Experimental model and study participant details

NOD mice were housed and inbred in the animal facility of KU Leuven (Leuven, Belgium).⁴⁸ Diabetes incidence is higher in NOD females than in male mice,⁴⁸ thus 5-week-old female NOD mice were used for the study. C57BL/6N and NOD/Severe-Combined-Immunodeficiency (SCID) mice were housed and inbred in the animal facility of ULB (Brussels, Belgium). 8-10-week-old male and female C57BL/6N mice were used for pancreatic islet isolation. 8-10-week-old female NOD/SCID mice were used for human islet-like cell transplantation. All mice were housed under semi-barrier conditions, and animals were fed sterile food and water ad libitum. NOD mice were screened for the onset of diabetes by evaluating glucose levels in urine (Diastix Reagent Strips; Bayer, Leverkusen, Germany) and venous blood (AccuCheck; Roche Diagnostics, Vilvoorde, Belgium). Animals were maintained by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experimental procedures were approved and performed following the Animal Ethics Committee of the KU Leuven (P131/2019) and the Commission d’Ethique du Bien-être Animal of the ULB (reference 732N). Body weights and food intake were regularly monitored.

Mouse islets were isolated according to previously published protocol³⁰ and treated in vitro with acetate (1mM) and/ or butyrate (1mM) for 1h prior to a treatment of mIFN-γ (1000U/mL, PeproTech, London, UK) and hIL-1β (50U/mL, R&D Systems, Minneapolis, MN) for 24h. Cell death was measured using SYTOX green (ThermoFisher Scientific, Scientific, Gibco, UK).

The insulin-producing INS-1E β-cell line was cultured as described.⁴⁹ Cytokine concentration (rIFN-γ 1000U/mL R&D Systems and hIL-1β 50U/mL) were selected based on previous time course and dose-response studies.^29,49 Sodium acetate or sodium butyrate (Sigma-Aldrich) were added to dose response and according to previous established concentrations.^6,8,33

Human Embryonic Stem Cells (hESC) H1 (WiCell, Madison, WI) were cultured on plates coated with Matrigel (Corning BV, Amsterdam, the Netherlands) in Essential 8™ Medium (Life Technologies). hESC were differentiated into insulin-producing β-like cells (aggregates) in a 30-day protocol previously described.^29,30 NOD/SCID mice were anesthetized, and hESC-differentiated aggregates were implanted under the kidney capsule using a Hamilton syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland). 200mM acetate and 150mM butyrate was added in their drinking water for one week. Circulating human C-peptide was measured in mouse plasma by ELISA (Mercodia, Uppsala, Sweden).

Cultured cells were regularly checked for mycoplasma contamination with MycoAlert® PLUS Mycoplasma Detection Kit (#LT07-705, Lonza, Basel, Switzerland).