Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract believed to arise from an imbalance between its epithelial, immune and microbial components. It has been shown that biological differences (e.g. genetic, epigenetic, microbial, environmental) exist between patients with IBD. It is also known that there is important heterogeneity in the response to therapies that target very specific biological pathways (e.g. TNF‐alpha signaling, IL‐23R signaling, immune cell trafficking). The aim of this study was to identify potential biological differences associated with differential treatment response to the anti α4β7 integrin therapy known as vedolizumab. We performed targeted analyses of > 150 proteins and metabolites, and nontargeted analyses of > 1100 lipid entities in serum samples from 92 IBD patients (42 CD, 50 UC) immediately prior to initiation of therapy with vedolizumab (baseline samples) and at their first clinical assessment (week 14 samples). We detected that the baseline levels of multiple serum cytokines, amino acids, acylcarnitines and triglycerides were different between responders and nonresponders to treatment with vedolizumab. We also noted changes in serum analytes between baseline and week 14 samples that were different between these two groups of patients. Many of these serum analytes are markers of biological pathways that are involved in the activation, proliferation and metabolism of pro‐inflammatory cells. This study provides support for the hypothesis that biological differences between individuals not only impact the risk to develop IBD and IBD‐related clinical phenotypes but also an IBD patient's likelihood of responding to a biological therapy.

CD4(-)CD8(-) TCR alpha beta(+) (double-negative [DN]) T cells represent a rare T cell population that promotes immunological tolerance through various cytotoxic mechanisms. In mice, autologous transfer of DN T cells has shown protective effects against autoimmune diabetes and graft-versus-host disease. Here, we characterized human DN T cells from people living with type 1 diabetes (PWT1D) and healthy controls. We found that while DN T cells and CD8(+) T cells share many similarities, DN T cells are a unique T cell population, both at the transcriptomic and protein levels. We also show that by using various cytokine combinations, human DN T cells can be expanded in vitro up to 1,000-fold (mean >250-fold) and remain functional post-expansion. In addition, we report that DN T cells from PWT1D display a phenotype comparable to that of healthy controls, efficiently expand, and are highly functional. As DN T cells are immunoregulatory and can prevent T1D in various mouse models, these observations suggest that autologous DN T cells may be amenable to therapy for the prevention or treatment of T1D.

Sphingosine‐1‐phosphate receptor 1 (S1P 1 ) ligands effectively reduce immunopathological damage in viral pneumonia models. Specifically, S1P 1 ligands inhibit cytokine storm and help preserve lung endothelial barrier integrity. We recently showed that the S1P receptor ligand ozanimod can be safely administered to hospitalized patients with coronavirus disease 2019 (COVID‐19) exhibiting severe symptoms of viral pneumonia, with potential clinical benefits. Here, we extend on this study and investigate the impact of ozanimod on key features of the immune response in patients with severe COVID‐19. We quantified circulating cytokine levels, peripheral immune cell numbers, proportions and activation status; we also monitored the quality of the humoral response by assessing anti–severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) antibodies. Our findings reveal that patients receiving ozanimod during acute SARS‐CoV‐2 infection exhibit significantly reduced numbers of circulating monocytes compared with those receiving standard care. Correspondingly, in the ozanimod‐treated group, circulating levels of C–C motif ligand 2 (CCL2) were decreased. While treatment with ozanimod negatively impacted the humoral response to COVID‐19 in unvaccinated patients, it did not impair the development of a robust anti–SARS‐CoV‐2 antibody response in vaccinated patients. These findings suggest that ozanimod influences key immune mechanisms during the acute phase of SARS‐CoV‐2 infection.

Leigh Syndrome French Canadian (LSFC) is a rare autosomal recessive metabolic disorder characterized by severe lactic acidosis crises and early mortality. LSFC patients carry variants in the Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) nuclear gene, which lead to defects in the respiratory chain complexes and mitochondrial dysfunction. Mitochondrial respiration modulates cellular metabolic activity, which impacts many cell processes, including the differentiation and function of immune cells. The purpose of this study is to define the role of Lrpprc on immune cell function. As genetic deletion of Lrpprc is not viable, we generated two conditional mouse models: a model for systemic deletion of Lrpprc and a knock-in (KI) model carrying the most common LSFC pathogenic variant in Quebec, NM_133259.4(LRPPRC):c.1061C > T (p.Ala354Val). We demonstrate that Lrpprc is an essential gene even in adult mice, as systemic deletion of Lrpprc leads to prominent weight loss and mortality. We also find an increase in lactate levels, a symptom of metabolic crises in LSFC. Lrpprc deletion and pathogenic variant affect various immune cell subsets, with a strong impact on B cell development and proliferation. We generated a viable disease-relevant mouse model to study the role of Lrpprc in vivo and find that disruption of Lrpprc strongly impairs B cell development and proliferation.

Abstract NK cells participate in viral clearance and tumour control. They differentiate from bone marrow precursors and undergo a series of guided differentiation steps to acquire various functional properties. We previously showed that pre-mNK cells represent one of these precursors and that a locus on mouse chromosome 7 is linked to pre-mNK abundance. As pre-mNK cells are associated with a heightened anti-tumour activity, we sought to identify genes within this locus that influence their number, and could thus affect NK cell-mediated tumour growth. Through a candidate gene-based approach, we identify Itgad, encoding for CD11d, a member of the ß2 integrin family. We find that the absence of CD11d affects the expression of the other ß2 integrin subunits, particularly on NK cells. This led us to investigate the functional outcome of the loss of CD11d expression in NK cells. Although NK cells from CD11d-KO mice show unabated expression of effector proteins in vitro, their anti-tumour activity is impaired in vivo. Indeed, the growth of NK-sensitive RMA-S lymphoma is significantly accelerated in CD11d-KO mice. This increase in tumour growth was associated with a reduction in the number of NK cells in the tumours, suggesting a role for ß2 integrins in NK cell migration. Altogether, our candidate gene-based approach determined that expression of ß2 integrin subunits are co-regulated in NK cells and revealed an important role for CD11d in NK cell-mediated anti-tumour activity.

The humoral response is complex and involves multiple cellular populations and signaling pathways. Bacterial and viral infections, as well as immunization regimens, can trigger this type of response, promoting the formation of microanatomical cellular structures called germinal centers (GCs). GCs formed in secondary lymphoid organs support the differentiation of high-affinity plasma cells and memory B cells. There is growing evidence that the quality of the humoral response is influenced by genetic variants. Using 12 genetically divergent mouse strains, we assessed the impact of genetics on GC cellular traits. At steady state, in the spleen, lymph nodes and Peyer's patches, we quantified GC B cells, plasma cells and follicular helper T cells. These traits were also quantified in the spleen of mice following immunization with a foreign antigen, namely, sheep red blood cells, in addition to the number and size of GCs. We observed both strain- and organ-specific variations in cell type abundance, as well as for GC number and size. Moreover, we find that some of these traits are highly heritable. Importantly, the results of this study inform on the impact of genetic diversity in shaping the GC response and identify the traits that are the most impacted by genetic background. In this study, we investigated the impact of genetic variation on the germinal center response. Using a panel of genetically divergent strains, we uncover an important impact of genetics on the number, size and cellular composition of germinal centers, both at steady state and after immune challenge. image

The inflammatory bowel diseases (IBD) known as Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract believed to arise because of an imbalance between the epithelial, immune and microbial systems. It has been shown that biological differences (genetic, epigenetic, microbial, environmental, etc.) exist between patients with IBD, with multiple risk factors been associated with disease susceptibility and IBD-related phenotypes (e.g. disease location). It is also known that there is heterogeneity in terms of response to therapy in patients with IBD, including to biological therapies that target very specific biological pathways (e.g. TNF-alpha signaling, IL-23R signaling, immune cell trafficking, etc.). It is hypothesized that the better the match between the biology targeted by these advanced therapies and the predominant disease-associated pathways at play in each patient will favor a beneficial response. The aim of this pilot study was to identify potential biological differences associated with differential treatment response to the anti α4β7 integrin therapy known as Vedolizumab. Our approach was to measure a broad range of analytes in the serum of patients prior to initiation of therapy and at the first clinical assessment visit, to identify potential markers of biological differences between patients at baseline and to see which biomarkers are most affected by treatment in responders. Our focus on early clinical response was to study the most proximal effects of therapy and to minimize confounders such as loss of response that occurs further distal to treatment initiation. Specifically, we performed targeted analyses of >150 proteins and metabolites, and untargeted analyses of >1100 lipid entities, in serum samples from 92 IBD patients (42 CD, 50 UC) immediately prior to initiation of therapy with vedolizumab (baseline samples) and at their first clinical assessment (14-week samples). We found lower levels of SDF-1a, but higher levels of PDGF-ββ, lactate, lysine, phenylalanine, branched chain amino acids, alanine, short/medium chain acylcarnitines, and triglycerides containing myristic acid in baseline serum samples of responders as compared to non-responders. We also observed an increase in serum levels of CXCL9 and citrate, as well as a decrease in IL-10, between baseline and week 14 samples. In addition, we observed that a group of metabolites and protein analytes was strongly associated with both treatment response and BMI status, although BMI status was not associated with treatment response.

roflumilast alters immune cell recruitment and inflammatory signaling to reduce infectious injury.Conclusions: Alcohol reduces CFTR ion transport and decreases airway surface hydration, mucociliary transport, and antimicrobial defense.PDE4 inhibitors boost lung host defense against bacterial pathogens and improve infection-related outcomes in individuals with excessive alcohol use.Findings from this study offer insights into pathogenic mechanisms in adults with CF, who were recently reported to use alcohol more than the general population despite significant comorbidities, including advanced lung disease, lung transplantation, CF-associated liver disease, and CFrelated diabetes.

Background: Cystic fibrosis (CF) neutrophils fail to eradicate infection despite their massive recruitment into the lung. While studies mostly focus on pathogen clearance by normal density neutrophils in CF, the contribution of low-density neutrophil (LDNs) subpopulations to disease pathogenesis remains unclear.Methods: LDNs were isolated from whole blood donations of clinically stable adult CF patients and from healthy donors. LDN proportion and immunophenotype was assessed by flow cytometry. Associations of LDNs with clinical parameters were determined. Results: LDN proportion was increased in CF patients' circulation compared with healthy donors. LDNs are a heterogeneous population of both mature and immature cells in CF and in healthy individuals. Moreover, a higher proportion of mature LDN correlates with a gradual decline in lung function and repeated pulmonary exacerbations in CF patients.Conclusions: Collectively, our observations suggest that low-density neutrophils are linked to CF pathogenesis and underscore the potential clinical relevance of neutrophil subpopulations in CF.(c) 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Autoimmunity results from defects in immune tolerance pathways that impair the capacity of immune cells to distinguish between self and non-self. One of these immune tolerance processes involves immunoregulatory TCRαβ+CD4−CD8− double negative T cells (DN T). Indeed, DN T cells protect from autoimmune pathologies and graft rejection in mice. Similarly, high DN T cell numbers correlate with a low incidence of chronic graft-versus-host disease in humans, an autoimmune-like pathology. While the thymic differentiation process of DN T cells was recently described, the function of these unconventional T cells is still poorly documented. To characterize DN T cells, we performed bulk RNA sequencing on CD4, CD8, γδ and DN T cells isolated from mouse spleen. Comparison of the transcriptome profiles revealed that DN T cells express a unique signature. Moreover, following in vitro stimulation, we find that DN T cells also present a distinct cytokine secretion pattern. Interestingly, we observed a lower induction of CD69, LAG-3 and PD-1 on DN T cells in comparison to CD4 and CD8 T cells, whereas LCK phosphorylation is unabated. DN T cell proliferation in mixed lymphocyte reactions is also lower than that of CD4 and CD8 T cells. Overall, our transcriptomic and cytokine analyses clearly identify DN T cells as a unique T cell subset. Moreover, while DN T cells do not express an exhaustion profile, they are more refractory to in vitro T cell stimulation than conventional T cells. A better characterization of DN T cell function will help conceive novel immunotherapies to treat autoimmune diseases. Supported by the Canadian Institutes of Health Research - PJT 159603. S.P. was supported by a postdoctoral CIHR fellowship

BACKGROUND:Crohn's disease (CD) can affect any segment of the digestive tract but is most often localized in the ileal, ileocolonic, and colorectal regions of the intestines. It is believed that the chronic inflammation in CD is a result of an imbalance between the epithelial barrier, the immune system, and the intestinal microbiota. The aim of the study was to identify circulating markers associated with CD and/or disease location in CD patients.METHODS:We tested 49 cytokines, chemokines, and growth factors in serum samples from 300 patients with CD and 300 controls. After quality control, analyte levels were tested for association with CD and disease location.RESULTS:We identified 13 analytes that were higher in CD patients relative to healthy controls and that remained significant after conservative Bonferroni correction (P < 0.0015). In particular, CXCL9, CXCL1, and interleukin IL-6 had the greatest effect and were highly significant (P < 5 × 10-7). We also identified 9 analytes that were associated with disease location, with VEGF, IL-12p70, and IL-6 being elevated in patients with colorectal disease (P < 3 × 10-4).CONCLUSIONS:Multiple serum analytes are elevated in CD. These implicate the involvement of multiple cell types from the immune, epithelial, and endothelial systems, suggesting that circulating analytes reflect the inflammatory processes that are ongoing within the gut. Moreover, the identification of distinct profiles according to disease location supports the existence of a biological difference between ileal and colonic CD, consistent with previous genetic and clinical observations.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene ( CFTR ). Cystic fibrosis-related diabetes (CFRD) is the most common comorbidity, affecting more than 50% of adult CF patients. Despite this high prevalence, the etiology of CFRD remains incompletely understood. Studies in young CF children show pancreatic islet disorganization, abnormal glucose tolerance, and delayed first-phase insulin secretion suggesting that islet dysfunction is an early feature of CF. Since insulin-producing pancreatic β-cells express very low levels of CFTR, CFRD likely results from β-cell extrinsic factors. In the vicinity of β-cells, CFTR is expressed in both the exocrine pancreas and the immune system. In the exocrine pancreas, CFTR mutations lead to the obstruction of the pancreatic ductal canal, inflammation, and immune cell infiltration, ultimately causing the destruction of the exocrine pancreas and remodeling of islets. Both inflammation and ductal cells have a direct effect on insulin secretion and could participate in CFRD development. CFTR mutations are also associated with inflammatory responses and excessive cytokine production by various immune cells, which infiltrate the pancreas and exert a negative impact on insulin secretion, causing dysregulation of glucose homeostasis in CF adults. In addition, the function of macrophages in shaping pancreatic islet development may be impaired by CFTR mutations, further contributing to the pancreatic islet structural defects as well as impaired first-phase insulin secretion observed in very young children. This review discusses the different factors that may contribute to CFRD.

Leigh Syndrome French Canadian (LSFC) is a rare autosomal recessive metabolic disorder characterized by severe lactic acidosis crises and early mortality. LSFC patients carry mutations in the Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) gene, which lead to defects in the respiratory chain complexes and mitochondrial dysfunction. Mitochondrial respiration modulates cellular metabolic activity, which impacts many cell types including the differentiation and function of immune cells. Hence, we postulated that, in addition to neurological and metabolic disorders, LSFC patients may show impaired immune activity. To gain insight into the quality of the immune response in LSFC patients, we examined the response to the measles, mumps and rubella (MMR) vaccine by measuring antibody titers to MMR in the plasma. In a cohort of eight LSFC patients, the response to the MMR vaccine was variable, with some individuals showing antibodies to all three viruses, while others had antibodies to two or fewer viruses. These results suggest that the mutations in the LRPPRC gene present in LSFC patients may affect the immune response to vaccines. Monitoring vaccine response in this fragile population should be considered to ensure full protection against pathogens.

Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-13C6]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). These idiopathic and chronic diseases result from inflammation of the gastrointestinal tract and are mainly mediated by the immune system. Genome wide association studies link genes of the IL-12 and IL-23 biology to both CD and UC susceptibility. IL-12 and IL-23 cytokines share a functional subunit, p40, and their respective receptors also share a functional subunit, IL-12R beta 1. However, clinical trials targeting p40, and thus inhibiting both IL-12 and IL-23 pathways, provided mitigated effects on IBD, suggesting context dependent effects for each cytokine. In addition to IL-12 and IL-23, genetic deficiencies in IL-10 also result in severe IBD pathology. We generated various mouse models to determine how IL-12 or IL-23 interacts with IL-10 in IBD pathology. Whereas defects in both IL-10 and IL-12R do not impact the severity of the Dextran Sulfate Sodium (DSS)-induced colitis, combined deficiencies in both IL-10 and IL-23R aggravate the disease. In contrast to DSS-induced colitis, defects in IL-12R and IL-23R both protect from the spontaneous colitis observed in IL10(-/-) mice. Together, these studies exemplify the complexity of genetic and environmental interactions for identifying biological pathways predictive of pathological inflammatory processes.

Various signaling pathways have been identified in the heart as important players during development, physiological adaptation or pathological processes. This includes the MAPK families, particularly p38MAPK, which is involved in several key cellular processes, including differentiation, proliferation, apoptosis, inflammation, metabolism and survival. Disrupted p38MAPK signaling has been associated with several diseases, including cardiovascular diseases (CVD) as well as diabetes and its related complications. Despite efforts to translate this knowledge into therapeutic avenues, p38 inhibitors have failed in clinical trials due to adverse effects. Inhibition of MK2, a downstream target of p38, appears to be a promising alternative strategy. Targeting MK2 activity may avoid the adverse effects linked to p38 inhibition, while maintaining its beneficial effects. MK2 was first considered as a therapeutic target in inflammatory diseases such as rheumatoid polyarthritis. A growing body of evidence now supports a key role of MK2 signaling in the pathogenesis of CVD, particularly ischemia/reperfusion injury, hypertrophy, and hypertension and that its inhibition or inactivation is associated with improved heart and vascular functions. More recently, MK2 was shown to be a potential player in diabetes and related complications, particularly in liver and heart, and perturbations in calcium handling and lipid metabolism. In this review, we will discuss recent advances in our knowledge of the role of MK2 in p38MAPK-mediated signaling and the benefits of its loss of function in CVD and diabetes, with an emphasis on the roles of MK2 in calcium handling and lipid metabolism. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

RationaleLRPPRC (leucine‐rich pentatricopeptide repeat‐containing protein) is commonly described as a regulator of RNA metabolism in both mitochondria and nuclei, but the overall biological impact of this role remains unclear. Integrative genomics analyses have revealed that mutations in the LRPPRC gene were causing the cytochrome c oxidase defect in the French‐Canadian variant of Leigh Syndrome (LSFC) (Mootha et al. 2003). More recently, a targeted metabolomics study in LSFC patients highlighted specific markers reflecting mitochondrial metabolic perturbations for various nutrients including fatty acids (Thompson‐Legault et al. 2015). The role of LRPPRC in lipid metabolism remains, however, to be better deciphered.Goal & MethodsTo dissect the role of LRPPRC in lipid metabolism, we have analyzed plasma from LSFC patients as well as plasma and livers from hepato‐specific KO‐LRPPRC mice using a combination of mass spectrometry (MS)‐based approaches. These include: (1) an untargeted and comprehensive lipidomic workflow, which enables the coverage of > 1300 unique lipid entities, and (2) targeted lipidomic analyses to probe fatty acid (FA) metabolism through acylcarnitines (ACs) profiling (covering >100 species) or cholesterol catabolism into bile acids (BAs; 9 (un)conjugated species).ResultsThe lipidomic profile observed in LSFC patients is typically characteristic of peroxisomal dysfunction as revealed by a: (i) 2‐fold decrease in plasmalogens (Pls; p<0.01), (ii) 2‐fold decrease in conjugate BAs such as glyco‐(G), glycodeoxy‐(GD) (p<0.001) and tauro‐(T) (p<0.05) cholic acids (CA), and (iii) 1.5‐fold increase in specific AC species, namely those with odd chains or > 20 carbons (p<0.05). These results in humans were corroborated in transgenic mice. Indeed, in plasma, there is a 1.5‐fold decrease in Pls (p<0.05). In addition, in liver, several results point out to a remodeling of peroxisomal metabolism. These include: (i) an imbalance in BA conjugation (10‐fold increase in CA, p<0.01; 1.4‐fold decrease in GDCA, p<0.05) and a 4‐ to 10‐fold increase in odd chain and >20 carbons ACs (p<0.01). This notion is further substantiated by changes in gene and/or protein expression levels for: (i) catalase, a classical marker of peroxisome content (+16%; protein, p<0.001), (ii) ACOX1, marker of peroxisomal β‐oxidation (−40%; protein, p<0.05) and (iii) several peroxins (Pex) involved in peroxisome biogenesis and transport, namely Pex11β, Pex14, Pex19, Pex1 and Pex 10 (up to +60%; mRNA; p<0.05).ConclusionCollectively there results highlight a novel role for LRPPRC in the regulation of lipid metabolism beyond mitochondria, namely in peroxisomes. Whether peroxisomal lipid perturbations are specific to LRPPRC gene defect or represent a common feature of mitochondrial dysfunction remain, however, to be further ascertained.Support or Funding InformationThis work was supported by the “Fondation Grand défi Pierre Lavoie”.

Background & ObjectiveLSFC is a recessive mitochondrial disease caused by mutations in the LRPPRC gene resulting in a decrease in the LRPPRC protein. This leads to a tissue‐specific reduction of complex IV or cytochrome c oxidase (COX). Our previous work in LSFC fibroblasts demonstrated several mitochondrial functional abnormalities including reduced mitochondrial membrane potential, fragmentation of the mitochondrial network and impaired oxidative phosphorylation capacity. Despite these abnormalities, LSFC fibroblasts did not display any reduction in ATP levels, suggesting the activation of compensatory mechanisms. We hypothesized that mTORC1‐mediated glycolytic ATP production, also known as Warburg effect, might compensate for mitochondrial dysfunction. To address this question, we evaluated glycolytic flux and the Akt/mTORC1 signaling pathway, using LSFC and control skin fibroblasts as a model.ResultsOur results showed numerous adaptations of LSFC energy metabolism. Metabolic flux analysis revealed that glucose contribution to pyruvate, lactate and alanine formation was respectively increased by 41%, 61%, and 52% (p≤0.05) in LSFC fibroblasts compared to controls. At the molecular level, the Akt/mTORC1 pathway was up regulated in LSFC cells compared to controls (pAkt 111%, p≤0.05; Akt 44%, p≤0.001; mTOR 51%, p≤0.01). This includes enhanced phosphorylation of mTORC1 downstream targets p70S6K by 105% (p≤0.05); as well as an increase in the expression of the transcription factor HIF‐1α by 200% (p≤0.01). The level of pyruvate dehydrogenase kinase 1 (PDHK1), a HIF‐1α downstream target and an inhibitor of mitochondrial glucose oxidation via pyruvate dehydrogenase 1 (PDH1) phosphorylation, was also up regulated by 150% (p≤0.05) in LSFC cells. Consistent with these results, we observed an increase of PDH1 phosphorylation by 94% (p≤0.01) in LSFC cells compared to controls. To further delineate the role of mTORC1 pathway in these adaptations, we inhibited its activity with rapamycin. mTORC1 inhibition did not affect HIF‐1α or PDHK1 expression. However, rapamycin was associated with increased PDH1 phosphorylation (p≤0.05) by 37% and 42% in LSFC and control fibroblasts, respectively. Rapamycin also reduced ATP levels by 8% (p=0.07) in LSFC fibroblasts comparatively to 15% (p≤0.05) in controls. Interestingly, rapamycin decreased LRPPRC expression by 41% (p≤0.01) in LSFC cells compared to only 11% (p≤0.05) reduction in control. This was associated with a selective reduction of COX expression in LSFC (32%, p≤0.01) fibroblasts, while we did not observe any change in expression of the others mitochondrial complexes.ConclusionLSFC fibroblasts maintain ATP levels probably through a “Warburg‐like effect”; for which mTORC1 is not the primary regulator. mTORC1 activation, however, appears important for LRPPRC and therefore, COX expression in LSFC fibroblasts.Support or Funding InformationSupported by: Association de l'acidose lactique and CIHR Emerging Team Grant

Heart disease remains a major complication of diabetes, and the identification of new therapeutic targets is essential. This study investigates the role of the protein kinase MK2, a p38 mitogen-activated protein kinase downstream target, in the development of diabetes-induced cardiomyopathy. Diabetes was induced in control (MK2+/+) and MK2-null (MK2−/−) mice using repeated injections of a low dose of streptozotocin (STZ). This protocol generated in MK2+/+ mice a model of diabetes characterized by a 50% decrease in plasma insulin, hyperglycemia, and insulin resistance (IR), as well as major contractile dysfunction, which was associated with alterations in proteins involved in calcium handling. While MK2−/−-STZ mice remained hyperglycemic, they showed improved IR and none of the cardiac functional or molecular alterations. Further analyses highlighted marked lipid perturbations in MK2+/+-STZ mice, which encompass increased 1) circulating levels of free fatty acid, ketone bodies, and long-chain acylcarnitines and 2) cardiac triglyceride accumulation and ex vivo palmitate β-oxidation. MK2−/−-STZ mice were also protected against all these diabetes-induced lipid alterations. Our results demonstrate the benefits of MK2 deletion on diabetes-induced cardiac molecular and lipid metabolic changes, as well as contractile dysfunction. As a result, MK2 represents a new potential therapeutic target to prevent diabetes-induced cardiac dysfunction.