André Lambert

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.

Pronase Effect on Pancreatic Beta Cell Secretion and Morphology

Pronase at low concentration (4 micrograms per milliliter) produces a reversible increase of glucose-stimulated insulin release in isolated islets of Langerhans. Pronase also affects the ultrastructure of the beta cells by inducing extensive development of tight junctions as well as the accumulation of secretory product within the extracellular spaces.

Determination by latex immunoassay of protein 1 in normal and pathological urine.

A sensitive latex particle assay has been developed to study the occurrence of protein 1 in human urine. The coefficients of variation (CVs) of the method which is fully automated vary between 3 and 11.5%. The assayable concentration range is 0.3 to 40 micrograms/l. Protein 1 is clearly a sex-dependent protein. In contrast to urinary retinol-binding protein (RBP) which shows no variation with age or sex, protein 1 is excreted in greater amounts in males from the puberty. In adults, the mean concentration of protein 1 in urine of men is approximately 5 times that of women. In the urine from both sexes, protein 1 occurs as a single component with a Mr around 21,000 and an pI of about 4.8. Protein 1 is correlated with RBP in the urine from female or male patients with impaired proximal tubular function, which suggests that it is handled by the kidney in a similar manner as RBP. Diabetics, however, show elevations of urinary protein 1 which do not correlate with the RBP excretion but with the albuminuria. A competition between albumin and protein 1 for renal tubular uptake might explain this paradoxical behaviour of protein 1 in the course of diabetic nephropathy.

Cationic Environment and Dynamics of Insulin Secretion: II. Effect of a High Concentration of Potassium

The effects of 24 mM K+ upon the dynamics of immunoreactive insulin (IRI) release were studied in perifused rat islets in the absence of glucose and in its presence at concentrations of 50 and 300 mg. per 100 ml. Raising external K+ to 24 mM in the absence or in the presence of glucose (50 mg. per 100 ml.) was rapidly followed by a transient discharge of IRI. This rapid secretory phase was markedly inhibited in a medium containing 1 mM Na+ and was totally obliterated in a Ca2+-free medium. The early IRI response to a simultaneous increase of glucose to 300 mg. per 100 ml. and of K+ to 24 mM was enhanced while the late response was not significantly modified. In contrast, the same rise in glucose concentration after a twenty-five minute perifusion in a medium containing 24 mM K+ provoked markedly diminished early and late phases of IRI release. Under both experimental conditions, the effect of glucose was almost completely abolished in a medium containing 1 mM Na+. The stimulant action of glucose was not restored when K+ concentration was lowered from 24 to 6 mM at the time of glucose increase, but was normal if this diminution of K+ occurred ten minutes earlier. It is hypothesized that modifications in IRI secretion rate induced by 24 mM K+ result from changes in membrane permeability for Ca2+.

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...

Acid Phosphatase Activity in Secretory Granules of Pancreatic Beta Cells of Normal Rats

A cytochemical staining method for acid phosphatase combined with electron microscopy was applied to the islets of Langerhans of adult normal rats. In addition to dense bodies, some Golgi cisternae as well as a varying number of secretory granules of β cells were found to contain acid phosphatase. In view of the paucity of the knowledge concerning the role of acid phosphatase, the possible functional significance of its presence in the secretory granules of pancreatic β cells cannot be denned at the present time.

The beta cell and its responses: summarizing remarks and some contributions from Geneva.

In summarizing the proceedings concerned with the /? cell, it will be my aim to condense into short statements my necessarily prejudiced reactions and to underline newer developments or shifts in emphasis. Further, I shall try to indicate where work originating from our Geneva group either has led to slightly different conclusions or perhaps has added or emphasized specific points. Dr. Paul Lacys film provided a superb and coherent summary of the ultrastructural substrate of insulin biosynthesis, storage and release in the pancreatic ft cell. Surely the central aspect of his views of the secretory process remains that of emiocytosis of /? granules, although he would be the first to admit, I believe, that classical electron microscopy has experienced difficulties in quantifying this process or in even approaching an estimation of whether emiocytosis is the only quantitatively important mechanism of insulin release. Accordingly, I should like to present a sample of the work of Dr. Lelio Orci, from the Department of Histology and Embryology in Geneva. Dr. Orcis novel approach in its

Cationic environment and dynamics of insulin secretion III. Effect of the absence of potassium

SummaryThe absence of extracellular K+ modifies glucose-induced insulin secretion. The dynamics of these changes was studied in isolated rat islets perifused with K+-deprived media containing various Na+ concentrations. Omission of K+ from the perifusate during stimulation with 300 mg glucose/100 ml did not cause significant changes in IRI secretion, when Na+ concentration was normal (149 mM Na+). If external Na+ was partially substituted (24 mM Na+) by choline, a decrease in the amplitude of the second phase of release was noted. Both phases were markedly depressed, when Na+ was reduced to 2 mM. Removal of K+ for 25 min before glucose stimulation resulted in an acceleration and potentiation of the early phase of release, contrasting with the progressive inhibition of the late one. This inhibition was slowly reversible 10 min after K+ reintroduction. Under the same experimental conditions, decrease of Na+ concentration to 24 mM slowed and inhibited the first phase of secretion, and depressed further the second phase, without alteration of the dynamics of secretion. Glucose oxidation by islets was reduced by 35% after 2 h of incubation in a K+-free medium; this inhibition was more pronounced (65%) after 1 h, when the islets were previously perifused for 60 min in the absence of K+. From these results, it is suggested that changes in glucose-induced secretion observed in the absence of K+, are secondary to the intracellular modifications of Na+ and K+ concentrations due to the decreased activity of the Na+/K+) pump. They seem to be the result of two antagonistic effects: a facilitation of secretion by Na+ accumulation and an inhibition by K+ depletion.

Biphasic Insulin Release from Perifused Cultured Fetal Rat Pancreas: Effects of Glucose, Pyruvate, and Theophylline

Recent interest in the dynamics of insulin release in the adult has prompted the investigation of immunoreactive insulin (IRI) release profiles during perifusion of cultured fetal rat pancreas, a preparation previously studied extensively in a static incubation system. Glucose alone (16.4 mM) induced an early (primary) IRI release response and a minimal later (secondary) response. Pyruvate alone (16.4 mM) evoked no response above baseline. Addition of 2.5 mM theophylline to either of these substrates resulted in enhanced and biphasic IRI release, the theophylline effect occurring predominantly in the second phase. With glucose as substrate, increasing the theophylline concentration to 10 mM caused similar enhancement of the primary response, but less enhancement of the secondary response, by comparison with 2.5 mM theophylline. By contrast, 10 mM theophylline further enhanced the secondary response to pyruvate. Hence, this preparation of cultured fetal rat pancreas resembled adult rat pancreas with respect to the first phase of IRI release in response to glucose. However, the much smaller secondary response to glucose alone contrasted markedly with that of the adult, and appears to explain the relatively poor responsiveness to glucose of fetal and newborn rat pancreas during incubation.

Stimulation of Insulin Release in Vitro by Non-metabolized Amino Acid Analogues

Summary Two nonmetabolized amino acid analogues, α-aminoisobutyric acid (AIB) and 2-aminobicyclo |2,2,1| heptane-2-carboxylic acid (BCH) stimulated immunoreactive insulin (IRI) release from cultured fetal rat pancreas incubated in vitro. AIB was effective in the presence of either caffeine or glucose; whereas BCH was only active in the presence of the methylxanthine. AIB-induced IRI release together with glucose was inhibited by oligomycin or incubation at a low temperature. The uptake of 3H-AIB was demonstrated in this preparation. With changing experimental conditions, IRI release induced by AIB paralleled changes in AIB uptake (i.e., its active transport). The results suggest the existence of a relationship between the two phenomena.