Mitochondrial function is pivotal to β-cell competence. The mitochondrial life cycle balances mitochondrial biogenesis and turnover (mitophagy) to ensure optimal metabolic function. Human type 2 diabetic (T2D) β-cells are known to develop mitochondrial structural/functional defects, suggestive of a defective mitochondrial life cycle. However, it is unclear if these defects are a cause or consequence of T2D. Here, we observed that human T2D β-cells had reduced mitophagic flux, mtDNA content, and expression of mitochondrially encoded genes. To test the importance of the mitochondrial life cycle to drive β-cell failure in T2D, we developed 2 distinct β-cell specific mouse models: βTfam^KO (to deplete mtDNA) and βClec16a^ΚΟ (to impair mitophagy). We observed age dependent loss of glucose tolerance, glucose stimulated insulin secretion and β-cell mass in both models, which was exacerbated by obesity in βClec16a^ΚΟ mice. Loss of β-cell mass was largely independent of changes in proliferation or apoptosis but rather, due to an induction of β-cell immaturity. Using lineage tracing approaches, we confirmed that βClec16a^KO and βTfam^KO mice induce formation of both insulin-negative immature β-cells and β-to-α cell transdifferentiation. Further, high-throughput gene expression profiling, biochemical, and metabolic assays highlighted that either Clec16a or Tfam deficiency induces an aberrant retrograde signaling program, manifested by reductions in cellular ATP and activation of the integrated stress response (ISR). Importantly, inhibition of the ISR in vivo ameliorated glucose intolerance, defective insulin secretion, β-cell immaturity, and loss of β-cell mass, suggesting that aberrant retrograde signaling may directly lead to loss of β-cell identity. Taken together, our studies illustrate that a unified mitochondrial lifecycle is necessary to maintain β-cell mass and identity and may be targeted to prevent β-cell failure in T2D. Disclosure G. Pearson: None. N. Lawlor: None. J. Zhu: None. E. M. Walker: None. E. C. Reck: None. M. L. Stitzel: None. S. Soleimanpour: None. Funding American Diabetes Association (1-19-PDF-063 to G.P.)

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ∼20% of which are islet specific and occur mostly distal to known gene TSS. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validated the transcriptional enhancer activity for 2,279 of 3,378 (∼68%) tested islet CAGE elements (5% false discovery rate). TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting active enhancers at GWAS loci.

Pancreatic islet (dys)function and failure is central to both type 2 diabetes (T2D) genetic risk and pathogenesis. Islet cellular heterogeneity has impeded precise understanding of the specific gene(s) and pathway(s) in each cell type that are altered by and/or may contribute to pathophysiology in the prediabetic (PD) and type 2 diabetic (T2D) states. To identify cellular and molecular features of islet dysfunction in PD and T2D states, we completed single cell transcriptome profiling of 137k human islet cells from 11 ND, 6 PD, and 11 T2D donors (⋃5k/individual). Single cell transcriptomes aggregated into distinct clusters of endocrine (alpha, beta, delta, PP/gamma), exocrine (acinar, stellate, ductal), endothelial, and immune cell types. Targeted analysis of each endocrine cell type identified a proliferative alpha cell sub-population comprising ∼0.15-0.3% of endocrine cells and variable insulin production and endoplasmic reticulum stress beta cell states. We compared the proportion of islet endocrine cell type (sub)populations sampled between T2D, PD, and ND donors and found that T2D samples contained significantly fewer beta cells and more alpha cells than ND and, to a lesser degree, PD samples. Delta and PP/gamma cell proportions were similar between disease states. Importantly, no (sub)populations were ND, PD, or T2D-specific. We compared aggregate T2D, PD, and ND cell type-specific transcriptomes to identify differentially expressed genes in each cell type. Genes related to exocytic granule assembly/organization, including those induced by T2D risk variants (DGKB) or T2D state (GAP43), were among 99 genes induced in T2D beta cells. 103 repressed genes included those associated with glucose transport and implicated as T2D GWAS effector genes such as SLC2A2, SLC5A1, and STARD10, a gene whose deletion in mice impairs islet insulin secretion. Studies are ongoing to assess the functional role(s) of these and additional genes in islet (dys)function. Disclosure N. Lawlor: None. R. Kursawe: None. M.L. Stitzel: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-18-ACE-15 to M.L.S.)

Type 2 diabetes (T2D) is a complex disorder in which both genetic and environmental risk factors contribute to islet dysfunction and failure. Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs), most of which are noncoding, in >200 loci to islet dysfunction and T2D. Identification of the putative causal variants and their target genes and whether they lead to gain or loss of function remains challenging. Here, we profiled chromatin accessibility in pancreatic islet samples from 19 genotyped individuals and identified 2,949 SNPs associated with in vivo cis-regulatory element use (i.e., chromatin accessibility quantitative trait loci [caQTL]). Among the caQTLs tested (n = 13) using luciferase reporter assays in MIN6 β-cells, more than half exhibited effects on enhancer activity that were consistent with in vivo chromatin accessibility changes. Importantly, islet caQTL analysis nominated putative causal SNPs in 13 T2D-associated GWAS loci, linking 7 and 6 T2D risk alleles, respectively, to gain or loss of in vivo chromatin accessibility. By investigating the effect of genetic variants on chromatin accessibility in islets, this study is an important step forward in translating T2D-associated GWAS SNP into functional molecular consequences.

Molecular mechanisms remain unknown for most type 2 diabetes genome-wide association study identified loci. Variants associated with type 2 diabetes and fasting glucose levels reside in introns of ADCY5, a gene that encodes adenylate cyclase 5. Adenylate cyclase 5 catalyzes the production of cyclic AMP, which is a second messenger molecule involved in cell signaling and pancreatic β-cell insulin secretion. We demonstrated that type 2 diabetes risk alleles are associated with decreased ADCY5 expression in human islets and examined candidate variants for regulatory function. rs11708067 overlaps a predicted enhancer region in pancreatic islets. The type 2 diabetes risk rs11708067-A allele showed fewer H3K27ac ChIP-seq reads in human islets, lower transcriptional activity in reporter assays in rodent β-cells (rat 832/13 and mouse MIN6), and increased nuclear protein binding compared with the rs11708067-G allele. Homozygous deletion of the orthologous enhancer region in 832/13 cells resulted in a 64% reduction in expression level of Adcy5, but not adjacent gene Sec22a, and a 39% reduction in insulin secretion. Together, these data suggest that rs11708067-A risk allele contributes to type 2 diabetes by disrupting an islet enhancer, which results in reduced ADCY5 expression and impaired insulin secretion.

We used an unbiased genome-wide approach to identify exonic variants segregating with diabetes in a multigenerational Finnish family. At least eight members of this family presented with diabetes with age of diagnosis ranging from 18 to 51 years and a pattern suggesting autosomal dominant inheritance. We sequenced the exomes of four affected members of this family and performed follow-up genotyping of additional affected and unaffected family members. We uncovered a novel nonsynonymous variant (p.Trp314Arg) in the Wolfram syndrome 1 (WFS1) gene that segregates completely with the diabetic phenotype. Multipoint parametric linkage analysis with 13 members of this family identified a single linkage signal with maximum logarithm of odds score 3.01 at 4p16.2-p16.1, corresponding to a region harboring the WFS1 locus. Functional studies demonstrate a role for this variant in endoplasmic reticulum stress, which is consistent with the β-cell failure phenotype seen in mutation carriers. This represents the first compelling report of a mutation in WFS1 associated with dominantly inherited nonsyndromic adult-onset diabetes.