INGAP-PP up-regulates the expression of genes and proteins related to K+ ATP channels and ameliorates Ca2+ handling in cultured adult rat islets.
Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K(+)(ATP) channel, Ca(2+) handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca(2+) ([Ca(2+)](i)), static and dynamic insulin secretion, and (86)Rb efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 muM tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC(50) of 10.0+/-0.4 vs. 13.7+/-1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 microM tolbutamide and accelerated the velocity of glucose-induced reduction of (86)Rb efflux in perifused islets. These effects were accompanied by a significant increase in [Ca(2+)](i) and the maintenance of a considerable degree of [Ca(2+)](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K(+)(ATP) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca(2+)](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes.
Silva
Kelly E
KE
Departamento de Fisiologia e Biofísica, Instituto de Biologia Universidade Estadual de Campinas, 13083-970, Campinas-SP, Brazil.
Barbosa
Helena C
HC
Rafacho
Alex
A
Bosqueiro
José R
JR
Stoppiglia
Luiz F
LF
Carneiro
Everardo M
EM
Borelli
Maria I
MI
Del Zotto
Hector
H
Gagliardino
Juan J
JJ
Boschero
Antonio C
AC
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D054086:KATP Channels / Q000235:genetics / Q000378:metabolism*; D000073718:Pancreatitis-Associated Proteins; D010446:Peptide Fragments / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D046509:Tissue Culture Techniques; D014044:Tolbutamide / Q000494:pharmacology; D000099161:Kcnj11 Channel
D002118:Calcium; D016207:Cytokines; D005947:Glucose; D051542:Hepatocyte Nuclear Factor 3-beta; D007328:Insulin; D054086:KATP Channels; D000073718:Pancreatitis-Associated Proteins; D010446:Peptide Fragments; D024661:Potassium Channels, Inwardly Rectifying; D011189:Potassium Chloride; D014044:Tolbutamide; D000099161:Kcnj11 Channel; C496504:Foxa2 protein, rat; C500438:INGAP peptide; C000612141:REG3A protein, human