Robert D. Johnson

Method for Large-Scale Isolation of Pancreatic Islets by Tissue Culture of Fetal Rat Pancreas

Detailed studies of the maturation of stimulus-secretion coupling of the pancreatic B-cell requires a supply of isolated fetal islets, which has so far been difficult to obtain. To overcome this problem we have maintained minced and mildly collagenase-digested fetal rat pancreatic glands (21.5 days gestational age) in tissue culture to enable degeneration of the acinar part, leaving the endocrine cells in an isolated and surviving state. Indeed, after 1 wk in culture there was a complete separation between acinar and endocrine cells with the appearance of numerous discrete islets and the disappearance or dedifferentiation of the exocrine cells. Isolated islets were either free floating or attached on top of a monolayer of fibroblast-like cells. Their number after 1 wk in culture was estimated as about 90 per explanted fetal pancreas and a total yield of about 5000 isolated islets was easily achieved. Both light arid electron microscopic examinations showed an excellent structural preservation with a marked predominance of well-granulated B-cells. Numerous islets of the same weight as that measured in cultured islets of adult rats were regularly found after 1 wk in culture. The insulin concentration of the cultured fetal islets was related to the glucose concentration of the growth medium. A similar relationship was found with respect to the insulin release in response to glucose. Thus, fetal islets cultured for 8 days in growth media containing 11.1 or 22.2 mM glucose showed a marked and significant insulin response to glucose in batch-type incubations at the end of the culture period. By contrast, the glucose stimulation of insulin release was insignificant in islets cultured at 5.5 or 2.8 mM glucose. When the culture period was confined to 1 day, there were no effects of glucose on the insulin release irrespective of the glucose concentration of the growth medium. It is concluded that the present technique for tissue culture of fetal rat pancreas makes it possible to isolate substantial amounts of fetal islets predominantly composed of B-cells. The transition in vitro from a poor glucose sensitivity to an adult-type insulin response indicates that the technique can be used in further detailed studies of the molecular mechanisms involved in the growth and development of the pancreatic B-cell.

INTERMITTENT ALDOSTERONISM IN PERIODIC PARALYSIS: DEPENDENCE OF ATTACKS ON RETENTION OF SODIUM, AND FAILURE TO INDUCE ATTACKS BY RESTRICTION OF DIETARY SODIUM

WE have reported that spontaneous attacks of periodic paralysis are preceded by large increases of urinary aldo-sterone and by intense retention of sodium (Conn et al. 1956). This is followed shortly by sequestration of potassium within the body, both serum and urinary potassium falling abruptly and intensely. Serum-sodium often rises to abnormally high values as serum-potassium reaches its lowest ones. As the attack subsides a great diuresis of sodium occurs together with a less intense increase of urinary potassium. Serum-sodium and serum-potassium return to normal together. By this time urinary aldosterone has returned to base-line values. We have explored further the role of retention of sodium in setting off potassium sequestration-i.e., acute hypo-kalsemia and hypokaluria. Two young men with the familial type of periodic paralysis were the subjects of this study. One of them, living in our laboratory, submitted cheerfully to 11 months of a rigid metabolic balance regime. The other was studied for 30 days to test in a different person the applicability of the metabolic principles discovered in the first one. The subjects are not related. Fig. I-Induction of episode of periodic paralysis by 2-methyl-9-cx-fluorohydrocortisone and high sodium intake. The aim of this short communication is to disseminate quickly information which is of immediate practical value to patients with this disorder. Extensive data will be published later. At this time we wish merely to outline some of our results and conclusions. Fig. 2-Changes in electrolyte metabolism during episode of periodic paralysis induced by administration of glucose and insulin. Method Two levels of sodium intake were used-8 and 208 m.eq. per day. The composition of the daily ration was otherwise the same, the high sodium regime differing from the low by addition of sodium chloride to the basic diet. Chloride ion, the only other variable, was 27 m.eq. per day on the low, and 229 m.eq. per day on the high, sodium ration. Potassium intake was constant at 132 m.eq. per day. Results Under both of these dietary conditions a series of identical experiments were carried out, each designed to induce an episode of periodic paralysis. The following facts were disclosed : (1) When the diet contained the larger quantity of sodium the administration of glucose and insulin,, or of 2-methyl-9-a-fluorohydrocortisone (an extremely potent mineralocorticoid) produced complete (from the neck down) and prolonged (24-48 hours) paralysis (figs. 1 and 2). (2) When glucose and insulin induce an episode of …

Differential Effects of Age Versus Glycemic Stimulation on the Maturation of Insulin Stimulus-Secretion Coupling During Culture of Fetal Rat Islets

We have cultured islets from 21.5-day-old fetal rats for 1–7 days in RMP11640/10% fetal calf serum containing 2.8 or 11.1 mM glucose to evaluate the differential effects of age vis-à-vis glycemic stimulation on the maturation of selected components of stimulus-secretion coupling. After 1 day of culture in either media, acute stimulation with 3.0 mg/ml glucose during basal perifusion with 0.5 mg/ml glucose elicited only a small first phase of stimulated insulin secretion and no second phase. The acute exposure to 3.0 mg/ml glucose produced no change in the prevailing high rates of oxygen consumption (Q02) and caused only minor increments in phosphate efflux (i.e., peak values for phosphate flush of 126 ± 16% of baseline for islets that had been cultured in 11.1 mM glucose and 162 ± 30% for islets cultured in 2.8 mM glucose). After 7 days of culture in 11.1 mM glucose, acute stimulation with 3.0 mg/ml glucose increased QO2 (as in adult islets) and effected acute increases in the AT32P and GT32P content of prelabeled islets. The 3.0 mg/ml glucose also triggered phosphate flush to 599 ± 45% of baseline and elicited first as well as early second phases of stimulated insulin secretion that replicated the performance of adult islets. By contrast, after 7 days of culture in 2.8 mM glucose, similar acute exposures of fetal islets to 3.0 mg/ml glucose effected only a small first phase and a negligible second phase of stimulated insulin secretion despite the occurrence of the same increments in QO2 as after culture in 11.1 mM glucose, highly significant increases in AT32P and GT32P, and phosphate flushes that peaked at 306 ± 16% of basal values. Thus, the ontogeny of individual components of stimulus-secretion coupling may occur in asynchronous fashion and varyingly require glycemic stimulation in addition to aging per se. The capacities to augment efflux of orthophosphate, enhance respiration, and heighten nucleotide turnover in response to acute stimulation with glucose seem to mature in large measure in time-dependent fashion, whereas some chronic exposure to ambient glucose in excess of basal levels may be required to establish and/or maintain the other coupled components that underlie bi-phasic stimulated insulin release. However, we did not achieve full exocytotic maturation even after 7 days of culture with 11.1 mM glucose. When stimulatory perifusion of such islets was prolonged beyond 30 min, the adult-like second phase of stimulated insulin release began to diminish. The fall-off was not due to limitations in preformed islet stores of immunoreactive insulin and it was not prevented by supplementing the perifusates with secretory-enhancing amounts of theophylline. Our experiences indicate that controlled tissue culture of fetal islets may provide a useful approach for unmasking the relative interdependence and independence of individual steps in insulin stimulus-secretion coupling. They also underscore the multiplicity of potential sites through which arrest of functional maturation could result in faulty insulin release.

Mechanisms and Management of Hyperosmolar Coma Without Ketoacidosis in the Diabetic

The mechanisms involved in the production, maintenance and progression of the state of nonketotic, hyperosmolar coma of mild diabetic patients are discussed; and a method is outlined for both initial therapy (before insulin activity is sufficiently increased) and subsequent therapy. Factors involved in the development of the syndrome include: (1) Persistence of enough insulin production to prevent ketoacidosis but very severe suppression of insulin release in relation to the level of blood glucose. (2) Insidious development of extreme hyperglycemia, increasingly great osmotic diuresis, severe depletion of body water and large volumes of urine. (3) Lack of appropriate response of the thirst center as the sensorium becomes inadequate. Because the mortality of this condition now approaches 50 per cent, careful attention to details of therapy is imperative. Delay in recognition and, therefore, of therapy leads to fatalities.

METABOLIC EFFECTS OF SULFONYLUREAS IN NORMAL MEN and IN VARIOUS TYPES OF DIABETIC PATIENTS

The studies described in this report were initiated in an effort to elucidate the mechanism of action of the sulfonylurea compounds. It seemed to us that a broad clinical investigative approach, involving the study of a large number of conditions in which the metabolism of carbohydrate is disturbed, was most likely to eliminate quickly a number of possible modeg of action and to define more sharply the areas upon which further effort should be concentrated. Thus, extensive metabolic-balance studies and numerous individual testing procedures have been performed before, during, and following the administration of carbutamide (BZ-55) and/or tolbutamide (Orinasei) in the following subjects: (1) healthy young men; (2) three middle-aged, obese, stable diabetics; ( 3 ) an unstable diabetic of normal weight; (4) a patient with lipo-atrophic diabetes; ( 5 ) a totally depancreatized woman; (6) patients with coexisting diabetes mellitus and Addison’s disease, familial diabetes and Cushing’s syndrome, and diabetes and panhypopituitarism; and (7) an acromegalic with mild diabetes. Although the data reported below do not define a specific mode of action of the sulfonylurea compounds, they eliminate from consideration a number of important possibilities. Results

Management of Diabetic Ketoacidosis

Any condition which reduces available insulin activity in the diabetic may precipitate diabetic ketoacidosis.Important factors in prognosis include duration of ketoacidosis and coma, patients age and vascular status, and associated complications.The incidence of serious complications and the mortality in this condition have been reduced by earlier and individualized treatment and improved management of late complications of therapy.