Intracellular signals mediating the food intake-suppressive effects of hindbrain glucagon-like peptide-1 receptor activation

Abstract

Glucagon-like peptide-1 receptor (GLP-1R) activation within the nucleus tractus solitarius (NTS) suppresses food intake and body weight (BW), but the intracellular signals mediating these effects are unknown. Here, hindbrain (fourth i.c.v.) GLP-1R activation by Exendin-4 (Ex-4) increased PKA and MAPK activity and decreased phosphorylation of AMPK in NTS. PKA and MAPK signaling contribute to food intake and BW suppression by Ex-4, as inhibitors RpcAMP and U0126 (fourth i.c.v.), respectively, attenuated Ex-4's effects. Hindbrain GLP-1R activation inhibited feeding by reducing meal number, not meal size. This effect was attenuated with stimulation of AMPK activity by AICAR (fourth i.c.v.). The PKA, MAPK, and AMPK signaling responses by Ex-4 were present in immortalized GLP-1R-expressing neurons (GT1-7). In conclusion, hindbrain GLP-1R activation suppresses food intake and BW through coordinated PKA-mediated suppression of AMPK and activation of MAPK. Pharmacotherapies targeting these signaling pathways, which mediate intake-suppressive effects of CNS GLP-1R activation, may prove efficacious in treating obesity.

Figure 1

(A) Increased PKA activity (nmol/g/min) in tissue of the caudal DVC following 4^th icv Ex-4 (0.3μg) administration. * = P< 0.05 from aCSF. Cumulative chow intake (B) and 24h body weight change (C) following 4^th icv delivery of the PKA inhibitor RpcAMP (20μg) or Ex-4 (0.2μg) alone or in combination, counterbalanced with vehicle/vehicle (aCSF/aCSF). Data are mean ± SEM. * = P< 0.05 from vehicle/vehicle. † = P< 0.05 from vehicle/Ex-4.

Figure 2

(A) Increased phosphorylation of p44/42-MAPK in caudal DVC tissue lysates following 4^th icv Ex-4 (0.3μg) administration. Representative immunoblots for phosphorylated and total p44/42-MAPK are shown. * = P< 0.05 from aCSF. Cumulative chow intake (B) and 24h body weight change (C) following 4^th icv delivery of the MEK inhibitor U0126 (2μg) or Ex-4 (0.2μg) alone or in combination, counterbalanced with vehicle/vehicle (DMSO/aCSF). Data are mean ± SEM. * = P< 0.05 from vehicle/vehicle. † = P< 0.05 from vehicle/Ex-4.

Figure 3

(A) Decreased phosphorylation of AMPKα2 in caudal DVC tissue lysates following 4^th icv Ex-4 (0.3μg) administration. Representative immunoblots for pAMPKα2 and total AMPKα are shown. * = P< 0.05 from aCSF. Cumulative chow intake (B) and 24h body weight change (C) following 4^th icv delivery of the AMPK activity promoter AICAR (300μg) or Ex-4 (0.1μg) alone or in combination, counterbalanced with vehicle/vehicle (aCSF/aCSF). Chow intake recorded via automated feedometers that continuously record food intake to the nearest ±0.1g/m/24h. Data are mean ± SEM. * = P< 0.05 from vehicle/vehicle. # = P=0.065 from vehicle/Ex-4.

Figure 4

(A) 4^th icv Ex-4 (0.3μg)-driven decrease phosphorylation in AMPKα2 in caudal DVC tissue lysates is PKA dependent, as 4^th icv administration of the PKA inhibitor RpcAMP (20μg) attenuated the suppression in pAMPKα2 by Ex-4. Representative immunoblots for pAMPKα2 and total AMPKα are shown. * = P< 0.05 from aCSF/aCSF. † = P< 0.05 from aCSF/Ex-4. (B) RpcAMP administration attenuated the increased phosphorylation of p42-MAPK by Ex-4 administration, but did not alter the increased phosphorylation of p44-MAPK by Ex-4. Data are mean ± SEM. * = P< 0.05 from aCSF/aCSF. † = P< 0.05 from aCSF/Ex-4.

Figure 5

Cumulative chow intake following (A) hindbrain (4^th icv) delivery of Ex-4 (0.025μg and 0.05μg in 1μl), (B) unilateral intraparenchymal mNTS delivery of Ex-4 (0.025μg and 0.05μg in 100nl), (C) unilateral intraparenchymal DMV delivery of Ex-4 (0.05μg in 100nl), (D) intraparenchymal AP delivery of Ex-4 (0.025μg and 0.05μg in 100nl), counterbalanced with aCSF. (E) Functional verification of AP injections measured by cumulative 4h chow intake following AP administration of the amylin (calcitonin) receptor agonist, salmon calcitonin (sCT) (0.4μg in 100nl) counterbalanced with aCSF. Data are mean ± SEM. * = P< 0.05 from aCSF.

Figure 6

(A) Total cAMP levels in 3hr serum-starved GLP-1R-expressing GT1-7 neuronal cells following 15min treatment with Ex-4 (0.0, 0.01, 0.1, 1.0, 10 nmol). * = P< 0.05 from vehicle (0.0nmol Ex-4). (B) Ex-4 treatment (0.0, 1.0, 10, and 100 nmol; 15min) dose-dependently decreased pAMPKα2 and (C) increased phosphorylated p44/42 MAPK in the GLP-1R-expressing GT1-7 neurons. Data are mean ± SEM. * = P< 0.05 from vehicle (0.0nmol Ex-4).

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., 2009a) to engage cAMP-dependent increase in PKA activity. Increased PKA activity concomitantly increases phosphorylation of p44/42-MAPK and decreases phosphorylation of AMPK. The combined increase in PKA and p44/42-MAPK activity together with decreased AMPK activity is hypothesized to increase CREB-mediated nuclear transcription and protein synthesis. mTOR = mammalian target of rapamycin; CAMKK = Calmodulin-dependent protein kinase kinase; CAMKII = calcium/calmodulin-dependent protein kinase II; VGCC = voltage gated calcium channel; MEK = mitogen-activated protein kinase kinase.