The prismatic forms of diabetes in pediatrics : natural evolution, strategies for ß-cell replacement, and tolerance to physical activity

I. Title of the proposal: The prismatic forms of diabetes in pediatrics: natural evolution, strategies for β-cell replacement, and tolerance to physical activity. II. Results obtained a) Study of glycemic control and β-cell mass evolution after onset of T1D in pediatrics We characterized the principal determinants of partial remission (PR) in a pediatric cohort of patients with type 1 diabetes (T1D) 1. We developed a new formula (GTAA1C score) to predict PR in youngsters with T1D, based on routine clinical parameters of glycemic variability 2. More recently, we demonstrated the positive residual impact of PR on short-term post-PR glycemic control (i.e., at 6 months post-PR) in a cohort of children (n=189) with T1D 3. In our DIABHONEY study, while residual post-PR effects were correlated to PR duration, these were not sustained: long PR induced better glycemic control at 6 months post-PR (compared to short PR or PR- controls) but did not impact glycemic variability indexes one year after PR. Inside the DiaType consortium (Bridge 2017 – Innoviris), we evaluated the evolution of patients with atypical forms of diabetes to better understand genetic and metabolic determinants of residual β-cell function, and propose etiology-based, patient-centered therapeutic approaches. Our pediatric initiative, called the GENEPEDIAB study, gattered six diabetes convention centers inside the French-speaking part of Belgium. We created a new score (DIAMODIA), based on typical clinical features, to identify patients with atypical diabetes. So far, out of 1550 patients with “type 1 or type 2 diabetes”, we isolated a subgroup of 149 patients presenting atypical forms of diabetes (called “ADia cohort”). This ADia cohort was subjected to a stepwise genetic screening (routine gene panel → whole exome sequencing (WES) → genome sequencing) to screen for a potential molecular diagnosis. Routine MODY gene panel analysis identified 34 patients with class V variant (the “MODY” cohort). In-depth WES analysis identified 37 ADia patients with class III variant, providing our DIAMODIA score a yield of 31% for class V and 34% for class III variant positivity. Our DIAMODIA score was cross-validated on a MODY cohort (vs typical T1D cohort) to ensure the external validity and the predictive value of this new tool for the diagnosis of patients carrying a potential gene variant. These results were presented at ISPAD and ESPE 2022 Scientific Sessions 4,5 and were submitted for publication. Our group is also leading the DIATAG consortium study that gathers eight diabetes convention centers in Belgium. This study aims at identifying biomarkers of β-cell mass evolution in children and adolescents with new-onset T1D and to stratify patients in their capacity to enter or not in PR. In this cohort, we identified new determinants of PR by evaluating composite scores of serum biomarkers and parameters of glucose control. We observed that CGM metrics strongly correlated with clinical parameters and were sufficient to distinguish remitters from non-remitters. CGM metrics (or indexes) are thus minimal-invasive tools with strong potential to identify PR, as opposed to more demanding dynamic β-cell residual secretion testing. Also, thorough CGM analysis allowed us to identify four novel glucotypes that segregate patients into subgroups and mirror the evolution of remission after diabetes onset 6. This work was presented at ADA (poster) and ENDO (oral) in 2022. Also, we identified new makers of remission status using a comprehensive multi-parametric analysis of pancreatic structure of patients with new-onset T1D. Our current analyses confirm the significant decrease in volume (50%) and pancreatic ratio in these patients, compared to matched controls. We also observed a negative correlation of the pancreatic ratio with HbA1C levels at diagnosis. Correlation analyses with other parameters of disease controls and markers of β-cell function were performed and results were presented during ENDO 2022 7 and submitted for publication. In DIATAG, Proteomic analysis of the sera is being performed by collaboration with the MASSPROT platform at de Duve Institute (S. Pyr dit Ruys, D. Vertrommen, UCLouvain) for the setting-up of the assays (e.g. protein purification, annihilation of background noise) and for the running of experiments using 2D-LC-MS/MS without protein tagging. On a pilot series of plasmas from 6 DIATAG patients, using TMT labeling, we identified 1487 unique plasma proteins (FDR <0.01), covering a dynamic range of 107. When comparing remitters vs non-remitters to identify proteins exhibiting a different level of expression, we identified 11 candidate proteins, among those sCD14, thrombospondin-1, and gastric inhibitory polypeptide play a role in the pathophysiology of T1D. Achievement of the abundance ratio of sCD14 and GIP correlated with IDAA1C at three months (R=-0.94, p=0.005). We are now measuring the abundance of these proteins by targeted mass spectrometry in the entire DIATAG cohort. In our evaluation of markers of glycemic control in pediatric patients with T1D, we recently described the new concept of “post-hypoglycemic hyperglycemia” (PHH) (i.e. hypoglycemia that recover to hyperglycemia in any circumstance) and studied factors likely to influence PHH characteristics in a cohort of young patients with established T1D. Our EPHICA study 8 highlighted the importance of PHH as a prominent component of hyperglycemia in some children and adolescents with T1D. Factors associated with PHH features were age, BMI and parameters of glycemic control. Young and lean children were more prone to experience hypoglycemia that recovered with hyperglycemia, but adolescents and obese children tended to experience hyperglycemia of longer duration. This PHH parameter is now thoroughly evaluated by our team in the circumstance of PR in patients with new-onset T1D. b) Study of secondary forms of diabetes Secondary forms of diabetes mellitus are commonly understudied and underdiagnosed. For example, despite the evidence suggesting that glucocorticoids, asparaginase, radiotherapy, and immunosuppressants increase the risk of developing diabetes, its incidence, associated risk factors and biological prediction markers remain unknown. Yet it is important to study diabetes incidence and risk in contexts of organ transplantation and oncologic treatments, since developing diabetes in these settings is associated with unfavorable outcome and increase in cardiovascular events. The objectives of our DIABGRAFT and DIABONCO 9 studies are to identify early markers of glycemic dysregulation respectively in transplanted (liver and kidney) patients and cancer survivors, to eventually improve clinical management of these conditions, both in terms of treatment and follow-up. Results of our retro- and prospective DIABGRAFT study were submitted for publication and are currently under review. c) Types 1 diabetes and inflammation It is accepted that β-cell destruction occurs via cytokine secretion and activation of specific receptors and lymphocytes. A current objective of our research is to evaluate whether knockdown strategies, directed toward key protein components of islet inflammation 10, might be combined to a pharmacological alleviation of glucotoxicity in an effort to protect β-cell mass against destruction, during the early events leading to β-cell demise in T1D. In a subsidiary study of this research project 11, we evaluated whether glucotoxicity could contribute to β-cell mass destruction through participation to islet inflammation. We evaluated the potential of empagliflozin and GABA respectively to protect β-cell mass against glucotoxicity and to increase β-cell mass after diagnosis of T1D. In a streptozotocin-treated mouse model of T1D, we observed that empagliflozin and/or GABA had the potential to improve glucose homeostasis and pancreatic insulin content in treated animals, as compared to diabetic controls. We noticed that the effects of empagliflozin were associated with a reduction of islet ER stress and inflammation, whereas in mice co-treated with empagliflozin and GABA, β-cell mass increased after a first burst of proliferation of the α-cell compartment. Our next goal is to evaluate whether these effects of empagliflozin and GABA might be translated into longer-term protocols in the NOD mouse model. d) Diabetes cell therapy Our group has discovered pancreatic progenitor cells with the potential to produce new β cells in vitro and to decrease glucose levels of diabetic animals after transplantation 12-15. We developed a new system for overexpression of MAFA transcription factor using synthetic modified mRNA. We observed that the overexpression of MAFA mRNA was sufficient and efficient to drive our pancreas progenitors toward glucose-responsive β-like cells 13. e) Diabetes and exercise During physical activity, patients with T1D have to face various challenges to maintain blood glucose levels into the normal range. In the TREAD-DIAB trial 16, we evaluated the needs of children and adolescents in terms of insulin and carbohydrate modifications during sports. We developed an algorithm that helps us to precisely adapt insulin and carbs during exercise sessions, specifically for each individual patient. Our results showed the possibility to normalize blood glucose for patients under pump therapy. Because this was more challenging in young patients under insulin injections, we have now conducted a new study (CAR2DIAB), to evaluate whether we might provide fine-tuning of insulin injections and carb intake to every patient with diabetes. In this pediatric investigation, the application of algorithmic and individual adaptations of treatment globally improved glycemic control during 15h after exercises performed in real life by children and adolescents (n=12) with T1D 17. We are now extending this trial to a larger group of patients (CAR2DIAB-2 study). III. References 1. Pecheur A, Barrea T, Vandooren V, Beauloye V, Robert A, Lysy PA. Characteristics And Determinants Of Partial Remission In Children With Type 1 Diabetes using the Insulin-Dose-Adjusted A1C Definition. J Diabetes Res. 2014;2014:851378. 2. Nielens N, Pollé O, Robert A, Lysy PA. Integration Of Routine Parameters Of Glycemic Variability In A Simple Screening Method For Partial Remission In Children With Type 1 Diabetes. J Diabetes Res. 2018 Jan 17;2018:5936360. 2018 UCLouvain Medical School prize for best master thesis (N. Nielens). 3. Boutsen L, Costenoble E, Pollé O, Erdem K, Bugli C, Lysy PA. Influence of the Occurrence and Duration of Partial Remission On Short-term Metabolic Control in Type 1 Diabetes: the DIABHONEY Pediatric Study. Therapeutic Advances in Endocrinology and Metabolism. 2022, accepted for publication (IF: 4.435). 4. Welsch S, Gallo P, Beckers D, Lebrethon MC, Mouraux T, Seret N, Lysy PA. Etiology-based diagnosis of pediatric patients with atypical diabetes using routine and omic-based phenotyping and genotyping: results from the GENEPEDIAB study. Horm Res Paediatr 2022;95:1–616. https://doi.org/10.1159/000525606. Poster. 5. Welsch S, Gallo P, Beckers D, Lebrethon MC, Mouraux T, Seret N, Lysy PA. Etiology-based diagnosis of pediatric patients with atypical diabetes using routine and omic-based phenotyping and genotyping: results from the GENEPEDIAB study. Pediatric Diabetes. 20 October 2022. https://doi.org/10.1111/pedi.13399. Oral presentation. 6. Pollé OG, Delfosse A, Martin M, Louis J, Gies I, den Brinker M, Seret N, Lebrethon MC, Mouraux T, Gatto L, Lysy PA; DIATAG Working Group. Glycemic Variability Patterns Strongly Correlate With Partial Remission Status in Children With Newly Diagnosed Type 1 Diabetes. Diabetes Care. 2022 Oct 1;45(10):2360-2368. doi: 10.2337/dc21-2543. IF: 19.112. 7. Pollé, O. G., Delfosse, A., Michoux, N., Peeters, F., Duchene, G., Mouraux, T., Clapuyt, P., Louis, J., Gies, I., den Brinker, M., Lebrethon, M-C., Seret, N., & Lysy, P. A. (2022). Deep Characterization of Pancreas Volume of New-Onset Type 1 Diabetes Patients Reveals Puberty-Specific Patterns and New Topographic Correlations with Pancreatic Functions. Journal of the Endocrine Society, 6 (Supplement_1), 424. Poster with oral presentation. 8. Colinet V, Lysy PA. Characterization of Post-Hypoglycemic Hyperglycemia in Children and Adolescents With Type 1 Diabetes: The EPHICA Study. Front Endocrinol (Lausanne). 2022 Jun 27;13:887976. doi: 10.3389/fendo.2022.887976. PMID: 35832426; PMCID: PMC9272988. 9. Welsch S, Sawadogo K, Brichard B, de Ville de Goyet M, Van Damme A, Boulanger C, Lysy PA. Characterization and risk factors of hyperglycaemia during treatment of childhood hematologic malignancies. Diabet Med. 2021 Oct 15:e14720. doi: 10.1111/dme.14720. Epub ahead of print. PMID: 34652870. 10. Daems C, Vanderroost J, Sokal E, Lysy PA. Partial CRISPR/Cas9 IL1R1 & IFNGR1 Knock-Down Improves β-cell Survival And Function Under Cytokine-Induced Inflammation. Submitted as an abstract to the ESPE Congress 2019 (Vienna) and was awarded the Henning Andersen Prize, and the best award prize from the 2019 meeting. 11. Daems C, Welsch S, Boughaleb H, Vanderroost J, Robert A, Sokal E, Lysy PA. Early Treatment with Empagliflozin and GABA Improves β-Cell Mass and Glucose Tolerance in Streptozotocin-Treated Mice. J Diabetes Res. 2019;2019:2813489. doi:10.1155/2019/2813489. 12. Corritore E, Dugnani E, Pasquale V, Misawa R, Witkowski P, Lei J, Markmann J, Piemonti L, Sokal EM, Bonner-Weir S, Lysy PA. β-Cell Differentiation Of Human Pancreatic Duct-Derived Cells After In Vitro Expansion. Cell Reprogram, 2014 Dec;16(6):456-66. 13. Corritore E, Lee YS, Pasquale V, Liberati D, Hsu MJ, Lombard CA, Van Der Smissen P, Vetere A, Bonner-Weir S, Piemonti L, Sokal E, Lysy P. V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog A Synthetic Modified mRNA Drives Reprogramming of Human Pancreatic Duct-Derived Cells Into Insulin-Secreting Cells. Stem Cells Transl Med. 2016 Jul 12. pii: sctm.2015-0318. Stem Cells Translational Medicine Young Investigator Award for Best 2016 paper. 14. Yamada T, Cavelti-Weder C, Caballero F, Lysy P, Guo L, Sharma A, Li W, Zhou Q, Bonner-Weir S, Weir GC. Reprogramming Mouse Cells With a Pancreatic Duct Phenotype to Insulin-Producing β-Like Cells. Endocrinology, 2015, Apr 2:en20141987. 15. Yuan Y, Hartland K, Boskovic Z, Wang Y, Walpita D, Lysy PA, Zhong C, Young DW, Kim YK, Tolliday NJ, Sokal EM, Schreiber SL, Wagner BK, A small-molecule inducer of PDX1 expression identified by high-throughput screening. Chem Biol. 2013 Dec 19;20(12):1513-22. 16. Moniotte S, Owen M, Barrea T, Robert A, Lysy PA. Outcomes of algorithm-based modifications of insulinotherapy during exercise in MDI vs insulin pump-treated children with type 1 diabetes: results from the TREAD-DIAB study. Pediatr Diabetes 2017. doi: 10.1111/pedi.12509. 17. Lysy P.A., Absil H., Gasser E., Boughaleb H., Barrea T., Moniotte S. Combined algorithm-based adaptations of insulin dose and carbohydrate intake during exercise in children with type 1 diabetes: results from the CAR2DIAB study (submitted)