Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of langerhans

Abstract

A B S T R A C T Islets of Langerhans isolated from rat pancreas were incubated at 370C(95% 02/5% C02) in buffered medium containing 1.0 mg/ml glucose and leucine 8H for 1 hr (1st hr), washed, and incubated for an additional hr (2nd hr) in low glucose medium (0.5-1.0 mg/ml) containing unlabeled leucine. A portion of the islets was then extracted with acid-ethanol and the remainder were transferred to medium containing 3.0 mg/ml glucose and incubated for 2 hr (3rd and 4th hr) at 370C. The medium was exchanged at 30-min intervals and portions of the islets were extracted at the 3rd and 4th hr. The total amounts and specific activities of the proinsulin and insulin in the islet extracts and medium samples were determined after fractionation on Biogel P-30 columns in 3 M acetic acid.Maximal release of newly synthesized insulin occurred between the 3rd and 4th hr of incubation, confirming the results of Howell and Taylor (Biochem. J. 102: 922. 1967). The high glucose medium increased the secretion of insulin approximately three to fourfold. The ratio of the specific activities of the insulin in the medium to that in the islets was about 1/1 during incubation in low glucose, but it increased to 2.5/1 during incubation with high glucose. The peak occurred at the 3rd hr, i.e., 1 hr after exposure to high glucose. The ratio of labeled proinsulin to insulin was slightly lower in the medium than in the islets. Addition of sufficient cycloheximide after the 1st hr to inhibit protein synthesis did not inhibit these responses. The specific activity of the proinsulin in the medium was about the same as that in the islets, and both were about 10-fold higher than the specific activity of the insulin. High glucose did not alter the proinsulin specific activity, which tended to decline throughout the period of observation. With cycloheximide present, the concentration of proinsulin in the islets steadily declined while the specific activity of proinsulin remained high, indicating that the proinsulin pool is small and is turning over rapidly. In terms both of amount and radioactivity proinsulin amounted to 6-7% on a molar basis of the insulin in both the medium and the islets. Addition of dibutyryl cyclic 3',5'-adenosine monophosphate (DBCAMP) (0.002 M) with high glucose during the postlabeling period slightly increased the rate of insulin secretion (133% of control) but did not significantly alter the other parameters.The results suggest that while newly synthesized insulin and proinsulin may be preferentially secreted to a slight degree, about 90% of the insulin released during 3 hr in response to glucose, or to glucose and DBCAMP, is derived from pre-existing granule stores. There were no indications of the existence of independent or nongranule pathways of insulin or proinsulin secretion.

Analysis of insulin secretion based on changes in plasma insulin and C-peptide in man.

“…Glucose load has been widely applied as a basic tool for a stimulation test of insulin secretion. In the present study, the response of plasma CPR in the normal controls was identical to that of plasma IRI, except for a 30-min delay of the peak level, as reported by many authors (Sando et al 1972;Kaneko 1973; Heding and Rasmussen). Therefore, the results obtained in GTT of the present study are compatible with the theory that insulin and C-peptide are released from the B-cell of the islet of Langerhans in equimolar quantities (Rubenstein et al 1969;Heding et al 1974;Horwitz et al 1975).…”

Effects of streptozotocin in vitro on proinsulin biosynthesis, insulin release and ATP content of isolated rat islets of Langerhans

“…Proinsulin content thus appears to be as sensitive a marker of the rate of synthesis as incorporation of tritiated leucine, consistent with a small proinsulin pool turning over rapidly. Both the observation and our conclusions are in good agreement with those of Sando et al [44].…”

“…Variations in this ratio should not be af-fected by variation in the total IRI amount in any cell sample since proinsulin and insulin should be altered to the same extent. Indeed, it has been shown by Sando et al [44] that the percentage of secreted proinsulin to insulin remains the same under stimulated and non-stimulated conditions and, even assuming an increase in IRI content due to decreased release (rela-"i proinsulin ....... tive to controls), the rat o total IRI woma most nKely remain the same, since proinsulin retention would also increase. It seems most reasonable to assume that the proinsulin ratio reflects primarily a decrease in the total IRI decrease in the proinsulin content, and that this is most likely due to streptozotocin-induced decrease in proinsulin biosynthesis.…”

Effects of glucose on the ultrastructure and insulin biosynthesis of isolated mouse pancreatic islets maintained in tissue culture

“…The approximate specific radioactivity of the extracted insulin was eMculated by dividing the mean value for the radioactivity (.ep.m) incorporated into each of the four fraetions eompmsmg the maximum of the insulin peak with the mean value for their respective immunoreactive insulin content (el. Sando et al, [13]). …”