Degradation of cAMP-responsive element-binding protein by the ubiquitin-proteasome pathway contributes to glucotoxicity in beta-cells and human pancreatic islets.
OBJECTIVE
In type 2 diabetes, chronic hyperglycemia is detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The transcription factor cAMP-responsive element-binding protein (CREB) is crucial for beta-cell survival and function. We investigated whether prolonged exposure of beta-cells to high glucose affects the functional integrity of CREB.


METHODS
INS-1E cells and rat and human islets were used. Gene expression was analyzed by RT-PCR and Western blotting. Apoptosis was detected by cleaved caspase-3 emergence, DNA fragmentation, and electron microscopy.


RESULTS
Chronic exposure of INS-1E cells and rat and human islets to high glucose resulted in decreased CREB protein expression, phosphorylation, and transcriptional activity associated with apoptosis and impaired beta-cell function. High-glucose treatment increased CREB polyubiquitination, while treatment of INS-1E cells with the proteasome inhibitor MG-132 prevented the decrease in CREB content. The emergence of apoptosis in INS-1E cells with decreased CREB protein expression knocked down by small interfering RNA suggested that loss of CREB protein content induced by high glucose contributes to beta-cell apoptosis. Loading INS-1E cells or human islets with a cell-permeable peptide mimicking the proteasomal targeting sequence of CREB blocked CREB degradation and protected INS-1E cells and human islets from apoptosis induced by high glucose. The insulin secretion in response to glucose and the insulin content were preserved in human islets exposed to high glucose and loaded with the peptide.


CONCLUSIONS
These studies demonstrate that the CREB degradation by the ubiquitin-proteasome pathway contributes to beta-cell dysfunction and death upon glucotoxicity and provide new insight into the cellular mechanisms of glucotoxicity.
Costes
Safia
S
Institut National de la Santé et de la Recherche Médicale (INSERM), U661, Equipe Avenir, Institut de Génomique Fonctionnelle, Montpellier, France.
Vandewalle
Brigitte
B
Tourrel-Cuzin
Cécile
C
Broca
Christophe
C
Linck
Nathalie
N
Bertrand
Gyslaine
G
Kerr-Conte
Julie
J
Portha
Bernard
B
Pattou
François
F
Bockaert
Joel
J
Dalle
Stéphane
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001926:Brain Death; D050882:CREB-Binding Protein / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D051786:Cyclic AMP Response Element Modulator / Q000187:drug effects / Q000378:metabolism*; D053938:DNA Fragmentation; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D005947:Glucose / Q000633:toxicity*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000473:pathology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000473:pathology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025801:Ubiquitin / Q000378:metabolism*
D007328:Insulin; D025801:Ubiquitin; D051786:Cyclic AMP Response Element Modulator; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494365:Crebbp protein, rat; D046988:Proteasome Endopeptidase Complex; D005947:Glucose