Karl E. Sussman

Insulin Release After ACTH, Glucagon and Adenosine-3′-5′-Phosphate (Cyclic AMP) in the Perfused Isolated Rat Pancreas

An isolated rat pancreas preparation has been employed for the study of insulin Secretion. The preparation consists of the pancreas and the duodenum in which the thoracic aorta is cannulated for arterial inflow and the portal vein cannulated to collect the venous effluent. The administration of ACTH, glucagon and cyclic AMP will increase the secretion of insulin from the isolated perfused pancreas. Theophylline was infused into the pancreas with glucagon and compared with glucagon alone. Theophylline did not have any effect on altering insulin secretion from the pancreas nor did it potentiate the glucagon-induced insulin release. While glucagon, ACTH, and cyclic AMP all stimulate insulin secretion, there is no definitive evidence that the effect of glucagon or ACTH on the beta cell is due to intracellular generation of cyclic AMP.

Insulin and Glyburide Increase Cytosolic Free-Ca2+ Concentration in Isolated Rat Adipocytes

We investigated the effect of insulin and a hypoglycemic sulfonylurea agent glyburide on cytosolic free-Ca2+ concentrations ([Ca2+]i) in isolated ratadipocytes. Both insulin and glyburide increased [Ca2+]i in a dosedependent manner. Half-maximal effects were seen at 0.5 ng/ml of insulin and 0.5 μM glyburide. Nifedipine (25 μM), a Ca2+-channel blocker, inhibited the effectof both agents. The effect of insulin on [Ca2+]i was 40 and 70% potentiated by ambient glucose concentrations at 180 and 300 mg/dl, respectively. Depolarizingdoses of potassium (40 mM) induced an increase in cytosolic Ca2+ that was also inhibited by nifedipine. It is suggested that both insulin and glyburide increase cytosolic free Ca2+ levels at least in part by promoting Ca2+ influx through voltage-dependent Ca2+ channels.

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.

An in vitro method for studying insulin secretion in the perfused isolated rat pancreas

Abstract An isolated in vitro pancreas perfusion preparation is described which is suitable for study of insulin secretion. The surgery by which this tissue is removed from the animal is described as well as a chamber for perfusing the pancreas. The in vitro perfused pancreas demonstrates increased secretion of insulin with tolbutamide and glucose. In addition to d-glucose, d-fructose and d-mannose were demonstrated to stimulate the pancreas to secrete insulin, whereas 1-arabinose and d-galactose were without effect. The release of insulin after glucose, tolbutamide, mannose and fructose administration appears to occur immediately and probably involves the release of preformed insulin granules.

Effect of Diabetes on Cytosolic Free Ca2+ and Na+-K+-ATPase in Rat Aorta

We examined Na+-K+-ATPase activity and the levels of αI-, αII-, and β-subunit mRNA and protein in aortic cells of diabetic rats. Diabetes was induced by streptozocin. Na+-K+-ATPase activity was significantly reduced on the 2nd day of diabetes (9.4 ± 1.3 vs. 17.5 ± 2.1 μmol NADH · mg−1 protein · h−1, P < 0.05) and remained depressed on days 7 and 14. The levels of 5.3-kilobase (kb) mRNA band of the catalytic αII-subunit of Na+-K+-ATPase were also decreased on the 2nd day of diabetes, whereas the second band, 3.4 kb, was not affected. Both bands were significantly decreased on days 7 and 14. This was followed by a reduction in the levels of αII-protein (day 14). The levels of αI- and β-subunit mRNA and alpha I- protein were not affected by diabetes. A decrease in Na+-K+-ATPase activity was accompanied by a significant (P < 0.001) increase in the cytosolic free Ca2+ concentrations ([Ca2+]i) in diabetic aortic cells (221 α 18 nM on the 7th day and 242 ± 17 nM on the 14th day vs. 153 ± 7 nM in controls). These findings are consistent with the hypothesis that decreased Na+-K+-ATPase activity and gene expression in vascular smooth muscle cells with accompanied rises in [Ca2+]i may be an important pathogenetic factor in the development of hypertension and atherosclerosis in diabetes.

Differential Effects of Diabetes on Adipocyte and Liver Phosphotyrosine and Phosphoserine Phosphatase Activities

We examined the activities of particulate and cytosolic phosphotyrosine phosphatase (PTPase) and phosphoserine phosphatase (PSPase) in adipocytes and livers of diabetic rats. PTPase activity was assessed with [32P]tyrosine-phosphorylated insulin receptor (IR), whereas PSPase activity was assayed with [32P]serine-phosphorylated glycogen synthase. Diabetes increased adipocyte particulate PTPase activity and enhanced IR dephosphorylation by 75% on the 2nd, 93% on the 14th, and 108% on the 30th day. In contrast, cytosolic PTPase activity decreased by 78% on the 14th and 45% on the 30th day (no change on the 2nd day). Similar changes were observed with PSPase (increased activity in particulate and decreased in cytosolic). Insulin therapy for 14 or 30 days restored PTPase and PSPase activities in both fractions. Vanadate, despite rapid normalization of glycemia, restored these activities only after 30 days of therapy. Diabetes-related changes in liver PTPase activity were observed on the 14th day only. At this time, it was increased in both particulate and cytosolic fractions. There was spontaneous normalization of the liver PTPase activity at 30 days of diabetes. In contrast, liver cytosolic PSPase activity was significantly inhibited and not normalized by the 30th day of disease without therapy. In summary, diabetes appears to induce tissue-specific changes in PTPase and PSPase activities resulting in significant alterations in dephosphorylation of IR and glycogen synthase. Moreover, there appears to be a differential regulation of PTPase and PSPase activities in diabetes, particularly in the liver.

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.

Changes in glucose and insulin metabolism induced by dialysis in patients with chronic uremia

Abstract The effect of repeated hemodialysis on glucose metabolism was studied in 10 patients with severe uremia. Carbohydrate metabolism was evaluated during the first dialysis and again in a subsequent dialysis (second, third or fourth dialysis). Dialysis was performed using a high bath glucose concentration ( 1500 mg. 100 cc. ) in both study periods. Improvement in carbohydrate metabolism was demonstrated in the second dialysis study period by a lower mean glucose during dialysis and by more rapid disappearance rate of glucose in the immediate postdialysis period. This improvement in carbohydrate metabolism was associated with an increase in both mean and maximum insulin levels during the second study period in 8 of the 10 patients. It is concluded that the carbohydrate intolerance associated with uremia is improved by hemodialysis. In the majority of the patients studied, this improvement was associated with a significant increase in circulating insulin levels. The increase in insulin levels in response to hyperglycemic stimulation after only one or two dialyses suggests the removal of a dialyzable substance that either inhibits insulin release or increases insulin degradation.

Na+K+-ATPase activity and its αII subunit gene expression in rat skeletal muscle: Influence of diabetes, fasting, and refeeding ☆

We have examined the effects of diabetes, fasting, and refeeding on Na+/K(+)-adenosine triphosphatase (ATPase) activity and its catalytic alpha II subunit gene expression in skeletal muscle. Two hypoinsulinemic states, streptozotocin-induced diabetes and 48-hour fasting caused a significant decrease (P less than .05) in skeletal muscle Na+/K(+)-ATPase activity and a marked increase (P less than .01) in the levels of alpha II subunit mRNA. A decrease in enzyme activity was observed on the 2nd and the 14th day of diabetes, whereas an increase in alpha II mRNA levels was found only on the 14th day. The levels of alpha I mRNA were not affected, while the levels of mRNA of the structural beta subunit were decreased on the 14th day of diabetes. Correction of hyperglycemia with insulin restored enzyme activity and alpha II isoform mRNA levels toward normal in diabetic animals. Refeeding for 48 or 72 hours restored these parameters to normal in skeletal muscle of previously fasting rats. These observations suggest that a decrease in muscle Na+/K(+)-ATPase activity may lead to a compensatory increase in its alpha II subunit gene expression. The levels of insulin and not of glycemia appear to be critical in modulating Na+/K(+)-ATPase activity and gene expression.

Plasma Insulin Levels During Reactive Hypoglycemia

The plasma insulin response to an oral glucose load was studied in fourteen patients with reactive hypoglycemia—based on blood glucose values of 45 mg./100 ml. or below following 100 gm. of glucose. In reactive hypoglycemia, no single mechanism involving insulin secretion could explain the development of low blood glucose values. Six patients with reactive hypoglycemia demonstrated elevated plasma insulin values in association with normal blood glucose levels. In five patients the results of glucose tolerance tests suggested mild diabetes mellitus, and plasma insulin levels were increased. Three patients with reactive hypoglycemia had essentially normal glucose tolerance tests and no abnormality in plasma insulin response was noted.