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 Ser791 following β-adrenergic stimulation. Thus, we tested whether GLP-1R agonists similarly stimulate mTORC1 activity via PKA phosphorylation of Raptor at Ser791 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 Ser791 in response to liraglutide, we expressed myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant in hGLP1R-stably expressing CHO-K1 cells, treated them with vehicle or liraglutide, immunoprecipitated myc-WT or myc-Ser791Ala 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 Ser791Ala 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-Ser791Ala Raptor. In addition, knock-in mice expressing Ser791Ala 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 Ser791, which activates mTORC1 signaling in a non-canonical fashion to facilitate the weight lowering effects of liraglutide.