Studies of Insulin Antagonism: I. An Artifactual Antagonist to Insulin and Plasma Nonsuppressible Insulin-like Activity Occurring in Preparation of “Albumin”

Abstract

Because ILA was encountered in TCA-ethanol extracts of plasma dialyzed in boiled Visking casing which interfered with measurement of the "synalbumin insulin antagonist" on isolated muscle, a systematic study of the influence of the casing on these factors was undertaken. Plasma from healthy humans was extracted with TCA-ethanol and dialyzed in nonboiled and boiled Visking casing. The extract ("albumin") was tested by measurement of glucose uptake in rat hemidiaphragms, without and with insulin (1,000 /iU./ml.).. Significant amounts of both ILA and anti-insulin activity were detected in both "albumins" at 3 to 5 gm. per cent; however, more ILA and less insulin inhibition was found in "albumin" dialyzed in boiled casing. The magnitude of ILA could not be accounted for by immunoreactive insulin levels in the "albumin," and the ILA was not suppressed by insulin-specific antiserum (NSILA).From dose-response curves measuring glucose uptake and glycogen synthesis, more NSILA was detected in the "albumin" from boiled casing; however, at 3 to 5 gm. per cent concentration, "albumin" dialyzed in either casing caused inhibition of NSILA. Because partially purified NSILA was found to be retained equally by either boiled or nonboiled casing, an antagonist in Yisking casing was sought. A substance from Visking casing with small molecular weight was found to cause insulin inhibition. When nonantagonistic crystalline albumin was dialyzed in nonboiled casing, marked antagonism to both crystalline insulin and partially purified NSILA was demonstrated.It is concluded that an artifactual casing antagonist to both NSILA and insulin is bound to albumin and may contribute to the measurement of the "synalbumin insulin antagonist.

The nature of human serum insulin-like activity (ILA): characterization of ILA in serum and serum fractions obtained by acid-ethanol extraction and adsorption chromatography

“…In contrast to the evidence of inhibition of ILA with the above fractions, the ILA in the alkaline eluate protein did not appear to be inhibited since it achieved the maximum effect observed with 800 ,AU of insulin. That this lack of inhibition is not the result of lesser amounts of protein in the alkaline eluate is indicated by the observations that purified albumin at concentrations of 50 mg/ml does not inhibit the effect of insulin in the hemidiaphragm bioassay (28). Fig.…”

“…Extraction was then performed as described for serum (28). After passage of serum, the protein retained on the resin was eluted with 2 volumes of 0.02 N NH4OH into an equivalent quantity of 0.2 N sulfuric acid under constant stirring.…”

“…The rat hemidiaphragm bioassay described by Vallance-Owen and Hurlock was modified as detailed previously (28). Glucose was measured in a Technicon AutoAnalyzer by the method of Hoffman (49) and the glucose uptake expressed as micrograms per milligram of dry weight during 90 min incubation.…”

“…In addition, the ILA in these fractions was not neutralized by insulin specific antiserum. The low amounts of ILA detected in Fraction II in Experiments 1 and 2, in which the protein concentrations were 40 mg/ml, may be due to the presence of an inhibitor associated with albumin as described previously (28). Thus, nonsuppressible insulin-like activity in serum, in acid-ethanol extracts of serum ("albumin"), and in its concentrate in the supernatant fraction was eluted from DEAE cellulose under identical conditions.…”

“…These extracts have been prepared by the hydrochloric acid-ethanol method for extraction of insulin (25) and the trichloroacetic acid-ethanol procedure for extraction of albumin (26). Because TSIA stimulated glycogen synthesis in muscle, it has been considered to represent insulin (22,27), however, the levels of TSIA ranging from 0.8-7 mU/ml were inconsistent with the values of immunoassayable insulin in these extracts (28).…”