Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide Introduction Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists reduce food intake and body weight and are approved for weight loss (Drucker, 2022).Elucidating the mechanisms through which GLP-1R agonists regulate body weight could enhance the therapeutic potential of these compounds or identify novel targets for anti-obesity drug development.We have shown that the GLP-1R agonist Exendin-4 (Ex4) stimulates mechanistic Target of Rapamycin (mTOR) Complex-1 (mTORC1) signaling and that inhibition of mTORC1 signaling in the ventromedial hypothalamus (VMH) with rapamycin blocks the anorectic effect induced by Ex4 in this nucleus (Brown et al., 2018;Burmeister et al., 2017).The present studies aim to define the mechanism(s) by which GLP-1R agonism stimulates mTORC1 signaling and reduces body weight.
Here we demonstrate that GLP-1R activation induces mTORC1 signaling and Raptor phosphorylation in a PKA-dependent manner and show that this contributes to weight loss by the therapeutic GLP-1R agonist liraglutide.This reveals a novel weight-lowering GLP-1R-PKA-mTORC1 axis and broadly suggests that other G α s-coupled receptors may use this PKA-mTORC1 pathway for certain biological responses.

GLP-1R activation induces PKA phosphorylation of Raptor at Ser 791
The catalytic subunit of PKA directly phosphorylates Ser 791 of Raptor (Liu et al., 2016).We tested whether liraglutide promotes PKA phosphorylation of Raptor at Ser 791 by expressing Myc-tagged wild-type (WT) Raptor or Myc-tagged mutant Raptor with alanine replacing serine at the 791 st position (Ser 791 Ala Raptor) in CHO-GLP-1R cells.Using an antibody against the PKA motif RRXS*/T*, immunoblotting of immunoprecipitated Myc-proteins shows that liraglutide and forskolin increase PKA motif phosphorylation on Myc-WT Raptor in a PKA-dependent manner (blocked by H89), but liraglutide fails to increase PKA motif phosphorylation on Myc-Ser 791 Ala Raptor (Figure 2A and B).This was also observed in β-TC3 cells, an immortalized β-cell line endogenously expressing the GLP-1R (Figure 2C).We tested the physiological role of PKA phosphorylation of Raptor at Ser 791 by measuring body weight and glycemic responses to liraglutide in wild-type (WT) and knock-in mice in which endogenous Raptor is replaced by a PKA-resistant Raptor expressing alanine instead of serine at position 791 in all tissues (CMV-Ser 791 Ala Raptor).WT and CMV-Ser 791 Ala Raptor knock-in mice lost weight in response to liraglutide treatment, but liraglutide-induced weight loss was attenuated in the CMV-Ser 791 Ala Raptor knock-in mice (Figure 3A-D).This was primarily due to a lesser reduction in fat mass in liraglutidetreated CMV-Ser 791 Ala Raptor knock-in compared to WT mice (Figure 3E-F).There was no significant difference between genotypes in the ability of liraglutide to reduce lean (Figure 3G-H) or free fluid (not shown) mass.Metabolic cage experiments demonstrate that food intake was equally suppressed in WT and CMV-Ser 791 Ala Raptor knock-in mice during the first day of liraglutide treatment.However, food intake returned to pre-liraglutide treatment levels in CMV-Ser 791 Ala Raptor knock-in mice by the second day of treatment whereas they did not return to pre-liraglutide treatment levels in WT mice until the fifth day of dosing (Figure 3I-J).Energy expenditure (EE) decreased in response to liraglutide in both genotypes and remained significantly lower in CMV-Ser 791 Ala Raptor knock-in mice compared to their vehicle counterparts for up to 8 days of dosing (Figure 3K-L).In WT mice, EE only remained lower in liraglutide-treated mice for 3 days (Figure 3K-L).These data suggest that phosphorylation of Raptor at Ser 791 is required for the full body weight-lowering effect of liraglutide via regulation of both food intake and EE.
There was no difference between genotypes in fasting blood glucose prior to liraglutide treatment (Figure 3-figure supplement 1).Two weeks of liraglutide treatment lowered fasting glucose to similar degrees in both genotypes, and there was no difference in fasting plasma insulin levels (Figure 3-figure supplement 1).

PKA phosphorylation of Raptor at Ser 791 does not mediate melanocortin-4 receptor (MC4R) agonist-induced weight loss
To test whether Raptor phosphorylation at Ser 791 is required exclusively for the weight loss effect of liraglutide or is necessary for other weight loss drugs, particularly those targeting G α s-coupled receptors, we treated CMV-Ser 791 Ala Raptor knock-in mice with the MC4R agonist setmelanotide.Like the GLP-1R, the MC4R is also a G α s-coupled receptor, and setmelanotide is approved for weight loss in individuals with monogenic obesity caused by mutations in pro-opiomelanocortin, proprotein convertase subtilisin/kexin type 1, or leptin receptor deficiency and in individuals with Bardet-Biedl syndrome (Markham, 2021).As shown in Figure 5, CMV-Ser 791 Ala Raptor knock-in mice were not resistant to weight loss in response to setmelanotide.This suggests that Raptor phosphorylation at Ser 791 by PKA contributes to the weight loss effect of GLP-1R agonists but not another G α s-targeting pharmacological agent such as setmelanotide.

Discussion
GLP-1R-stimulated mTORC1 signaling in the VMH reduces food intake (Burmeister et al., 2017).This is consistent with studies showing that activation of hypothalamic mTORC1 reduces food intake and mediates weight lowering by leptin (Blouet et al., 2008;Cota et al., 2008;Cota et al., 2006) .We show that PKA phosphorylates the mTORC1-regulatory protein Raptor at Ser 791 in response to liraglutide and that this is responsible for ~50% of liraglutide-induced weight loss.Given the complexity of signaling mechanisms downstream of the GLP-1R (Fletcher et al., 2016), assigning specific pathways to a desirable phenotype could be leveraged towards more effective weight loss therapeutics.
The online version of this article includes the following source data for figure 2:  demonstrating that forskolin inhibits insulin-stimulated S6 phosphorylation and also subsequently shown by Liu and colleagues (Liu et al., 2016).
In vivo results using novel PKA-resistant Raptor knockin mice suggest that PKA phosphorylation of Raptor contributes to the weight loss effect of liraglutide in lean but not in obese mice.One limitation of these in vivo studies is that PKA-resistant Raptor is expressed in all tissues.Loss of PKA phosphorylation of Raptor in one or multiple tissues may contribute to our observation that CMV-Ser 791 Ala mice gained significantly more weight on the HFD compared to control mice.This difference in starting body weight could explain why liraglutide-induced weight loss was not attenuated in obese CMV-Ser 791 Ala mice, as in lean mice, since heavier mice tend to be more responsive to liraglutide (Figure 4-figure supplement 1).This could offset any reduction in liraglutide responsiveness resulting from expression of PKA-resistant Raptor.We support this by showing that CMV-Ser 791 Ala mice with lower initial body weights display a tendency towards reduced responsiveness to liraglutide.Future studies will circumvent the limitations of the CMV-Ser 791 Ala model by introducing the Ser 791 Ala mutation to specific tissues/cell types that specifically mediate liraglutide-induced weight loss.The brain is a key target for the weight-lowering effects of GLP-1R agonists (Adams et al., 2018;Sisley et al., 2014;Varin et al., 2019).Regulation of energy balance by mTORC1 appears to be most critical in hypothalamic regions such as the arcuate (ARC), paraventricular hypothalamus (PVH), VMH, and suprachiasmatic nucleus (Cota et al., 2006;Inhoff et al., 2010;Proulx et al., 2008), where the GLP-1R is highly expressed (Jensen et al., 2018;Cork et al., 2015).The ARC mediates weight loss effects of liraglutide (Secher et al., 2014) and is engaged by systemic liraglutide (Salinas et al., 2018).Hindbrain regions are also engaged by systemic liraglutide and mediate its weight-lowering effects (Salinas et al., 2018;Fortin et al., 2020).Inhibition of PKA attenuates the anorectic effect of hindbrain-targeted Ex4 in rats (Hayes et al., 2011).Future studies expressing Ser 791 Ala Raptor in a brain region/cell-type specific manner will enable identification of neuronal population(s) where a PKA-mTORC1 interaction mediates the anorectic effect of GLP-1R agonists.
The observation that CMV-Ser 791 Ala Raptor knock-in mice were not resistant to the weight loss effect of the MC4R agonist setmelanotide suggests that PKA-mediated phosphorylation of Raptor at Ser 791 is not a general mechanism for all G α s-coupled receptor-targeting weight loss drugs.However, as with our results using liraglutide in HFD-fed mice, a caveat to interpreting our findings with setmelanotide is the use of the whole-body CMV-Ser 791 Ala mouse model.Since the weight loss effect of setmelanotide is primarily mediated via activation of the MC4R in the PVH, future studies will test the hypothesis that the weight loss effect of setmelanotide will be blunted in PVH MC4R-specific Ser 791 Ala Raptor knock-in mice.This could establish a role for PKA-mediated regulation of mTORC1 as a key        Our results suggest that liraglutide does not regulate mTORC1 via Akt, as was observed for βAR signaling in adipocytes (Liu et al., 2016).Inhibition of Akt activity in the hindbrain (Rupprecht et al., 2013) or hypothalamus (Yang et al., 2017) attenuates the anorectic effect of Ex4, so identifying targets of Akt mediating the effects of GLP-1R agonists requires further study.AMP-activated protein kinase (AMPK) is also implicated in the metabolic actions of GLP-1R agonists.Studies from our lab and others show that GLP-1R agonists reduce food intake by inhibiting AMPK (Burmeister et al., 2017;Hayes et al., 2011;Burmeister et al., 2013;Sandoval et al., 2012).Since AMPK suppresses mTORC1 by phosphorylating TSC2 (Inoki et al., 2003) or Raptor (Gwinn et al., 2008), GLP-1R activation could stimulate mTORC1 by reducing AMPK activity.AMPK can also be inhibited by mTORC1 signaling (Dagon et al., 2012), so GLP-1R-mediated inhibition of AMPK may be downstream of mTORC1.
In summary, we have uncovered a novel signaling mechanism downstream of the GLP-1R characterized by PKA-mediated phosphorylation of Raptor and increased mTORC1 signaling that contributes to the weight-lowering effect of liraglutide.Given the variability in response and adverse effects (e.g.nausea) of current GLP-1-based drugs, the identification of therapeutically relevant signaling profiles downstream of the GLP-1R holds promise for improving efficacy and reducing side effects of these drugs.Furthermore, our findings provide a new framework for future investigations into the significance of the PKA-mTORC1 interaction to other beneficial metabolic effects of GLP-1R agonists.GLP-1R agonists continue to be used as glucose-lowering agents for type 2 diabetes based on their insulinotropic effect via pancreatic β-cells (Drucker, 2022).The PKA-mTORC1 interaction could contribute to this or another β-cell phenotype associated with GLP-1R agonists (e.g.proliferation/ cytoprotection).Furthermore, GLP-1R agonists also display cardioprotective effects that could be mediated directly via effects on the heart and vasculature and/or be secondary to the ability of these drugs to improve glycemia, body weight, lipid profiles, and inflammation (Ussher and Drucker, 2023).This provides an opportunity to investigate whether the PKA-mTORC1 interaction contributes to the cardioprotective effects of GLP-1R agonists via direct or indirect mechanisms.

Immunoprecipitation
Myc-tagged wild-type (WT) and PKA-resistant mutant (Ser 791 Ala) Raptor vectors (Liu et al., 2016) were transfected into CHO-GLP-1R cells using lipofectamine-3000 (Invitrogen).Forty-eight hours later, cells were washed 2 X and maintained in serum-free media for 3 hr.Cells were treated with DMSO, H89 or MK-2207 for 30 min and treated with 1 X PBS, liraglutide, forskolin, or insulin for 1 hr at concentrations stated in the figure legend.Cells were washed once with ice-cold 1 X PBS and lysed in lysis buffer (25 mM Tris HCl, 131 150 mM NaCl, 1 µM EDTA, 1 µM EGTA, 2.5 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM sodium orthovanadate) containing 1 X protease and phosphatase inhibitors.After centrifugation of cell homogenates (17,000xg, 10 min, 4 °C) supernatant protein concentration was determined by BCA protein assay.A total of 200 μg protein was incubated with anti-c-Myc affinity gel (EZview Red Anti-c-Myc Affinity Gel, #E6654, Sigma-Aldrich) overnight.Beads were washed in lysis buffer and immunoblotting was done as described above using a PKA substrate monoclonal antibody (Cat#9624, Cell Signaling Technology).

Animals
Mice were kept on a 12 hr/12 hr light/dark cycle with ad libitum access to water and chow (5L0D, LabDiet) from weaning and were studied at 12-16 weeks old.Procedures were approved by the Institutional Animal Care and Use Committee at Vanderbilt University (Protocol #M1500158).Ser 791 Ala Raptor knock-in mice were generated by inGenious Targeting Laboratory Inc using the targeting vector shown in Figure 3-figure supplement 2. The indicated loxP sites were engineered into the genomic DNA and a two base pair point mutation was introduced into a second inverted exon 20 of the Raptor coding sequence to change the codon for Ser at position 791 to Ala.Raptor fl/fl mice (013188, The Jackson Laboratory) were crossed to mice expressing the Ingenious construct, and offspring were then crossed to CMV-Cre (006054, The Jackson Laboratory) mice to generate hemizygous wild-type mice or hemizygous mice with global knock-in of Raptor Ser 791 Ala.This strategy was chosen because the Ingenious construct in homozygosity prior to Cre recombinase inadvertently created a hairpin loop in the transcript between the two exon 20 regions that was spliced out.Genotyping and cDNA sequencing confirmed expression of Ser 791 Ala Raptor in place of endogenous Raptor (Figure 3, Figure 3-figure supplement 2).Male mice were switched to either a low-fat diet (D12450J, Research Diets, Inc) for 4 weeks or a 60% HFD (D12492, Research Diets, Inc) for 8-10 weeks prior to initiation of dosing.Mice were maintained on the low-fat diet and were administered vehicle (0.9% saline), liraglutide (200 μg/kg body weight, BID), or setmelanotide (4 mg/kg body weight once daily) subcutaneously for 9-14 days.Body weight was measured daily, and body composition was obtained by NMR (Minispec 235 LF90II-TD NMR Analyzer, Bruker) prior to and on the last day of dosing.Four hours fasting blood glucose and insulin levels were measured using a Contour Next EZ glucose meter (Cat#BD15789, Bayer) and an Ultra Sensitive Mouse Insulin ELISA kit (Crystal Chem), respectively.A separate cohort of male mice was acclimated to a Promethion System (Sable Systems International, Inc) for one week prior to undergoing dosing with vehicle or liraglutide as described above for 10 days.Food intake and energy expenditure were measured continuously.

Data analysis
Data were analyzed using Prism-9 (GraphPad Software, Inc).One-way or two-way ANOVA or mixedeffects analysis followed by Tukey's, Holm-Sidak's, or Brown-Forsythe multiple comparisons was used when appropriate and indicated in the figure legends.Linear regression analysis was also used as indicated in the figure legends.Sample size was determined based on the variability of the primary measurements of interest (phosphorylation of S6 for cell culture studies and body weight for in vivo studies) and the objective of detecting significance (p<0.05) with 90% certainty.Treatments were randomized and blinded to the experimenter when possible.

Figure supplement 1
Figure supplement 1-source data 1.Quantification of absolute values from data in Figure 1-figure supplement 1 and raw data.

Figure supplement 2 .
Figure supplement 2. Dose response and time course of absolute pS6 and pCREB expression in CHO-K1 cells stably expressing the hGLP1R and treated with liraglutide.

Figure
Figure supplement 2-source data 1. Figure 1-figure supplement 2 and raw blots and raw data for Figure 1-figure supplement 2.

Figure 2 ,
raw blots from Figure 2, and raw data for Figure 2.

Figure 3
and raw data for Figure 3.

Figure supplement 1 .
Figure supplement 1. Blood glucose prior to drug treatment and blood glucose and insulin post-treatment in male wild-type (WT) and Ser 791 Ala Raptor knock-in mice (Resubmission Rev 2 Figure 3-figure supplement 1source data 1).

Figure supplement 1
Figure supplement 1-source data 1.Figure 3-figure supplement 1 and raw data for Figure 3-figure supplement 1.
Figure supplement 1-source data 1.Figure 3-figure supplement 1 and raw data for Figure 3-figure supplement 1.

Figure 4
and raw data for Figure 4.

Figure supplement 1 .
Figure supplement 1. Relationship between baseline body weight and liraglutide-induced weight loss in male wild-type (WT) and Ser 791 Ala Raptor knock-in mice (Resubmission Rev 2 Figure 4-figure supplement 1-source data 1).

Figure supplement 1
Figure supplement 1-source data 1.Figure 4-figure supplement 1 and its raw data for Figure 4-figure supplement 1.
Figure supplement 1-source data 1.Figure 4-figure supplement 1 and its raw data for Figure 4-figure supplement 1.

Figure 5 .
Figure 4 continued Figure 5 and raw data for Figure 5.