Myosins of secretory tissues.

Myosin has been purified from the principal pancreatic islet of catfish, hog salivary gland, and hog pituitary. Use of the protease inhibitor Trasylol (FBA Pharmaceuticals, New York) was essential in the isolation of pituitary myosin. Secretory tissue myosins were very similar to smooth muscle myosin, having a heavy chain of 200,000 daltons and light chains of 14,000 and 19,000 daltons. Salivary gland myosin cross-reacted with antibodies directed toward both smooth muscle myosin and fibroblast myosin, but not with antiskeletal muscel myosin serum. The specific myosin ATPase activity measured in 0.6 M KCl was present. Tissues associated with secretion of hormone granules contained substantial amounts of this ATPase, rat pancreatic islets having 4.5 times that of rat liver. Activation of low ionic strength myosin ATPase by actin could not be demonstrated despite adequate binding of the myosin to muscle actin and elution by MgATP. The myosins were located primarily in the cytoplasm as determined by cell fractionation and were quite soluble in buffers of low ionic strength.

Muscle actin filaments bind pituitary secretory granules in vitro.

Hog anterior pituitary secretory granules sediment at 3,000 g. When rat or rabbit skeletal muscle actin filaments are present with the granules, the sedimentation decreases markedly. Depolymerized actin or viscous solutions of Ficoll and collagen have no effect on granule sedimentation. With this assay, actin filaments bind secretory granules (consisting of the proteinaceous core plus limiting membrane), secretory granule membranes, mitochondria, artificial lecithin liposomes, and styrene-butadiene microspheres, but have little or no interaction with membrane-free secretory granule cores and albumin microspheres. A secretory granule-actin complex sedimentable between 3,000 g and 25,000 g can be isolated. Metal ions, nucleotides, salts, dithiothreitol, or pretreatment of the granules with trypsin do not destroy the binding, which appears to be a lipophilic interaction.

Physiological regulation of total tubulin and polymerized tubulin in tissues.

Polymerized and depolymerized forms of tubulin were measured in rat and mouse liver, rat islets, human lymphocytes, and platelets. The percent of the total tubulin present in the polymerized form varied from 30.3 +/- 1.5% in the liver of the fed rat to 89.2 +/- 0.2% in human platelets. Fasting decreased the total tubulin and to a greater extent the polymerized form of tubulin in both rat and mouse liver. Glucose feeding increased the polymerized tubulin without affecting the total tubulin content in rat liver. Phytohemagglutinin-stimulated lymphocytes exhibited at least a three-fold increase in total tubulin (expressed in terms of DNA content), which during the initial 48 h of incubation was accounted for in toto by an increase in polymerized tubulin. It is suggested that the lectin not only accelerates tubulin synthesis but also stimulated the polymerization process. Storage of platelets at 4 degrees C for 6 days resulted in a marked decrease in total tubulin and an even greater reduction in the polymerized form. It is concluded that both the total tubulin content and its degree of polymerization can be modulated independently by a wide variety of physiological factors.

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues.

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule-stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.

Regulation of intracellular sodium concentrations in rat diaphragm muscle.

The concept is suggested that the sodium pump mechanism is influenced by the transmembrane electrical potential, and that the pump acts to maintain a constant electrochemical gradient for sodium. Evidence leading to this suggestion was obtained in rat diaphragm muscle by altering systematically the transmembrane chemical gradient for sodium ions and the transmembrane voltage. The voltage changes were produced by varying the extracellular and intracellular potassium ion concentrations. In each case the intracellular sodium concentration changed, presumably by activity of the sodium pump, so that the total electrochemical gradient for sodium was restored.

Microtubule assembly and the intracellular transport of secretory granules in pancreatic islets.

Polymerized and depolymerized tubulin were measured in pancreatic islets under various physiological and pharmacological conditions. Variations in insulin release from islets of fed or fasted rats were accompanied by concomitant changes in tubulin polymerization. Glucose induced the formation of microtubules in vitro independent of extracellular calcium. Total and polymerized tubulin content were decreased by fasting and restored by glucose feeding.

Plasma insulin responses to oral and intravenous glucose: studies in normal and diabetic sujbjects.

The plasma insulin responses of normal weight and obese, diabetic, and nondiabetic subjects to intravenous glucose was only 30-40% of that seen after oral glucose, indicating that alimentary mechanism(s) in addition to the arterial blood sugar concentration regulate insulin secretion. Observations made in subjects with diverted portal circulation indicate that the alimentary insulinogenic mechanism is located in the intestinal tract. The insulinogenic potency of the alimentary and glycemic stimuli expressed in terms of insulin secretion per gram of glucose were remarkably similar within each group of individuals. Between these groups, however, there were considerable differences. Obesity, with or without associated diabetes, was associated with a true hypersecretory responsiveness, whereas diabetes was characterized, with or without obesity, by a marked impairment in insulin secretion. The experimental design used in these studies permitted quantitation of the magnitude of the glycemic component of an oral glucose load. As a consequence of impaired insulin secretion, a greater than normal proportion of the oral glucose load escapes initial hepatic extraction in the maturity-onset diabetic and enters the peripheral circulation. Therefore, in the noninsulin-requiring maturity-onset diabetic, the glycemic insulinogenic stimulus for a given oral glucose load is significantly greater than in normal subjects and accounts for the excessive plasma insulin responses observed late in the course of an oral glucose tolerance test.

Radioimmunoassay of beta lipoprotein-protein of rat serum.

A double antibody radioimmunoassay for rat serum beta lipoprotein-protein (beta Lp-protein) is described. The protein was purified by ultracentrifugation, selective heparin-manganous precipitation, and gel filtration on Sephadex G-200. Antiserum was prepared in rabbits by biweekly immunization and absorbed with nonbeta lipoprotein containing rat serum. Iodination with (125)I and purification by gel filtration provided a radiolabeled protein which was > 98% displaced by purified beta lipoprotein in the immunoassay. The radioimmunoassay was sensitive to beta Lp-protein concentrations from 0.1 to 1.5 mug. Specificity of the immunoassay for beta Lp-protein was established by comparison of the displacement curves obtained with serum very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and density (d) > 1.21 fractions and with the beta and alpha migrating lipoproteins eluted from paper electrophoretograms. Suitability of the assay for measuring beta Lp-protein in serum was established by demonstrating 100% recovery of beta lipoprotein added to whole serum and by the absence of immunoreactive beta Lp-protein in serum of orotic acid-treated rats. Examination of sera from six other vertebrates species revealed partial cross-reactivity. Normal rat serum was found to contain 0.25+/-0.01 mg/ml of beta Lp-protein and hepatic production by an isolated perfused rat liver system was determined as 0.145 mg/hr.

Growth hormone secretion during sleep.

Plasma growth hormone (GH), insulin, cortisol, and glucose were measured during sleep on 38 nights in eight young adults. Blood was drawn from an indwelling catheter at 30-min intervals; EEG and electrooculogram were recorded throughout the night. In seven subjects, a plasma GH peak (13-72 mmug/ml) lasting 1.5-3.5 hr appeared with the onset of deep sleep. Smaller GH peaks (6-14 mmug/ml) occasionally appeared during subsequent deep sleep phases. Peak GH secretion was delayed if the onset of sleep was delayed. Subjects who were awakened for 2-3 hr and allowed to return to sleep exhibited another peak of GH secretion (14-46 mmug/ml). Peak GH secretion was not correlated with changes in plasma glucose, insulin, and cortisol. The effects of 6-CNS-active drugs on sleep-related GH secretion were investigated. Imipramine (50 mg) completely abolished GH peaks in two of four subjects, whereas chlorpromazine (30 mg), phenobarbital (97 mg), diphenylhydantoin (90 mg), chlordiazepoxide (20 mg), and isocarboxazid (30 mg) did not inhibit GH peaks. Altered hypothalamic activity associated with initiation of sleep results in a major peak of growth hormone secretion unrelated to hypoglycemia or changes in cortisol and insulin secretion.

The effect of fasting, diet, and actinomycin D on insulin secretion in the rat.

The present studies were performed to elucidate the mechanisms responsible for the impairment of glucose-stimulated insulin secretion observed in fasting. Rats fasted for 48 hr displayed marked impairment in their insulin secretory response to both oral and intravenous glucose. Glucose-stimulated insulin secretion was restored within 24 hr by refeeding; actinomycin D given before refeeding blocked the expected return of normal glucose-stimulated insulin secretion despite adequate food intake. Fasted rats refed a diet devoid of carbohydrate failed to display a return of normal insulin secretory responsiveness to oral glucose in contrast to rats fed isocalorically a high carbohydrate diet. Differences in insulin secretion in fed, fasted, and fasted-refed rats could not be attributed to changes in pancreatic insulin content. There was no significant difference in the insulin secretory response to aminophylline of fed, fasted, or fasted-refed rats. The intermittent pulsing of fasted rats with hyperglycemic episodes by the injection of small amounts of glucose (500 mg) intraperitoneally every 8 hr ameliorated the impairment of glucose-stimulated insulin secretion characteristic of the fasting state. These results suggest that the impairment of glucose-stimulated insulin secretion during fasting and its restoration by refeeding are regulated by changes in a glucose-inducible enzyme system in the pancreatic beta cell.

Effects of reduced renal mass and dietary protein intake on amino acid release and glucose uptake by rat muscle in vitro.

Epitrochlearis muscles obtained from normal male Holtzman rats used as controls (C) and rats with reduced renal mass (Nx) fed isocaloric diets of varying protein content were incubated in Krebs-Ringer buffer containing 5 mM glucose for 1 or 3 h with or without insulin. Alanine (ALA) release rates from muscles of Nx rats were increased 40% above C values after 1 h of incubation regardless of protein intake. Addition of insulin decreased the ALA release from muscles of Nx rats to C values in animals fed 10 and 20% casein and chow but did not in rats fed 40% casein. After 3 h of incubation, all ALA release rates decreased by congruent with40%. The ALA release from muscles of Nx rats fed 10% casein was comparable to C values and decreased further with the addition of insulin. On the other hand, ALA release from muscles of Nx rats fed 20 and 40% casein as well as chow remained significantly elevated above C values, but responded to the addition of insulin with a reduction in release rates to C values, except from the muscles of Nx animals fed 40% casein. Tyrosine (TYR) and phenylalanine (PHE) release rates also were increased in muscles from Nx rats compared with C after 1 h of incubation. Release rates were highest in the Nx group fed 10% casein and decreased with increasing protein intake. Addition of insulin decreased the release rates of Nx rats to C values in each group. After 3 h of incubation, release rates of TYR and PHE in muscles from Nx rats remained significantly above C values for all groups, but responded to the addition of insulin with a decrease to C values. Glutamine and glutamate release were not significantly affected by reduction in renal mass.Base-line glucose uptake by all groups of muscles from Nx rats was significantly greater than corresponding C values, but maximal insulin-stimulated glucose uptake was comparable in all groups. Tissue pool sizes for glycogen, ATP, phosphocreatine, ALA, glutamate, and glutamine were unaffected by reduction in renal mass. The results indicate that Nx is associated with accelerated ALA, TYR, and PHE release from muscle. ALA release rose with increasing protein intake and decreased to values observed from C muscles after addition of insulin except in Nx animals fed 40% casein. TYR and PHE release decreased with increasing protein intake and also decreased to C values with the addition of insulin. The data also suggest that ALA release is not dependent upon glucose uptake in muscles from either C or Nx rats.

The role of adrenergic mechanisms in the substrate and hormonal response to insulin-induced hypoglycemia in man.

Sequential determinations of glucose outflow and inflow, and rates of gluconeogenesis from alanine, before, during and after insulin-induced hypoglycemia were obtained in relation to alterations in circulating epinephrine, norepinephrine, glucagon, cortisol, and growth hormone in six normal subjects. Insulin decreased the mean (+/-SEM) plasma glucose from 89+/-3 to 39+/-2 mg/dl 25 min after injection, but this decline ceased despite serum insulin levels of 153+/-22 mul/ml. Before insulin, glucose inflow and outflow were constant averaging 125.3+/-7.1 mg/kg per h. 15 min after insulin, mean glucose outflow increased threefold, but then decreased at 25 min, reaching a rate 15% less than the preinsulin rate. Glucose inflow decreased 80% 15 min after insulin, but increased at 25 min, reaching a maximum of twice the basal rate. Gluconeogenesis from alanine decreased 68% 15 min after insulin, but returned to preinsulin rates at 25 min, and remained constant for the next 25 min, after which it increased linearly. A fourfold increase in mean plasma epinephrine was found 20 min after insulin, with maximal levels 50 times basal. Plasma norepinephrine concentrations first increased significantly at 25 min after insulin, whereas significantly increased levels of cortisol and glucagon occurred at 30 min, and growth hormone at 40 min after insulin. Thus, insulin-induced hypoglycemia in man results from both a decrease in glucose production and an increase in glucose utilization. Accelerated glycogenolysis produced much of the initial, posthypoglycemic increment in glucose production. The contribution of glycogenolysis decreased with time, while that of gluconeogenesis from alanine increased. Of the hormones studied, only the increments in plasma catecholamines preceded or coincided with the measured increase in glucose production after hypoglycemia. It therefore seems probable that adrenergic mechanisms play a major role in the initiation of counter-regulatory responses to insulin-induced hypoglycemia in man.

Hepatic fructose-1,6-diphosphatase deficiency. A cause of lactic acidosis and hypoglycemia in infancy.

An 8-month-old female, maintained on breast feeding for 6 months, experienced numerous attacks of hyperventilation when weaned to baby food and was admitted with severe lactic acidosis (20 mM) and hypoglycemia. Physical examination was negative except for hepatomegaly. Fasting (18 hr) after stabilization on a high carbohydrate diet resulted in hypoglycemia (plasma glucose 40 mg/100 ml), lactic acidosis (6-10 mM), and a rise in plasma alanine. Glucagon produced a glycemic response after 6 hr, but not after 18 hr fasting. Intravenous galactose increased plasma glucose (Delta 45 mg/100 ml) but intravenous fructose, glycerol, and alanine caused a 40-50% fall in plasma glucose and a significant rise in lactate (Delta 3-4 mM). Liver biopsy showed fatty infiltration. Liver slices incubated with galactose, lactate, fructose, alanine, or glycerol converted only galactose to glucose. Hepatic glycolytic intermediates were increased below the level of fructose-1,6-diphosphate and decreased above. Hepatic phosphorylase, glucose-6-phosphatase, amylo-1,6-glucosidase, phosphofructokinase, fructose-1-phosphate aldolase, and fructose-1,6-diphosphate aldolase levels were normal, but no fructose-1,6-diphosphatase (FDPase) activity was detected. Further studies on the liver homogenate of this patient revealed the presence of an acid-precipitable activator of FDPase. Normal plasma glucose and lactate levels were maintained on an 800 cal diet of 66% carbohydrate (sucrose and fructose excluded). 5% protein, and 20% fat. When carbohydrate was reduced to 35% and protein or fat increased to 23 and 53% respectively, lactic acidosis and hypoglycemia recurred. These studies show that a deficiency of FDPase produced infantile lactic acidosis and hypoglycemia and can be controlled by an appropriate diet.

Hypoalaninemia: a concomitant of ketotic hypoglycemia.

The cause of of ketotic hypoglycemia, the commonest form of hypoglycemia in childhood, is not known. The present study was undertaken to determine whether the primary defect in this condition is a deficiency of gluconeogenic precursor(s) or an abnormality in the hepatic gluconeogenic enzyme system. Plasma glucose, alanine, and insulin and blood beta-hydroxybutyrate (beta-OHB), pyruvate, and lactate levels were determined in eight ketotic hypoglycemic children and seven agematched controls maintained on a normal diet and after being fed a provocative hypocaloric low-carbohydrate diet (1200 kcal/1.73 m(2), 15% carbohydrate, 17% protein, and 68% fat). On a normal diet, overnight fasting plasma alanine (211+/-10 muM) and glucose (68+/-4 mg/100 ml) were significantly lower and blood beta-OHB (1.22+/-0.37 mM) significantly higher in ketotic hypoglycemic children than in controls (alanine, 315+/-15 muM; glucose, 81+/-3 mg/100 ml; beta-OHB, 0.18+/-0.08 mM). All ketotic hypoglycemic children developed symptomatic hypoglycemia (33+/-3 mg/100 ml) and ketosis (beta-OHB, 3.70+/-0.32 mM) 8-16 hr after starting the provocative diet and these changes were associated with a further decline in plasma alanine (155+/-17 muM). Normal children, even after 36 hr on this diet, maintained higher plasma glucose (48+/-2 mg/100 ml) and alanine (225+/-5 muM) and lower beta-OHB levels (2.56+/-0.44 mM). Intravenous infusions of alanine (250 mg/kg) uniformly restored the hypoglycemic plasma glucose levels of ketotic hypoglycemic children to normal. Cortisone acetate (300 mg/m(2)), given orally in three divided doses during feeding of the provocative diet, produced a 3- to 4-fold increase in plasma alanine within 4-6 hr after beginning steroid therapy and completely prevented the development of hypoglycemia and ketosis. Quantitative amino acid profiles were performed and demonstrated that alanine was the only gluconeogenic amino acid which differed significantly between the two groups. Plasma insulin and blood lactate and pyruvate levels did not differ significantly between normal and ketotic hypoglycemic children under all conditions examined. These results support the hypothesis that a deficiency in gluconeogenic precursor (e.g., alanine) rather than a defect in the hepatic gluconeogenic enzyme apparatus represents the most likely factor in the pathogenesis of ketotic hypoglycemia.

Incorporation of 75Se-selenomethionine into human apoproteins. III. Kinetic behavior of isotopically labeled plasma apoprotein in man.

The metabolism of lipoprotein-apoprotein was examined in four subjects with normal lipid metabolism and in one subject with type II hyperlipemia by means of isotopic tracer methodology. Studies were performed after intravenous injection of a radioactive amino acid precursor for apoprotein synthesis (75Se-selenomethionine), in both the basal state and following the acute injection of intravenous heparin. Computer technics were used to evaluate a series of multicompartmental models, and a general model is proposed that yields optimum fitting of experimental data for serum free amino acid precursor, very-low-density lipoprotein-apoprotein (VLD-apoprotein), and low-density lipoprotein-apoprotein (LDL-apoprotein) in man. The analysis demonstrates that approximately half of the transport of 75Se-apoVLDL from the plasma VLDL pool is converted to 75Se-apoLDL. The acute injection of heparin in two normal subjects results in a two-and-a-half-fold increase in this rate of conversion of 75Se-apoVLDL to 75Se-apoLDL. 75Se-apoLDL is metabolized by rapid transport into a recycling extravascular pool and by irreversible catabolism. The fractional rate of recycling is large relative to the fractional rate of catabolism of apoLDL (3.7:1.0), suggesting extravascular recycling as a potential site of regulation of the plasma concentration of apoLDL. In a patient with type II hyperlipemia, the extravascular recycling pathway is reduced and is not corrected with D-thyroxine therapy. However, this therapy did reduce conversion of apoVLDL to apoLDL in this type II patient. The kinetic data support the validity of the compartmental model in simulating both normal and pathologic apoprotein metabolism and that perturbation of physiology seen with heparin injection and D-thyroxine therapy. These data support a quantitative role of apoVLDL as a precursor of apoLDL and identify an important recycling pathway of apoLDL metabolism in addition to that of catabolism.

Physiologic mechanisms in the development of starvation ketosis in man.

The present study was undertaken to determine whether alterations in ketone body utilization and hepatic production, independent of the FFA load, were also involved in the development of fasting ketosis. Plasma Beta-OH butyric acid (Beta-OHB) increased to 2.5-4.5 mM and plasma FFA to 1,000-1,400 muEq/L. in normal weight individuals after five to seven days' starvation and in obese subjects after ten to fourteen days' fasting. Acute elevations fo the plasma FFA greater than 1,500 muEq/L. for sixty minutes in fed normal weight and obese subjects with a fat meal-heparin regimen resulted in peak elevations of plasma Beta-OHB (0.25-0.45mM), only 10 percent of that seen during fasting. When plasma FFA were lowered acutely during fasting with the antilipolytic agent Pyrazole to control levels (400-600 muEq/L.), plasma Beta-OHB decreased 35 plus or minus 5 per cent. Comparable lowering of plasma FFA in normal weight or obese starved subjects given dexamethasone to maintain elevated fasting plasma insulin levels resulted in an 87 plus or minus 3 per cent decrease in plasma Beta-OHB. Similar studies in obese fasted subjects pretreated with an intravenous infusion of insulin (1.0 U/hr. for eight hours) before receiving Pyrazole resulted in a 65 plus or minus 5 per cent decrease in plasma Beta-OHB. Plasma Beta-OHB half-life, determined after injections of 12 gm. Beta-OHB, increased significantly during fasting (110 plus or minus 15 minutes) and was decreased when the fasting subjects were maintained on dexamethasone (65 plus or minus 7 minutes). These studies indicate that accelerated hepatic ketogenesis during starvation is a result of both enhanced activity of the enzymatic system(s) involved in ketone body production as well as an increased FFA load. The increase in plasma Beta-OHB during fasting reflects not only an accelerated rate of hepatic ketogenesis but also an impairment of peripheral utilization, both processes apparently being sensitive to insulin. Diabetes 24:10-16, January, 1975.

Effect of lectins on hormone release from isolated rat islets of langerhans.

Lectins from Agaricus bisporus and Agaricus campestris stimulate insulin and glucagon release from isolated rat islets in the presence of 2 mM glucose. In the case of insulin release, maximal stimulation was observed at lectin concentrations above 58 mug. per milliliter (approximately 1 muM).A. bisporus PHA-B-stimulated insulin release was independent of a source of metabolic energy but was abolished by deuterium oxide. The lectin did not alter islet glucose oxidation to CO2 or incorporation of [3H] leucine into trichloracetic acid-precipitable material nor did it modify rates of insulin secretion induced by 20 mM glucose. None of nine other lectins tested stimulated insulin release, whereas stimulation of fat cell glucose oxidation was a general property of the lectins. Binding of 125I-labeled A. bisporus PHA-B to islets increased with time up to one hour and after attainment of equilibrium was very slowly reversible. Binding was directly proportional to islet number and the estimated Kdiss of the binding reaction was 17 mug per milliliter. The total number of A. bisporus PHA-B binding sites per islet was approximately 2 times 10(10). Binding of A. bisporus PHA-B to the islets and A. bisporus PHA-B-stimulated insulin release were inhibited in parallel by a glycopeptide containing the oligosaccharide receptor for the lectin, suggesting that lectin binding is essential for the expression of insulin-releasing activity. It is proposed that the specific interaction between mushroom lectin and its receptors may lead to conformational changes in the structure of the membranes of the islet A2- and B-cells that facilitate exocytosis.

Hyperinsulinemia and enlarged adipocytes in patients with endogenous hyperlipoproteinemia without obesity or diabetes mellitus.

Studies of adipocyte metabolism were performed in twelve male subjects with normal plasma lipids and eleven male patients with Type IV or Type V hyperlipoproteinemia. Patients with obesity or diabetes mellitus were excluded from the study. Although all patients had normal glucose tolerance tests, the blood glucose levels during these tests were higher in the hyperlipoproteinemic patients than in the normal control subjects and the plasma insulin responses were even more strikingly elevated in the hyperlipemic group. Adipocytes isolated from hypertriglyceridemic subjects were larger than those obtained from normal individuals and exhibited increased activities of both Type I and Type II hexokinase and increased rates of glucose oxidation and lipogenesis from glucose. Cell size, hexokinase isoenzyme activities and rates of lipogenesis from glucose were all strongly correlated with each other, but none of these measurements were correlated with glucose oxidation. It is not known how the adipocyte abnormalities are related to the lipid transport disorder.

Incorporation of 75Se-selenomethionine into human apoproteins. II. Characterization of metabolism of very-low-density and low-density lipoproteins in vivo and in vitro.

This investigation is designed to explore the potential role of apo VLDL as a precursor of a polypeptide component of human LDL. Attention was directed to the chromatography-defined Sf-I polypeptide fraction of apo VLDL, which has been previously shown to be immunologically and chemically indistinguishable from the major component of apoLDL.1-3 In VLDL isolated from bloow drawn within two hours following 75Se-SM injection, the Sf-I polypeptide fraction of apo VLDL was highly enriched with isotope, providing an appropriate preparation for in-vitro tracer studies. Conversion of 75Se-VLDL to 75Se-LDL occurred in vitro in the presence of normal plasma at 37 degrees C., and this conversion was augmented by post-heparin plasma. No conversion to HDL lipoproteins could be detected. Injection of heparin in vivo resulted in acute reciprocal changes in the radioactivity contained within serum apo VLDL and apoLDL. These findings suggest that a component of the Sf-I polypeptide fraction of apo VLDL can be metabolized into the apoprotein of LDL in man. Thus, the biochemical and immunologic similarities between the Sf-I fractions of apoVLDL and apoLDL may result from a physiologic "precursor-product" relationship between the apoprotein moieties of these two lipoprotein species. A method for further investigation of the metabolism of human apoprotein is suggested.

The effect of hyperglucagonemia on blood glucose concentrations and on insulin requirements in insulin-requiring diabetes mellitus.

The effect of elevated glucagon concentrations on insulin requirements and on blood glucose concentrations was studied in five insulin-requiring diabetic subjects during feedback control of hyperglycemia with an automated glucose-controlled insulin infusion system (artificial endocrine pancreas) for six to eight hours. Two levels of hyperglucagonemia were induced by means of constant intravenous infusion. Raising plasma glucagon concentrations to levels reported in poorly controlled diabetics (450 to 665 pg. per milliliter) did not alter total insulin requirements or blood glucose concentrations. Higher glucagon concentrations (850 to 1,050 pg. per milliliter) caused a modest (26 per cent) increase in insulin requirements and only a slight increase in mean blood glucose concentrations. These studies demonstrate that the degree of hyperglucagonemia found most frequently in insulin-requiring diabetics does not increase insulin requirements or decrease insulin effectiveness in patients given insulin in amounts appropriate to maintain euglycemia.