"Big insulin": a new component of plasma insulin detected by immunoassay.

Abstract

Since the advent of radioimmunoassay, I the concentration of insulin in plasma of normal and diabetic subjects in response to many stimuli has been studied extensively.2 However, the chemical nature of the circulating hormone is not entirely clear. We find that endogenous plasma insulin has two components.One is very similar to pancreatic insulin. The other component is larger in size and immunologically less reactive than pancreatic insulin. With our antiserum, it accounts for up to 50 per cent of plasma insulin measured by immunoassay.Materials and Methods.-Purified crystalline porcine and bovine insulin were gifts of Eli Lilly. Highly purified A-chain and B-chain sulfonates of bovine insulin were gifts of Dr. Arnold Marglin.' Cytochrome c, pancreatic ribonuclease, and porcine adrenocorticotropin (ACTH) were purchased from Mann, crystalline human serum albumin from Armour, rabbit fraction II and normal guinea pig serum from Pentex, guinea pig fraction II from Hyland, and NaI'25 and NaI"11 from Union Carbide. Guinea pig antiporcine insulin serum was a gift of Drs. A. Kagan and S. M. Glick. Antibody to guinea pig gamma globulin was generated in sheep by repeated injection of guinea pig fraction II in complete Freund's adjuvant (Difco). Albumin and ACTH were labeled with 112 and insulin with I'll by the chloramine T method at specific activities of 5, 5, and 450 uc/igg, respectively.4 The albumin-I125 was freed of radioactive contaminants by batch adsorption with Dowex 1X10. The ACTH-I'25 was purified by adsorption to silica and elution with acetic acid and acetone.' The insulin-I131, prepared fresh for each assay, was purified on a cellulose column.1 The normal subjects for the experiments were healthy male volunteers in their early twenties without a family history of diabetes. For the standard oral glucose tolerance tests 100 gm of glucose was administered in the morning after an overnight fast; for 3 days prior to the studies the subjects received a normal diet that included 300 gm of carbohydrate daily. For the starvation tests, food was withheld until the urine reacted strongly positive for acetone; the fast, about 36 hr in duration, was terminated by the administration of glucose, 100 gm orally. Patient MIT is an obese adult female with long-standing glucose intolerance, delayed hyperinsulinism following oral glucose, and retinal microaneurysms typical of diabetes. Plasma that was essentially free of insulin was obtained after a 72-hr fast from a patient who had had an islet-cell adenoma removed several years earlier. None of the subjects had ever been treated with insulin.Blood was drawn into heparin, refrigerated, and centrifuged, and the plasma stored at -20°. Plasma, 1-3 ml, was enriched with albumin-I125 and iodidel25 and applied to a 1 X 50-cm column of Sephadex G-50 fine (Pharmacia). Shorter columns or coarser grades of gel were unsatisfactory. Since the results of radioimmunoassay can be affected by variations in the salt content of the samples,' the column was equilibrated and developed in the same...

Studies on the biological activity of porcine proinsulin

“…Clark and Steiner in a prior publication reported such an in vitro action for glucose (8). A "big insulin" in human and animal plasmas has been described (9). Though not conclusively proven, it seems likely that this material is indeed proinsulin.…”

“…Though not conclusively proven, it seems likely that this material is indeed proinsulin. Efforts to specifically identify and measure proinsulin in the plasma have been Received for publication 8 September 1969 and in revised form 16 October 1969. hampered by technical problems such as lack of human proinsulin standards and the likelihood that the antigenic "connecting peptide" of proinsulin is also in the circulation and must be differentiated from intact proinsulin (8)(9)(10).…”

Comparison of impedance to insulin-mediated glucose uptake in normal subjects and in subjects with latent diabetes

“…This subject was critically reviewed by Berson and Yalow in 1966 (14), and they concluded that there was very little to support these alternative views. However, since 1966 two laboratories (15,16) have published evidence for substances of greater molecular weight than insulin, which react with anti-insulin antibody, but which appear to have a lessened biological activity. In addition, several groups of workers (17)(18)(19) (17,18) or the fasting insulin level (19).…”

The Metabolism of Proinsulin and Insulin by the Liver

“…Proinsulin, the single-chain polypeptide precursor of insulin, has been identified in human (1)(2)(3)(4)(5)(6), bovine (7,8), transaminase was assayed (18) in the initial and final perfusate sample from each experiment. The values were below 10 IU and did not increase significantly in the latter samples.…”