Neil J. Elgee

Specificity of Insulin Degradation Reaction

In a previous publication, inferential evidence was presented indicating that the distribution of the radioactivity of injected insulin labeled with radioactive iodine (I) is probably representative of the distribution of unlabeled insulin. In the present report, further evidence of the Specificity of insulin-I is presented in the demonstration that the degradation of a trace amount of insulin-I is competitively lessened by a load of either labeled or nonlabeled insulin, and not by other proteins of similar size. The distribution and degradation of ribonuclease, a nonhormonal protein of comparable molecular weight to insulin and labeled with I, is, however, shown to be very similar to that of insulin-I.

Studies of Radioactive Insulin

Highly purified crystalline insulin was labeled (and hereinafter noted as insulin* ) by iodination with radioactive iodine (I), presumably on the tyrosine rings, by the method of Ferrebee et al. The material was hypoglycemic to rabbits and assayed according to prediction by the rat diaphragm-glucose uptake technic. The radioactivity stayed with the protein fraction in trichloracetic acid precipitation, in dialysis and in chromatography, and had the same mobility as unlabeled insulin in paper electrophoresis. Therefore the label did not alter certain measurable properties of the insulin. Distribution studies in the rat fifteen minutes after intravenous administration of insulin* showed the greatest concentration to be in the renal cortex. Thyroid (presumably largely free I), liver and stomach also showed concentrations higher than plasma. Muscle concentration was not striking but total muscle mass was such that 30 to 50 per cent of the material was in that locus. Brain showed a notably low concentration, and red blood cells none at all. There was very little in the urine. It concentrated in the placenta and a small amount entered the fetus. A small but significant fraction was absorbed into the blood when insulin* was injected into the small intestine. Studies of the subcellular fractions of liver, kidney and muscle showed that insulin* had entered the cell. It was distributed in the cytostructural elements of the liver in a characteristic manner and fixation on the subcellular particles was shown to depend on cellular

Degradation of Insulin-I131 by Liver and Kidney in vivo.

Summary Nephrectomy or physiological hepatectomy decreased the rate of degradation of insulin-I131 in rats. There was some degradation even when both operations had been performed. Kidney and liver therefore are major, but not the only, sites of insulin-I131 degradation in the rat.

The Distribution and Metabolism of Insulin Labeled with Radioactive Iodine (I131) in Normal and Diabetic Subjects

To investigate possible differences in the distribution and metabolism of insulin in diabetics and normals, insulin labeled with radioactive iodine (I131) was given intravenously to 40 diabetics of varying type and severity, 15 healthy controls, and 11 other nondiabetics with various illnesses. The labeled insulin was given in tracer amounts (50 to 100 microcuries) and test doses varied between 0.5 and 5.0 units of insulin. The dose was diluted in normal physiologic saline solution and injected slowly into fasting subjects over a two-minute period. In most cases therapeutic insulin had been withheld from diabetics for 24 to 96 hours. Plasma samples were drawn at predetermined times, and the amount of protein-bound radioactivity was determined by precipitation with trichloroacetic acid (TCA). Since labeled insulin is virtually completely precipitated with TCA, the protein-bound radioactivity was assumed to represent undegraded labeled insulin, whereas the TCA-soluble radioactivity was assumed to be comprised of the degradation products of the labeled hormone. Total urinary radioactivity, of which an average of 95 per cent is TCA-soluble, was also measured during the test period. In some subjects surface measurement of radioactivity over various organ areas, such as the thyroid, liver, kidney, and muscle masses, was made.

Further investigations relative to studies with I131-labeled proteins, insulin in particular.

Summary An appreciable portion of inorganic I113 was found to be precipitated by TCA in the presence of red blood cells. This was not the case with serum or other tissues studied. In evaluating the degradation of I131-labeled proteins by measuring the TCA precipitability of the blood radioactivity, it should, therefore, be appreciated that the degradation product, inorganic I131, may show the phenomenon of “re-entry,” and be precipitated by TCA. Findings, somewhat similar to those with inorganic I131 were also obtained with I131-labeled diiodotyrosine.

Inhibition of degradation of insulin-I131 in vivo.

Summary Insulin-I131 degradation in rats in vivo was not inhibited by the administration of zinc in the diet, or by treatment with the substance, polyphloretin phosphate. Marked inhibition was obtained by the intravenous or prolonged oral administration of 5-isopropylidene-2,4-dithiohydantoin and it was suggested that this may explain the protection afforded against diabetes by this substance in the partially depancreatized rat.

Influence of altered states of carbohydrate metabolism on distribution and degradation of insulin-I131.

Summary 1. The distribution and breakdown of insulin-I131 in rats under various changes in carbohydrate metabolism has been studied. 2. An intravenous glucose load exerted no detectable effect other than to reduce renal radioactivity. 3. Intravenous fructose markedly reduced renal precipitable radioactivity, and increased hepatic and muscle radioactivity. 4. Intraperitoneal glucose or fructose has the same effects as intravenous fructose but to a lesser degree. 5. Alloxan diabetic animals concentrated more precipitable radioactivity in the liver and blood. 6. Phloridzin exerted no effect on insulin distribution and degradation. 7. Fasting effected little change; there was some decrease in renal and some increase in hepatic precipitable radioactivity. 8. The limitations and implications of the study are discussed.