Assay of Insulin Antagonism by Serial Incubation of Paired Rat Hemidiaphragms

Abstract

A method utilizing serial incubations of paired rat hemidiaphragms has been developed. In this technic, two basal glucose uptakes and two uptakes in the presence of insulin can be determined in a single rat, thus eliminating rat to rat variations when comparing two insulin effects. This system, particularly useful for the study of insulin antagonism, was applied to the assay of the albumin-associated antagonist in various human and commercial preparations. The presence of higher concentrations of this antagonist in untreated adult-onset diabetes was confirmed in two out of three diabetic patients tested. Albumin from a third diabetic patient was stimulatory. Varying amounts of antagonism were found in the commercial albumin preparations. Evidence is presented suggesting that total bound FFA do not play an important role in the albumin-associated insulin antagonism. DIABETES 15: 380-87, June, 1966. The use of the isolated rat diaphragm to study the effects of insulin was first introduced in 1940 by Gemmill. 1 Since then many workers have adapted this technic to the quantitative measurement of insulin in plasma and various tissues. It is generally agreed that whereas this method is a relatively sensitive one, it is not very precise because of the marked variation in the behavior of diaphragms from different rats. 2 ' 3 These difficulties are compounded if the rat diaphragm is utilized for the study of insulin antagonism since four bits of information (1. basal glucose uptake with buffer alone, 2. with just insulin added, 3. with just the antagonist added, and 4. in the presence of both insulin and antagonist) are necessary whereas in the simple assay system, only three bits of information (1. basal glucose uptake, 2. uptake in the presence of insulin, and 3. uptake in the presence of the unknown) are needed.One antagonist has been described by Vallance-Owen 4 who has shown that albumin in a concentration of 1.25

Insulin Dose-Response Characteristics Among Individual Muscle and Adipose Tissues Measured in the Rat In Vivo with 3(H)2-Deoxyglucose

“…The relative maximal response to insulin for isolated muscle preparations incubated in vitro ranges from two-to fivefold. 16 The overall response of the rat hindquarter reaches fivefold in normal animals of comparable age to our test animals, 18 while the muscle tissues of the human forearm perfused in vivo display an 8-to 11-fold relative insulin response to intra-arterial administration of insulin. 15 The maximal response for the three skeletal muscles examined in the present study ranged from 6.8-to 15-fold, while the maximal response by heart muscle was 10-fold over basal.…”

“…Insulin responsiveness in vitro may be influenced by the conditions of incubation, 16 the method of tissue collection and preparation, 17 and the absence of normal circulatory relationships. Further, if one wishes to study the effects of nutritional, environmental, neural, or endocrine influences on insulin sensitivity, the use of in vitro methods depends on the assumption that such influences are fully expressed in tissues after removal from the test animal.…”