1. Cell Biology

β-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveal their essential role as nutrient sensors for the KATP channel

  1. Hannah R Foster
  2. Thuong Ho
  3. Evgeniy Potapenko
  4. Sophia M Sdao
  5. Shih Ming Huang
  6. Sophie L Lewandowski
  7. Halena R VanDeusen
  8. Shawn M Davidson
  9. Rebecca L Cardone
  10. Marc Prentki
  11. Richard G Kibbey
  12. Matthew J Merrins  Is a corresponding author
  1. University of Wisconsin-Madison, United States
  2. Massachusetts Institute of Technology, United States
  3. Yale University, United States
  4. University of Montreal, Canada
https://doi.org/10.7554/eLife.79422
Share this article
Cite this article
  1. Hannah R Foster
  2. Thuong Ho
  3. Evgeniy Potapenko
  4. Sophia M Sdao
  5. Shih Ming Huang
  6. Sophie L Lewandowski
  7. Halena R VanDeusen
  8. Shawn M Davidson
  9. Rebecca L Cardone
  10. Marc Prentki
  11. Richard G Kibbey
  12. Matthew J Merrins
(2022)
β-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveal their essential role as nutrient sensors for the KATP channel
eLife 11:e79422.
https://doi.org/10.7554/eLife.79422
  2. Download BibTeX
  3. Download .RIS

Abstract

Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking β-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism generates both glycolytic and mitochondrially-derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially-derived ATP. Following membrane depolarization, the PEP cycle is also involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and β-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the β-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.

Data availability

Datasets generated or analyzed in this study are included in the manuscript and supporting files. Source data files are provided for Figures 1-6 and the associated figure supplement files.

Article and author information

Author details

  1. Hannah R Foster

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Thuong Ho

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Evgeniy Potapenko

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Sophia M Sdao

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Shih Ming Huang

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Sophie L Lewandowski

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Halena R VanDeusen

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Shawn M Davidson

    Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Rebecca L Cardone

    Department of Internal Medicine, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Marc Prentki

    Department of Nutrition, University of Montreal, Montreal, Canada
    Competing interests
    The authors declare that no competing interests exist.

  11. Richard G Kibbey

    Department of Internal Medicine, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.

  12. Matthew J Merrins

    Department of Medicine, University of Wisconsin-Madison, Madison, United States
    For correspondence
    merrins@wisc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1599-9227

Funding

National Institutes of Health (R01DK113103)

  • Matthew J Merrins

National Institutes of Health (R01DK113103)

  • Matthew J Merrins

U.S. Department of Veterans Affairs (I01B005113)

  • Matthew J Merrins

Health Resources and Services Administration (T32HP10010)

  • Hannah R Foster

National Institutes of Health (T32AG000213)

  • Hannah R Foster

National Institutes of Health (T32DK007665)

  • Sophie L Lewandowski

American Diabetes Association (1-17-PDF-155)

  • Halena R VanDeusen

National Institutes of Health (R01AG062328)

  • Matthew J Merrins

National Institutes of Health (R01DK127637)

  • Richard G Kibbey

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

Ethics

Animal experimentation: Animal experiments were conducted under the supervision of the IACUC of the William S. Middleton Memorial Veterans Hospital (Protocol: MJM0001).

Copyright

© 2022, Foster 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

  • 2,150
    views
  • 464
    downloads
  • 40
    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. Hannah R Foster
  2. Thuong Ho
  3. Evgeniy Potapenko
  4. Sophia M Sdao
  5. Shih Ming Huang
  6. Sophie L Lewandowski
  7. Halena R VanDeusen
  8. Shawn M Davidson
  9. Rebecca L Cardone
  10. Marc Prentki
  11. Richard G Kibbey
  12. Matthew J Merrins
(2022)
β-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveal their essential role as nutrient sensors for the KATP channel
eLife 11:e79422.
https://doi.org/10.7554/eLife.79422

Share this article

https://doi.org/10.7554/eLife.79422

Categories and tags

Research organism

Further reading

    1. Cancer Biology
    2. Cell Biology

    Changes in local mineral homeostasis facilitate the formation of benign and malignant testicular microcalcifications

    Ida Marie Boisen, Nadia Krarup Knudsen ... Martin Blomberg Jensen
    Research Article

    Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.

    1. Cell Biology
    2. Immunology and Inflammation

    RAS–p110α signalling in macrophages is required for effective inflammatory response and resolution of inflammation

    Alejandro Rosell, Agata Adelajda Krygowska ... Esther Castellano Sanchez
    Research Article

    Macrophages are crucial in the body’s inflammatory response, with tightly regulated functions for optimal immune system performance. Our study reveals that the RAS–p110α signalling pathway, known for its involvement in various biological processes and tumourigenesis, regulates two vital aspects of the inflammatory response in macrophages: the initial monocyte movement and later-stage lysosomal function. Disrupting this pathway, either in a mouse model or through drug intervention, hampers the inflammatory response, leading to delayed resolution and the development of more severe acute inflammatory reactions in live models. This discovery uncovers a previously unknown role of the p110α isoform in immune regulation within macrophages, offering insight into the complex mechanisms governing their function during inflammation and opening new avenues for modulating inflammatory responses.

    1. Cell Biology

    Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

    Affiong Ika Oqua, Kin Chao ... Alejandra Tomas
    Research Article

    G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.