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

  1. Thao DV Le
  2. Dianxin Liu
  3. Gai-Linn K Besing
  4. Ritika Raghavan
  5. Blair J Ellis
  6. Ryan P Ceddia
  7. Sheila Collins
  8. Julio E Ayala

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    Evaluation Summary:

    This manuscript examines the importance of PKA-dependent mTORC1 activation for the weight-loss effects of liraglutide. The work has the potential to provide important insights, but at present is deemed preliminary as it lacks details on the mouse model and control data and needs a more in-depth analysis of the metabolic phenotype.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

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 Ser 791 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 Ser 791 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 (Ser 791 Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser 791 Ala 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 Ser 791 Ala Raptor in CHO-Glp1r cells. Lean Ser 791 Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser 791 Ala 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 Ser 791 Ala 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 Ser 791 contributes to liraglutide-induced weight loss.

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  1. Version published to 10.7554/elife.80944 on eLife
  2. Version published to 10.1101/2022.04.21.489078v3 on bioRxiv
  3. Version published to 10.1101/2022.04.21.489078v2 on bioRxiv
  4. eLife

    Author Response

    Reviewer #3 (Public Review):

    The manuscript by Le T.D.V. et al used in vitro cell culture and inhibitors for cellular signaling molecules and found that GLP-1 receptor activation stimulated the phosphorylation of Raptor, which was PKA-mediated and Akt-independent. The authors reported the physiological function of this GLP-1R-PKA-Raptor in liraglutide stimulated weight loss. This timely study has high significance in the field of metabolic research for the following reasons.

    (1) The authors' findings are significant in the field of obesity research. GLP-1 receptor (GLP-1R) is a successful target for diabetes (and weight loss) therapeutics. However, the mechanisms of action for the weight-loss effect of GLP-1 agonists are not fully understood. Therefore, mechanistic studies to elucidate the signaling pathways of GLP-1 receptors pertaining to weight loss at the cellular level are timely.

    (2) G protein-coupled receptors (GPCRs) induces various signaling activities, which could be cellular and tissue specific. As these are an important protein family for drug targeting, understanding the basic biology of these receptors is of interest to a broad readership.

    (3) The authors have made important discoveries that Exendin-4 stimulated mTORC1 signaling was essential for the anorectic effect induced by Exendin-4. The study reported in this current manuscript provides more details of brain GLP-1R signaling pathways and is innovative.

    Overall, the authors have presented sufficient background in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, took potential caveats into consideration, and made a justified conclusion.

    Recommendations for the authors:

    The manuscript can be further strengthened with more clarification on the following points.

    1. In Figure 1 panels B and C, please provide the quantification for pCREB/CREB. In Figure 1 panel D, please provide the quantification for pAkt/Akt.

    We thank the Reviewer for this suggestion. We now provide quantification of pCREB and pAkt expression in Supp. Fig. 1.

    1. The western blots to assess the signaling activities revealed the phosphorylation status of the key signaling molecules at a single time point. Whether the overall signaling dynamics have been affected is unclear.

    We agree with the reviewer on this point. We conducted initial time course experiments to identify a suitable time point for the subsequent experiments conducted in the present studies. The 1h time point presented in our results was chosen because it was the earliest time point at which both liraglutide stimulated mTORC1 signaling and this effect was inhibited by the various pharmacological inhibitors. We agree with the reviewer that at this point it is not clear whether the various inhibitors or the Ser791Ala mutation in Raptor modifies the dynamics of mTORC1 signaling. Although we have preliminary data in CHO-K1 cells suggesting that the temporal dynamics of these signaling events are not affected, this does not necessarily translate to the in vivo setting. Once we identify the key target tissue/cell type(s) mediating the weight loss effect of liraglutide via the PKA-Raptor interaction and generate the necessary mutant mice, we will test whether this affects signaling dynamics in vivo.

    1. Figure 3 panels A and B demonstrated the remarkable importance of the Ser791 Raptor. However, this PKA-resistant mutant did not completely abolish the weight loss effect of liraglutide. The authors pointed out the importance of AMPK in mTORC1 signaling. Other pathways that may complement GLP-1R-PKA-Raptor signaling can be further discussed.

    We agree with the reviewer that other signaling pathways are likely involved that contribute to the remaining weight-lowering effect of liraglutide. Besides AMPK, we have also included a discussion of Akt being a potential molecule that interacts with these pathways in vivo (lines 218-225). The word limitations of a Short Communication prevent us from further expanding on these possible mechanisms.

    1. Food intake was decreased on day 2 in Figure 3D but became comparable between WT and S791A Raptor groups on the following days. Could this be due to some compensatory mechanisms?

    This pattern of food intake response to GLP-1R agonists has been previously reported by our group and others (please see Brown JD et al. Am J Physiolo Regul Integr Comp Physiol 2018 and Adams JM et al. Diabetes 2018). The reason for this is unclear at this moment, but we can speculate that the rebound in food intake is a compensatory mechanism to prevent the organism from continuously losing weight. We now also present also showing an initial drop in energy expenditure with liraglutide treatment that progressively increases to pre-treatment levels.

  5. eLife

    Evaluation Summary:

    This manuscript examines the importance of PKA-dependent mTORC1 activation for the weight-loss effects of liraglutide. The work has the potential to provide important insights, but at present is deemed preliminary as it lacks details on the mouse model and control data and needs a more in-depth analysis of the metabolic phenotype.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  6. eLife

    Reviewer #1 (Public Review):

    This is an interesting manuscript that uses cell culture models to demonstrate the activation of mTORC1 by GPCR (GLP1R) PKA signaling and then goes on to use a PKA-insensitive mutant raptor-expressing mouse like to imply the importance of this PKA-dependent mTORC1 signaling for GLP1R agonist-dependent weight loss.

    There are some important weaknesses in the manuscript as it currently stands, however:
    1. There is no information on the mouse model, other than statements about the model expressing the mutant raptor in all cells and tissues. It is impossible to evaluate the results of this manuscript without some information on the genetics of the model, and some data showing the expression of the mutant, however.

    2. The in vivo (mouse data) doesn't show the specificity of the weight-loss effects of GPCR-PKA signaling.

    3. The cell culture data appear not to permit the direct comparison of results among conditions - is there no attenuation of Insulin-mediated pS6 by KT and no attenuation of Lira-mediated pS6 by MK? Relatedly, why does MK block FSK-mediated pS6?

  7. eLife

    Reviewer #2 (Public Review):

    In this study, Le and coauthors have investigated the role of the mTOR-regulating protein Raptor in determining the body weight loss effect of the GLP1-R agonist liraglutide. Here the authors have used cell culture to demonstrate that GLP1-R activation leads to stimulation of the mTOR pathway by phosphorylating the mTOR regulating protein raptor at Ser791 through a protein kinase A (PKA)-dependent mechanisms. The authors have then generated a specific PKA-resistant Raptor mutant. This mouse model shows a blunted weight loss response to 2 weeks of peripheral administration of Liraglutide.

    Strengths:

    There is a major interest in the field of metabolism in pinpointing the exact mechanisms of action of GLP1-R agonists at the molecular level as this evidence may pave the way towards better treatments for obesity. This study suggests that the raptor may be an interesting molecular mechanism to follow upon.

    Weaknesses:

    The work carried out with the use of the mouse mutant model does not allow pinpointing the exact mechanisms leading to impaired weight loss. The evidence provided by the authors, although of interest, remains preliminary and incomplete.

    Liraglutide is used in patients suffering from obesity to help decrease weight. Here the mouse model used has not been made obese by diet before starting the treatment. Therefore, differently from the claim of the authors, we cannot conclude that raptor is relevant for the therapeutic action of Liraglutide. The data rather show that treatment with Liraglutide prevents weight gain when animals are switched to a high-fat diet and that this effect is partly obliterated by the PKA-resistant Raptor mutant.

    The statistical analysis used is not the most appropriate. The authors should consider carrying out statistical analyses in which both the treatment and the genotype are included as independent variables.

  8. eLife

    Reviewer #3 (Public Review):

    The manuscript by Le T.D.V. et al used in vitro cell culture and inhibitors for cellular signaling molecules and found that GLP-1 receptor activation stimulated the phosphorylation of Raptor, which was PKA-mediated and Akt-independent. The authors reported the physiological function of this GLP-1R-PKA-Raptor in liraglutide stimulated weight loss. This timely study has high significance in the field of metabolic research for the following reasons.

    (1) The authors' findings are significant in the field of obesity research. GLP-1 receptor (GLP-1R) is a successful target for diabetes (and weight loss) therapeutics. However, the mechanisms of action for the weight-loss effect of GLP-1 agonists are not fully understood. Therefore, mechanistic studies to elucidate the signaling pathways of GLP-1 receptors pertaining to weight loss at the cellular level are timely.

    (2) G protein-coupled receptors (GPCRs) induces various signaling activities, which could be cellular and tissue specific. As these are an important protein family for drug targeting, understanding the basic biology of these receptors is of interest to a broad readership.

    (3) The authors have made important discoveries that Exendin-4 stimulated mTORC1 signaling was essential for the anorectic effect induced by Exendin-4. The study reported in this current manuscript provides more details of brain GLP-1R signaling pathways and is innovative.

    Overall, the authors have presented sufficient background in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, took potential caveats into consideration, and made a justified conclusion.

    The manuscript can be further strengthened with more clarification on the following points.

    1. In Figure 1 panels B and C, please provide the quantification for pCREB/CREB. In Figure 1 panel D, please provide the quantification for pAkt/Akt.

    2. The western blots to assess the signaling activities revealed the phosphorylation status of the key signaling molecules at a single time point. Whether the overall signaling dynamics have been affected is unclear.

    3. Figure 3 panels A and B demonstrated the remarkable importance of the Ser791 Raptor. However, this PKA-resistant mutant did not completely abolish the weight loss effect of Liraglutide. The authors pointed out the importance of AMPK in mTORC1 signaling. Other pathways that may complement GLP-1R-PKA-Raptor signaling can be further discussed.

    4. Food intake was decreased on day 2 in Figure 3D but became comparable between WT and S791A Raptor groups on the following days. Could this be due to some compensatory mechanisms?

  9. Version published to 10.1101/2022.04.21.489078v1 on bioRxiv