231-OR: Reduced NKX2-2 Expression Enhances Insulin Secretion by Primary Human ß-Cells

Pancreatic islet endocrine cell phenotype and function are tightly regulated by transcription factor (TF) regulatory networks. In a mouse model of pancreas organogenesis, the key islet-enriched TF NKX2.2 governs both β and α cell specification. Further, loss of NKX2.2 in adult mouse β cells results in glucose intolerance, reduced insulin content, and polyhormonal β cells, demonstrating its importance in the maintenance of β cell phenotype. To understand the undefined role of NKX2.2 in adult human islets, we utilized adenoviral delivery of CRISPR and shRNA constructs in our primary human pseudoislet system to knockout (gNKX2-2) or knockdown (shNKX2-2) NKX2-2 in islet cells, respectively (n=4-6 donors without diabetes). Immunofluorescence analyses of gNKX2-2 pseudoislets demonstrated loss of NKX2.2 signal across β (81 ± 2.0%; p<0.001), α (58 ± 5.5%; p=0.001), and δ cells (83 ± 2.2%; p<0.001) compared to scrambled controls (gSCR). Surprisingly, functional assessment by dynamic perifusion revealed increased insulin secretion in gNKX2-2 versus gSCR pseudoislets measured as area under the curve in response to 16.7 mM glucose (6.64 ± 1.53 vs. 3.55 ± 1.53 ng/100 islet equivalents (IEQs); p=0.007) and cAMP-evoked potentiation with 16.7 mM glucose + 100 μM isobutylmethylxanthine (IBMX) (5.43 ± 0.50 vs. 3.15 ± 0.59 ng/100 IEQs p=0.003) without changes in total insulin content (gNKX2-2 vs. gSCR: 3.64 ± 0.75 vs. 4.78 ± 0.87 ng/IEQ, p=0.45). Furthermore, these findings were recapitulated in shNKX2-2 versus shSCR human pseudoislets (16.7 mM glucose: 10.17 ± 2.46 vs. 6.50 ± 2.51 ng/100 IEQs, p=0.02; 16.7 mM glucose + IBMX: 8.43 ± 1.31 vs. 4.21 ± 1.33 ng/100 IEQs, p=0.0002; insulin content: 6.18 ± 1.05 vs. 7.53 ± 1.26 ng/IEQ, p=0.36), where we observed a reduction in NKX2.2hi β (65 ± 10%; p=0.008), α (52 ± 5.5%; p=0.003), and δ cells (83 ± 4.6%; p=0.003). Collectively, our data indicate a critical role of NKX2-2 in the maintenance of primary adult human β cell function and suggest differential roles in islet function across species.