Glucagon-Like Peptide-1 Receptor Activation Stimulates PKA-Mediated Phosphorylation of Raptor and this Contributes to the Weight Loss Effects of Liraglutide

Objective: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists are approved for obesity management, yet surprisingly little is known about the signaling mechanisms mediating GLP-1R agonist-induced anorexia and weight loss. We have shown that GLP-1R activation in the ventromedial hypothalamus (VMH) stimulates mechanistic Target of Rapamycin Complex 1 (mTORC1) activity, and this is necessary for the ability of VMH GLP-1R agonism to reduce food intake and body weight. Here, we aim to define the signaling mechanisms by which GLP-1R activation induces mTORC1 activity to regulate body weight. Although mTORC1 is classically activated by Akt, previous studies show that the canonical target of the GLP-1R, Protein Kinase A (PKA), stimulates mTORC1 signaling by phosphorylating the mTOR-regulating protein Raptor at Ser 791 following β-adrenergic stimulation. Thus, we tested whether GLP-1R agonists similarly stimulate mTORC1 activity via PKA phosphorylation of Raptor at Ser 791 and whether this phosphorylation event is relevant to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. Methods: We treated Chinese Hamster Ovary (CHO-K1) cells stably expressing the human GLP-1R ( hGLP1R ) with the GLP-1R agonist liraglutide in combination with either of the PKA inhibitors H89 or KT5720 and measured phosphorylation of the mTORC1 signaling target ribosomal protein S6. To test whether PKA phosphorylates Raptor at Ser 791 in response to liraglutide, we expressed myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser 791 Ala) Raptor mutant in hGLP1R -stably expressing CHO-K1 cells, treated them with vehicle or liraglutide, immunoprecipitated myc-WT or myc-Ser 791 Ala Raptor, and immunoblotted for phosphorylation of the PKA substrate RRXS*/T* motif, as previously described by Liu et al. 1 . To assess the physiological relevance of GLP-1R agonist-induced PKA phosphorylation of Raptor, we measured the body weight response to vehicle or liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser 791 Ala Raptor. Results: In CHO-K1 cells stably expressing the hGLP1R , liraglutide significantly increased phosphorylation of S6 and PKA phosphorylation of myc-tagged WT Raptor, both of which were blocked by PKA inhibition. Liraglutide also failed to stimulate phosphorylation of the PKA-targeting motif in cells expressing myc-Ser 791 Ala Raptor. In addition, knock-in mice expressing Ser 791 Ala Raptor were partially resistant to GLP-1R agonist-induced weight loss. Conclusion: Our results demonstrate that PKA activation downstream of the GLP-1R leads to phosphorylation of Raptor at Ser 791 , which activates mTORC1 signaling in a non-canonical fashion to facilitate the weight lowering effects of liraglutide. GLP-1 and GLP-1R agonists. In nutrient-deficient states, AMPK suppresses mTORC1 activity by phosphorylating TSC2 37 or Raptor 38 . Prior studies from our lab and others suggest that AMPK may mediate the anorectic effects of GLP-1R activation. 11,31,34,39,40 We show that Ex4 inhibits AMPK and that the AMPK activator AICAR delivered intracerebroventricularly (ICV) 28 or stereotaxically to the VMH 11 blunts the anorectic effect of Ex4. This suggests that GLP-1R agonists reduce food intake and body weight, at least in part, by inhibiting AMPK. Since