Pharmacological agents targeting the peripheral GLP-1 system are used to treat T2DM, and may also prove to be efficacious in obesity treatment (Hayes et al., 2010a
; Holst, 2007
; Larsen et al., 2003
; Lovshin and Drucker, 2009
). A variety of evidence indicates that activation of central GLP-1Rs engages a behavioral and physiological profile of responses that is similar to that observed following activation of peripheral GLP-1Rs and therefore may provide another pharmacological strategy to regulate blood glucose levels and reduce excessive adiposity by decreasing energy intake. Of the various brain regions containing GLP-1R-expressing neurons, previous reports establish those in the NTS as a physiologically relevant population of central GLP-1Rs in food intake control (Hayes et al., 2009a
; Hayes et al., 2008c
; Kinzig et al., 2002
). The intracellular signaling pathways mediating food intake suppression following NTS GLP-1R activation have not been previously established. Here, we show that the suppression of food intake and body weight following activation of hindbrain GLP-1Rs involves a coordinated PKA-mediated suppression in AMPK activity and activation of p44/42-MAPK signaling in the NTS.
Our working model [; modified from (Hayes et al., 2010a
)] derives from the current data, as well as previous reports examining the intracellular signaling cascades following GLP-1R activation in pancreatic β-cells (Gomez et al., 2002
; Perfetti and Merkel, 2000
), from known signaling pathways downstream of cAMP/PKA activation in neuronal cells (Dugan et al., 1999
; Vossler et al., 1997
), and from recent reports showing that increased AMPK mRNA in the hypothalamus following food deprivation is attenuated by GLP-1R activity (Seo et al., 2008
). It is likely that the intake suppressive effects following NTS GLP-1R activation, that involve increased PKA and MAPK signaling and decreased AMPK signaling, engage additional intracellular signaling cascades, as well as neuronal projections to other brain regions relevant to energy balance control. In addition to the intake inhibitory responses produced by 4th
icv Ex-4 administration, hindbrain GLP-1R signaling also triggers energy expenditure responses including hypothermia and tachycardia (Hayes et al., 2008c
). Interestingly, inhibition of hindbrain AMPK activity also increases heart rate and locomotor activity in rats. Therefore, it is plausible that energy expenditure responses produced by hindbrain GLP-1R activation would involve AMPK signaling (Hayes et al., 2009b
). However, it remains to be determined whether energetic responses mediated by hindbrain GLP-1R activation involve the same PKA, MAPK, and AMPK intracellular signaling cascades as described here for food intake.
Figure 7 Proposed signaling pathways in NTS-GLP-1R-expressing neurons mediating suppression of intake by central GLP-1. Gastric vagal afferent signaling increases endogenous NTS-derived GLP-1 which activates endemic NTS GLP-1R-expressing neurons (Hayes et al., (more ...)
Previous reports show that the GLP-1R-expressing NTS neurons are the likely mediators of the intake suppressive effects of hindbrain ventricular Ex-4 administration (Hayes et al., 2009a
; Hayes et al., 2008c
). Direct pharmacological blockade of mNTS GLP-1Rs increases food intake (Hayes et al., 2009a
), indicating that endogenous NTS GLP-1R activation contributes to the physiological control of food intake. Vagal afferent signals arising from distension of the stomach appear to engage NTS GLP-1R signaling (Hayes et al., 2009a
) following activation of the proglucagon-expressing neurons of the NTS (Vrang et al., 2003
) which increases endogenous CNS GLP-1 ligand availability. Data presented in this report show that the food intake suppressive effects of DVC parenchymal application of a GLP-1R ligand is mediated by mNTS neurons, as a significant suppression of intake was observed following Ex-4 delivery to the mNTS at a 4th
icv sub-threshold dose (0.025μg); effects not observed following administration of Ex-4 at the same dose or even at a ventricular-effective dose (0.05μg) to other hindbrain GLP-1R-expressing nuclei (i.e. AP or DMV). Moreover, using a combination of in vivo
and in vitro
techniques with the GLP-1R ligand Ex-4, we demonstrate that the intake suppressive effects of NTS-GLP-1R activation occur through a coordinated PKA-mediated suppression of AMPK activity and activation of p44/42 MAPK signaling. These effects may promote Ca+
-dependent depolarization of the GLP-1R expressing neurons and longer-term cAMP response element-binding protein (CREB)-mediated transcriptional effects. The present findings further suggest that the intake suppressive effects of gastric distension may also be mediated in part, by an increase in PKA, MAPK, and possibly CREB signaling, as well as inhibition of AMPK signaling within NTS neurons. It may also be the case that NTS mediation of the intake inhibitory responses of other anorectic signals [e.g. cholecystokinin, melanocortin, leptin] that engage these intracellular signaling pathways (Hayes et al., 2009b
; Hayes et al., 2010b
; Sutton et al., 2005
; Sutton et al., 2004
)] could potentially reduce food intake in a synergistic fashion with either CNS GLP-1R ligands and/or gastric distension. Given that PKA inhibition did not completely reverse the 24h intake suppression by 4th
icv Ex-4, it is also reasonable to assume that other non-PKA-dependent signaling pathways may mediate the suppression of intake by hindbrain GLP-1R activation.
While caudal brainstem processing (in the absence of forebrain communication) is sufficient to mediate the food intake suppressive effects following 4th
icv Ex-4 administration (Hayes et al., 2008c
), the NTS is not the only CNS GLP1-R-expressing nucleus relevant to energy balance control (e.g. PVH; central nucleus of the amygdala, CeA; dorsal medial hypothalamus, DMH). Indeed, parenchymal application of GLP-1(7-36) in the PVH suppresses food intake in rats (McMahon and Wellman, 1997
). Whether the intracellular signaling cascades for PVH or other GLP-1R-expressing neurons are identical to those reported here for the DVC GLP-1R remains to be determined. It may be possible that in GLP-1R-expressing nuclei outside the NTS, different intracellular signaling pathways may mediate different behavioral/physiological responses produced by GLP-1R activation. Such a case has been made previously for the angiotensin receptor (Daniels et al., 2005
). Nonetheless, it is reasonable to assume that the signaling cascades for GLP-1R-expressing neurons are similar throughout the brain, as the ex vivo
signaling responses reported here for the DVC tissue lysates were also found in vitro
in immortalized hypothalamic GLP-1R-expressing GT1-7 cells. It is virtually impossible to rule out the possibility that intracellular signaling inhibitors/activators (e.g., U0126/AICAR) attenuate the behavioral intake suppression of 4th
icv Ex-4 by acting on both GLP-1R- and non-GLP-1R-expressing neurons.
Hindbrain Ex-4 administration rapidly increases NTS PKA activity, with a peak in activity occurring 10min-post administration and a return to baseline (vehicle) levels seen at 30min-post administration. Yet, behavioral analyses show that the suppression of food intake by 4th
icv Ex-4 is observed by the 3rd
hour post-administration, an effect dependent upon an increase in PKA and MAPK activity, as well as a decrease in AMPK activity. This distinction in the time-course effects for the intracellular signaling cascades from tissue lysates and those for the food intake response have been reported previously for other anorectic ligands and intracellular signals acting within the brain (Morton et al., 2009
; Niswender et al., 2001
; Sutton et al., 2005
). These observations can be interpreted to suggest that although PKA-, MAPK-, and AMPK-signaling are required to mediate the suppression of intake by NTS GLP-1R activation, other downstream signaling cascades and recruitment of neurons of other CNS nuclei are also required to mediate the intake-suppressive action of hindbrain Ex-4. These additional signaling cascades and neuronal projections arising from NTS GLP-1R-expressing neurons remain to be identified.
That suppression in meal number accounts for the reduced food intake resulting from hindbrain
Ex-4 supports the notion that the central GLP-1 system is different, but complementary to the peripheral GLP-1 system. This conclusion is based in part on data presented here, as well as previous observations (Ruttimann et al., 2009
; Scott and Moran, 2007
) that show that the short term intake inhibitory effects of peripheral
GLP-1R activation result from a reduction in meal size, with minimal effect on meal number. It may be possible that peripheral administration of Ex-4 or other DPP-IV-resistant GLP-1R ligands with longer half-lives than GLP-1(7-36) (e.g., liraglutide) may reduce food intake across a longer period of time by simultaneous activation of both peripheral and central GLP-1 systems.
In conclusion, current findings demonstrate that the food intake- and body weight-suppressive effects of hindbrain GLP-1R activation are dependent upon an increase in PKA activity and subsequent simultaneous phosphorylation of p44/42-MAPK and inhibition of AMPK activity in NTS neurons. Ex-4 activation of the same signaling cascades in GT1-7 neuronal cells and in NTS lysates supports the view that these pathways occur in GLP-1R-expressing neurons. Together with our previous reports (Hayes et al., 2009a
; Hayes et al., 2008c
), current data demonstrate that NTS GLP-1R-expressing neurons play a physiological role in control of food intake and energy balance regulation. These data provide clues to understanding how NTS neurons mediate anorectic actions of other stimuli involved in meal-to-meal food intake control. Future pharmacotherapies targeting not only NTS GLP-1R, but the CNS GLP-1 system as a whole, may prove to be efficacious in ameliorating chronic hyperphagia associated with obesity.