Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix.
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.
Perfetti
R
R
Diabetes Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Henderson
T E
TE
Wang
Y
Y
Montrose-Rafizadeh
C
C
Egan
J M
JM
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
D018076:DNA, Complementary; D004338:Drug Combinations; D007328:Insulin; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D013004:Somatostatin; D003094:Collagen; D005934:Glucagon