Werner Stauffacher

Diabetogenic action of streptozotocin: relationship of dose to metabolic response

The relationship between the dose of intravenously administered streptozotocin (a N-nitroso derivative of glucosamine) and the diabetogenic response has been explored by use of the following indices of diabetogenic action: serum glucose, urine volume, and glycosuria, ketonuria, serum immunoreactive insulin (IRI), and pancreatic IRI content. Diabetogenic activity could be demonstrated between the doses of 25 and 100 mg/kg, all indices used showing some degree of correlation with the dose administered. Ketonuria was only seen with the largest dose, 100 mg/kg. The most striking and precise correlation was that between the dose and the pancreatic IRI content 24 hr after administration of the drug, and it is suggested that this represents a convenient test system either for both related and unrelated beta cytotoxic compounds or for screening for modifying agents or antidiabetic substances of a novel type. Ability to produce graded depletion of pancreatic IRI storage capacity led to an analysis of the relationship between pancreatic IRI content and deranged carbohydrate metabolism. Abnormal glucose tolerance and insulin response were seen when pancreatic IRI was depleted by about one-third, while fasting hyperglycemia and gross glycosuria occurred when the depletion had reached two-thirds and three-quarters, respectively. The mild yet persistent anomaly produced by the lowest effective streptozotocin dose, 25 mg/kg, exhibits characteristics resembling the state of chemical diabetes in humans and might thus warrant further study as a possible model. Finally, the loss of the diabetogenic action of streptozotocin by pretreatment with nicotinamide was confirmed and was shown to be a function of the relative doses of nicotinamide and streptozotocin and of the interval between injections.

Insulin and Glucagon Release from Monolayer Cell Cultures of Pancreas from Newborn Rats

Abstract. Monolayer culture of pancreatic cells from newborn rats has been shown to yield ultrastructurally normal endocrine cells, virtual absence of differentiated exocrine cells, and the maintenance of the capacity to synthesize immuno‐reactive insulin (IRI) and glucagon (ERG). Such a preparation has potential advantages for the study of mechanisms of hormone synthesis and release. Therefore, a survey of factors influencing hormone content and release from cultured cells was undertaken. The following general features were demonstrated: (1) highly reproducible responses within a given preparation of cells despite (2) some variations of absolute hormone content and release between preparations, (3) suitability for preparing sufficient quantities of cells to permit the simultaneous comparison of several factors within a given preparation, and (4) persistence of physiological responses to known modulators of IRI and IRG release. Thus, IRI release was stimulated in concentration‐related fashion by glucose. Amino acids, tolbutamide and glucagon augmented release, and 2‐deoxy‐D‐glucose, mannoheptulose and diazoxide inhibited glucose‐induced release. Epinephrine inhibited glucose‐induced IRI release through stimulation of an α‐adrenergic receptor mechanism, and the presence of probable β‐receptor stimulation of release was also demonstrated. Agents affecting the microtubular‐microfilamentous system exerted effecte similar to those demonstrated in other preparations. Ouabain, as well as the absence or augmented potassium in the medium increased IRI release in the presence of non‐stimulatory 2.75 mM glucose, whereas absence of calcium inhibited the response to 11 mM glucose without affecting baseline, non‐stimulated release. IRG release was shown to be inversely related to the glucose concentration in the medium. It was stimulated by epinephrine, arginine, alanine, lactate and pyruvate, and inhibited by β‐hydroxybutyrate. Diazoxide alone had no effect on IRG release. The monolayer culture employed in these studies provides a convenient, reproducible system for the further study of adult‐type IRI and IRG secretory behaviour. In addition to acute or short‐term regulation it may be especially suited for the study of long‐term modulating effects during the culture period.

Ocular Lesions in Rats Rendered Chronically Diabetic with Streptozotocin

The first results of a long-term study on rats rendered chronically diabetic with streptozotocin are presented. Besides well-known ocular changes (formation of cataracts, refractive changes, neovascularisation of iris, loss of retinal capillary endothelial and mural cells; variations in capillary diameter due to fusiform capillary microaneurysms), the formation of typical saccular microaneurysms, qualitative and quantitative changes of the capillary basement membrane ultrastructure have been observed. The basement membranes of diabetic animals showed focal thickenings containing basement membranelike and/or fibrillar material with the periodicity of collagen, but an increase of mean basement membrane width was not found compared with the controls.

Spontaneous Hyperglycemia and/or Obesity in Laboratory Rodents: An Example of the Possible Usefulness of Animal Disease Models with Both Genetic and Environmental Components

Publisher Summary This chapter provides an overview of spontaneous hyperglycemia and/or obesity in laboratory rodents. In considering the spontaneously occurring hyperglycemic syndromes of laboratory animals as they relate to understanding the pathogenesis of human diabetes, it is emphasized that no single animal syndrome can yet be considered the exact or even the best model for human diabetes. Further extensive study of the available animal strains may prove to be extremely useful. Observations related to the heredity of the syndromes emphasize the need for caution in considering any theory of the pathogenesis of diabetes based on a single and genetically simple anomaly. The secretion of excessive amounts of insulin appears to be the earliest discernible anomaly. Insulin resistance may be the cause of the enhancement of insulin release in those animal strains in which environmental manipulation has resulted in an increase in caloric intake.

Insulin Biosynthesis and Secretion — A Still Unsettled Topic

Abstract The mechanism of insulin biosynthesis and secretion is only partially understood, and new evidence suggests that its complexity is greater than previously thought. Thus, ultrastructural evidence indicates the presence of a discontinuous, microvesicular transfer of secretory material between rough endoplasmic reticulum and Golgi complex, and further suggests that more than one morphologic mode of insulin release must be considered. Similarly, biochemical evidence suggests that stimulation of insulin release involves a multicomponent system including cyclic 3′, 5′ AMP, α and β autonomous nervous receptors, Ca++ and at least two types of substrate effects, one of them specific for certain substrates only. The complexity of the stimulatory mechanism involved is particularly well illustrated by studies with metabolic inhibitors — for example, mannoheptulose inhibits glucose-induced stimulation of insulin release, as expected, but enhances insulin release induced by tolbutamide. D-2-deoxyglucose inhibi...

Streptozotocin diabetes: time course of irreversible B-cell damage; further observations on prevention by nicotinamide.

Summary Further studies on the mode of action of streptozotocin and the prevention of its effects by nicotinamide have yielded the following results: 1. The injection of nicotinamide at varying time intervals after streptozotocin afforded partial protection against its β-cytotoxic effects, decreasing with the length of the intervals but still significant when the interval was 2 hr. 2. Manno-heptulose failed to suppress the insulin release occurring between 6 and 10 hr after the injection of streptozotocin. 3. Nicotinic acid administered by gastric tube failed to protect against the diabetogenic effect of streptozotocin. 4. Nicotinamide injected 10 min after alloxan afforded no protection against the diabetogenic effect of the latter. It is concluded (1) that the β-cytotoxic effect of streptozotocin is not immediately irreversible or that it does not affect all B-cells simultaneously; (2) that the insulin release observed between 6 and 10 hr after the injection of streptozotocin occurs by way of leakage from damaged B-cells; (3) that the ineffectiveness of nicotinic acid in preventing the β-cytotoxic effect of streptozotocin is probably not the consequence of its insufficient conversion to nicotinamide after parenteral administration; and (4) that streptozotocin and alloxan differ in their mechanism of action on the pancreatic B-cell.

COMPARATIVE STUDIES OF MUSCLE AND ADIPOSE TISSUE METABOLISM IN LEAN AND OBESE MICE

The study of insulin action in vitro has revealed many similarities between the effect of insulin on striated muscle and on adipose tissue, Yet a number of physiological observations indicate the existence of a mechanism by which the metabolic fate of glucose can be directed either toward storage as fat or toward its more rapid disposal through oxidation in muscle. Since insulin is a major, possibly the major, factor controlling the deposition of fat in adipose tissue, it would seem likely tha t such regulation might be achieved by a mechanism which antagonizes the action of insulin on contracting muscle. The postulation of such icsulin-antagonizing factors, most effective on muscle, underlies many of the more recent studies concerned with obesity and diabetes, as has been amply evidenced by several discussions during this conference. (See, in particular, the contributions of Vallance-Owen and Recant and Alp.) Some years ago Rabinowitz and Zierler’ measuring insulin effects on forearm metabolism concluded tha t in obesity, both adipose tissue and muscle displayed resistance to insulin, and that this resistance was likely to be a consequence rather than a cause of obesity. Conversely, workers such as Laplane and collaborators,2 who studied glucose tolerance in obese children using capillaro-venous differences, interpreted their results as indicating a primary resistance of muscular tissue towards insulin. Animal tissue studies in vitro are complicated by the foreign environment into which the tissues are transferred. Accordingly we have considered i t of interest to study some genetic and experimental obesities in mice by means of the technique recently described by RafaelsenJ and subsequently modified by Rafaelsen, Lauris and R e n ~ l d , ~ a technique which permits the localized, yet in uiuo, estimation of insulin action in rats, after its intraperitoneal injection in quantities insufficient to produce systemic effects. We have adapted this technique for use in mice, and have begun a series of experiments which will ultimately include the New Zealand strain of obese mice, the Bar Harbor obese hyperglycemic mice, and mice made obese by the injection of gold thioglucose. Although this series is still incomplete the results obtained thus far seem of sufficient interest to warrant discussion a t this time.

Defective Immunoreactive Insulin Secretion in the Acomys Cahirinus

In vivo studies of immunoreactive insulin (IRI) secretion and electron microscopic studies of pancreatic morphology have been performed in the spiny mouse, acomys cahirinus. A defective IRI release was demonstrated in response to glucose 1.0 gm./kg., arginine 200 mg./kg., glucagon 1 mg./kg., isoprenaline 20 μg./kg., aminophylline 240 mg./kg. and dibutyryl cyclic AMP 60 mg./kg., administered intraperitoneally when compared to normal Swiss white mice. The defect appeared to involve both phases of IRI release. Electron microscopic evidence supported the concept of defective IRI release in this species. Of particular interest was the frequent occurrence in B cells of autophagic vacuoles comparable to those described for other cells undergoing secretory arrest. It is suggested that the defect of IRI release involves some basic mechanism concerned with the transport of insulin out of the B cell.

Adrenergic modification of glucose-induced biphasic insulin release from perifused rat pancreas.

Immediate and deferred effects of agents stimulating or inhibiting α or β adrenergic receptors have been studied in a perifusion preparation of rat pancreas in which glucose induces a biphasio pattern of IRI release. Epinephrine directly inhibited both the primary and the secondary phases of IRI release during the period of glucose stimulation. Propranolol inhibited only the secondary response. Low concentrations of isoproterenol stimulated, yet high concentrations inhibited both primary and secondary glucose‐induced IRI responses. Prestimulation with either epinephrine or nor‐epinephrine, followed by replacement of the adrenergic agent with stimulating concentrations of glucose, produced a marked enhancement of the glucose‐induced primary response. This deferred enhancement was abolished by the further addition of either α or β receptor blocking agents during prestimulation. — These results suggests that adrenergic stimulation may both enhance and depress glucose‐stimulated IRI release and that the suppressive α‐type effect is exerted at a point distal to the enhancing β‐type effect in the chain of events leading to IRI release; that α and β types of effects are not mutualy exclusive; that adrenergic suppression of IRI release may act to ‘prime’ the B‐cell such that subsequent immediate responses become more marked. Since adrenergic ‘priming’ of B‐cells selectively affects the primary phase of glucose‐stimulated IRI release, the possibility that inadequate function of this priming mechanism may contribute to the delayed response in diabetes must be considered.

Comparative Study of Early Metabolic Events Resulting from the Administration of the Two Diabetogenic Agents Alloxan and Streptozotocin

Abstract In this study it was demonstrated that comparable doses of streptozotocin and alloxan produced similar degrees of pancreatic insulin depletion 48 hours after intravenous injection into rats. The metabolic status of animals injected with either streptozotocin (65 mg/kg) or alloxan (60 mg/kg) was assessed prior to and 24 and 48 hours after injection. Metabolic changes consistent with the induction of acute diabetes were observed after both agents. No qualitative differences were revealed between the responses to the two drugs as reflected by plasma glucose, FFA, glycerol and triglyceride responses and by changes in FFA and glycerol release in vitro. This applied whether the animals were fed ad libitum or were restricted to glucose as the sole caloric source. These results indicate that potential extrapancreatic toxic effects of alloxan and streptozotocin do not significantly interfere with the acute diabetic metabolic changes induced by these agents and that these changes, over the first 48 hours, were primarily a consequence of β‐cell destruction and insulin deficiency. —‐ Comparison of the effects of the differing dietary regimes indicates that both endogenous and exogenous triglyceride contribute to the hypertriglyceridaemia of the acute diabetes induced by both agents. Higher plasma triglyceride concentrations were observed in streptozotocin than in alloxan treated animals fed a free diet but this difference was not evident when the diet was replaced by glucose. Such quantitative differences in the metabolic responses to the two agents could be explained on the basis of the observed variation in the rate of pancreatic insulin depletion and/or variations in food intake.