Hyperglycemia promotes p53-Mdm2 interaction but reduces p53 ubiquitination in RINm5F cells
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The apoptosis of β cells induced by hyperglycemia has been associated with p53 mobilization to mitochondria and p53 phosphorylation. Murine double minute 2 (Mdm2) induces the degradation of p53 and thereby protects cells from apoptosis. We studied the effect of glucose at high concentration on the ability of Mdm2 to ubiquitinate p53 and promote its degradation. RINm5F cells were grown in RPMI-1640 medium with 5 or 30 mM glucose for varying periods of time. After this treatment, the expression of Mdm2 was measured using real-time PCR. The phosphorylation of Mdm2 at Ser166, p53 at Ser15, and the kinases Akt and ATM were measured by Western blotting. The formation of the p53-Mdm2 complex and p53 ubiquitination was assessed by p53 immunoprecipitation and immunofluorescence. Our results showed that high glucose reduced Mdm2 mRNA expression and protein concentration and increased Mdm2 and Akt phosphorylation, albeit with slower kinetics for Akt. It also promoted p53-Mdm2 complex formation, whereas p53 ubiquitination was suppressed. Furthermore, phosphorylation of both p53 Ser15 and ATM was increased in the presence of 30 mM glucose. These data indicate that high concentration glucose decrease the mRNA expression and cytosolic concentration of Mdm2. However, although the increase in glucose promoted the phosphorylation of Mdm2, it also decreased p53 ubiquitination, thus avoiding p53 degradation. In hyperglycemic conditions, such as diabetes mellitus, the reduction of pancreatic β cells mass is favored by stabilization of p53 in association with low p53 ubiquitination and reduced expression of Mdm2.
KeywordsMdm2 p53 Beta cells Ubiquitin pMdm2 Ser166 Hyperglycemia
This work was sponsored by the Fund for Research in Health (Fondo de Investigación en Salud, FIS), Mexican Social Security Institute (Instituto Mexicano del Seguro Social, IMSS) (FIS/IMSS/PROT/G11 968 2012). We extend our thanks to the IMSS Foundation A.C. and the Rio Arronte Foundation I.A.P. for their support in equipping the Medical Research Unit in Biochemistry. We would also like to thank the National Council for Science and Technology (CONACYT) and the Coordination for Research in Health, IMSS (R Barzalobre-Geronimo, scholarship).
Conflict of interest
The authors declare no conflict of interest.
- 7.Ortega-Camarillo C, Guzmán-Grenfell AM, García-Macedo R, Rosales-Torres AM, Ávalos-Rodríguez A, Duran-Reyes G, Medina-Navarro R, Cruz M, Díaz-Flores M, Kumate J (2006) Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells. Mol Cell Biochem 281:163–170CrossRefPubMedGoogle Scholar
- 8.Flores-López LA, Díaz-Flores M, García-Macedo R, Ávalos-Rodríguez A, Vergara-Onofre M, Cruz M, Contreras-Ramos A, Konigsberg M, Ortega-Camarillo C (2013) High glucose induces mitochondrial p53 phosphorylation by p38 MAPK in pancreatic RINm5F cells. Mol Biol Rep 40:4947–4958CrossRefPubMedGoogle Scholar
- 14.Meek DW, Knippschild U (2003) Posttranslational modification of MDM2. Mol Cancer Res 1:1026–1071Google Scholar
- 21.Lablanche S, Cottet-Rousselle C, Lamarche F, Benhamou P-Y, Halimi S, Leverve X, Fontaine E (2011) Protection of pancreatic INS-1 β-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin. Cell Death Dis 2(3):e134. doi: 10.1038/cddis.2011.15 CrossRefPubMedCentralPubMedGoogle Scholar
- 28.Tornovsky-Babeay S, Dadon D, Ziv O, Tzipilevich E, Kadosh T, Haroush RS-B, Hija A, Stolovich-Rain M, Furth-Lavi J, Granot Z, Porat S, Philipson LH, Herold KC, Bhatti TR, Stanley C, Ashcroft FM, Veld P It, Saada A, Magnuson MA, Glaseremail B, Doremail Y (2014) Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in β-cells. Cell Metab 19:109–121CrossRefPubMedGoogle Scholar