P. H. Wright

Insulin Immunoassay by Back-Titration; Some Characteristics of the Technic and the Insulin Precipitant Action of Alcohol

Precipitation of insulin by alcohol and some aspects of the “back-titration” method of insulin immunoassay were investigated. In the absence of insulin antibodies, alcohol has a definite nonspecific precipitant action on labeled insulin, an action which is most marked when alcohol is added in high concentration or when the medium contains much protein. This nonspecific precipitation of radio-iodinated insulin is not affected by the presence of high concentrations of unlabeled insulin; this is not explained but has no effect upon the assay system. The “back-titration” method of insulin assay is more sensitive and reproducible than the “competitive” technic when the latter is carried out under similar brief conditions of incubation. The “back-titration” method is shown to be specific for insulin, proinsulin being the only other hormonal substance tested and found to interfere with the assay system. The assay is not affected by small changes in pH about neutrality, can be carried out with pooled anti-porcine or anti-bovine insulin serum, and is not species-specific. Results obtained with human plasma are comparable with those found by the method of Morgan and Lazarow. Expected amounts of insulin are recovered over a wide range of plasma dilutions. For routine purposes, illustrated in a series of glucose tolerance tests, the “backtitration” method has proved practical, rapid, and reproducible.

Insulin Secretion in vitro by Pancreatic Tissue from Normal, Adrenalectomized, and Cortisol-Treated Rats.

Summary Insulin secretion provoked by glucose in the pancreas of the rat is not modified by addition of methylprednisolone to the incubation medium. Secretion by pancreatic tissue of the normal rat is reduced (to 60%) by prior adrenalectomy and increased (by 50%) after treatment with cortisol for 2 or 5 days. Treatment with cortisol for 3 days prevents the effect of adrenalectomy. These alterations are not associated with significant changes in the insulin content of the pancreas, and suggest that gluco-corticoids increase the sensitivity of the insulin secretory mechanism of the beta cells to glucose either directly or indirectly in a chronic process.

Insulin Immunoassay by Back-titration Using Alcohol Precipitation of Insulin-Antibody Complexes

A method is described in detail for the assay of insulin in plasma or serum. It involves titration with labeled insulin of reactive insulin antibodies remaining after preincubation of the sample under assay or of standard solutions of insulin with an excess of guinea pig anti-insulin serum. Labeled insulin bound by antibodies in the assay is precipitated in the presence of alcohol (75 per cent, v/v) at room temperature, unbound labeled insulin then remaining in solution. The technic is simple and assays can be carried out in a few hours. It is shown that individual results are amenable to statistical assessment and suggested that the sensitivity of the method is only limited by the specific activity of the labeled insulin used.

Control of gluconeogenesis by acetyl CoA in rats treated with glucagon and anti-insulin serum

Abstract Glucagon has recently been shown to have a direct lipolytic action on liver (Bewsher and Ashmore 1966). Increased hepatic lipolysis, plus enhanced availability of plasma fatty acids, causes an elevation of acetyl CoA and fatty acyl CoA levels (Williamson, Herczeg, Coles and Danish 1966). Such elevated levels of acetyl CoA presumably account for the increased rate of ketogenesis observed with glucagon. Acute effects of insulin deficiency, induced by administration of guinea pig anti-insulin serum (AIS) to rats, are similar to effects produced by glucagon: elevation of plasma glucose, free fatty acid, and ketone levels, plus an increased rate of gluconeogenesis (Wagle and Ashmore, 1963 ; Wagle and Ashmore, 1964 ; Tarrant, Mahler, and Ashmore, 1964) . These findings have led to the suggestion that the early metabolic changes in experimental insulin deficiency may be induced by hormones with actions normally counterbalanced by insulin (Wright, 1965) . Such hormones are glucagon, epinephrine, ACTH and corticosteroids (Mahler, Stafford, Tarrant and Ashmore, 1964 ; Jungas and Ball, 1963) . Data presented in this paper supports the concept that the lipolytic, ketogenic, and gluconeogenic effects of glucagon are revealed in the insulin-deficient rat. It is suggested that stimulation of gluconeogenesis, both with glucagon and with AIS, is secondary to enhanced lipolysis and is mediated through facilitation of pyruvic carboxylase by elevated hepatic levels of acetyl CoA.

Inhibition by Halothane of Glucose-stimulated Insulin Secretion in Isolated Pieces of Rat Pancreas

In isolated pieces of rat pancreas addition of 300 mg glucose/100 ml bathing medium resulted in a marked and significant increase in insulin secretion. This glucose-stimulated insulin secretion is reversibly inhibited by halothane in a concentration-dependent fashion (0.63, 1.25, and 1.88 MAC halothane produced depressions of 8.4, 18.9, and 37.0 per cent, respectively). Halothane, 1.88 MAC, had no effect on the basal or nonglucose-mediated insulin release.

Guinea Pig Anti-Insulin Serum: Adjuvant Effect of H. Pertussis Vaccine

After three injections at weekly intervals of insulin in a water-in-oil emulsion containing H. pertussis vaccine, guinea pigs were given the same inoculum containing no vaccine at intervals of four weeks. At the first bleeding after eight weeks, most animals yielded serum binding more than 1.0 U. insulin per milliliter; the mean binding capacities of sera from animals in four out of seven groups exceeded 4.5 U. insulin per milliliter. Over eighteen months, five groups totalling 136 animals yielded 3,000 ml. serum, the pooled sera from each group binding 2.7 to 4.8 U. insulin per milliliter. It is concluded that use of H. pertussis vaccine offers a reliable method for the production of large volumes of potent anti-insulin serum.

Hepatic effect of sulfonylureas

Abstract The sulfonylureas, tolbutamide, tolazamide and acetohexamide significantly diminish uptake of insulin by the isolated perfused rat liver, whereas the nonhypoglycemic metabolite of tolbutamide, carboxytolbutamide, has no such effect. These results suggest that the sustained action of the sulfonylureas may in part be due to reduced hepatic uptake of endogenously secreted insulin.

A Method for the Immunoassay of Insulin

A method is described for the assay of insulin. It involves pre-incubation of insulin under assay with an excess of guinea pig anti-insulin serum and subsequent incubation of the partially neutralized serum with an excess of unlabeled and/or I-131-labeled bovine insulin. If no more than about half of the antibodies in the serum are neutralized during pre-incubation, then the amount of bovine insulin bound during incubation decreases linearly and in proportion to the amount of pre-incubated insulin. Such a linear relationship was obtained with nine samples of pooled serum from uve groups of guinea pigs and was shown to be applicable over a wide range of serum and insulin concentrations. Since neutralized acid-alcohol affected the assay only when present in relatively high concentrations, the method has been applied to neutralized acid-alcohol extracts of pancreatic tissue without removal or excessive dilution of the alcohol. It is applicable at concentrations of insulin to be found in blood and is therefore a potentially simple and rapid method which would not require the use of labeled insulin of high specific activity. The uses and limitations of the method are discussed.

Studies on glucagon secretion using isolated islets of langerhans of the rat

SummaryGlucagon secretion and its control have been studied in perifused isolated islets of Langerhans of the rat. It was shown that a low concentration of glucose per se does not cause increased glucagon secretion, but that at low glucose concentrations the amino acid arginine stimulates a biphasic secretory response. Such amino acid stimulated glucagon secretion can be suppressed by increasing the glucose content of the perifused media from 1.67 to 5.5 or 16.7 mM; insulin secretion is also then increased. Since high concentrations of added porcine insulin (10 mU/ml) did not affect amino acid stimulated glucagon secretion at low glucose concentration, it was concluded that high concentrations of glucose and not insulin secreted in response to that glucose are probably responsible for suppression of glucagon secretion. At low concentrations of glucose, epinephrine (2.5 × 10−7 M) also stimulated glucagon secretion. It is concluded that isolated rat islets of Langerhans can be used for the study of glucagon secretion in vitro, and that substances appearing in the blood in vivo at low glucose concentrations are probably responsible for increased glucagon secretion under conditions associated with hypoglycemia.

Effects of halothane on glucose-stimulated insulin secretion and glucose oxidation in isolated rat pancreatic islets.

Previous studies have shown that halothane inhibits glucose-stimulated insulin secretion. This study was designed to determine whether the mechanism of inhibition involves a reduction in glucose metabolism. The effects of halothane on glucose (16.7 mM)-stimulated insulin secretion and glucose oxidation were studied in isolated rat pancreatic islets. Halothane, 0.11 mM (0.5 MAC), 0.22 mM (1.0 MAC), and 0.33 mM (1.5 MAC), inhibited glucose-stimulated insulin release in a dose-related manner by 5.2 per cent (NS), 21.0 per cent (P < 0.05), and 32.6 per cent (P < 0.01), respectively. At the 0.33 mM (1.5 MAC) concentration, halothane did not significantly inhibit the oxidation of 6-14C-glucose to 14CO2, although higher concentrations of halothane did result in significant inhibition. The data suggest that halothanes inhibitory effect on glucose-stimulated insulin secretion is not due to interference with glucose oxidation.