Glucose is a key physiological driver of adaptive β-cell mass expansion, promoting β-cell proliferation both in vitro and in vivo. Carbohydrate response element binding protein (ChREBP) is required for glucose-stimulated β-cell proliferation in both rodent and human β-cells. ChREBP has two splice isoforms - α and β. Translation of the ChREBPβ mRNA generates a truncated protein, resulting in a potent, constitutively active isoform. To study the role of the 2 splice isoforms in β-cell biology, we performed numerous experiments including immunostaining and chromatin immunoprecipitation with antibodies that recognize only ChREBPα or both isoforms. We found that ChREBPα is only transiently expressed in the nucleus. By contrast, ChREBPβ, once expressed at high enough levels to detect, is constitutively nuclear. Our data indicate that mice fed on high fat diet and diabetic db/db mice have abundant ChREBPβ localized in the nucleus while control mice have mostly cytoplasmic ChREBPα. We generated Ins-1 cell lines where we can distinguish between the splice isoforms using CRISPR/Cas9: 1. The C-terminus of ChREBPα and ChREBPβ was labeled with eGFP; 2. The N-terminus of ChREBPα was labeled by mCherry; and 3. ChREBPα was labeled with mCherry and eGFP on the N- and C- terminus, respectively and ChREBPβ is labeled with eGFP on the C-terminus. We validated the three cell lines by sequencing, ChIP, qRT-PCR and also showed that the glucose response is not altered in these cells. We found that in low glucose, ChREBPβ is present at very low levels in the nucleus. By contrast, in high glucose, ChREBPβ is present at high levels in the nucleus. Our study suggests that in response to glucose, ChREBPα, which is mostly cytoplasmic, transiently enters the nucleus, binds to a carbohydrate response element (ChoRE), and induces ChREBPβ expression, which is mostly nuclear. We propose that while the induction of ChREBPβ drives adaptive beta cell expansion, ChREBPβ contributes to glucose toxicity after prolonged exposure to high glucose and in diabetes.
L.S. Katz: None. A.K. Laxman: None. P. Zhang: None. D. Scott: None.