Deficiency of transcription factor Nkx6.1 does not prevent insulin secretion in INS-1E cells

Abstract

Pancreatic-β-cell-specifying transcription factor Nkx6.1, indispensable for embryonic development of the pancreatic epithelium and commitment to β-cell lineage, directly controls the expression of a glucose transporter (Glut2), pyruvate carboxylase (Pcx), and genes for insulin processing (endoplasmic reticulum oxidoreductase-1β, Ero1lb; zinc transporter-8, Slc30a8). The Nkx6.1 decline in aging diabetic Goto-Kakizaki rats contributes to β-cell trans-differentiation into δ-cells. Elucidating further Nkx6.1 roles, we studied Nkx6.1 ablation in rat INS-1E cells, prepared by CRISPR/Cas9 gene editing from single colonies. INS-1ENkx6.1–/– cells exhibited unchanged glucose-stimulated insulin secretion (GSIS), moderately decreased phosphorylating/non-phosphorylating respiration ratios at high glucose; unchanged but delayed ATP-elevation responses to glucose; delayed uptake of fluorescent glucose analog, but slightly improved cytosolic Ca2+-oscillations, induced by glucose; despite approximately halved Glut2, Pcx, Ero1lb, and Slc30a8 expression, and reduced nuclear receptors Nr4a1 and Nr4a3. Thus, ATP synthesis was time-compensated, despite the delayed GLUT2-mediated glucose uptake and crippled pyruvate-malate redox shuttle (owing to the PCX-deficiency) in INS-1ENkx6.1–/– cells. Nkx6.1 thus controls the expression of genes that are not essential for acute insulin secretion, the function of which can be compensated for. Considerations that Nkx6.1 deficiency is an ultimate determinant of β-cell pathology beyond cell trans-(de-)differentiation or β-cell identity are not supported by our results.

Pitfalls of Mitochondrial Redox Signaling Research

“…Studying pancreatic β-cells [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ], we have encountered situations upon the glucose-stimulated insulin secretion (GSIS) [ 18 , 19 ] and fatty acid- (FA)-stimulated insulin secretion (FASIS) [ 12 , 22 ], whereby a distinct direction of pro-oxidant vs. more reduced states occur in cytosolic vs. mitochondrial matrix compartments ( Figure 1 ). For both, upon GSIS and FASIS, the cytosolic compartments became more oxidized via H 2 O 2 release, whereas the mt matrix superoxide release was indicated to decline [ 12 , 13 , 14 , 18 , 19 , 22 , 23 ].…”

The transcription factors ISL-1 and MAFA, but not NKX6-1 Characterize a Stem-cell Derived Population of Endocrine Pancreatic Cells Capable of Controlling blood Glucose in Rodent Models of Diabetes

“…al. demonstrated that NKX6-1 expression was found to be dispensable for glucose sensitive insulin secretion in rat insulinoma cells 16 . Furthermore, NKX6-1 also has an important role in the specification of the enterochromaffin population of serotonin-producing enteroendocrine cells of the gut 17 .…”