figure 2

Results indicate that 39% of mNTS leptin responsive neurons are stimulated by gastric distension (Figure 2A).  To functionally evaluate whether NTS LepRb signaling amplifies the intake inhibitory effect of GI satiation signals, a dose a leptin and a level of gastric distension were selected from pilot studies for having no effect on feeding (see dark and light grey bars versus the control black bar in Figure 2B). When hindbrain-delivered leptin was combined with gastric distension (white bar), the food intake-suppressive effects of the distension were significantly greater (amplified) than in the distension-vehicle condition (Figure 2B).   

 

Complementing the NTS leptin delivery studies that examined the effect of increased LepRb signaling, the functional effect of reducing NTS LepRb signaling was assessed using a short-hairpin RNA delivered to NTS in an adeno-associated virus (AAV).

 

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Figure 3. (A)  NTS LepRb mRNA expression was reduced by approximately 40% . (B) This reduction in NTS LepRb signaling resulted in significantly greater weight gain for LepRb knockdown (KD) than for control rats (shCtrl). The weight gain effect was observed when rats were maintained on chow and was magnified when rats were switched to a high fat diet.  The weight gain was associated with greater adiposity (inset) as assessed by postmortem fat pad dissection and weight). 

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Figure 4 (A) The weight gain is explained by an increase in food intake observed during chow and high fat diet maintenance phases and not by a reduction in energy expenditure (B)
 

To pursue the hypothesis that the increased feeding and associated weight gain resulting from targeted reduction in NTS LepRb signaling (resulting from LepRb-KD) is explained in part by an attenuation of the intake suppressive effects of GI satiation signals, two experiments were performed. In the first experiment, the effect of NTS LepRb knockdown on the intake suppressive response to CCK injection was examined.  In the second (Figure 5B) NTS LepRb knockdown reduced the intake suppressive effects of intra-duodenal nutrient infusion.

 

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Figure 5 (A) CCK reduced intake in rats prior to AAV treatment (Figure 5A) but 4 weeks post AAV delivery CCK no longer reduced feeding in rats with NTS LepRb-KD versus sh-Ctrl rats (Hayes, et al., 2010).  (B) The intake suppression associate with different caloric loads infused directly into the duodenum was reduced by the decrease in NTS LepRb signaling achieved by AAV-shRNA treatment (Kanoski et al, 2012).
 

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Figure 5

Both studies show that reduced NTS LepRb signaling reduces the intake inhibitory effects of GI satiation signals. Kanoski et al. (2012) also showed that the average meal size of rats with NTS LepRb knockdown was significantly larger than that of controls.  This result is interesting as it shows that targeted reduction in CNS LepRb signaling confined to the dorsal medulla yields hyperphagia associated with increased meal size. That result is congruent with the hyperphagia associated with increased meal size in the obese Koletsky rat (f/f) with a nonsense leptin receptor mutation.

 

Figure 6 (A) Average meal size during the dark cycle was greater in rats with reduced NTS LepRb signaling (Kanoski et al., 2012).  (B)  Average meal size of Koletsky rats lacking LepRb signaling (fak/fak) is significantly greater than lean controls (Fa/Fa) (Morton et al., 2005).
 

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A role for the pAMPK (energy status correlate) in NTS in mediating the effects of leptin receptor signaling
In related studies, we showed that the increase in NTS LepRb signaling associated with hindbrain leptin delivery reduces the phosphorylation of the cellular energy gauge AMPK in NTS enriched tisues.  Furthermore, we found that rats pre-treated (hindbrain ventricle) with a drug (AICAR - increases levels of pAMPK) have an attenuated response to leptin.

 

Figure 7 (A) Food intake measured at 2 and 4h post-injection in response to one of four treatments (in figure).  Hindbrain leptin treatment reduced intake but failed to affect feeding when rats were pretreated with a dose of AICAR that had no feeding effect of its own (Hayes et al., 2009).  (B) The associated western blot results from NTS-enriched tissue shows the leptin-reduced pAMPK level but in combination with a dose of AICAR that had no effect of its own, leptin had no effect on pAMPK level.  (C) The associated western blot results from hypothalamic tissue showed no effects on pAMPK levels following leptin deliverey to the 4th ventricle.  
 

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We also examined the complementary question of whether chronic reduction of NTS LepRb signaling using AAV-shRNA would result in an increase in basal pAMPK level and whether such a change could contribute to the hyperphagia observed in the NTS LepRb knockdown rat. 

 

Figure 8. (A) Western blots and quantitative results show that NTS LepRb knockdown significantly elevates pAMPK levels compared to shCtrl rats.  (B)  LepRb-KD and shCtrl rats were treated with vehicle and with compound C an agent that decreases AMPK phosphorylation.  The dose of compound C was selected to be subthreshold for affecting the food intake of shCtrl rats but that dose applied to the LepRb-KD rats significantly reduced 24 h food intake bringing it to a level that was not significantly different from that of shCtrl rats.

 

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