Donald B. Martin

Glucagon Secretion from the Perfused Pancreas of Streptozotocin-treated Rats

One hour following intravenous streptozotocin, rat pancreases were perfused in situ, and , in contrast to saline-injected controls a marked decrease of insulin secretion was observed. In these streptozotocin-treated animals, baseline glucagon secretion was enhanced when the perfusate glucose concentration was either 80 mg./100 ml. or 300 mg./100 ml. In addition there was hypersecretion of glucagon in response to arginine. Exogenous insulin (20,000 μU./ml.) could suppress glucagon secretion when endogenous secretion was plentiful. Baseline and arginine-stimulated glucagon secretion of the streptozotocin treated animals was not suppressed by large amounts of glucose and insulin to the degree seen in control animals. The glucagon rise in response to an abrupt fall of glucose from 80 mg./100 ml. to 25 mg./100 ml. was not significantly higher in the control group than in the streptozotocin group. The results seen with epinephrine were in sharp contrast to those found with arginine. Epinephrine-stimulated glucagon secretion was not enhanced in the streptozotocin group. In addition, epinephrine-induced secretion could be suppressed by exogenous insulin in both the control and streptozotocin groups. The differences may be secondary to differences of endogenous insulin secretion. The present results are compatible with the hypothesis that local insulin secretion can exert a significant suppressive effect upon the alpha cell and that the inhibition of glucagon secretion by glucose is partially mediated by this mechanism. Furthermore, anomalous local insulin secretion may contribute to the abnormal glucagon secretion of diabetes mellitus.

Glucagon Secretion from the Perfused Rat Pancreas STUDIES WITH GLUCOSE AND CATECHOLAMINES

The isolated in situ perfused rat pancreas was used to study glucose and catecholamine control of glucagon secretion, and to investigate the possible role of endogenous cyclic AMP as a mediator of this secretory process. When perfusate glucose was acutely dropped from 100 to 25 mg/100 ml, glucagon was released in a biphasic pattern with an early spike and a later plateau-like response. 300 mg/100 ml glucose suppressed glucagon secretion to near the detection limit of the radioimmunoassay (15 pg/ml). When perfusate glucose was dropped from 300 to 25 mg/100 ml, a delayed, relatively small peak occurred suggesting persisting alpha cell suppression by prior high glucose exposure. 2-Deoxy d-glucose stimulated glucagon secretion and inhibited insulin secretion. Glucagon was secreted in a biphasic pattern in response to both 2.7 x 10(-7) M epinephrine and norepinephrine. The glucagon response to epinephrine was markedly suppressed by glucose at 300 mg/100 ml, and the biphasic response pattern was obliterated. Glucose evoked a two-phase insulin secretory pattern, and the second phase was markedly and rapidly inhibited by epinephrine. Pancreases were perfused with glucose at 300 mg/100 ml which was then lowered to 80 mg/100 ml. 5 min later, epinephrine was infused and definite blunting of the first-phase spike occurred. 10 mM theophylline produced modest rapid uniphasic stimulation of glucagon release, and, in addition, caused enhancement of epinephrine-stimulated glucagon release. An inhibitory influence upon epinephrine-stimulated glucagon was observed as well. Insulin secretion was stimulated by 10 mM theophylline, and this stimulation was inhibited by epinephrine.

MEASUREMENT OF SMALL QUANTITIES OF INSULIN-LIKE ACTIVITY USING RAT ADIPOSE TISSUE. II. EVALUATION OF PERFORMANCE*

In the preceding paper (1), a biological assay method was proposed which utilized the increased production of CO2 from glucose in response to minute quantities of insulin by epididymal adipose tissue of rats. Since the quality of an assay method is best revealed by its performance over a period of time, the present report will present the results obtained during 25 months when this assay procedure was applied to insulin solutions and was tested on more complex material such as human blood serum. The quantitative evaluation of the data was carried out to a large extent independently from the actual performance of the assays, although consultations between the statistical and the biological teams occurred at irregular intervals of a few months.

The effect of sulfhydryl blockade on insulin action and glucose transport in isolated adipose tissue cells

Abstract The effects of sulfhydryl blockade on glucose metabolism have been investigated using isolated adipose tissue cells from the rat and the specific sulfhydryl binding agent, N -ethylmaleimide. According to the conditions of the experiment, there can be (1) an insulin-like effect, (2) inhibition of insulin-stimulated glucose metabolism, and (3) inhibition of basal glucose metabolism. At least the first two effects appear to occur at the level of membrane transport.

Glucose transport by trypsin-treated red blood cell ghosts

Abstract 1. Glucose transport has been studied in preparations of human erythrocyte ghosts which have been treated with trypsin (10μg/ml for 30 min). [ 3 H] d -Glucose uptake by this preparation has been measured using a Millipore filter technique, with [ 14 C] l -glucose serving as a marker for non-specific glucose uptake. Membrane vesicles prepared from erythrocyte ghosts did not preferentially take up d -glucose without prior treatment with trypsin. 2. This trypsin-treated erythrocyte ghost preparation demonstrates many of the characteristics of glucose transport: (1) the d -isomer is both taken up and released more rapidly than the l -isomer; (2) countertransport of d -glucose can be demonstrated; (3) competitive inhibition of d -glucose uptake is observed with certain sugars; (4) known inhibitors of glucose uptake, phlorizin and N-ethylmaleimide , inhibit d -glucose uptake. 3. Using very low concentrations of trypsin (1 μg/ml), the onset of glucose transport has been correlated with the pattern of cleavage of membrane peptides (assessed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate). The appearance of glucose transport is temporally associated with the hydrolysis of two major membrane peptides. Specific d -glucose uptake is not inhibited by extensive proteolysis (trypsin, 100 μg/ml) or selective elution of specific peptides (by manipulation of ionic strength).

Effect of low levels of trypsin on erythrocyte membranes

Abstract The effects of trypsin at concentrations of 1 μg/ml on red blood cell membranes (ghosts) have been investigated. Tryptic digestion induced a gross morphologic change as observed by phase microscopy with the formation of small close vesicles by a process of exocytosis. These vesicles were more tightly sealed (less leaky) to hexoses than the parent ghosts. Coincident with vesiculation there occurred a decrease in particle density from 1.06 to 1.01 as determined by equilibrium density gradient centrifugation in Dextran. Since the equilibrium density in sucrose gradients was much less affected, the creation of new charge groups on the internal membrane surface was suggested. Continued accessibility of all sialic acid residues to attack by neuraminidase after tryptic digestion indicated that the exocytotic vesicles were in fact “right-side-out”. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed that two of the major membrane peptides (mol. wt of 89000 and 77500) were selectively attacked. Under these conditions, proteolysis did not cause the release of protein from the membrane. We suggest that the vesiculation and alteration in membrane permeability seen in association with the cleavage of these two peptides indicates that one or both are critical to the maintenance of erythrocyte membrane structure, probably through their association with lipid.

Insulin-Stimulated Glucose Uptake by Subcellular Particles from Adipose Tissue Cells

Microsomal particles prepared from isolated rat adipose cells take up D-glucose more rapidly than L-glucose. The rate of D-glucose uptake, but not that of L-glucose, is stimulated by incubation of the intact cells with insulin at concentrations as low as 10 microunits per milliliter before disruption and preparation of the microsomes.

Nesidioblastosis Associated with Insulin-mediated Hypoglycemia in an Adult

Nesidioblastosis, the process of differentiation of pancreatic islets from ductular epithelium, is a well-described cause of insulin-mediated hypoglycemia in neonates and infants, but not in adults. A 58-yr-old woman with characteristic clinical features of fasting hypoglycemia had inappropriately elevated plasma immunoreactive insulin levels during symptomatic episodes of fasting hypoglycemia. Angiography, palpation at laparotomy, and resection of the distal three-quarters of the pancreas provided no evidence of a tumor. Pathologic examination of the resected pancreas revealed the findings of nesidioblastosis, i.e., budding of islets from the wall of ductules, and also increased number and size of islets and abnormal shape and location of islets. An entire spectrum of islet cell abnormalities including nesidioblastosis can cause insulin-mediated hypoglycemia in adults, as it does in neonates and infants.

Insulin-stimulated plasma membranes from rat adipocytes: their physiological and physicochemical properties

Abstract Using methods previously described, we have isolated plasma membranes from insulin-treated adipocytes of the rat epididymal fat pad. Such membranes showed an accelerated uptake and release of d -glucose when compared with similar preparations from cells not exposed to the hormone. The only change observed was in the rate of d -glucose uptake; at equilibrium, d -glucose space was identical in both preparations. Vigorous alkaline hydrolysis of the insulin resulted in loss of its typical effect on both intact cells and derived plasma membranes. Anti-insulin antiserum, potent enough to inhibit the effect of the hormone when both were added to intact cells, did not prevent the insulin effect when added to plasma membranes prepared from insulin-treated cells. Addition of insulin directly to plasma membranes was without effect; exposure of cells prior to rupture was required. Studies of infrared spectra, native membrane protein fluorescence, and fluorescence of 8-anilino-1-naphthalene sulfonate added to the membranes, showed no differences between control plasma membranes and those prepared from insulin-treated cells. We conclude that: (1) plasma membranes can be prepared from insulin-treated fat cells which retain an enhanced glucose transport; (2) the effect of insulin on glucose transport does not involve large scale changes in the structure of the plasma membrane; (3) the insulin unresponsiveness of isolated plasma membranes, as well as the resistance to anti-insulin serum of membranes prepared from insulin-treated cells, appears to result from an uncoupling of insulin binding from glucose transport, the basis of which requires further exploration.

Glucagon Secretion From the Perfused Rat Pancreas Following Acute and Chronic Streptozotocin

C O N T R O V E R S Y over the mechanism of the anomalous glucagon secretion of diabetes as a pr imary or secondary alpha cell defect 1 prompted studies in the perfused rat pancreas which support the concept that local insulin secretion within the islet exerts an important suppressive effect, 2 and demonstrates enhanced glucagon secretion following acute t reatment with streptozotocin. Pagliara et al. performed a similar study but used rats with chronic streptozotocin and alloxan diabetes, and found reduced glucagon secretion. 3 The present study is an effort to clarify the differences between our studies in acutely diabetic rats and those in the chronically diabetic rats of Pagliara et al.