1. Cell Biology
  2. Medicine

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  Is a corresponding author
  8. Julio E Ayala  Is a corresponding author
  1. Vanderbilt University, United States
  2. Vanderbilt University Medical Center, United States
https://doi.org/10.7554/eLife.80944
Share this article
Cite this article
  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
(2023)
Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of raptor and this contributes to the weight loss effect of liraglutide
eLife 12:e80944.
https://doi.org/10.7554/eLife.80944
  2. Download BibTeX
  3. Download .RIS

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 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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

Article and author information

Author details

  1. Thao DV Le

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6242-2562

  2. Dianxin Liu

    Department of Medicine, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Gai-Linn K Besing

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Ritika Raghavan

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Blair J Ellis

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ryan P Ceddia

    Department of Medicine, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Sheila Collins

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    For correspondence
    sheila.collins@vumc.org
    Competing interests
    The authors declare that no competing interests exist.

  8. Julio E Ayala

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    For correspondence
    julio.e.ayala@vanderbilt.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3224-2365

Funding

National Institutes of Health (R01DK097361)

  • Julio E Ayala

National Institutes of Health (R01DK116625)

  • Sheila Collins

National Institutes of Health (S10OD028455)

  • Julio E Ayala

National Institutes of Health (R01DK132852)

  • Julio E Ayala

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Procedures were approved by the Institutional Animal Care and Use Committee at Vanderbilt University (Protocol #M100158).

Copyright

© 2023, Le et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,092
    views
  • 167
    downloads
  • 4
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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
(2023)
Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of raptor and this contributes to the weight loss effect of liraglutide
eLife 12:e80944.
https://doi.org/10.7554/eLife.80944

Share this article

https://doi.org/10.7554/eLife.80944

Categories and tags

Research organism

Further reading

    1. Cell Biology

    High-throughput expansion microscopy enables scalable super-resolution imaging

    John H Day, Catherine M Della Santina ... Laurie A Boyer
    Tools and Resources

    Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

    1. Cell Biology
    2. Developmental Biology

    The exocyst complex controls multiple events in the pathway of regulated exocytosis

    Sofía Suárez Freire, Sebastián Perez-Pandolfo ... Mariana Melani
    Research Article

    Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.

    1. Cell Biology

    Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation

    Yue Miao, Yongtao Du ... Mei Ding
    Research Article

    The spatiotemporal transition of small GTPase Rab5 to Rab7 is crucial for early-to-late endosome maturation, yet the precise mechanism governing Rab5-to-Rab7 switching remains elusive. USP8, a ubiquitin-specific protease, plays a prominent role in the endosomal sorting of a wide range of transmembrane receptors and is a promising target in cancer therapy. Here, we identified that USP8 is recruited to Rab5-positive carriers by Rabex5, a guanine nucleotide exchange factor (GEF) for Rab5. The recruitment of USP8 dissociates Rabex5 from early endosomes (EEs) and meanwhile promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In USP8-deficient cells, the level of active Rab5 is increased, while the Rab7 signal is decreased. As a result, enlarged EEs with abundant intraluminal vesicles accumulate and digestive lysosomes are rudimentary. Together, our results reveal an important and unexpected role of a deubiquitinating enzyme in endosome maturation.