Glucose Regulates Steady-state Levels of PDX1 via the Reciprocal Actions of GSK3 and AKT Kinases

Abstract

The pancreatic beta cell is sensitive to even small changes in PDX1 protein levels; consequently, Pdx1 haploinsufficiency can inhibit beta cell growth and decrease insulin biosynthesis and gene expression, leading to compromised glucose-stimulated insulin secretion. Using metabolic labeling of primary islets and a cultured ␤ cell line, we show that glucose levels modulate PDX1 protein phosphorylation at a novel C-terminal GSK3 consensus that maps to serines 268 and 272. A decrease in glucose levels triggers increased turnover of the PDX1 protein in a GSK3-dependent manner, such that PDX1 phosphomutants are refractory to the destabilizing effect of low glucose. Glucose-stimulated activation of AKT and inhibition of GSK3 decrease PDX1 phosphorylation and delay degradation. Furthermore, direct pharmacologic inhibition of AKT destabilizes, and inhibition of GSK3 increases PDX1 protein stability. These studies define a novel functional role for the PDX1 C terminus in mediating the effects of glucose and demonstrate that glucose modulates PDX1 stability via the AKT-GSK3 axis.The homeodomain protein PDX1 has been shown to be a critical regulator of pancreatic development, in both humans and in mice (1, 2). Whereas genetic ablation or the total functional inhibition of PDX1 results in pancreatic agenesis, haploinsufficiency of PDX1 in humans leads to diabetes (3) and is associated with diminished glycemic control in mice (4, 5). Loss of a single allele of Pdx1 results in increased beta cell death (6) and a diminished capacity to mount a compensatory response in some models of insulin resistance (7). Thus, the beta cell is highly sensitive to total cellular levels of PDX1 protein.The mechanisms by which PDX1 may regulate glucose homeostasis have been widely examined. In addition to regulating the insulin gene (8, 9), PDX1 has been shown to activate Glut2 and glucokinase genes, two key players required for glucose sensing (10, 11). PDX1 can also impact glucose sensing by influencing expression of mitochondrial metabolic pathways (12, 13). Accordingly, proteins important in glucose sensing are down-regulated in Pdx1 ϩ/Ϫ mice (5, 7). In the beta cell, PDX1 is thought to be a direct mediator of glucose. Several studies have suggested that glucose-stimulated phosphorylation of PDX1 impacts DNA binding (14, 15) and the nucleocytoplasmic shuttling of PDX1 (16). Glucose-dependent phosphorylation has also been suggested to increase the transactivation potential of PDX1 by increasing recruitment of chromatin-modifying proteins such as p300 (17,18) or by decreasing binding with histone deacetylases (19). Other studies suggest direct, glucose-dependent phosphorylation of PDX1 by specific kinases, including ERK 2 1 and 2 (20), and perhaps p38 MAPK (14, 16). However, to date, the specific amino acid residues at which glucose-induces PDX1 phosphorylation, within the context of the cell, are not known.The N terminus of PDX1 harbors a strong transactivation domain, flanked by the DNA-binding homeodomain (8,21,22). The C terminus of PDX1 ...

Methylglyoxal impairs insulin signalling and insulin action on glucose-induced insulin secretion in the pancreatic beta cell line INS-1E

“…In addition, insulin modulates PDX-1 expression and action [27]. Moreover, it was recently proposed that glucose regulates the levels of PDX-1 via the reciprocal actions of GSK-3 and PKB kinase [28]. To investigate whether MGO modulates gene expression, INS-1E cells were incubated with 0.5 mmol/l MGO for 30 min and then stimulated with insulin.…”

Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice

“…Upstream regulatory elements have been described that temporally and spatially control Pdx1 expression and are required for outgrowth and differentiation of pancreatic progenitors (8,(15)(16)(17)(18)(19)(20); however, posttranslational regulation of Pdx1 via phosphorylation has also been proposed to have diverse functional effects, including regulation of Pdx1 transactivation and protein stability (13,(21)(22)(23)(24)(25). We previously described Pdx1 C terminus-interacting factor 1 (Pcif1, also known as SPOP), a broad complex, tramtrack, bric-a-brac (BTB) domain-containing protein that interacts directly with the C terminus of Pdx1 (26,27).…”

Loss of TRB3 Alters Dynamics of MLK3-JNK Signaling and Inhibits Cytokine-activated Pancreatic Beta Cell Death

“…However, AKT also indirectly regulates protein stability via GSK3 to impact beta cell function and survival. GSK3-mediated phosphorylation and proteasomal degradation is a prevalent mechanism (46) and reportedly regulates beta-cell specific transcription factors PDX1 (47,48) and MAF-A (49, 50), as well as MCL-1 (51,52), an antiapoptotic BCL family protein.…”

“…Cycloheximide Chase, Western Blotting, and Insulin Stimulation-Experiments were performed as described (47).…”