Showing papers on "Insulin published in 1972"

Insulin: The Structure in the Crystal and its Reflection in Chemistry and Biology by

Abstract: Publisher Summary This chapter reviews the physical, chemical, and biological properties of insulin in the light of the atomic arrangement found in insulin crystals. It also describes the relation of the three-dimensional arrangement of the atoms in the molecule of 2-zinc insulin crystal to the solution properties of insulin (particularly its states of aggregation), to the chemical reaction and chemical modification of the molecule, and to its primary biological activity. Normally the insulin crystals contain two zinc ions to every six molecules of insulin—a hexamer. The slow solution of the crystals provides a method of delaying the action of insulin that closely parallels the methods adopted in the pancreas itself for the storage and release of insulin. Within many β granules, grains can be seen that almost certainly contain zinc insulin hexamers packed in a crystalline array, and in experimental animals diabetes has been induced by chelating agents, such as EDTA, perhaps simply by interfering with normal insulin storage. It, therefore, seems plausible that ready crystallization of insulin in the presence of zinc is a reflection of the storage processes in the β cell.

Determination of total serum insulin (IRI) in insulin-treated diabetic patients

Abstract: A routine method is described for the determination of total IRI (imraunoreactive insulin) in insulintreated diabetics. The method involves an easy acid ethanol extraction, whereby antibody-bound IRI is dissociated and separated, together with the “free” IRI from the serum proteins and the antibodies. The recovery of IRI in this procedure is about 80%. After the separation, the isolated total IRI is measured in an immunoassay, using ethanol for the separation of free and antibody bound125I-insulin. In 169 diabetic patients treated with insulin in doses of from 6 to 120 units/day, the fasting serum total IRI was between 6 and 4374 μU/ml, with a mean of 392 μU/ml. During treatment with insulin, the level of total IRI increased from normal values, registered during the first two months, to a higher level which became stable after about 5 months of treatment. The increase in IRI occurred simultaneously with the formation of antibodies. Insulin-resistant patients showed very high IRI levels.

A threshold distribution hypothesis for packet storage of insulin and its mathematical modeling

Abstract: Phases of insulin release were studied in the perfused pancreas during a variety of glucose stimulation patterns. Patterns included staircase stimulations, constant prolonged single steps, restimulations, and ramp functions. Except at low concentrations, prolonged single steps of glucose elicited early spikes of insulin and a slowly rising second phase. Total insulin in the initial spikes increased with higher glucose concentrations. However, the time-related pattern of these spikes was similar in all cases; ratios of initial secretion rate to total insulin released were constant. Total insulin released in this early phase approximated a sigmoidal function of glucose concentration; mathematical differentiation of this function gave a skewed bell-shaped distribution curve. Staircase stimulations caused insulin to be released as a series of transient spikes which did not correlate with the increment of glucose but rather to the available insulin for a given glucose concentration minus that released in previous steps. The sum of total insulin released as spikes in a staircase series leading to a given glucose concentration was the same as when that concentration was used as a single step. Interrupted prolonged glucose infusions indicated the second phase of insulin release could prime the pancreas and that the first and second phases were interrelated. When glucose was perfused as ramp functions of slow, increasing, concentration, phasic response disappeared.A previous two-compartmental model was expanded to include a threshold or sensitivity distribution hypothesis. This hypothesis proposes that labile insulin is not stored in a homogeneous form but as packets with a bell-shaped distribution of thresholds to glucose. These packets respond quickly when their threshold levels to glucose are reached or exceeded. Data from single step stimulations were utilized for constructing a mathematical model which simulated satisfactorily the various stimulation patterns.

Insulin and Obesity in the Zucker Genetically Obese Rat “Fatty”

Abstract: Insulin status and functional activity have been studied during the life of the Zucker “fatty” rat (an obese mutant). Serum immunoreactive insulin (IRI) is normal at 2 weeks when fattening is beginning, rises to a peak of 400 μ-U/ml at 15 weeks while fattening proceeds rapidly, then drops to near 200 μU/ml in older rats. This was observed with two normal diets and a high protein diet. When fattening is reversed by fasting adult “fatties,” IRI remains abnormally high until body lipid has returned to normal (79 days). Blood sugar, basally and after a glucose load, is normal in young and old “fatties,” and is barely above normal at peak IRI. Sugar falls to below 50 mg/100 ml with prolonged fasting. Amino nitrogen tends to be slightly low, free fatty acids (FFA) are very high, in “fatties.” There is a developing resistance of diaphragm to glucose uptake in vivo, both basally and after injected insulin. The role of elevated FFA in this resistance to glucose uptake by muscle is not known. Basal glucose uptake i...

Isolation of the Insulin Receptor of Liver and Fat-Cell Membranes

Abstract: Extraction of liver and fat-cell membranes with the nonionic detergent Triton X-100 prevents specific binding of 125I-labeled insulin to these membranes. This loss of binding to particulate material is quantitatively recovered in a high-speed (300,000 × g, 2 hr) supernatant of the extract. Specific and reversible insulin binding to soluble proteins is readily demonstrable by gel filtration. A simple and sensitive assay for detection of specific macromolecule-insulin complexes has been developed based on the selective precipitation of the complex by polyethylene glycol. Extraction of membrane lipids with organic solvents or by phospholipase digestion does not impair the subsequent extraction of the insulin-binding protein with detergent. Binding of insulin to the soluble protein is a saturable and dissociable process having a dissociation constant of about 100 nM. Derivatives of insulin compete for binding in direct proportion to their biological activity; other peptide hormones are without effect. The quantitative features of the detergent extractions and the specific insulin-binding properties of the material so obtained indicate that the protein solubilized is the biologically significant insulin receptor, whose insulin-binding function is essentially unaltered.

Pancreatic Beta-Cell Web: Its Possible Role in Insulin Secretion

Abstract: A cortical band of fine microfilaments is consistently observed in the beta cells of the rat pancreas. Alteration of this cell web by cytochalasin B is associated with an enhancement of glucose-induced secretion of insulin by isolated islets. The microfilamentous web of the beta cell may play an important role in the emiocytosis of insulin secretory granules, by controlling their access to the cell membrane.

Perifusion of Isolated Rat Islets in Vitro: Participation of the Microtubular System in the Biphasic Release of Insulin

Abstract: A simple perifusion system for in vitro studies on the rate of insulin secretion of isolated rat islets is described. A biphasic pattern of insulin secretion was produced by glucose whereas tolbutamide stimulated only the first phase of insulin secretion. The perifusion system was used to determine the effect of anti-mitotic agents on the biphasic pattern of insulin secretion. Yinblastine and colchicine destroy microtubules whereas deuterium oxide (D2O) produces stabilization and interference with the function of microtubules. Vinblastine (10-4 M) and D2O (100 per cent) inhibited completely the first and second phase of glucose-induced insulin secretion. The inhibitory effect of D2O was reversible and a biphasic pattern of secretion occurred following the replacement of D2O with water. Colchicine (10-3 M) produced significant inhibition of only the second phase of glucose-induced insulin secretion. The first phase of tolbutamide-induced insulin secretion was inhibited by vinblastine and D2O and the inhibitory effect of D2O was reversible. Colchicine did not inhibit the first phase of tolbutamide-induced insulin release. These findings indicate that the microtubular system is involved in both phases of insulin secretion. The first phase of secretion may be due to the release of beta granules already associated with the microtubular system and the second phase could be the result of stored and newlysynthesized granules becoming associated with the system.

Splanchnic and peripheral glucose and amino acid metabolism in diabetes mellitus

Abstract: Splanchnic and leg exchange of glucose, lactate, pyruvate, and individual plasma amino acids was studied in diabetics 24 hr after withdrawal of insulin and in healthy controls. Measurements were made in the basal postabsorptive state and during the administration of glucose at a rate of 2 mg/kg per min for 45 min. In the basal state, net splanchnic glucose production did not differ significantly between diabetics and controls. However, splanchnic uptake of alanine and other glycogenic amino acids was 1½-2 times greater in the diabetics, while lactate and pyruvate uptake was increased by 65-115%. Splanchnic uptake of these glucose precursors could account for 32% of hepatic glucose output in the diabetics, as compared to 20% in the controls. This increase in precursor uptake was a consequence of a two- to threefold increment in fractional extraction of these substrates inasmuch as arterial levels of alanine, glycine, and threonine were reduced in the diabetics, while the levels of the remaining substrates were similar in the two groups. Peripheral output of alanine and other glycogenic amino acids as reflected in arterio-femoral venous differences was similar in both groups. An elevation in arterial valine, leucine, and isoleucine was observed in the diabetics, but could not be accounted for on the basis of alterations in splanchnic or peripheral exchange of these amino acids. Administration of glucose (2 mg/kg per min) for 45 min resulted in an 80% reduction in splanchnic glucose output in controls, but failed to inhibit hepatic glucose release in the diabetics despite a twofold greater increment in arterial glucose levels. In both groups no consistent changes in arterial glucagon were observed during the infusion. It is concluded that in nonketotic diabetics (a) total splanchnic output of glucose is comparable to controls, but the relative contribution of gluconeogenesis may be increased by more than 50%; (b) accelerated splanchnic uptake of glucose precursors is a consequence of increased hepatic extraction of available substrates rather than a result of augmented substrate supply; and (c) the failure of glucose infusion to inhibit hepatic glucose output suggests that the exquisite sensitivity of the liver to the infusion of glucose in normal man is a consequence of glucose-induced insulin secretion.

The pentose cycle and insulin release in mouse pancreatic islets

Abstract: 1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.

Decreased sensitivity of the pancreatic beta cells to glucose in prediabetic and diabetic subjects. A glucose dose-response study.

Abstract: The dose-response curve for glucose-induced insulin release obtained by infusing glucose at four to five different rates to normal subjects was sine-shaped, the insulin secretion starting at a blood glucose concentration of about 100 to 125 mg. per 100 ml. The steepest rise in plasma insulin occurred between blood glucose levels of 200 and 350 mg. per 100 ml. When blood sugar was increased further, the plasma insulin curve started to level off. There were no differences in the dose-response characteristics of the early and late insulin responses to glucose infusion. In prediabetic subjects variations in blood glucose concentration between the fasting level and about 300 mg. per 100 ml. were accompanied by only a minor insulin response. Further eleva-ation of blood glucose to 450 to 650 mg. per 100 ml. induced a normal initial insulin response in two out of the eight prediabetics, while the late response was normalized in almost all of them. Thus, the insulin-glucose dose-response curves in the prediabetics seemed to be similar to those of the normals but shifted toward higher blood glucose concentrations. In subjects with mild maturity-onset diabetes or glucose intolerance only, there seemed to exist a further shift to the right of the dose-response curves. These findings suggest that (1) the mechanisms governing the early and late insulin responses to glucose are of identical nature, and (2) the decreased insulin response to glucose in prediabetics and mild diabetics is a relative one, very high glucose concentrations being capable of eliciting normal responses. These findings are compatible with our hypothesis that the defective insulin release in prediabetes and diabetes is due to a decrease in the sensitivity of the glucose receptor of the pancreatic β cell which transmits the glucose signal for insulin release.

Nerve Growth Factor and Insulin

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The immediate effects of insulin and fructose on the metabolism of the perfused liver. Changes in lipoprotein secretion, fatty acid oxidation and esterification, lipogenesis and carbohydrate metabolism

Abstract: 1. When livers from fed rats were perfused with blood containing elevated concentrations of rat insulin or blood to which fructose was added, the oxidation of free fatty acids was depressed and their esterification was increased. 2. Raised concentrations of insulin or addition of fructose increased secretion of triglyceride in very-low-density lipoproteins, but only insulin caused more of the free fatty acids taken up by the liver to be incorporated into very-low-density lipoproteins. 3. When insulin and fructose were added together the combined effect on oxidation and esterification of free fatty acids and on secretion of very-low-density lipoproteins was equal to the sum of the effects of either alone. No statistically significant interaction between the effects of fructose and insulin was found for any of the parameters investigated. 4. Bovine insulin had similar effects, in most respects, to comparable studies with raised concentrations of rat insulin. 5. Lipogenesis was increased in the livers treated with fructose plus bovine insulin. 6. A significant proportion of the fatty acids in very-low-density lipoproteins were derived either from the liver triglyceride pool or from lipogenesis. This fraction was increased both by treatment with insulin or fructose, and was augmented further when both insulin and fructose were present together. 7. The uptake of fructose by the perfused liver was similar to that found in vivo. It was unaffected by the presence of insulin. 8. Addition of fructose to the perfused liver caused perfusate lactate concentrations to increase, as a result of diminished hepatic uptake of lactate. 9. The uptake of free fatty acids by the perfused liver was unaffected by the addition of either insulin or fructose. 10. The distribution among the various lipid classes in plasma lipoproteins of label arising from the hepatic uptake of [(14)C]oleate was unaltered by the addition of either fructose or insulin. 11. It is suggested that the effects described are due principally to control of the balance between esterification of fatty acids and lipolysis of the ensuing triglyceride, fructose enhancing esterification and insulin inhibiting lipolysis.

Insulin receptors in human circulating cells and fibroblasts.

Abstract: Human lymphocytes obtained from fasted adult subjects and cultured human tumor lymphocytes were investigated for specific insulin receptors. By use of monoiodoinsulin, specific insulin binding sites were demonstrated in peripheral human lymphocytes, cultured human lymphocytes, and in other types of human circulating cells. Insulins and insulin derivatives that varied in their potency to stimulate glucose oxidation in the fat cell and to inhibit binding of [125I]insulin to purified plasma membranes, varied in an analogous fashion in their ability to inhibit the binding of labeled insulin to human lymphocytes. Hormones that had no effect on the binding of insulin to fat cells or liver membranes also had no effect on the binding of insulin to lymphocytes. Binding was time and temperature dependent; dissociation of [125I]insulin was rapid upon addition of 10 μM insulin. These findings afford a direct approach to the study of endocrine disorders in man.

Modulation of adenylate cyclase activity in liver and fat cell membranes by insulin.

Abstract: Insulin depresses both the activity of adenylate cyclase stimulated by glucagon, epinephrine, and sodium fluoride in liver cell membranes and the activity of adenylate cyclase stimulated by epinephrine and adrenocorticotropin in particulate preparations from homogenates of isolated fat cells. Significant inhibition is detected with very low concentrations (10 -11 molar) of insulin but not with unphysiologically high (10 -9 molar) concentrations of the hormone. These direct effects of insulin on an enzymatic system in broken-cell -preparations suggest a fundamental role of adenylate cyclase activity and of cyclic adenosine monophosphate in the mechanism of action of insulin.

Carbohydrate and lipid metabolism in middle-aged, physically well-trained men

Abstract: Body composition, maximal oxygen uptake, plasma lipids, glucose and lipid tolerance, and plasma insulin were examined in middle-aged, physically well-trained men in comparison with randomly selected men of the same age. The well-trained men were characterized by a small adipose tissue consisting of small fat cells, and probably by an increased muscle mass. They had an elevated maximal oxygen uptake. Fasting plasma lipids were low. Assimilation of 100 g glucose perorally was very rapid and occurred while insulin concentrations in plasma were much lower than in controls. Fasting plasma insulin values were also low. Intravenous lipid tolerance test showed a rapid removal rate of triglycerides. Analyses of glucose metabolism in vitro in muscle biopsies from these men showed an increased activity in several metabolic pathways. Succinic oxidase activity, as a marker of aerobic capacity as well as glycogen contents, was also increased. These results indicate that physical training is a potent factor for regulation of plasma insulin levels. It was suggested that qualitative and quatitative changes in muscle capacity to metabolize glucose are in some way involved in this regulation.

The Stimulus-Secretion Coupling of Glucose-Induced Insulin Release. VII. A PROPOSED SITE OF ACTION FOR ADENOSINE-3′,5′-CYCLIC MONOPHOSPHATE

Abstract: Glucose-induced insulin release is thought to result from the following sequence of events in the beta cell: glucose metabolism leading to the production of a metabolic signal, net calcium uptake by the beta cell in response to the signal, and interaction between calcium and a microtubular-microfilamentous system, leading to emiocytosis of the secretory granules. Dibutyryl-cyclic AMP (db-cAMP) and theophylline are known to potentiate glucose-induced insulin release, their insulinotropic action being most marked at high glucose concentrations. Based on the above mentioned concepts, it was considered in the present experiments that the primary site of action of cAMP in the beta cell could correspond to either a facilitation of glucose metabolism, a modification of calcium distribution, or an interaction with the microtubular-microfilamentous system. The first of these hypotheses appeared unlikely because db-cAMP and theophylline, in sharp contrast with other agents known to affect glucose metabolism in the beta cell, did not modify glucose-induced calcium uptake by isolated islets incubated at high glucose concentrations. The last hypothesis also appeared unlikely since theophylline did not interfere with the deleterious effect of colchicine on the microtubular system, and since vincristine or colchicine did not differentially affect the respective insulinotropic action of glucose and theophylline. An effect of cAMP upon calcium distribution in the beta cell was suggested by the following findings. Whereas glucose and leucine were unable to promote insulin release in the absence of extracellular calcium, the addition of db-cAMP or theophylline to the calcium-depleted media partially restored theinsulinotropic action of glucose and leucine. Moreover, theophylline caused a dramatic increase in 45Ca efflux from perifused islets, even in the absence of glucose. It is concluded that the insulinotropic action of cAMP could be due to a glucose-independent translocation of calcium within the beta cell, from an organelle-bound pool to a cytoplasmic pool of ionized calcium readily available for transport across the cell membrane.

Studies on oral glucose intolerance in fish

Abstract: The oral glucose tolerance test, a diagnostic procedure used in the detection of human diabetes, was used to study carbohydrate metabolism in rainbow trout, Salmo gairdneri (Richardson). Fish exhibited pronounced and persistent hyperglycaemia on oral glucose administration. Hyperglycaemia was accompanied by decrease in blood amino acids, serum free fatty acids and cholesterol and marked increase in hepatic storage of glycogen. The incidence of oral glucose intolerance results, at least in part from insufficient circulating insulin. Exogenous insulin exerts a hypoglycaemic action and effectively abolishes the hyperglycaemia resulting from glucose administration. Tolbutamidc, the sulphonylurea hypoglycaemic drug, is without effect. Possibly as an indirect result of hyperadreno-corticism, oral glucose tolerance is markedly improved in the pre-spawning female. Long-term feeding of high carbohydrate diet to goldfish Carassius auratus (L.) resulted in gross hepatomegaly due to excessive hepatic glycogen accumulation and, possibly, fatty change of the liver. Protein metabolism was impaired as evidenced by protein depletion. Such degenerative changes in liver metabolism are probably a direct result of oral glucose intolerance and reflect a metabolism adapted to diets normally low in available carbohydrate.

The Metabolism of Proinsulin and Insulin by the Liver

Abstract: A B S T R A C T The removal of bovine proinsulin by the isolated perfused rat liver has been studied and the results compared with the removal of insulin. At high concentrations of insulin (> 180 ng/ml) the removal process was saturated and the ti varied between 35 and 56 min. With low initial insulin levels the disappearance followed first-order kinetics, the mean regression coefficient being -0.022, to 13.8 min, and the hepatic extraction 4.0 ml/min. The results with proinsulin were in striking contrast to these findings. At both high and low concentrations the hepatic removal of proinsulin was considerably slower, averaging 10-15 times less than that of insulin. Specific immunoassay techniques and gel filtration of samples taken from perfusions to which both labeled and unlabeled proinsulin had been added did not show conversion to either insulin or the C-peptide. Bovine and rat 'I-labeled proinsulins were degraded more slowly than bovine insulin-'1I by bovine and rat liver homogenates. Both proinsulin and insulin inhibited the degradation of insulin-tI, equimolar quantities of proinsulin being 2-5 times less effective than insulin. These results indicate significant differences in the capacity of the liver to remove and degrade insulin and proinsulin. The low hepatic extraction of proinsulin may account for its prolonged half-life in vivo and contribute to its relatively high plasma concentration in the fasting state. Furthermore this finding will have to be taken into account in the interpretation of changes in the proinsulin: insulin ratios in peripheral blood in a variety of metabolic situations.

Influence of insulin deprivation on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction.

Abstract: Summary The present study investigates the possibility that insulin dependence, a property exhibited in organ culture by a majority of the 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas, might also be operating in vivo. It was found that induction of alloxan diabetes 3 or 4 weeks after 7,12-dimethylbenz(a)anthracene administration completely prevented mammary tumor formation. Moreover, induction of alloxan diabetes in tumor-bearing rats produced the rapid regression of 90% of the tumors, with a time course similar to that observed after oophorectomy or hypophysectomy. In contrast to what occurs after oophorectomy but in accordance with what is observed after hypophysectomy, administration of estradiol benzoate failed to prevent the tumor regression produced by alloxan diabetes. Because induction of alloxan diabetes involves a considerable loss of body weight, the effect on the mammary tumors of a severe food restriction, leading to an even greater loss of body weight, was also studied. It was found that food restriction resulted in a rapid regression partially counteracted by estradiol benzoate. Tumors regressing as a result of alloxan diabetes, those progressing (about 10%) in spite of diabetes, and those that progressed under the stimulating effect of estradiol benzoate in rats subjected to food restriction were investigated with respect to their insulin dependence in organ culture. It was observed that the tumors regressing in alloxan-diabetic rats were, by and large, markedly insulin dependent in vitro, whereas those growing despite diabetes were little- or noninsulin dependent. Eventually, most tumors that were stimulated to grow by estradiol benzoate in food-restricted rats proved very insulin dependent in vitro. These observations suggest that the tumors that are insulin dependent in organ culture similarly have a stringent requirement for insulin to grow in vivo and that they regress after induction of alloxan diabetes as a consequence of insulin deprivation. This would explain why only tumors that are insulin independent in vitro are able to grow despite the insulin deprivation of alloxan diabetes. It is conceivable that food restriction results in tumor regression also as a consequence of a decreased rate of insulin secretion. However, this decrease would be smaller than in the diabetic state, and the available insulin would be present in amounts large enough to allow insulin-dependent tumors to grow, provided other limiting factors such as estrogens or estrogen-stimulated hormones are restored.

Emergence of Insulin Receptors on Human Lymphocytes During In Vitro Transformation

Abstract: Essentially no specific binding sites for insulin are detected in small lymphocytes freshly isolated from human blood. Insulin-binding sites appear on the lymphocyte surface during transformation in vitro with concanavalin A, and the number of these receptors increases sharply to reach a maximum between 24 and 46 hr after exposure to the mitogen. The appearance of de novo binding sites for insulin coincides with the increase in [3H]thymidine uptake into nuclear DNA and clearly precedes the appearance of enlarged, morphologically transformed cells. No changes in insulin-binding are detected in unstimulated control cultures. A maximum of about 350 molecules of insulin can bind per transformed lymphocyte, while less than six insulin molecules bind to an untransformed cell. Circulating human leukemic lymphoblasts bind about as much insulin as the lymphocytes transformed in vitro. Giant, polynucleated, transformed lymphocytes cultured in the presence of cytochalasin B bind about 10 times more insulin than transformed lymphocytes, which is in harmony with a 10-fold increase in cell-surface area in these cells. Specific binding of insulin is a saturable process in transformed lymphocytes but not in the untransformed cells. In transformed cells, [125I]-insulin is displaced by as little as 2 ng/ml of native insulin, while in untransformed cells no significant displacement is observed with native insulin. Digestion of transformed cells with phospholipase C (EC 3.1.4.3.) enhances the specific binding of [125I]insulin 3-fold, but no effect occurs with untransformed cells. These observations indicate a possible functional role of insulin and of adenylate cyclase in cell growth and division.

The Role of Insulin in the Regulation of α‐Amylase Synthesis in the Rat Pancreas

Abstract: . Changes in pancreatic exocrine enzyme activities were studied in different forms of experimental diabetes in rats. The effects of adrenalectomy on these changes were measured. The early actions of insulin on pancreatic enzyme activities and on incorporation of (4,5-3H)-leucine into amylase were also determined. The main results are: 1) Alloxan-diabetes leads to a decrease in amylase activity, a decreased rate of amylase synthesis and an increase in the activities of trypsinogen and chymotrypsinogen as described by Palla et al. [9]. Only the decrease in amylase activity correlates with the increase in blood glucose concentration. 2) Adrenalectomy does not reverse the changes in the activities of exocrine pancreatic enzymes induced by diabetes. 3) Diazoxide-diabetes is accompanied by an increase in all pancreatic enzyme activities, most probably due to the inhibition of enzyme secretion. 4) After treatment with streptozotozin a significant decrease in pancreatic amylase activity appears after 36 h. Amylase activity continues to fall during the following days, in contrast the increase in the activities of proteolytic enzymes is not significant until the 4th day. 5) Insulin treatment of severely diabetic, non-ketotic rats leads, as early as 90 min. after injection, to a significant increase in pancreatic amylase activity with no change in the other exocrine enzyme activities. A decrease in all enzyme activities, seen 6 h after insulin, is due to enhanced enzyme secretion. 6 As soon as 2 h after injection insulin leads to a significant increase in the incorporation of (4,5-3H)-leucine into pancreatic amylase but not into total pancreatic protein. This increase is completely abolished by actinomycin D. 7) Short-term effects of adrenalectomy (20 h) and effects of substitution with 6-α-fluoro-16α-methyl-prednisolon on the incorporation of (4,5-3H)-leucine into pancreatic amylase and total pancreatic protein result mainly from changes in the size of the cold leucine pool. According to these results insulin regulates pancreatic amylase synthesis mainly at the level of transcription. Insulin plays a permissive role in pancreatic amylase synthesis and is not involved in short-term regulation of amylase synthesis in the non-diabetic state. Clinical findings in juvenile diabetics indicate a similar type of regulation for amylase synthesis.

Circulating C-peptide immunoreactivity. Studies in normals and diabetic patients.

Abstract: The discovery of proinsulin and recognition of its intracellular conversion to insulin and C-peptide, which are stored and subsequently secreted together, have provided a simple method for monitoring β-cell function in insulinrequiring diabetics in whom insulin antibodies hinder measurement of immunoreactive insulin. Using an antiserum which reacts with antigenic determinants in human Cpeptide and this region in proinsulin and its intermediates we have measured C-peptide immunoreactivity (CPR) in unextracted serum and characterized its components. During oral glucose tolerance tests (OGTT) in nine healthy subjects, fasting serum CPR was 1.3 ± 0.3 ng./ml. and peaked at 4.4 ± 0.8 ng./ml. (60 min.). These values were positively correlated with immunoreactive insulin levels (IRI). CPR was undetectable in five newly diagnosd, untreated diabetics with fasting blood sugars above 200 mg./100 ml. and unmeasurable IRI. Similarly, serum CPR could not be measured in five ketosis-prone juvenile diabetics who had received insulin for longer than five years.9 In contrast, fasting CPR was 2.7 ± 0.7 ng./ml. in twelve insulin-treated adult onset diabetics and rose to 5.0 ± 1.1 ng./ml. two hours after glucose. Several of these sera were extracted in acid-ethanol, gel-filtered. and measured against human proinsulin and C-peptide standards. Fasting proinsulin levels were markedly elevated, while C-peptide levels were comparatively low. Both components increased after glucose administration. As these diabetics had circulating insulin antibodies it was postulated that the elevated proinsulin levels represented circulating proinsulininsulin antibody complexes. Evidence favoring this possibility was derived from in vitro experiments in which human I-125-proinsulin was incubated with diabetic sera and the samples analyzed by gel filtration, paper electrophoresis and immune precipitation. Similar experiments with 1-125-C-peptide showed no evidence for preferential binding to plasma proteins in diabetic sera. Residual β-cell secretory capacity is thus present in many insulin-treated adult onset diabetics. The C-peptide immunoassay should facilitate the prospective study of β-cell function in these patients.