Targeting Glutamine Metabolism to prevent Diabetic Cardiovascular Complications – GlutaDiab

GLUTADIAB addresses the hypothesis that decreased glutamine blood concentration in diabetes reflects enhanced glutaminolysis in blood monocytes and tissue resident macrophages, thus promoting a switch of monocyte/macrophage polarization through modifications of the gene expression program. The immune-inflammatory response is exacerbated increasing the risk of developing cardiovascular complications.
To address this hypothesis, we developed three main work packages.

WP1: Assess the causal association of glutamine with risk of cardiovascular events in diabetes
1.1- Validate and identify new genetic loci associated with glutamine plasma concentration in 39 563 participants from three independent cohorts -> GWAS analyses
1.2- Associate genetic scores for glutamine with cardiovascular events in diabetes -> Mendelian randomization

WP2: Quantification of glutamine metabolism in serum and monocytes of T1D and T2D patients with low vs high risk of cardiovascular events in relation to their metabolic reprogramming – clinical study
2.1- recruit patients into four groups according to their cardiovascular and diabetic status: T1D/T2D/non-diabetic + high, or low risk of complications/ no complications/ history of cardiovascular disease
2.2- Identify glutamine metabolites in blood monocytes of recruited patients and associate glutamine metabolites with monocytes activation status and transcriptomic profile.
2.3- Characterize the transcriptomic program through modification gene expression and epigenetic changes related to KDM6B and TET2 activity in blood monocyte of study population.

WP3: To elucidate the molecular determinants involved in sensing glutamine pathways that orchestrate reprogramming of monocytes/macrophages polarization
3.1- Demonstrate that specific deficiency of GLS1, TET2 and KDM6B in myeloid cells interferes with the development in atherosclerosis in mice with diabetes (models of T1D and T2D).

To date, we have made important progress in addressing several of the initial objectives.

1-The consortium agreement (between APHP and Inserm) overseeing GLUTADIAB was signed on February 5, 2021.

2-Regarding the clinical study WP2: we obtained approval of the ethical committee on July 9, 2020 (CPP: 54/20_3, N° dossier SI: 20.06.08.73925). Also, GLUTADIAB now has a ClinicalTrials.gov Identifier: NCT04353869. The electronic Case Report Form was setup by the Clinical Research Unit of Bichat hospital and is currently used in the recruiting centers. Recruitment for the study was impacted by the COVID19 crisis but has been back on track since the end of lockdown. Inclusions started in Lariboisière hospital on February 2, 2021. To date, there are 89 T2D and 11 T1D patients included. At Bichat hospital, recruitment started on November 16, 2020, with 108 T2D and 4 T1D patients to date (WP2.1). Analyses have been on-going since to identify glutamine metabolites in blood monocytes of recruited patients (WP2.2).

3-in vivo experiments using unique mouse models have been performed in the context of diabetes and atherosclerosis. Interestingly, KDM6B in macrophages seems to play a key function in the polarization of perivascular macrophages. The loss of KDM6B in macrophages may favor atherosclerotic plaque formation and inadequate adipose tissue expansion upon diet-induced obesity and diabetes. Molecular mechanisms involved in the reprogramming of perivascular macrophages in absence of KDM6B are ongoing. In the second part of the preclinical studies, we made the surprising observation that Gls1 deficiency in macrophages can be compensated by a change in the glutamine flux mediated by glutamine synthase (GS). Based on this, we performed RNA sequencing in Gls1 deficient macrophages in presence or absence of a GS inhibitor named methionine sulfoximine (MSO). We found that both Gls1 deficiency and GS inhibition limited mitochondrial oxidative phosphorylation gene signature and these effects were not additive. We confirmed these findings using Seahorse extracellular flux analyses. However, in term of macrophage functions, we found that Gls1 deficiency reduced macrophage polarization and efferocytosis but not GS inhibition. GS inhibition rather modified membrane organization and survival process. This later mechanism appears to correlate with reduced Tet2 activity. We are currently investigating how modulation of glutamine flux in macrophages differently impacts their functions. To address the in vivo relevance of these observations, diabetic mice control or deficient for Gls1 in macrophages were treated for 3 weeks w/ or w/o MSO. Both Gls1 deficiency in macrophages and GS inhibition accelerated pancreatic inflammation, an effect synergized with reduced activity of the two enzymes. Our findings suggest a fine-tune modulation of glutamine flux by Gls1 and GS that have complementary roles in preventing diabetes-induced pancreatic macrophage inflammation.

- Identification of an unexpected function of KDM6B in controlling perivascular macrophage function and activation.
- Identification of a crucial role Gls1 and GS in modulating glutamine flux in macrophages with different but complementary role in diabetes-induced pancreatic macrophage inflammation.

Despite COVID-19 pandemic, we managed to include more than 200 diabetic patients and performed key in vivo experiments in our different mouse models. New concepts seems to emerge from our preliminary results that need to be confirmed in the second part of the project.

Clinical and fundamental publications are expected in 2022.

A high percentage of patients with diabetes develop macro-vascular diseases such as atherosclerosis. This complication is associated with exacerbated inflammation, which is characterized by activation of blood monocytes and tissue macrophages into a pro-inflammatory status. More and more evidence suggests that the changes in intracellular metabolic pathways in monocytes and macrophages influence their fate and functionality. Among the metabolic pathways, the TCA cycle (or Krebs cycle) is a key regulatory checkpoint that contributes to immune cell activation. Gutamine-derived glutamate (called glutaminolysis) is a critical fuel for the TCA cycle to generate TCA intermediates that orient monocyte activation status. In our current project, we hypothesize that the documented-decreased glutamine blood concentration in diabetes (preliminary results) reflects enhanced glutaminolysis of blood monocytes and tissue resident macrophages, which promotes a switch of monocyte/macrophage polarization through modifications of the gene expression program. Consequently to this metabolic rewiring, the immune/inflammatory response is exacerbated increasing the risk of developing CV complications in diabetic subjects. Targeting glutamine-dependent pathways in monocytes/macrophages may limit the inflammatory phenotype and CV events in diabetes. We propose here to test this hypothesis using the combination of unique mouse models, genome-wide molecular and epigenomic analyses and human studies to dissect the glutamine metabolism in monocytes and macrophages, aiming at demonstrating that this pathway is involved in the development of cardiovascular complications in diabetes. Our 3 mains objectives are:

1/ To assess the causal association of glutamine with the risk of CV events in type 1 and type 2 diabetes:
We will perform a meta-anaysis of genome-wide association studies for glutamine in 39 563 participants (Mendelian Randomization approach) and investigate the causality between glutamine and cardiovascular events in diabetes cohorts of 9 130 participants (Among 9130 participants, 2262 are cases (i.e, prevalent and incident of CV events).

2/ To quantify glutamine metabolism in serum and monocytes of type 1 and type 2 diabetic patients with low versus high risk of CV events:
The main objective of this task will be to correlate the plasma glutamine levels with the glutamine metabolites within blood monocytes (CD14+ cells: main monocyte population) of T1D or T2D subjects with incremental risk of cardiovascular complications (low versus high). Additionally, we will measure the transcriptomic program through gene expression modifications and epigenetic changes of blood monocyte from diabetic populations.

3/ To elucidate the molecular determinants involved in sensing glutamine pathways that orchestrate reprograming of monocytes/macrophages polarization:
The functional characterisation of GLS1, TET2, and KDM6B (known to be sensitive to glutamine intermediates) function in macrophage polarisation in the context of diabetes is not very well defined. Using unique mouse models developed by the consortium, we aim at demonstrating that specific deficiency of GLS1, TET2 and KDM6B in myeloid cells interfere with the development in atherosclerosis in diabetic mice.