The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin (CsA). Voclosporin (VCS), a next generation calcineurin inhibitor is reported to cause fewer incidences of NODAT but the reason is unclear. Whilst calcineurin signaling plays important roles in pancreatic β-cell survival, proliferation, and function, its effects on human β-cells remain understudied. In particular, we do not understand why some calcineurin inhibitors have more profound effects on the incidence of NODAT. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, programmed cell death rate, and the transcriptomic profile of human islets. We studied two clinically relevant doses of TAC (10 ng/ml, 30 ng/ml) and VCS (20 ng/ml, 60 ng/ml), meant to approximate the clinical trough and peak concentrations. TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the distal stages of exocytosis after voltage-gated Ca 2+ entry. No significant effects on islet cell survival or total insulin content were identified. RNA sequencing showed that TAC significantly decreased the expression of 17 genes, including direct and indirect regulators of exocytosis (SYT16, TBC1D30, PCK1, SMOC1, SYT5, PDK4, and CREM), whereas VCS has less broad and milder effects on gene expression. Clinically relevant doses of TAC, but not VCS, directly inhibit insulin secretion from human islets, likely via transcriptional control of exocytosis machinery

27Context. The incidence of new onset diabetes after transplant (NODAT) has increased over the 28 past decade. It has been suggested that NODAT is caused by exposure to calcineurin inhibitors, 29 particularly tacrolimus (TAC). Voclosporin (VCS), a next generation calcineurin inhibitor is 30 reported to cause fewer incidences of NODAT. 32Objective. Whilst calcineurin plays important roles in pancreatic -cell survival, proliferation, and 33 function, the effects of calcineurin inhibitors on human -cells remain understudied. In particular, 34 we do not understand why some inhibitors have more profound effects on the incidence of 35 NODAT. 37Design. Here, we compared the effects of TAC and VCS on the dynamics of insulin secretory 38 function, the programmed cell death rate, and the transcriptomic profile of human islets. We 39 studied two clinically relevant doses of TAC (10 ng/ml, 30 ng/ml) and VCS (20 ng/ml, 60 ng/ml), 40 meant to approximate the clinical trough and peak concentrations. 42Results. TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated insulin 43 secretion and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the 44 distal stages of exocytosis after voltage-gated Ca2+ entry. No significant effects on islet cell 45 survival or total islet insulin content were identified. RNA sequencing showed that TAC, and to a 46 lesser extent VCS, decreased the expression of genes that regulate insulin exocytosis, trafficking, 47 and processing, including SYT16, SYT5, PITPNM1, and PAM. 49Conclusions. TAC directly inhibits insulin secretion, likely via transcriptional control of exocytosis 50 machinery. VCS was found to be gentler on isolated human islets than TAC. 51 54 steroids or high dose calcineurin inhibitors, particularly tacrolimus, on cell types that are critical 55 for the maintenance of glucose homeostasis (1). Tacrolimus (TAC; also known as FK506) is a 56 mainstay for prevention of transplant rejection, but effective immunosuppression targeting a 57 concentration range of 4-11 ng/mL still results in NODAT in > 20% of patients (2). There is an 58 unmet clinical need for calcineurin inhibitors that do not cause diabetes in this already vulnerable 59 patient population. 60Voclosporin (VCS) is a next-generation calcineurin inhibitor that is structurally related to 61 cyclosporine A (CsA) with the addition of a carbon molecule at amino acid-1 of (CsA). This 62 modification results in enhanced binding of the VCS-cyclophilin complex to calcineurin and shifts 63 metabolism, resulting in increased potency and a consistent pharmacokinetic-pharmacodynamic 64 profile as compared to (CsA). VCS was previously evaluated in a Phase IIb, multi-center, open-65 label, concentration-controlled study in patients undergoing de novo renal transplantation, 66 evaluating the efficacy and safety of three doses of VCS (0.4, 0.6, and 0.8 mg/kg BID) as 67 compared to TAC (3). In that study, the low-dose VCS group had similar efficacy to tacrolimus in 68 controlling acute rejection w...

Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition.We now define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. We leverage the insulin response heterogeneity and use transcriptomics and proteomics to identify molecular pathways of specific nutrient responsiveness. Surprisingly, we find robust insulin secretion to fatty acid stimulus in ~8% of donors, challenging the idea that fat has negligible effects on insulin release. By comparing human islets to human embryonic stem cell-derived islet clusters, we show that, unlike glucose-responsiveness, fat hyper-responsiveness is equivalent and may be a hallmark of functionally immature cells. Our study represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition.

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