Exp Clin Endocrinol Diabetes 2004; 112 - P165
DOI: 10.1055/s-2004-819284

Rescue of the β-cell phenotype of a CaM-kinase IIδ2 deficient rat insulinoma cell line INS δ-W12 by re-transfection of human CaM-kinase IIβ1

MA Osterhoff 1, S Bessenyei 1, A Sharma 1, M Moehlig 1, AFH Pfeiffer 1
  • 1German Institute of Human Nutrition, Potsdam-Rehbruecke, Charité – Campus Benjamin Franklin, Berlin

Background: The expression of CaMK IId2 was reduced in INS-1 rat insulinoma cells by approx. 80% by a retroviral antisense mRNA approach (INS d-W12). This resulted in a partial loss of the b-cell phenotype of INS-1 cells. In the present work we investigated if a re-transfection of the human CaMK IIb1 into INS d-W12 cells is able to restore the b-cell phenotype of INS-1 cells.

Materials: INS-1 and INS d-W12 cells were cultured in RPMI 1640 media. A construct coding for the hCaMK IIb1 was transfected into INS d-W12 cells by use of FuGene 6 (Roche). The gene-expression of b-cell genes was determined by quantitative real-time PCR (RotorGene, Corbett Research) using alpha-tubulin as a standard.

Results: The reduction of the CaMK IId2-expression in INS-1 cells (INS d-W12) leads to a markedly decreased expression of the b-cell genes insulin, IAPP, GLUT-2 and glucokinase while the expression of hexokinase I increases. Metabolic assays show a right-shift of the half-maximum glucose-induced metabolic activity from 5mmol/l (Km of glucokinase) to 0.5mmol/l (Km of hexokinase). After re-transfection of hCaMK IIb1 into INS d-W12 cells, several clones were investigated by quantitative PCR concerning the above mentioned b-cell genes. We could show that the gene-expression of insulin, glucokinase and GLUT-2 doubles while the expression of hexokinase I is reduced to 50%, confirming that the transfection of hCaMK IIb1– at least in part – is able to restore the b-cell phenotype of INS-1 cells.

Conclusion: The rescue of the b-cell phenotype of INS-1 cells after CaMK IId2 suppression by re-transfection of human CaMK IIb1 not only proves the specifity of the retroviral antisense mRNA approach to selectively reduce the CaMK IId2 expression. It also shows clearly that CaMK II plays a major role in controlling the expression of b-cell genes. Possibly, CaMK II is a mediator of glucotoxicity since high intracellular Ca2+-levels lead to deactivation of CaMK II and by this to a decreased expression of b-cell genes and finally to dedifferentiation of b-cells