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An integrated multi-organ analysis of glutamate dehydrogenase

English title An integrated multi-organ analysis of glutamate dehydrogenase
Applicant Maechler Pierre
Number 192486
Funding scheme Project funding (Div. I-III)
Research institution Département de physiologie cellulaire et métabolisme Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Endocrinology
Start/End 01.04.2020 - 31.03.2024
Approved amount 800'000.00
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All Disciplines (4)

Discipline
Endocrinology
Neurophysiology and Brain Research
Biochemistry
Physiology : other topics

Keywords (6)

liver; metabolism; beta-cell; glutamate dehydrogenase; insulin; mitochondria

Lay Summary (French)

Lead
Modélisation de l’enzyme glutamate déshydrogénase dans un système multi-organes
Lay summary

La glutamate déshydrogénase (GDH) une protéine spécialisée dans la catalyse d’une réaction biochimique essentielle au bon fonctionnement de plusieurs organes. Ce type de protéines accélérant des réactions est appelé enzymes. Elles permettent de contrôler la vitesse de flux métaboliques. Comme toutes les protéines, la GDH est codée par un gène, appelé Glud1, qui peut être muté chez certaines personnes. Toutes les mutations connues du gène Glud1 résultent en une hyperactivité de l’enzyme GDH. Les conséquences sont sévères et les patients souffrent d’un syndrome d’hyperinsulinémie/hyperammonémie (HI/HA) qui se traduit par de dangereuses hypoglycémies (baisse du glucose sanguin) et des troubles du système nerveux central, tel que des crises d’épilepsie. Les organes impliqués dans ces syndromes sont le pancréas endocrine, le foie, et le cerveau. Dans le pancréas endocrine, la cellule bêta est un gluco-senseur couplé à un fournisseur d’insuline, analysant en temps réel toute modification du taux de glucose dans le sang et traduisant immédiatement ces changements en ajustant la sécrétion d’insuline. Trop d’insuline produite provoque une hypoglycémie, conséquence de mutations de la GDH. Dans le foie, les mêmes mutations participent à la surproduction de l’ammonium, potentiellement toxique pour le système nerveux. Le projet soutenu par le FNS a pour but de comprendre les spécificités tissulaires de la GDH, dans sa forme normale et mutée. Au final, nous souhaitons construire un modèle qui permette d’anticiper les conséquences de mutations spécifiques et ainsi offrir des traitements plus ciblés à chaque patient porteur d’une des mutations associées au syndrome HI/HA.

Direct link to Lay Summary Last update: 27.03.2020

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
166625 Glutamate pathways and metabolic stresses in energy homeostasis 01.09.2016 Project funding (Div. I-III)

Abstract

Background and rationale: Glutamate dehydrogenase (GDH) is a mitochondrial enzyme expressed in most tissues but it fulfills distinct and specific functions according to the organ where it operates. Closely associated with GDH, glutamate pathways are crucial for several organs, again with tissue-specific functions. On a biochemical basis, GDH catalyzes the same reaction in every tissue. However, the flux direction and the response to substrate concentrations vary according to the organ. In the brain, GDH recycles the neurotransmitter glutamate. In pancreatic ß-cells, GDH participates to the regulation of insulin secretion. Hepatic GDH controls metabolism of most amino acids and plays a key role in gluconeogenesis and ammonia homeostasis. These dedicated functions require highly specific sensitivity and responses to local changes. Alterations in such adaptations may result in severe diseases. This is illustrated by gain of function mutations of GDH, resulting in a syndrome of hyperinsulinemia/hyperammonia (HI/HA), often accompanied with epilepsy.Overall objectives: Although the basics of GDH biochemistry have been described long ago, the mechanisms conferring tissue specificity are still obscure. The main objective of the present project is to uncover the molecular basis for such specificities, in health and disease situations. We also aim at the development of an online Flux Balance Analysis platform offering visualization of GDH-related pathways in various conditions.Specific aims: The first part of the project revisits basics of GDH biochemistry in tissue-specific and in situ-like conditions. We will monitor key parameters reflecting GDH activity in primary cells isolated from pancreatic islets, liver, and brain. The tissues will also be isolated from corresponding tissue-specific GDH knockout mice and the activating mutation GDH-S445L will be compared to normal human GDH. GDH activity will be assessed in living cells, not in cell extracts, in order to preserve the specificity of the mitochondrial environment. In particular, we will monitor NADH/NAD+ ratio, mitochondrial pH, glutamate/glutamine levels, and oxygen consumption rate. Collectively, these data will be used to generate metabolic networks for Flux Balance Analysis. In the pancreatic ß-cell, we will investigate the apparent paradox of a cataplerotic function for GDH in energized mitochondria, in normal and HI/HA situations. In the brain, the GDH-S445L mutation will be studied in mice following adenovirus-mediated expression using stereotaxy. Astrocytes will be isolated for the measurements of metabolic fluxes at the cellular level. In vivo, metabolic profiling and sensitivity to epilepsy will be assessed. As a putative central amino acid sensor for energy homeostasis, brain GDH will also be investigated through its potential regulatory function on hepatic glucose production. In the liver, we will study the effects of the GDH-S445L mutation on gluconeogenesis and ammonia production. Regarding the latter parameter, we will use complementary approaches to generate a model of global ammonia homeostasis. Practically, we will use isolated hepatocytes, ex vivo liver perfusion, and in vivo vessel-specific blood sampling for an exhaustive overview.Expected impact: Overall, the proposed project aims at providing a novel comprehensive integration of GDH regulation, that takes into account three levels of specificities: the tissue, the metabolic state, and the mutations giving rise for instance to HI/HA. The ultimate goal is to generate an in silico FBA model that should be flexible enough to ensure continuous implementation, including by other researchers (open platform) and after the completion of the present project.
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