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
- University of Wisconsin-Madison, United States
- Massachusetts Institute of Technology, United States
- Yale University, United States
- University of Montreal, Canada
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
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).
Reviewing Editor
- Lori Sussel, University of Colorado Anschutz Medical Campus, United States
Publication history
- Preprint posted: February 10, 2022 (view preprint)
- Received: April 12, 2022
- Accepted: August 23, 2022
- Accepted Manuscript published: August 23, 2022 (version 1)
- Version of Record published: September 5, 2022 (version 2)
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.
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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
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Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r2 > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.
