Early loss of mammalian target of rapamycin complex 1 (mTORC1) signalling and reduction in cell size during dominant-negative suppression of hepatic nuclear factor 1-α (HNF1A) function in INS-1 insulinoma cells

Abstract

Aims/hypothesis Mutations in the HNF1A (previously known as TCF1) gene encoding hepatocyte nuclear factor-1α (HNF1A) lead to the development of maturity-onset diabetes of the young, type 3 (HNF1A-MODY), characterised by impaired insulin secretion and a reduction in beta cell mass. HNF1A plays an important role in pancreatic beta cell differentiation and survival. The mammalian target of rapamycin (mTOR) is a central growth factor-and nutrientactivated protein kinase controlling cell metabolism, growth and survival. We investigated the role of mTOR inactivation in the decline in beta cell mass in a cellular model of HNF1A-MODY. Methods Previously we showed that suppression of HNF1A function via expression of a dominant-negative mutant (DN-HNF1A) decreases insulin gene transcription in insulinoma (INS-1) cells. We investigated the signalling of two distinct mTOR protein complexes, mTORC1 and mTORC2, in response to DN-HNF1A induction.Results We observed delayed inactivation of mTORC2 48 h after DN-HNF1A induction, evidenced by a reduction in serine 473 phosphorylation of thymoma viral protooncogene 1 (AKT1). We also observed an early inactivation of mTORC1 24 h after DN-HNF1A induction, which was detected by decreases in threonine 389 phosphorylation of p70 ribosomal protein S6 kinase (S6K1) and serine 65 phosphorylation of translational inhibitor eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Flow cytometry and gene expression analysis demonstrated a preapoptotic decrease in INS-1 cell size in response to DN-HNF1A induction, and an increase in the level of the mTORC1-regulated cell-cycle inhibitor, cyclin-dependent kinase inhibitor 1B p27. Conclusions/interpretation Our data suggest that mTOR kinase and signalling through mTORC1 are highly sensitive to suppression of HNF1A function, and may contribute to disturbance of cell-size regulation and cell-cycle progression in HNF1A-MODY.

AMP-activated Protein Kinase Mediates Apoptosis in Response to Bioenergetic Stress through Activation of the Pro-apoptotic Bcl-2 Homology Domain-3-only Protein BMF

“…1, A and B). Previously, we demonstrated a reduction in expression of pyruvate kinase over this time scale after dominant-negative suppression of HNF1A function (8). Indeed, numerous other downstream target genes involved in energy metabolism have been shown to be downregulated 24 h after suppression (9).…”

“…In particular, the expressions of the Glut2 glucose transporter and pyruvate kinase have been consistently shown to be reduced by dominant-negative suppression of HNF1A function (2,3,8,9). Mitochondrial targets have also been identified, including decreased expression of the E1 subunit of 2-oxoglutarate dehydrogenase and increased levels of the uncoupling protein UCP2 (2,3).…”

Bone Morphogenetic Protein 3 Controls Insulin Gene Expression and Is Down-regulated in INS-1 Cells Inducibly Expressing a Hepatocyte Nuclear Factor 1A–Maturity-onset Diabetes of the Young Mutation

“…Among the genes identified in this microarray study were several known target genes of HNF1A including Pklr, Foxa3, and Pdk-1, and all were found to be down-regulated in response to Pro291fsinsC-HNF1A (16,17,26,27). Foxa2, which is subject to transcriptional repression by HNF1A (28), was found to be up-regulated.…”

Rapamycin protects against dominant negative-HNF1A-induced apoptosis in INS-1 cells

“…mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) are two structurally and functionally distinct protein complexes that share mTOR as their catalytic subunit. We have shown that the DN-HNF1A-induced decrease in mTOR signalling correlated with decreased activity of the mTORC1 targets, ribosomal p70S6 kinase and 4E-BP1, and the mTORC2 target, Akt [13].…”

“…In addition, we have previously reported that the activity of mammalian target of rapamycin (mTOR), which is negatively regulated by AMPK [12] is suppressed following DN-HNF1A overexpression [13]. mTOR is a serine/threonine protein kinase that integrates nutritional, physiological and hormonal signals within a cell [14,15].…”