title: Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix.
abstract: Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glucagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, beta cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 +/- 2.5 vs. 37.1 +/- 2.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single beta cell was reduced by 40-60% (p < 0.02) compared to that released from isolated beta cells derived from a 3-day culture of islets. Finally, there was a 35-55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal beta-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.
authors.affiliation: Diabetes Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: R
authors.initials: R
authors.lastname: Perfetti
authors.forename: T E
authors.initials: TE
authors.lastname: Henderson
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Montrose-Rafizadeh
authors.forename: J M
authors.initials: JM
authors.lastname: Egan
mesh_terms: D000818:Animals; D001483:Base Sequence; D003094:Collagen; D046508:Culture Techniques / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D004338:Drug Combinations; D005109:Extracellular Matrix; D005934:Glucagon / Q000235:genetics; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D007797:Laminin; D008297:Male; D011509:Proteoglycans; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013004:Somatostatin / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D004338:Drug Combinations; D007328:Insulin; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D013004:Somatostatin; D003094:Collagen; D005934:Glucagon