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

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 β 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.     

Glycogen synthase kinase 3 beta is a natural activator of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1)

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1.     

Glycogen synthase kinase 3beta is a novel regulator of high glucose- and high insulin-induced extracellular matrix protein synthesis in renal proximal tubular epithelial cells

High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.     

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.     

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.