Aims and hypothesis Loss-of-function mutations in SLC30A8, encoding the zinc ion (Zn2+) transporter ZnT8 in pancreatic beta cells, lower type 2 diabetes risk dose-dependently, but the underlying mechanisms remain unclear. Here, we combine proteomic, transcriptomic and functional approaches in human stem cell-derived islet-like clusters bearing common alleles or the inactivating variant R138X. We hypothesized that this variant protects against the deleterious effect of Zn2+ depletion on cell survival and function.

Methods Human embryonic stem cells INS(GFP/w) (MEL1), and CRISPR/Cas9-derived heterozygous or homozygous R138X lines were differentiated into stem cell-derived islet-like clusters. Intracellular Zn2+ levels were reduced using the chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,2-ethanediamine (TPEN). Apoptosis was assessed by TUNEL staining and protein expression by immunofluorescence. Glucose-stimulated calcium (Ca2+) dynamics were measured using the intracellular probe (Cal590) and insulin secretion by homogenous time-resolved fluorescence. Transcriptomic profiling was performed by bulk mRNA sequencing and proteomics by liquid chromatography-tandem mass spectrometry.

Results Intracellular Zn2+ depletion increased apoptosis in wild-type islet-like clusters, whereas R138X clusters were protected. R138X heterozygous clusters showed a mild increase in GCG+ cells and R138X homozygous clusters exhibited increased NKX6.1+ cells, without affecting polyhormonal populations. These changes were reversed under Zn2+ depletion. Transcriptomic and proteomic analyses, assessing genotype effects while accounting for Zn2+ depletion, showed that R138X clusters (versus wild-type) exhibited upregulation of genes and proteins involved in vesicle trafficking, secretion, Ca²⁺ signaling and mitochondrial metabolism, consistent with enhanced glucose-stimulated insulin secretion in homozygous clusters. Conversely, genes and proteins associated with extracellular matrix remodeling, metal-ion handling, apoptosis and cellular stress were downregulated. R138X clusters displayed altered Ca2+ signaling, with decreased area under the curve and oscillation amplitude, but increased frequency. These differences were reversed by TPEN, while Zn2+ depletion impaired Ca2+ response in wild-type clusters. Despite lowered overall activity, R138X homozygous clusters showed enhanced overall cell-cell connectivity, reversed by TPEN treatment. The opposite effects were observed in R138X heterozygous clusters, showing improved connectivity and activity under Zn2+ depletion.

Conclusion and interpretation Intracellular Zn2+ depletion compromises islet-like cluster identity and function, while the R138X variant confers protection against these effects. Under Zn2+-depleted conditions, ZnT8 deficiency promotes a more mature and metabolically active state of the R138X clusters, with enhanced Ca2+ signaling and insulin secretion, supported by a structural remodeling and the downregulation of apoptosis and cellular stress. These findings highlight the therapeutic potential of targeting ZnT8 in type 2 diabetes and support its relevance for further improving cell-based therapies.

What is already know about this subject?

Rare inactivating mutations in the insulin granule-associated zinc transporter gene, SLC30A8/ZnT8, drive lowered type 2 diabetes risk.

Previous studies have indicated that apoptosis is lowered, and glucose-stimulated insulin secretion enhanced, after ZnT8 inactivation.

The molecular mechanisms underlying these changes are unclear.

What is the key question?

How do inactivating mutations in SL30A8/ZnT8 lead to lowered apoptosis and enhanced insulin secretion from stem cell-derived islet-like clusters, and is altered susceptibility to intracellular zinc depletion involved?

What are the new findings?

The rare inactivating R138X mutation in SLC30A8 leads to gene dose-dependent changes in the transcriptome and proteome of islet-like clusters.

Changes include upregulation of maturity and downregulation of immaturity genes.

Depletion of intracellular Zn2+ exaggerates the protective effects of the inactivating mutation on apoptosis and insulin secretion

How might this impact on clinical practice in the foreseeable future?

Our findings suggest that careful monitoring of both dietary zinc intake and of circulating levels of zinc ions, whose effects are mitigated in SLC30A8 mutation carriers, may be helpful in some populations to lower diabetes risk.

GAR has served as a consultant for, and has received funding from, Sun Pharmaceuticals Inc.

MG is supported by a Postdoc Mobility Fellowship from the Swiss National Science Foundation (#225305). GO is the recipient of a postdoctoral FRQS scholarship (https://doi.org/10.69777/333390, https://doi.org/10.69777/376237). G.A.R. was supported by a Wellcome Trust Investigator (w212625/Z/18/Z) Award, and MRC Programme grant (MR/R022259/1), Diabetes UK (BDA 16/0005485), an NIH-NIDDK project grant (R01DK135268) a CIHR-JDRF Team grant (CIHR-IRSC TDP-186358 and JDRF 4-SRA-2023-1182-S-N), CRCHUM start-up funds, and Innovation Canada John R. Evans Leader Awards (CFI 42649, CFI 46539). M.F. was supported by a Canadian Institutes of Health Research project grant (PJT-175025).

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Bulk mRNA sequencing data will be deposited in the NCBI Sequence Read Archive (SRA). The mass spectrometry proteomics data will be deposited at the ProteomeXchange Consortium via the PRIDE (DOI: 10.1093/nar/gkab1038).