A loss of function variant in SLC30A8/ZnT8 drives proteomic changes associated with lowered apoptosis in human stem...

(1)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.

(2)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.

(3)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.

(4)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.