Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix

Pancreas. 1996 Jul;13(1):47-54. doi: 10.1097/00006676-199607000-00006.

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.

MeSH terms

  • Animals
  • Base Sequence
  • Collagen
  • Culture Techniques / methods
  • DNA, Complementary / genetics
  • Drug Combinations
  • Extracellular Matrix
  • Glucagon / genetics
  • Insulin / genetics*
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / metabolism*
  • Laminin
  • Male
  • Proteoglycans
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • Rats
  • Rats, Wistar
  • Somatostatin / genetics

Substances

  • DNA, Complementary
  • Drug Combinations
  • Insulin
  • Laminin
  • Proteoglycans
  • RNA, Messenger
  • matrigel
  • Somatostatin
  • Collagen
  • Glucagon