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The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.

Effects of isometric contraction and catecholamines on glucose and glycogen metabolism in skeletal muscle were investigated with the in vitro rat epitrochlearis preparation. Mechanical performance and glycogenolysis exhibited two phases. During the initial 30 min, muscle work was 30% greater and glycogenolysis 8- to 10-fold faster than the steady-state values in the subsequent 3–4 h of contraction. Glucose uptake was increased by contraction and remained relatively constant during stimulation. Epinephrine (10(-9) to 10(-6) M) and norepinephrine (10(-7) to 10(-5) M) produced inotropic and glycogenolytic effects blocked by propranolol but not phentolamine. Chemical sympathectomy and propranolol blocked the initial glycogenolytic and inotropic effects produced by isometric contraction, suggesting that they were caused by the release of endogenous catecholamines. Net lactate production in resting muscles accounted for > 50% of total glucosyl units utilized. During contraction net lactate production accounted < 10–15% of total glycosyl flux indicating that rat fast-twitch pale muscle is capable of significant rates of aerobic glucose oxidation. Oleate and caprylate did not affect mechanical performance, glycogen, or glucose metabolism in resting or contracting muscles.

Regulation of plasma insulin levels in the sheep, cow, rat, rabbit and pig was investigated utilizing a double antibody radio-immunoassay. In the sheep, infusion of the rumen metabolites, propionate and butyrate, increased plasma insulin levels approximately 4-fold and 14-fold, respectively, without affecting plasma glucose. All fatty acids containing 3–8 carbon atoms stimulated insulin production, valerate being the most effective. Acetate, aceto-acetate and beta-hydroxybutyrate had no effect on plasma insulin. Infusion of glucose was much less effective than propionate, butyrate or valerate and there was no correlation between fasting glucose levels and fasting plasma insulin. Infusion of propionate into rats, rabbits or pigs had no effect on the plasma insulin, whereas both propionate and butyrate provoked a rise in plasma insulin in the cow. It was concluded that propionate and butyrate are stimulators of insulin secretion in the sheep and cow but not of nonruminant species. (Endocrinology 83: 118, 1968)

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 mμg/ml) lasting 1.5-3.5 hr appeared with the onset of deep sleep. Smaller GH peaks (6-14 mμg/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 mμg/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.

Over 50 years ago, Benedict (2) published his extensive monograph on the metabolism of fasting in man, in which he demonstrated that carbohydrate stores provide a small but significant component of the body's fuel for only the first few days. Thereafter, protein and fat are the sole sources of fuel, the former contributing 15% of the calories and the latter the balance. The primary role of fat as fuel was apparent to Benedict and his contemporaries; it is plentiful and expendable. The significance of the protein requirement, however, was less clear; in fact, it was not fully understood until nearly 20 years later when the obligatory dependence of the central nervous system on glucose was firmly established (3). Since glycogen stores in man were known to approximate only 200 g, it was readily apparent that glucose has to be derived from protein in order to maintain cerebral metabolism during a prolonged fast. More recently, our understanding of the fasted state has been further clarified by the demonstration that free fatty acid is both the major transport form of lipid leaving adipose tissue (4, 5) and a substrate that is

A BST R A C T 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.

A method is described for separation of polyribosomes from as few as 25 isolated Islets of Langerhans, representing about 250 μg of pancreatic tissue. Islets are labeled with [ 3 H]leucine and polysomes are isolated with liver polyribosomes, which serve as carrier and inhibitor of ribonuclease activity. Islets incubated at 37°C for 45 min in 15.5 mM glucose, then pulsed with [ 3 H]leucine, incorporated about 2-3 times more label into nascent peptides on islet polysomes than islets incubated in 2.8 mM glucose. Sucrose gradient analysis of the labeled polysomes indicated that raising the glucose concentration preferentially stimulated synthesis of peptides on trisomes and larger polyribosomes. Islets incubated with [ 3 H]leucine for 15 min incorporated two-thirds of the label into proteins on membrane-bound polysomes. At least 85% of the proinsulin synthesis during this time occurs on membrane-bound polysomes.

Ruminants meet their energy requirements primarily from volatile fatty acids produced by rumen micro—organisms. It has been shown that several of these fatty acids are potent insulin secretagogues both in vivo and in vitro. In the present studies the influence of the plasma free fatty acid level on growth hormone (GH) secretion was investigated in sheep using a variety of antilipolytic agents such as 3,5–dimethyl pyrazole, nicotinic acid, salicylate, and insulin. Inhibition of fat mobilization by these compounds was invariably associated with highly significant elevations of plasma GH levels. This response was correlated with the degree of starvation and the accompanying elevated plasma free fatty acid level and was inhibited by the intravenous infusion of acetate, propionate or a combination of triglyceride and heparin. Dimethyl pyrazole or nicotinic acid failed to elicit a rise in plasma GH during the intravenous infusion of epinephrine (0.5 μg/kg/min) despite a precipitous fall in FFA levels, confirmin...

SummaryA simple reproducible assay for mitochondrial α-GPD has been described. It takes advantage of the ability of INT to accept directly electrons from the dehydrogenase. The assay as described measures accurately enzyme activity using 20 μg of mitochondrial protein, but it could be scaled down for smaller quantities of protein by using smaller reaction volumes and microcuvettes.

SummaryThe effect of intravenous infusion of epinephrine was studied in pigs. FFA increased 8–10-fold within an hour, and this high level was maintained throughout a 6-hour infusion period. During the infusion of epinephrine, the mean plasma insulin concentration remained below 2 μU/ml, despite marked severe hyperglycemia. Within 30 minutes after the withdrawal of epinephrine there was a 4–6-fold increase of plasma insulin. This was accompanied by a gradual decrease of glucose and FFA. By the time blood sugar and FFA fell to preinfusion levels insulin also returned to normal. It appears that epinephrine blocks insulin release normally induced by high blood sugar levels.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.

The synthesis and release of alanine and glutamine were investigated with an intact rat epitrochlaris muscle preparation. This preparation will maintain on incubation for up to 6 hours, tissue levels of phosphocreatine, ATP, ADP, lactate, and pyruvate closely approximating those values observed in gastrocnemius muscles freeze-clamped in vivo. The epitrochlaris preparation releases amino acids in the same relative proportions and amounts as a perfused rat hindquarter preparation and human skeletal muscle. Since amino acids were released during incubation without observable changes in tissue amino acids levels, rates of alanine and glutamine release closely approximate net amino acid synthesis. Large increases in either glucose uptake or glycolysis in muscle were not accompanied by changes in either alanine or glutamine synthesis. Insulin increased muscle glucose uptake 4-fold, but was without effect on alanine and glutamine release. Inhibition of glycolysis by iodacetate did not decrease the rate of alanine synthesis. The rates of alanine and glutamine synthesis and release from muscle decreased significantly during prolonged incubation despite a constant rate of glucose uptake and pyruvate production. Alanine synthesis and release were decreased by aminooxyacetic acid, an inhibitor of alanine aminotransferase. This inhibition was accompanied by a compensatory increase in the release of other amino acids, such as aspartate, an amino acid which was not otherwise released in appreciable quantities by muscle. The release of alanine, pyruvate, glutamate, and glutamine were observed to be interrelated events, reflecting a probable near-equilibrium state of alanine aminotransferase in skeletal muscle. It is concluded that glucose metabolism and amino acid release are functionally independent processes in skeletal muscle. Alanine release reflects the de novo synthesis of the amino acid and does not arise from the selective proteolysis of an alanine-rich storage protein. It appears that the rate of alanine and glutamine synthesis in skeletal muscle is dependent upon the transformation and metabolism of amino acid precursors.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Alanine release from skeletal muscle was increased by fasting (65%), cortisone (145%), thyroxine (200%), and diabetes (185%). Glutamine release was decreased by cortisone (37%) and diabetes (23%) but not significantly affected by fasting or thyroxine. Tissue levels of alanine were unchanged but tissue glutamine levels were markedly reduced (30 to 60%) in all treatment groups. Insulin added in vitro did not affect amino acid release even with preparations obtained from diabetic animals. Inhibition of glycolysis with 0.2 mM iodoacetate had no effect on the rate of alanine and glutamine formation in any treatment group. Pyruvate generation was increased by all treatments even in the presence of the inhibitor. Total skeletal muscle alanine, aspartate, and branched chain aminotransferase, glutamate dehydrogenase, and malic enzyme activities were not significantly altered in any treatment groups. The addition of 10 mM aspartate, cysteine, branched chain amino acids, and serine significantly increased alanine formation, whereas the maximal rate of glutamine formation in the presence of stimulating amino acids was reduced in each treatment groups--the most marked effects were noted with cortisone and diabetic preparations. Although accelerated muscle proteolysis is an important factor regulating alanine formation in skeletal muscle, the redirection of carbon flow from glutamine toward alanine formation observed in fasting, cortisone, thyroxine-treated, and diabetic rats, indicates that factors other than proteolysis also participate in the control of amino acid release from muscle.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Epinephrine reduced the release of alanine and glutamine in a concentration-dependent manner. Measurable inhibition was observed at 10(-9) M epinephrine, and maximal inhibition was obtained at 10(-5) M. Norepinephrine also reduced alanine and glutamine formation and release but the concentration required for maximal inhibition was approximately 100-fold greater than for epinephrine. Isoproterenol (beta agonist), but not phenylephrine (alpha agonist), reproduced the effects of epinephrine, and propranolol (beta antagonist), but not phentolamine (alpha antagonist), blocked the effect of the catecholamine. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate reproduced the effects of epinephrine and theophylline potentiated the effect of submaximal concentrations of the hormone. Glucagon and prostaglandin E2 had no observable effect on amino acid release. Insulin did not modify the inhibition of alanine and glutamine release produced by epinephrine. Alanine and glutamine formation from added precursor amino acids was unaffected by epinephrine or cyclic adenosine 3':5'-monophosphate. Epinephrine reduced alanine formation in muscles obtained from diabetic rats or animals treated with thyroxine or cortisone. These findings indicate that physiological levels of catecholamines reduce alanine and glutamine formation and release from skeletal muscle. This effect is mediated by a beta-adrenergic receptor and the adenylate cyclase system and can be accounted for by an inhibition of muscle protein degradation.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.

The effects of insulin and contraction on glucose transport and metabolism were investigated in rat epitrochlearis muscles in vitro. Insulin dose-response curves showed a threshold (approximately 50 microunits/ml) and saturation-type (approximately 1 mU/ml) kinetics, whereas isometric contraction activated glucose transport and metabolism in a linear fashion with no evidence of a threshold. Insulin and contraction increased the apparent maximal rate of uptake of the hexose transport system with minimal effect on its apparent Km. The stimulatory effects of insulin and contraction were additive; similar results were obtained with 2-deoxy-D-glucose. Contraction stimulated glucose transport in three different preparations of muscles depleted of insulin: 1) exhaustively washed for 2 h, 2) rats infused with anti-insulin serum, and 3) chronically (streptozotocin-induced) diabetic rats. Prostaglandin E2 augmented the effect of a submaximal concentration of insulin on glucose transport without exerting any effect by itself but had no effect on contraction-augmented glucose transport. It is concluded that insulin and contraction activate glucose transport and metabolism via independent mechanisms.

An in vitro rat muscle preparation is described that can contract at rates of 12–240 twitches/min. Maximum dF/dt paralleled maximum twitch tension, their ratio being constant at approximately 8 ms for contraction rates of 12-120 twitches/min. Time to peak tension was 8–13 ms, time to peak dF/dt 5–8 ms, and half-relaxation time 4 ms. These parameters were unaffected by rate of contraction or duration of isometric work. Differential ATPase staining demonstrated that 60–65% of the fibers were fast-twitch white, 20% fast-twitch red, and 15% slow-twitch red. The preponderance of fast-twitch fibers correlated with the observed mechanical performance of the muscle. Muscles contracting for 60 min at rates up to 48 twitches/min maintained total adenine nucleotide content (ATP, ADP, AMP) at near resting levels. At higher twitch rates (72–240 twitches/min), total adenine nucleotide content decreased 40%, reflecting exclusively a fall in ATP in the presence of adequate phosphocreatine stores. Adequate oxygenation was reflected by lactate-to-pyruvate ratios in the range of 11–15 at all rates of contraction.

Studies are presented which support the concept that a cell membrane localized glucoreceptor system is involved in the insulin secretory response to glucose and that the specific stimulation of insulin synthesis by glucose reflects effects at both the transcriptional and posttranscriptional level. After 48 hours of fasting the insulin secretory response to glucose is markedly reduced. This reduction is overcome by 24 hours of refeeding carbohydrate, but notprotein or fat, and is blocked by refeeding in the presence of an inhibitor of RNA synthesis. Although these studies clearly demonstrate an inducible glucoreceptor system, they do not permit conclusions regarding either its composition or location in the beta-cell. Phloridzin, a glycoside with a high affinity for glucose-carrier systems in plama membranes, stimulated basal insulin secretion sixfold. A large number of plant lectins were tested for ability to stimulate insulin release from isolated islets, and only mushroom lectin did so. The lectin concentration producing half-maximal hormone releaseis 3 x 10-7, a value in good agreement with the dissociation constant forlectin binding. Glucose stimulated insulin sythesis in isolated rat islets was determined to be partially inhibited by actinomycin D. The posttranscriptional effect was determined to be increased initiation of total islet mRNAas well as proinsulin mRNA. To futher quantitate the effect of glucose on proinsulin mRNA, immunoprecipitation of proinsulin synthesizing polysomes was accomplished. It appeared that proinsulin is synthesized on a mRNA accommodating six to eight ribosomes, and the size of the proinsulin mRNA is 10-11 S on sucrose gradients. This unexpectedly large size of the proinsulin mRNA is discussed.

A classification of genetic diabetes mellitus based on abnormalities of carbohydrate metabolism is given below. Progression or regression from one stage to the next may never occur, may proceed slowly over many years or may be very rapid. This classification does not consider the presence or absence of vascular disease, for patients with minimal glucose intolerance or even normal tolerance may have angiopathy.

Untersuchungen uber die insulinahnliche Aktivitat im Blut (ILA = insulin like aktivity) mit Hilfe der von Martin und Renold entwickelten biologischen Bestimmungsmethode am epididymalen Fettanhang der Ratte fuhrten zu Ergebnissen, die keine Ruckschlusse auf den wahren Gehalt des peripheren Blutes an Insulin zuliesen und Wiederspruche aufwiesen, wenn die Untersuchungen unter den verschiedensten Stoffwechselbedingungen durchgefuhrt wurden. Wie jede biologische Bestimmungsmethode, so ist auch der sog. „fat pad assay“ fur Insulin unspezifisch und mit einer betrachtlichen Fehlerbreite behaftet. Diese Tatsache wird auch von den Arbeitsgruppen, die sich mit dieser Methode befassen, hervorgehoben, jedoch nicht selten bei der Bewertung der erhaltenen Ergebnisse die erforderliche kritische Betrachtung auser acht gelassen.

A method is described for separation of polyribosomes from as few as 25 isolated Islets of Langerhans, representing about 250 mug of pancreatic tissue. Islets are labeled with [(3)H]leucine and polysomes are isolated with liver polyribosomes, which serve as carrier and inhibitor of ribonuclease activity. Islets incubated at 37 degrees C for 45 min in 15.5 mM glucose, then pulsed with [(3)H]leucine, incorporated about 2-3 times more label into nascent peptides on islet polysomes than islets incubated in 2.8 mM glucose. Sucrose gradient analysis of the labeled polysomes indicated that raising the glucose concentration preferentially stimulated synthesis of peptides on trisomes and larger polyribosomes. Islets incubated with [(3)H]leucine for 15 min incorporated two-thirds of the label into proteins on membrane-bound polysomes. At least 85% of the proinsulin synthesis during this time occurs on membrane-bound polysomes.