Effects of tri-iodothyronine (T3), insulin, insulin-like growth factor I (IGF-I) and transforming growth factor beta1 (TGFβ1) on the proportion of insulin cells in cultured embryonic chick pancreas

Abstract

With a view to ultimately identifying factors involved in the development of pancreatic insulin cells, we have cultured dorsal pancreatic buds from 5-day chick embryos on a basement membrane matrix (Matrigel) in a serum-free medium supplemented with selected factors. The endodermal components of the buds were freed of almost all the mesenchyme so as to eradicate as much as possible of this source of some such factors. In 7-day cultures, insulin and glucagon cells were demonstrated immunocytochemically; numbers of insulin cells were expressed as a percentage of insulin plus glucagon cell counts. Our standard medium contained insulin. Addition of tri-iodothyronine to this medium did not increase the proportion of insulin cells, but in combination with raised concentrations of glucose and essential amino acids it improved somewhat the marked increase previously recorded for these nutrient conditions. Omission of insulin from the standard medium greatly reduced the proportion of these cells; substitution of insulin by insulin-like growth factor I increased the proportion considerably more than did insulin. To test for an overall effect of growth factors, explants were cultured in standard medium on Matrigel containing reduced amounts of growth factors: the proportion of insulin cells proved to be increased over that reached on normal Matrigel. The suspicion that transforming growth factor beta1, a component of Matrigel, might act to reduce the proportion of insulin cells was tested and found to be correct. It is suggested that the different factors studied here may affect either or both of proliferation and determination in the differentiation pathway of insulin vis-à-vis glucagon cells.

Regulation of Embryonic Chick Insulin Cells: Effect of Retinoic Acid and Insulin-Like Growth Factor 1

“…Increased proportions of insulin cells have been achieved by the manipulation of the culture medium and the extracellular matrix [Andrew et al, 1994;Rawdon andAndrew, 1997, 1998;Mngomezulu and Kramer, 2000;Penny and Kramer, 2000]. RA alone (10 -6 M) was shown to increase the proportion of insulin cells by a factor of 3 [Penny and Kramer, 2000]; the addition of nicotinamide increased the proportion of insulin cells by a factor of 2.5 [Mngomezulu and Kramer, 2000] and IGF-1 by a factor of 1.7 [Rawdon and Andrew, 1998]. None of the results of these studies have approached the in vivo proportion of 34% for insulin cells from 12-day splenic lobe of intact embryos [Andrew et al, 1994].…”

“…The replacement of insulin by IGF-1 in F12.ITS resulted in a 29% increase in the proportion of insulin cells (17.3% with F12.TS versus 22.3% with F12.TS + IGF-1) [Rawdon and Andrew, 1998]. There is further evidence for the involvement of IGF-1 during pancreatic development; mRNAs for IGFs are expressed within the rat pancreas with distinct ontogenetic patterns during fetal and neonatal life [Hogg et al, 1994], and receptors for IGF-1 have been localized on both rat pancreatic insulin and glucagon cells [Peng et al, 1977].…”

“…When dorsal pancreatic buds were grown on Matrigel in Ham's F12 serum-free medium containing insulin, transferrin and selenium (F12.ITS), a proportion of 17.3% of insulin cells was obtained [Rawdon and Andrew, 1998]. When insulin was omitted from the medium (F12.TS), there was a 24% reduction in the proportion of insulin cells (from 17.3% with F12.ITS to 13.1% with F12.TS) [Rawdon and Andrew, 1998]. …”

“…Insulin-like growth factor-1 (IGF-1), which plays a role in differentiation and proliferation in general [Hansson et al, 1988], appears to be more effective in promoting insulin cell proliferation than is insulin [Rawdon and Andrew, 1998]. The replacement of insulin by IGF-1 in F12.ITS resulted in a 29% increase in the proportion of insulin cells (17.3% with F12.TS versus 22.3% with F12.TS + IGF-1) [Rawdon and Andrew, 1998].…”

“…In an attempt to increase the proportion of insulin cells over that shown independently for RA [Penny and Kramer, 2000] and IGF-1 [Rawdon and Andrew, 1998], we cultured 5-day-old embryonic chick pancreas in a serumfree medium supplemented with a combination of RA and IGF-1. The number of insulin and glucagon cells was quantitated and the proportion of insulin cells was determined.…”