Scintigraphic distribution of 123 I labelled proinsulin, split conversion intermediates and insulin in rats

Abstract

Summary. Insulin, biosynthetic human proinsulin and 2 human proinsulin conversion intermediates, des (64, 65) human proinsulin and des (31, 32) human proinsulin, were labelled with 123 I and the derivatives monosubstituted on Tyr A14 were purified by reverse phase high performance liquid chromatography. The four tracers were injected into anaesthetized rats via a jugular or a portal vein and time activity curves were generated for the liver and kidneys using a gamma camera and an online computer. Liver extraction coefficients varied in the order insulin (38%), des (64, 65) human proinsulin (11.7%), des (31, 32) human proinsulin (3.2%), human proinsulin (1.6%); whereas half-life of hepatic activity varied in the reverse order, from 6 rain for insulin, to 45 min for human proinsulin. As expected for a non-receptor mediated process, kidney extraction varied conversely to liver extraction, being highest for human proinsulin and lowest for insulin. It is concluded that the kinetics of human proinsulin conversion intermediates depends upon the site of cleavage and deletion and is intermediate between those of insulin and intact human proinsulin.Key words: Biosynthetic human proinsulin, conversion intermediates, 123-I-labelling, scintillation scanning.Most of proinsulin is converted to insulin inside the B cells of the pancreas, a process resulting from the interplay of several endopeptidases, different from trypsin itself [1][2][3]. Recently, two distinct site-specific endopeptidases regulated by calcium concentration and pH were identified in a B granule fraction of rat insulinoma [4]. During proinsulin processing, conversion intermediates are formed, in particular the split products des (64, 65) human proinsulin (HPI) and des (31, 32) HPI. These intermediates and intact HPI have been found in human sera [5], in insulin producing islet cell tumours [6-81 and in familial hyperproinsulinaemia [9-111. Conversion to intermediates or insulin does not seem to occur after proinsulin enters the blood stream; partial conversion to intermediates might occur in the skin after subcutaneous injection [121.From the standpoint of biological activity, proinsulin has remarkable characteristics. Compared to insulin, HPI has a low biological potency, a low avidity for insulin receptors and a prolonged half-life [13][14][15][16][17]. Moreover, the proinsulin conversion intermediates have been shown to exhibit greater biological activity than proinsulin itself, and this is particularly true for the conversion intermediate in which the C peptide/A chain bond has been cleaved. Peavy et al. demonstrated that cleavage and/or dibasic amino acid deletions in the connecting peptide region of proinsulin enhance the receptor binding and hypoglycaemic activities of these compounds. In these studies, C peptide/A chain split products exhibited greater receptor affinity and hypoglycaemic activity than B chain C peptide split intermediates [18]. These observations prompted us to investigate the hepatic extraction and processing of HPI and its conver...

Cell Itinerary and Metabolic Fate of Proinsulin in Rat Liver: In Vivo and in Vitro Studies

“…after injection of peptides labeled with 123 I at the A14 position, insulin uptake attained 32% of injected dose after 3 min and declined with a half-life of 6 min, whereas proinsulin uptake reached 18% at 1 min and decreased with a halflife of 45 min (16). Our estimates of liver uptake of [ 125 I]-proinsulin and [ 125 I]-insulin (1.6 and 5.2% of injected dose per gram of liver, respectively) and half-lives (28 and 6 min) compare favorably with these results.…”

“…The patterns of in vivo liver uptake of injected proinsulin and insulin have been compared in two previous studies (15,16). In one study, in which the liver was removed after injection of [ 131 I]-labeled peptides, insulin uptake was maximal at 1 min and rapidly declined over 30 min, whereas proinsulin uptake was stable up to 10 min and declined more slowly; at maximum, 29% and 6% of the injected dose, respectively, were recovered in the liver (15).…”

“…As expected from its low affinity for the liver insulin receptor (11,12), proinsulin displays a reduced extraction by the isolated perfused liver (13,14) and liver in vivo (15,16), which accounts for its prolonged half-life in serum. Furthermore, after in vivo uptake into the liver, proinsulin is more slowly cleared than insulin, suggesting a slower intracellular processing (15,16).…”

“…Furthermore, after in vivo uptake into the liver, proinsulin is more slowly cleared than insulin, suggesting a slower intracellular processing (15,16). However, although proinsulin has been shown to be less efficiently degraded than insulin by isolated liver subcellular fractions (11,17) and purified liver insulindegrading enzyme (IDE) (18), studies on proinsulin processing in intact cells have been limited to adipocytes (19) and fibroblasts overexpressing the insulin receptor (20).…”

“…Male Sprague Dawley rats, weighing 200 Ϯ 20 g, were obtained from Charles River (St. Aubin les Elbeufs, France) and Elevage Janvier (Le Genest-St-Isle, France) and were fasted for 16 h before killing. When indicated, porcine [ 125 I]-labeled peptides (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) or native human peptides (0.06 -13 nmol), diluted into 0.5 ml 0.15-m NaCl, were rapidly injected into the penis vein under ether or pentobarbital anesthesia. At the time indicated, the liver was rapidly removed, minced in ice-cold 0.25 m sucrose, and immediately homogenized.…”