Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum.

Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000-400,000 with a peak at about MW 200,000. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000-600,000 (77% of the fraction NSILA or 28% of total serum NSILA). Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000-200,000 and 35,000-100,000 fractions and corresponded to 37% of total serum NSILA. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000-100,000. Low MW NSILA (less than 15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000-600,000. This complex was not bound by Con-A Sepharose.

New powerful insulin-like protein from human promyelocytic leukemia cells.

Potent insulin-like activity was found in the conditioned medium of human promyelocytic leukemia (HL-60) cells. The conditioned medium of HL-60 cells at high density stimulated [3H]glucose incorporation into lipids in rat adipocytes in a time- and dose-dependent manner. The dose-response curve for this factor was not parallel to that for insulin, and the maximal effect achieved was much greater than reached by insulin or multiplication-stimulating activity. Moreover, the maximal effect reached by either insulin or the conditioned medium was additive. The insulin-like activity was not suppressed in the presence of antiinsulin antibody. Insulin-like activity was not detectable by radioreceptor assay for insulin, suggesting that the factor does not act through the insulin receptor. The factor in the conditioned medium of HL-60 cells was heat stable and sensitive to trypsin. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, the major part of insulin-like activity eluted in the position corresponding to an apparent molecular weight between RNAase and insulin markers. The remaining activity, approximately 10% of the total, appeared with a larger molecular weight species. On isoelectric focusing of the smaller molecular species, insulin-like activity was largely focused in the position corresponding to pI 7.8-8.2.

Evidence for a biologically active, carrier-bound form of non-suppressible insulin-like activity (NSILA) in human serum.

1. Quantitative gel filtration of Cohn fraction IV-1 has shown that high MW NSILA recovered under "neutral" conditions (pH 5.5) was greater in magnitude (approximately X 2) than acid-stable high MW NSILA obtained under acid conditions. 2. Recombination, at neutral poH, of high and low MW NSILA, obtained by previous acid gel filtration, gave quantitative recovery of activity which was solely of high MW. 3. These data indicate that a biologically active, and dissociable high MW form of NSILA exists in serum. 4. Con A-Sepharose affinity chromatography gave one unbound NSILA fraction and two bound fractions, which both gave high and low MW activity after acid chromatography. 5. This study provides the first evidence for a carrier-bound form of low MW NSILA which retains biological activity.

Responses of rat and chick chondrocytes and rate hepatoma cells to plasma fractions with insulin-like and growth-promoting activities.

Plasma protein Cohn fraction IV-1 was extracted and fractionated according to the method of Van Wyk et al.[1], with the omission of the Sephadex G-75 step. The fractionated polypeptides were investigated for insulin-like activity (lipid synthesis and lactate production) and growth-stimulatory activity (DNA synthesis) in three bioassay systems: rat hepatoma cells, and rat and chick chondrocyte suspensions. Binding of each fraction to insulin receptors in isolated plasma membranes was also determined. Three peptide subfractions, with molecular weights ranging from 400 to 20 000, stimulated lipid synthesis, lactate production and DNA synthesis in all three bioassay systems. There were distinct species- and cell-type-specific variations in the quantitative patterns produced by each subfraction. In contrast, authentic insulin (0.005-0.1 ng/ml) had no effect on DNA and lipid synthesis anad lactate production in hepatoma cells and chondrocytes. One of the three active subfractions and two relatively inactive fractions displaced insulin from its receptors. These observations indicate that acid-ethanol extracts of plasma contain a variety of peptide factors with insulin-like and growth-promoting effects whose expression in different cell types is modulated by inherent properties of each cell type which are determined genetically presumably. Surface receptors for authentic insulin do not appear to play an essential role in the insulin-like activities of the isolated plasma fractions.

Partial purification of low molecular weight non-suppressible insulin-like activity from human plasma: demonstration of the presence of multiple forms.

An alternative procedure for the separation of insulin-like growth factors from plasma, avoiding the harsh conditions of acid-ethanol extraction, suggested that several factors which have activity in the isolated rat adipocyte assay may be present. The initial step consisted of ion-exchange chromatography of an enriched Cohn fraction (IV-1) on SP-Sephadix using ammonium acetate buffers. The major activity appeared in a fraction eluted at pH 7.1-9.4. When this fraction was subjected to gel filtration of Sephadix G-75 in 1% formic acid, most of the activity was recovered in a fraction of molecular weight of 5000-8000, but up to 25% of the activity applied remained in a much higher molecular weight form. The active material of low molecular weight was recovered at a considerably greater yield compared with that obtained by the more conventional acid-ethanol extraction followed by gel filtration of Sephadix G-75. The fraction was further purified by DEAE-Sephadix chromatography and isoelectric focussing. Non-suppressible insulin-like activity (NSILA) of pI 4.8 and 6.0 were the major species detected. The results were in contrast with those obtained using acid-ethanol extraction of the Cohn fraction, where the major species were of pI 6.0 and 8.0. The presence of multiple forms of NSILA would thus appear to justify the use of alternative methods of purification.

The occurrence of a distinct high molecular weight form of serum non-suppressible insulin-like activity.

Gel filtration of acromegalic or normal serum at acid pH gave two distinct species of non-suppressible insulin-like activity (NSILA), one of high MW and the other of low MW (approximately 7000 daltons). The acid-stable high MW form remained high MW on rechromatography in acid. Gel filtration of serum at neutral pH however, gave only high MW activity, which remained high MW when rechromatographed under neutral conditions but split into both high and low MW forms when rechromatographed in acid. These results indicate that there are at least two circulating forms of NSILA--a low MW form which circulates in serum bound to a carrier protein in an acid-labile high MW complex and a species which circulates only as a stable, discrete high MW protein.

Purification of a basic somatomedin, from human plasma Cohn fraction IV-1, with physicochemical and radioimmunoassay similarity to somatomedin-C and insulin-like growth factor.

A basic somatomedin (SM) was purified from human plasma Cohn fraction IV-1 using an initial acid--ethanol--acetone extraction procedure followed by alternating molecular size or charge protein separation techniques. The final recovery of SM bioactivity was approximately 2% of that present in the starting Cohn fraction. The purified SM has an approximate molecular weight of 7500, pI 8.6, 4000 SM bioactivity units per milligram (as measured by a hypophysectomized rat bioassay) and a parallel approximately equipotent radioimmunoassay dose--response curve to SM-C and insulin-like growth factor-1 (IGF-I). Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of this purified SM revealed a single protein band. The preliminary determination of the amino acid sequence of the N terminus suggested that this SM preparation was over 75% pure and the first five N-terminal amino acids were identical with those of IGF-I.