Search

Because of the strong augmen-tary action of gastric inhibitory polypeptide (GIP) on insulin secretion, we studied in foxhounds the effect on GIP secretion of several factors known to affect insulin secretion and plasma glucose. Growth hormone infusion decreased plasma GIP, yet increased insulin; no change in plasma glucose was found. Growth hormone inhibited oral glucose-induced increments in plasma GIP and glucose. Prednisolone did not affect levels of GIP or insulin, whether given alone or preceding oral glucose. Diazoxide decreased glucose-induced GIP and insulin secretion, but increased plasma glucose. Fasting for 112 hr decreased GIP but did not decrease the glucose-induced GIP rise. Infusion of glucagon or insulin in amounts sufficient to produce high physiologic or pharmacologic plasma levels did not change GIP concentration, nor did they alter GIP rises with glucose. In conclusion, GIP was not inhibited by insulin or glucagon but was decreased by growth hormone, fasting, or diazoxide. Whereas fasting increased glucose-stimulated insulin secretion, diazoxide decreased it. The insulin-releasing activity of a number of factors does not correlate with increases in plasma GIP, and increases in plasma insulin or glucagon may not suppress increases in plasma GIP. There was not a good correlation of the plasma levels of GIP, insulin, glucose, and glucagon.I am deeply indebted for supplies from Drs. John Brown, Werner Creutzfeldt, and Roger Unger, and for aid from Ms. Patricia Huling.

Physiologic amounts of GIP stimulated the secretion of IRI and IRG by monolayer cultures of neonatal rat pancreatic islets. Localization of exogenous GIP to a minority subpopulation of the islet cells was observed. The results may be interpreted to indicate that the effect of GIP on IRI release by the B cell is not direct, but rather, is mediated through its action on another islet cell type. Conversely, the action of GIP on IRG release may be directly mediated through an effect of GIP on the A cell. The action of GIP on IRG release apparently overrides any sup pressive effect that high glucose levels may exert. The stimulation by GIP of both IRI and IRG release was greater when a mixture of amino acids was omitted.

Using an in vitro islet cell culture assay system, GIP and CCK-OP appear to be about equally potent insulinotropic agents. Secretin has only a very slight stimulatory effect on insulin release, and only at a very high peptide concentration. Furthermore, GIP plus CCK-OP and GIP plus secretin appear to have synergistic insulinogenic actions. If supraphysiologic GIP concentrations are tested, glucose is not necessary at an insulin stimulatory concentration for GIP to stimulate insulin secretion.Glucagon release is enhanced only by GIP at physiologic peptide concentrations. Both secretin and CCK-OP have only very slight glucagonotropic actions even at high concentrations.

In studies pertinent to pathophysiologic changes in diabetes and reactive hypoglycemia, we have investigated, in dogs, the effect of gastroenteropancreatic hormones upon gastric inhibitory polypeptide (GIP), since it is a potent insulin-releasing hormone. Intravenous infusion of pharmacologic doses of vasoactive inhibitory polypeptide (VIP), acetylcholine, isoproterenol, epinephrine (with or without phentolamine or propranolol), serotonin, or pancreatic polypeptide (PP) did not change fasting plasma GIP levels, despite the fact that several of them increased plasma insulin. However, glucose-induced increase in GIP was inhibited by acetylcholine, iosproterenol, and epinephrine (aloninhibitory polypeptide (GIP), since it is a potent insulin-releasing hormone. Intravenous infusion of pharmacologic doses of vasoactive inhibitory polypeptide (VIP), acetylcholine, isoproterenol, epinephrine (with or without phentolamine or propranolol), serotonin, or pancreatic polypeptide (PP) did not change fasting plasma GIP levels, despite the fact that several of them increased plasma insulin. However, glucose-induced increase in GIP was inhibited by acetylcholine, iosproterenol, and epinephrine (alone or plus phentolamine or plus propranolol), but was not affected by gastrin, VIP, or PP. Corn oil-stimulated GIP rise was inhibited by acetylcholine but not by isoproterenol. By infusing the hormones systemically we were testing an endocrine effect, but their endogenous release in the gut would conceivably promote neurocrinic and /or paracrinic actions. Their release in close proximity to the K cells (GIP-producing) would presumably provide higher concentrations and stronger actions on these cells.We are deeply indebted to: Drs. Joel Brodsky, Gerald Van Belle, and C. B. Bell for statistical analyses; Drs. John Brown, Werner Creutzfeldt, Roger Unger, Ronald Chance, Morton Grossman, and Sami Said for special hormones and/or antibodies; James Champagne (research technician); Patricia Hulin (secretary).

In the isolated perfused rat liver the biological half-life for pork immunoassayable insulin was determined to be 17 min. The corresponding figures for beef-pork, rat pancreatic, and rat gut, immunoassayable glucagon were 19, 23, and 27 min, respectively. The data are in keeping with the liver, which is a major site for degradation of insulin and glucagon.

Respiration in the perfused hearts of vitamin E-deficient and -sufficient rats was studied and compared in the control, oligomycin, and cyanide treated hearts. No effect of vitamin E deficiency was noted in the total qO2, phosphorylated respiration or nonmitochondrial oxygen fixation. Studies of lipid peroxidation, as tested by the thiobarbituric acid (TBA) method, demonstrated high in vitrolipid peroxidation in the homogenates of hearts and livers of the vitamin E-deficient rats compared to the control. The TBA test in the heart perfusate of the two groups was the same.

Undiluted sera of new, untreated diabetics and diabetics in ketoacidosis have been shown to exhibit antagonism to added insulin when assayed by the rat diaphragm technic. One acidotic serum showed unusually high insulin activity after hanging curtain electrophoresis. The possible significance of these data is discussed.We wish to thank Miss Ruth Miller for invaluable technical assistance in this work, and Drs. Joseph Crampton and Robert Reeves, Virginia Mason Hosp. for aid in obtaining suitable sera.

An appreciable portion of inorganic I113was found to be precipitated by TCA in the presence of red blood cells. This was not the case with serum or other tissues studied. In evaluating the degradation of I131-labeled proteins by measuring the TCA precipitability of the blood radioactivity, it should, therefore, be appreciated that the degradation product, inorganic I131, may show the phenomenon of “re-entry,” and be precipitated by TCA. Findings, somewhat similar to those with inorganic I131were also obtained with I131-labeled diiodotyrosine.

Using a radioimmunoassay for measuring guanosine 3,5′-phosphate (c-GMP) and adenosine 3,5′-phosphate (c-AMP), the effect of intravenous infusion of hormones and other compounds upon the urinary excretion of these cyclic nucleotides was tested in young healthy adult males. Glucagon, parathyroid hormone, and isoproterenol increased the excretion of c-GMP and c-AMP. The action of isoproterenol was more prolonged than that of the other two hormones. No definite effect was produced by epinephrine, ACTH, hydrocortisone hemisuccinate, vasopressin, triiodothyronine, or aminophylline. Vigorous exercise slightly increased total c-AMP excretion.The methodologic advantages of measuring c-AMP and c-GMP in urine are presented, and some implications of the significance of our studies and others are discussed.

Nephrectomy or physiological hepatectomy decreased the rate of degradation of insulin-I131in rats. There was some degradation even when both operations had been performed. Kidney and liver therefore are major, but not the only, sites of insulin-I131degradation in the rat.

When phenethyldiguanide (PEDG) is administered to guinea pigs, it produces hypoglycemia accompanied by depletion of liver glycogen with no increase in muscle glycogen. PEDG causes decreased blood urea formation and decreased non-protein nitrogen excretion in the urine. It also produces an early and significant rise in blood lactic acid. From studies made on surviving slices of guinea pig liver, it is evident that PEDG increases lactic acid formation and increases glycogen depletion, but it does not affect glucose output. PEDG also exerts an effect on isolated rat diaphragm, increasing glucose uptake, increasing lactic acid production, decreasing glycogen formation, and decreasing oxygen uptake.

1. The distribution and breakdown of insulin-I131in rats under various changes in carbohydrate metabolism has been studied. 2. An intravenous glucose load exerted no detectable effect other than to reduce renal radioactivity. 3. Intravenous fructose markedly reduced renal precipitable radioactivity, and increased hepatic and muscle radioactivity. 4. Intraperitoneal glucose or fructose has the same effects as intravenous fructose but to a lesser degree. 5. Alloxan diabetic animals concentrated more precipitable radioactivity in the liver and blood. 6. Phloridzin exerted no effect on insulin distribution and degradation. 7. Fasting effected little change; there was some decrease in renal and some increase in hepatic precipitable radioactivity. 8. The limitations and implications of the study are discussed.

Insulin-I131degradation in rats in vivowas not inhibited by the administration of zinc in the diet, or by treatment with the substance, polyphloretin phosphate. Marked inhibition was obtained by the intravenous or prolonged oral administration of 5-isopropylidene-2,4-dithiohydantoin and it was suggested that this may explain the protection afforded against diabetes by this substance in the partially depancreatized rat.

(1) I131-labeled thyroxine was administered intravenously to normal and thyroidectomized rats and the animals sacrificed at varying time intervals thereafter. Specimens of liver, kidney and muscle were homogenized, extracted with butanol and the extracts were subjected to kieselguhr chromatographic analysis. Small amounts of triiodothyronine were recovered from all 3 tissues in about 20% of the analyses carried out. (2) When triiodothyronine was administered and the tissues subjected to the same conditions, no thyroxine was recovered. (3) When the tissues of rats given thyroxine were homogenized and subjected to fractionation into their subcellular components, triiodothyronine was more readily recovered than was the case where the whole homogenate was used. A possible explanation for this is discusses.

A pooled design for adipose tissue bioassay measuring 14CO2production permitted an index of precision of 0.041-0.091. Comparison of dose-response curves at various glucose concentrations from 25 to 300 mg%, showed very similar classical sigmoid-shaped curves, with similar slopes. The insulin sensitivity of rat epididymal fat, in terms of the oxidation of glucose-1-14C to 14CO2, is independent of glucose concentration (25-300 mg%).We are very grateful to Ruth Miller and Lindsay Fiske for expert technical assistance.

In the isolated rat liver perfusion system, with a blood flow of 25-30 ml / minute, the biological half-life of crystalline pork insulin was determined to be 14 minutes and pork nonsuppressible insulin-like activity (NSILA) 63 minutes. The prolonged half-life of NSILA is consistent with the apparent lack of change in serum concentration in clinical situations and under physiologic experimentation.

In an in vitrosystem using in-sulin-I131and a special liver preparation which degrades insulin, it has been shown that certain amino acids and related compounds inhibit insulin-I131degradation, as measured by the accumulation of trichloracetic acid soluble radioactivity. Marked inhibition was demonstrated using cystine, ergothioneine, 3, 4-dihydroxyphenylalanine, isoleucine, tryptophane, indole-3-acetic acid, and casein hy-drolysate (Amigen). In the concentrations tested no significant inhibition was exerted by glutamine, glycine, β-alanine, asparagine, arginine, serine, threonine, leucine, methionine, histidine, hydroxyproline or tyrosine.

(1) Enlargement of rat adrenal and thyroid glands following the administration of Amphenone “B”, observed by Hertz, Allen, and Tullner, was confirmed. (2) The adrenal enlargement was associated with an increase in adrenal cholesterol concentration. At the same time, the adrenals remained capable of secreting sufficient steroid to produce a marked fall in circulating eosinophiles. Amphenone “B” may exert its adrenomegalic effect by direct stimulation of the adenohypo-physis to produce ACTH rather than indirectly as seen, for example, in thiouracil-induced goiter. (3) In addition to thyroid enlargement, a marked decrease in I131 concentration in the gland was observed. This could be due to a thiouracil-type of action, although the effects noted within the time relationships of our experiments were similar to those produced by TSH and hence possibly the result of increased secretion of radioiodine rather than a decreased uptake. (4) Decrease in size of the testes was observed in animals given Amphenone “B”. It is postulated that this may be due to a diminished production of FSH brought about either by elevated blood levels of Amphenone “B” itself or by an increase in adrenal corticoids as a result of adrenal stimulation.

Investigations with isolated lipocytes were conducted concerning the effects of Mg, Ca, Zn, and K upon the formation of c-AMP-14C from ATP-14C. The Mg markedly increased the production of c-AMP-14C. The Ca stimulated a smaller increment, but reduced that caused by Mg. When the cells were preincubated with Ca, and then repeatedly washed with Tris-glycerophosphate buffer, no effect was produced by Mg unless EGTA was also used, suggesting a stronger affinity of the lipocytes for Ca than for Mg. A similar level of c-AMP-14C was found whether Ca was used alone, or in each combination tested with Mg, isopropylnorepinephrine, K, or insulin. The Zn markedly reduced the increments of c-AMP-14C when used alone or with Mg. The K alone had no effect and influenced the results with Ca and/or Mg relatively little. The NaF, used alone or with Ca, produced no changes, but caused tenfold increments with Mg. Isopropylnorepinephrine had no effect alone, or with Ca or Zn, but it produced marked additional increments when used with Mg. Insulin reduced very slightly the Mg action but did not affect the action of the other metals.

1. Glutathione degrades in-sulin-l131and renders it more soluble in tri-chloroacetic acid. Evidence is presented that this is not the result of direct deiodination. 2. Rat liver extracts, even after exposure to a boiling water bath, can degrade insulin-l131. This heat stable activity could be explained by the presence of sulfhydryl compounds in liver. 3. Reductive as well as proteolytic processes must be considered when analyzing the degradation of insulin by crude liver extracts.The authors are indebted to Miss Ruth Miller for valuable technical assistance.

It was attempted to inhibit the selective destructive effect of alloxan on islet cells and the resultant blood sugar changes by administration of substances that combine chemically with alloxan or that inhibit some of its chemical actions. Of these substances 3,4-diaminotoluene, orthophenylenediamine, and sodium bisulfite exhibited definite inhibitory effects.

1. Administration of PAS to rats over a 2-week period produced goiter associated with diminished I131uptake by the thyroid gland. 2. No effect on the pituitary-adrenal axis was observed. 3. Elevation of liver glycogen concentration was noted in all PAS-treated animals.

Injection of glucagon and insulin in rats stimulates the adrenal medullary enzyme, phenylethanolamine-N-methyl transferase (PNMT), with evidence of this being apparent within 2 hours. The highest level of stimulation results when glucagon (1 mg) and insulin (6 units) are injected simultaneously. In hypophysectomized rats, there is a marked decrease in the enzyme activity. ACTH only partially corrects this, but insulin andACTH increase the enzyme activity to normal levels. It is suggested that, in the intact rats, insulin and glucagon may exert a stimulatory effect on PNMT activity independent of hypoglycemia or increased glucocorticoid production.

Guanyl cyclase, adenyl cyclase, cyclic 3′,5,′-adenosine monophosphate phosphodiesterase (cAMP PDE), and cyclic 3′,5′-guanosine monophosphate phosphodiesterase (cGMP PDE) activities in heart, lung, liver, and skeletal muscle of rats 26, 45, 70, and 300 days of age were measured. Decreases in each of these enzyme activities, except for that of cAMP PDE in lung, occurred with aging.3The most rapid declines in activity occurred within 70 days. The activities of the cyclases decreased more rapidly than those of the phosphodiesterases. Therefore, in our experiments, the rates of synthesis of the cyclic nucleotides declined faster than the rates of degradation with aging. Guanyl cyclase activity had greater decreases in lung homogenate than in other tissues, and adenyl cyclase activity decreased much faster in heart and lung than in liver and skeletal muscle. cGMP PDE and cAMP PDE activities were much greater in lung than in other tissues at all ages. It is not known how or to what extent cyclic nucleotide metabolism might contribute to aging, or by what mechanisms alterations in the metabolism of such compounds are produced with, or by, aging. However, numerous changes in metabolic and endocrine activities are known to occur at different intervals of the lifespan, especially during gestation, maturation, and senescence, and may be related to the decline in activity of these cyclic nucleotide synthetic and degradative enzymes.

Total quantities of immunoreactive insulin and glucagon in human fetal pancreases (aged 25–135 days) were determined. Small amounts of these hormones were demonstrable by the 50th day, larger amounts by the 90th day and relatively large quantities by the 110th day. Arginine (5 mM) produced a very significant increase in the secretion of each hormone from slices of pancreas, while glucose (300 mg/100 ml) did not affect glucagon release and had relatively little effect upon insulin release. Using Sephadex gel filtration, we have found a fraction with a molecular weight three times that of glucagon which combines with glucagon antibody. Studies are being performed to determine if this larger molecular compound contains glucagon.

1. When labeled 1-thyroxine was given to rats, the concentration of “hormonal” (TCA-precipitable) radioactivity in liver was significantly greater in the female than in the male. 2. Castration did not alter these findings in either the male or female. The administration of estrogens to normal males and to castrated females, and the administration of androgens to normal females and castrated males were also without effect. However, hypophysectomy in females lowered values to those in normal or hypophysectomized males. 3. No significant difference in the radioactivity of kidney, blood, skeletal muscle and G.I. tract were found after 1-thyroxine administration, nor were urinary, fecal, or bile excretion rates of radioactivity significantly different. 4. Following administration of labeled 3,5,3' 1-triiodothyronine, no consistent differences in liver conncentration between male and female were found. 5. The mechanism responsible for the difference in the “thyroxine” concentration in the liver of male and female rats remains unknown.

Palmitic-1-C14and linoleic-1-C14acids were infused into fasted dogs, and fractionation of the serum lipids carried out at time intervals up to 72 hours. Although many similarities were found in the metabolism of these 2 fatty acids, it was observed that linoleic-C14acid appeared to 1) persist longer in the plasma as the FFA and 2) to be incorporated preferentially into cholesterol esters and phospholipids.The authors gratefully acknowledge the technical assistance of Miss Claudine Shepard and Miss Inez Winslow.

Immunoreactive glucagon was detected in the pancreas and the small and large intestines of 4 different animals. Immunological differences existed between pancreatic and jejunal glucagon in the human and dog, but pancreatic and rectal glucagon appeared similar. A rise in serum immuno-reactive glucagon was demonstrated in unanaesthetized dogs after oral glucose. Basal serum glucagon levels in dogs fell only slightly after pancreatectomy. A marked rise in serum immunoreactive glucagon occurred after intrajejunal glucose in pancreatectomized dogs, suggesting its enteric source. An insulinogenic role for intestinal glucagon is considered.

(1) The difference between the rat and guinea pig with respect to thiouracil goitrogenesis was investigated using the binding of I131to surviving thyroid slices as a measure of thyroid hormone production. (2) Propylthiouracil in concentrations ranging from 10-7M through 10-5M inhibited hormone synthesis to an equal extent in both rat and guinea pig thyroid slices. In vivostudies have indicated the compound to concentrate in the gland to a comparable degree in both species. On the other hand, in the absence of propylthiouracil, hormone synthesis in rat thyroid slices occurred at 5 times the rate for guinea pig thyroid slices. (3) In view of these observations it is suggested that the species difference in thiouracil goitrogenesis can be accounted for by differences in the rates of peripheral utilization of circulating thyroid hormone.

Cholesterol-esterifying activity was demonstrated in homogenates of rat, rabbit and guinea pig adrenals. Cholesterol-esterifying activity was found to be deficient in homogenates of bovine adrenal tissue. This suggests that the amount of cholesterol esters in the adrenal gland is dependent on the level of cholesterol-esterifying activity in the gland, rather than on blood levels of cholesterol, free or esterified.

The effect of PEBG on triglyceride synthesis has been studied in vitro.In intact tissue PEBG interferes with the conversion of diglyceride to triglyceride. In homogenates, PEBG increases the incorporation of labeled fatty acid into triglyceride. It thus differs from insulin in its effect on triglyceride synthesis. An explanation for this is presented.

Serum protein-bound gluco-samine and protein-bound hexose were measured during prolonged glucose infusions in non-diabetic and diabetic subjects. Negligible changes in either of these substances occurred in either group.

These observations suggest, then, that there is no impairment in ability of fat stores in obese patients to release NEFA in response to epinephrine.

The effect within 2 hours of adrenocorticotropin and adrenal hormone administration on the free amino acid content of rat tissues was determined. Liver and kidney showed a significant increase following administration of either hormone preparation and the adrenals showed an increase following adrenocorticotropin administration.