Gene silencing of phogrin unveils its essential role in glucose-responsive pancreatic beta-cell growth.
OBJECTIVE
Phogrin and IA-2, autoantigens in insulin-dependent diabetes, have been shown to be involved in insulin secretion in pancreatic beta-cells; however, implications at a molecular level are confusing from experiment to experiment. We analyzed biological functions of phogrin in beta-cells by an RNA interference technique.


METHODS
Adenovirus-mediated expression of short hairpin RNA specific for phogrin (shPhogrin) was conducted using cultured beta-cell lines and mouse islets. Both glucose-stimulated insulin secretion and cell proliferation rate were determined in the phogrin-knockdown cells. Furthermore, protein expression was profiled in these cells. To see the binding partner of phogrin in beta-cells, coimmunoprecipitation analysis was carried out.


RESULTS
Adenoviral expression of shPhogrin efficiently decreased its endogenous expression in pancreatic beta-cells. Silencing of phogrin in beta-cells abrogated the glucose-mediated mitogenic effect, which was accompanied by a reduction in the level of insulin receptor substrate 2 (IRS2) protein, without any changes in insulin secretion. Phogrin formed a complex with insulin receptor at the plasma membrane, and their interaction was promoted by high-glucose stimulation that in turn led to stabilization of IRS2 protein. Corroboratively, phogrin knockdown had no additional effect on the proliferation of beta-cell line derived from the insulin receptor-knockout mouse.


CONCLUSIONS
Phogrin is involved in beta-cell growth via regulating stability of IRS2 protein by the molecular interaction with insulin receptor. We propose that phogrin and IA-2 function as an essential regulator of autocrine insulin action in pancreatic beta-cells.
Torii
Seiji
S
Secretion Biology Lab, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan.
Saito
Naoya
N
Kawano
Ayumi
A
Hou
Ni
N
Ueki
Kohjiro
K
Kulkarni
Rohit N
RN
Takeuchi
Toshiyuki
T
D000818:Animals; D001323:Autoantibodies; D002455:Cell Division; D020868:Gene Silencing; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics; D011817:Rabbits; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001323:Autoantibodies; C096322:ICA512 autoantibody; D007328:Insulin; D008565:Membrane Proteins; D012320:RNA Polymerase III; C516863:Ptprn protein, mouse; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; C533193:phogrin protein, mouse; D005947:Glucose