Abstract
Type 2 diabetes (T2D) and Alzheimer disease are degenerative diseases that may share common pathophysiologic mechanisms. Neuronal dysfunction in Alzheimer patients has been linked to overactivity of the cyclin-dependent kinase 5 (CDK5) and its activator p35. Both of these proteins are expressed in the insulin-producing beta cells of the pancreas. Further, glucose enhances p35 gene expression, promoting the formation of active p35/CDK5 complexes that regulate the expression of the insulin gene. In T2D, chronic elevations of glucose, glucotoxicity, impair beta cell function. We therefore postulated that CDK5 and p35 may be responsible for this beta cell impairment and that inhibition of CDK5 might have a beneficial effect. To test this hypothesis, the pancreatic cell line INS-1 was selected as a known in vitro model of glucotoxicity, and roscovitine (10 mum) was used as a CDK5 inhibitor. Chronic exposure of INS-1 cells to high glucose (20-30 mm) reduced both insulin mRNA levels and the activity of an insulin promoter reporter gene. Inhibition of CDK5 prevented this decrease of insulin gene expression. We used DNA binding (gel shift) assays and Western immunoblots to demonstrate that cellular levels of the transcription factor PDX-1, normally decreased by glucotoxicity, were preserved with CDK5 inhibition, as was the binding of PDX-1 to the insulin promoter. Analyses of nuclear and cytoplasmic PDX-1 protein levels revealed that CDK5 inhibition restores nuclear PDX-1, without affecting its cytoplasmic concentration, suggesting that CDK5 regulates the nuclear/cytoplasm partitioning of PDX-1. Using a Myc-tagged PDX-1 construct, we showed that the translocation of PDX-1 from the nucleus to the cytoplasm during glucotoxic conditions was prevented when CDK5 was inhibited. These studies indicate that CDK5 plays a role in the loss of beta cell function under glucotoxic conditions and that CDK5 inhibitors could have therapeutic value for T2D.
Highlights
Because intracellular overactivation of cyclindependent kinase-5 (CDK5) is a component of the pathophysiology of Alzheimer disease (8 –10) and hyperglycemia results in overactivation of CDK5 in beta cells [1], we investigated the possibility that CDK5 signaling may be involved in Type 2 diabetes (T2D)
We show that overstimulation of CDK5 in an experimental model of glucotoxicity is involved in the cytoplasmic translocation of PDX-1 during glucotoxicity
CDK5 inhibition on insulin gene expression, we studied the effect of the CDK5 inhibitor roscovitine on the glucotoxicity-induced down-regulation of insulin mRNA
Summary
Chemicals and Antibodies—Tissue culture media, serum, and other cell culture reagents were purchased from Invitrogen. Immunoprecipitation and Kinase Assay—Determination of CDK5 activity in cellular extracts from INS-1 cells grown in normal (5 mM) and high (30 mM) concentration of glucose was achieved as reported previously [1]. These data corroborated previous findings in cultured beta cell lines [11] We confirmed that this reduction of insulin mRNA levels was the result of a transcriptional response by measuring the activity of a luciferase reporter construct driven by the insulin promoter sequence (Ϫ410 of the rat insulin-1 gene). We determined that in these experimental conditions, sustained elevation of glucose concentration produces an increase of CDK5 activity that remains elevated over the 48 h duration of these experiments (Fig. 1C). Cific inhibition of CDK5 activity [1] Under these conditions, roscovitine almost completely prevented the down-regulation of insulin mRNA expression induced by high glucose (Fig. 1D). Densitometry analysis and statistical comparison of this set of data demonstrate that the effect of roscovitine is statistically significant (Fig. 1E)
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