Albert E. Renold

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.

Blood glucose and the liver

Abstract A graphical summary of those aspects of carbohydrate metabolism herein discussed, which contribute to the regulation of blood glucose under normal and pathological conditions, is presented in Figure 9. The rate-limiting steps are drawn in a schematic liver cell (A). The normal animal with a blood glucose concentration of 100 mg. per cent (B) slowly mobilizes glycogen to increase the concentration of glucose-6-phosphate (G6P) which in turn is depleted by the prevailing, relatively low level of blood glucose; thus glucose is produced by the liver. At 150 mg. per cent (C) there is no net flow of glucose into or out of the liver since both glucokinase (1) and glucose-6-phosphatase (2) are respectively metabolizing glucose and glucose-6-phosphate at the same rate. At 200 mg. per cent (D) glucokinase (1) phosphorylates more glucose molecules than are cleaved by glucose-6-phosphatase (2), the concentration of glucose-6-phosphate increases and glycogen is deposited. Thus glucose is cleared by the liver. In prolonged fasting states (E) glucokinase (1) falls by 50 per cent and glucose-6-phosphatase (2) increases. Thus glucose production is facilitated without a marked fall in blood glucose concentration. Intracellular glucose-6-phosphate concentration falls, effecting mobilization of glycogen. Fructose-1,6-diphosphatase activity (3) also increases and precursors are mobilized from the periphery under the influence of the adrenal cortex. In the diabetic liver (F) glucokinase is markedly decreased and glucose-6-phosphatase (2) is doubled. In spite of the alteration of both enzymes in favor of hepatic glucose production, the circulating hyperglycemia and the large inflow of precursors are able to keep the concentration of glucose-6-phosphate near normal, and therefore this animal has near normal glycogen reserves. In the adrenal-ectomized-diabetic animal (G), the supply of precursors is interrupted. Glucokinase (1) continues at the diabetic level, but glucose-6-phosphatase (2) is diminished to near normal values. Nevertheless, since the latter is still in relative excess, the concentration of glucose-6-phosphate is markedly reduced and is reflected by the limited reserve of hepatic glycogen. Adrenalectomy in the animal with normal islet tissue (H) reduces the ability to mobilize precursors from peripheral tissues and hence to supply them to the liver. Glucokinase (1) and glucose-6-phosphatase (2) remain relatively normal. Thus this animal can function in normal carbohydrate balance as long as adequate dietary carbohydrate is available, but is incapable of prolonged fasting since its only source of blood glucose is the limited supply of hepatic glycogen. The steroid diabetic (1) exhibits increased gluconeogenesis from peripheral precursors. Since there is adequate insulin, glucokinase (1) remains normal. Glucose-6-phosphatase (2) and fructose-1,6-diphosphatase (3) are increased. The marked difference between glucokinase and glucose-6-phosphatase characteristic of the diabetic state is not present in this animal, and the tremendous inflow of carbohydrate due to gluconeogenesis increases the concentration of glucose-6-phosphate, resulting in an extreme degree of glycogen deposition. Exposure of the liver cell to epinephrine or glucagon (J) increases phosphorylase activity (4) which in turn causes a rapid breakdown of glycogen to glucose-6-phosphate. As a result of the increased glucose-6-phosphate pool free glucose is rapidly produced by glucose-6-phosphatase action. Since glucose-6-phosphate is also a non-competitive inhibitor of glucokinase, the relative activity of glucose-6-phosphatase is increased. Entry of sodium ions into the liver cell due to trauma or anoxia also activates phosphorylase and results in the same metabolic pattern. Glucose-6-phosphatase (2) is absent in the liver cell of the child with glycogen storage disease (K) and consequently there is no glucose production. Lactate is produced when carbohydrate uptake exceeds its metabolism or the ability to deposit more glycogen. Hepatoma (L) has such a rapid glycolytic rate due to the absence of fructose-1,6-diphosphatase (3) that once glucose is phosphorylated it is rapidly metabolized to lactate. Glucose-6-phosphatase (2) also is absent in this tissue. In conclusion, it would appear that hepatic glucose metabolism is not limited by cellular permeability as is the metabolism of glucose in peripheral tissues, but is largely controlled by the activities of the enzymes concerned with the entry and release of glucose into and from the pool of metabolic intermediates. Three separate mechanisms appear to be involved. The first is concerned with the routine day-to-day deposition of glycogen postprandially and its release between meals, and is associated with no major change in enzyme activity. The second mechanism is concerned with sustained alterations of enzymatic levels or activities secondary to metabolic changes of the animal. These alterations occur in diabetes, prolonged fasting states, as a result of special diets, adrenalectomy, and like conditions. They condition the establishment of that blood glucose concentration whereby a steady state with glucose-6-phosphate is obtained. The third mechanism effects immediate glucose production through the medium of glucagon, epinephrine or intracellular entry of sodium ions, as a result of rapid glycogenolysis secondary to a rise in phosphorylase activity.

Studies on Serum Insulin-like Activity (ILA) In Prediabetes and Early Overt Diabetes

Diabetes mellitus is characterized by impaired carbohydrate tolerance. It would seem reasonable, therefore, that the definition of the prediabetic state should include a normal glucose tolerance, even under stress. At the present time, no characteristic deviation from normal is known in the prediabetic state, and we agree with Jackson that the diagnosis of prediabetes can be made with certainty only in retrospect, once diabetes has declared itself. However, a prospective diagnosis of diabetes may be made with a high degree of probability on genetic grounds. As reviewed by Steinberg, susceptibility to diabetes is likely to be associated with a recessive gene. Children of both a diabetic father and diabetic mother must be considered homozygous for the diabetic gene, i.e., potential diabetics or prediabetics, and we have selected this criterion for the selection of prediabetics in this study of serum insulin-like activity (ILA) in prediabetes. Patients with early overt diabetes were selected on clinical grounds, rather than on genetic.

The Roles of Intracellular and Extracellular Ca++ in Glucose-Stimulated Biphasic Insulin Release by Rat Islets

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of (45)Ca(++) efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with (45)Ca(++) during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 muM Verapamil had no effect upon Ca(++) uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 muM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. (45)Ca(++) efflux was inhibited during both phases of the insulin release response. The results show that increased calcium uptake in response to glucose is not involved in the mechanism of first phase insulin release but is required for the full development and maintenance of the second phase release. It seems possible that intracellular calcium is the major regulatory control for first phase insulin release and that intracellular calcium and increased uptake of extracellular calcium contribute almost equally to the second phase of glucose-induced release.

The Adrenal and Diabetes: Some Interactions and Interrelations: The Banting Memorial Lecture 1959

* Presented at the Nineteenth Annual Meeting of the American Diabetes Association in Atlantic City on June 6, 1959, by Dr. George W. Thorn, Hersey Professor of the Theory and Practice of Physic, Harvard Medical School, and Physician in Chief at the Peter Bent Brigham Hospital, Boston, Massachusetts, who was awarded the Banting Medal for 1959. I appreciate, deeply, the honor which has been given to me in being chosen to present this—the Ninth Annual Banting Memorial Lecture of the American Diabetes Association. The list of distinguished investigators which precedes me is evidence of the esteem and admiration in which Banting is held. For the younger members of the Association, who perhaps never knew Sir Frederick Banting, I have reproduced a photograph (figure 1) , and a short biographical sketch. It should be appreciated that this young mans career was interrupted by military service (a frequent experience nowadays); nevertheless, he ultimately returned to the laboratory in a department of physiology to pursue an idea—this after completing his clinical training and engaging temporarily in private practice (figure 2 ) .

Somatostatin- and epinephrine-induced modifications of 45Ca++ fluxes and insulin release in rat pancreatic islets maintained in tissue culture.

The effects of somatostatin and epinephrine have been studied with regard to glucose-induced insulin release and (45)Ca(++) uptake by rat pancreatic islets after 2 days in tissue culture and with regard to (45)Ca(++) efflux from islets loaded with the radio-isotope during the 2 days of culture. (45)Ca(++) uptake, measured simultaneously with insulin release, was linear with time for 5 min. (45)Ca(++) efflux and insulin release were also measured simultaneously from perifused islets. Glucose (16.7 mM) markedly stimulated insulin release and (45)Ca(++) uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 ng/ml) but was without effect on the glucose-induced stimulation of (45)Ca(++) uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of (45)Ca(++) efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and (45)Ca(++) uptake by glucose. Both phases of insulin release were inhibited by epinephrine and marked inhibition could be observed with no change in the characteristic glucose-evoked pattern of (45)Ca(++) efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on (45)Ca(++) uptake and insulin release appeared to be mediated via an alpha-adrenergic mechanism, since is was abolished in the presence of phentolamine. Somatostatin inhibits insulin release without any detectable effect upon the handling of calcium by the islets. In contrast, inhibition of insulin release by epinephrine is accompanied by a partial inhibition of glucose-induced Ca(++) uptake.

Specific Enzymatic Determination of Glucose in Blood and Urine Using Glucose Oxidase

Adequate methods for the determination of glucose in biologic fluids based upon its reducing ability are well established. These methods, however, lack specificity since they will detect reducing substances other than glucose. Improved specificity has resulted from procedures designed to remove interfering reducing substances. These measures have been applied most successfully to blood and plasma. The procedure for the precipitation of proteins introduced by Somogyi eliminates the great majority of interfering reducing substances likely to be present in blood, such as proteins, amino acids, glutathione, phosphorylated hexoses, and others. This, however, does not make it possible to distinguish glucose from other hexoses (such as fructose or galactose) or from pentoses and from such substances as glyceraldehyde. For fluids such as urine where many of the interfering reducing substances cannot be removed by available technics the need for a specific determination is obvious. Fermentation, formation of osazones, and chromatography have proved their usefulness, but being rather cumbersome and difficult to quantitate, have not found access to routine laboratories. Muller in 1928 and Coulthard and co-workers in 1942 isolated an enzyme from Aspergillus nlger and Penicillum notatum which had the unique property of specifically catalyzing the conversion of beta-glucose to gluconic acid. This enzyme, which was given the name of glucose oxidase, has been studied extensively by Bentley and Neuberger5 and by Keilin and Hartree. Glucose oxidase was shown to be a flavoprotcin with a molecular weight of 152,000 and an alloxazine-adenine dinucleotide as a prosthetic group.

Localized Intraperitoneal Action of Insulin on Rat Diaphragm and Epididymal Adipose Tissue in Vivo

1. The intraperitoneal injection of 100 μU. of insulin together with a tracer amount of glucose-U-C-14, also injected imraperitoneally, resulted over a period of two hours in a three- to fivefold increase in the incorporation of labeled glucose carbon into the glycogen of diaphragm-or epididymal adipose tissue. When the dose of insulin injected was 10,000 μU., the incorporation of glucose carbon into diaphragm was increased twenty-five- to fiftyfold, and that into epididymal fat glycogen ten- to twentyfold. These doses of insulin injected intraperitoneally were without effect upon tissues not in direct contact with the peritoneal cavity, such as heart muscle, trapezius muscle, and subscapular brown fat, while incorporation of labeled glucose into liver was either unaffected or decreased. These results were qualitatively and quantitatively similar, whether the trace amount of labeled glucose was injected intraperitoneally or intravenously. 2. When the same amounts of insulin were injected intravenously instead of intraperitoneally, together with a Jrace amount of labeled glucose, the pattern of tissue responsiveness was totally different, a small effect upon glucose carbon incorporation into glycogen being observed in only two tissues, the rhythmically contracting diaphragm and heart muscle tissues. 3. When the animals were fasted prior to the intraperitoneal injections of glucose without or with insulin, the responsiveness to insulin of diaphragm muscle was either unchanged or increased, while the response of epididymal adipose tissue to insulin decreased, this decrease being quite marked after a seventy-two-hour period of fasting. 4. These results obtained in vivo tend to support the hypothesis of the importance of insulin “binding” to tissues as a factor controlling the responsiveness of individual tissues to insulin. The results also indicate that in vivo rat diaphragm muscle is at least as sensitive to insulin as rat epididymal adipose tissue, if not more so.

Advances in the diagnosis of altered states of adrenocortical function.

GREAT advances in the diagnosis of altered states of adrenocortical function have been made possible by improvements in methods of steroid analysis and by the availability of purified preparations of ACTH and adrenocortical hormones. The development of simple methods for the measurement of blood and urinary 17-hydroxycorticoids has facilitated the estimation of the quantity of hydrocortisone secreted by the human adrenal cortex.1 , 2 The availability of ACTH has opened the way to the exploration of adrenocortical reserve in a wide variety of physiologic and pathologic states. It now appears that the intravenous administration of ACTH with the determination of 17-hydroxycorticoids and .xa0.xa0.

Diabetes mellitus and Addison's disease: a report on eight patients and a review of 55 cases in the literature.

DURING the past few years cases of associated diabetes mellitus and Addisons disease have been observed with increasing frequency despite a decreasing incidence of tuberculosis in diabetic patients. Eight patients with both Addisons disease and diabetes mellitus have been seen at this hospital since 1942, 5 within the last three years. The 2 earlier cases have been reported in some detail elsewhere,1 2 3 but a final report on the first case and a six-year follow-up report on the second are included in this survey. Six cases are described for the first time. The 56 cases previously reported in the world literature .xa0.xa0.