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Culture of rat pancreatic islets with interleukin-1 (IL-1) results in up-regulation of the inducible isoform of nitric oxide synthase and overproduction of nitric oxide (NO). This is associated with reversible inhibition of both glucose-induced insulin secretion and islet glucose oxidation, and these effects are prevented by the inducible nitric oxide synthase inhibitorN G-monomethylarginine. IL-1 also induces accumulation of nonesterified arachidonic acid in islets by an NO-dependent mechanism, and one potential explanation for that effect would involve an IL-1-induced enhancement of islet glycolytic flux. We have therefore examined effects of IL-1 on islet glycolytic utilization of glucose and find that culture of islets with IL-1 in medium containing 5.5 mm glucose results in suppression of islet glucose utilization subsequently measured at glucose concentrations between 6 and 18 mm. The IL-1-induced suppression of islet glucose utilization is associated with a decline in islet glucokinase mRNA content, as determined by competitive reverse transcriptase-polymerase chain reaction, and in glucokinase protein synthesis, as determined by immuoprecipitation experiments, and all of these effects are prevented byN G-monomethylarginine. These findings suggest that IL-1 can down-regulate islet glucokinase, which is the primary component of the islet glucose-sensor apparatus, by an NO-dependent mechanism. Because reductions in islet glucokinase levels are known to cause a form of type II diabetes mellitus, these observations raise the possibility that factors which increase islet NO levels might contribute to development of glucose intolerance.

D-chiro-inositol is a rare inositol isomer present in inositol phosphoglycans which are proposed mediators of insulin action. To study D-chiro-inositol metabolism in diabetes mellitus, a sensitive and specific assay was developed using negative-ion chemical ionization gas chromatography/mass spectrometry. Median urinary D-chiro-inositol excretion, which was 2.1 mumol/day in nondiabetics, was substantially increased to 12 mumol/day in non-insulin-dependent diabetes (P < 0.0001) and to 74 mumol/day in insulin-dependent diabetes (P < 0.0001). Urinary D-chiro-inositol was strongly correlated with fasting plasma glucose (r = 0.568, P < 0.0001), glycated hemoglobin (r = 0.529, P < 0.0001), and urinary glucose (r = 0.368, P = 0.01). The renal clearance of D-chiro-inositol was selectively elevated in both non-insulin-dependent and insulin-dependent diabetes when compared with the clearances of L-chiro-inositol or myo-inositol and exceeded the glomerular filtration rate in 71% of the diabetics but in none of the nondiabetics. In poorly controlled diabetic patients insulin treatment reduced urinary D-chiro-inositol losses by 63% and increased plasma levels by 8.8-fold. The metabolism of D-chiro-inositol is abnormal in diabetes and appears to be influenced by short- and long-term metabolic control.

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 40%. 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.

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 μg. per milliliter (approximately 1 μM). 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-Iabeled 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 μg per milliliter. The total number of A. bisporus PHA-B binding sites per islet was approximately 2 × 1010. 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.

The intraportal injection of 350 to 1,000 isolated islets into streptozotocin-diabetic rats immediately normalized (approximately 24 hours) fasting plasma glucose and insulin levels. Polyuria, polydipsia, and hyperglucagonemia disappeared more gradually over a 2-to-12-week period--the time required for normalization varying with the severity of the diabetes and the number of islets transplanted. In long-term islet-transplanted rats (greater than five months), the hepatic insulin and glucagon reserves averaged 50 per cent and 25 per cent, respectively, of the corresponding normal pancreatic hormone content. Glucagon was increased slightly in the pancreas of streptozotocin-diabetic rats and decreased considerably in transplanted animals. However, total pancreatic glucagon (i.e. pancreatic and hepatic reserves) in transplanted animals was the same as the pancreatic content of normal control rats, indicating the presence of feedback control mechanism(s) in the regulation of pancreatic glucagon reserves. Long-term transplanted islets demonstrated well-granulated A-, B-, and D-cell movement out of the vascular space and the formation of narrow intercellular spaces and junctional complexes with surrounding hepatocytes.

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.

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.

A simple, sensitive method for measuring N τ -methylhistidine in biological samples using a deuterated internal standard and methane chemical ionization gas chromatography-mass spectrometry is described. After sample preparation, a single analysis can be completed in 3 min; analysis in duplicate, including sample preparation for 40 samples, can be completed at a rate of 15 min per sample. Nanomole amounts of N τ -mmethylhistidine in urine or plasma samples are determined with a precision of 0.5%. Picomole amounts, released during in vitro rat epitrochlaris muscle incubations, are measured with a precision of 10%.

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.

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.

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.

This investigation is designed to explore the potential role of apoVLDL as a precursor of a polypeptide component of human LDL. Attention was directed to the chromatography-defined Sf-I polypeptide fraction of apoVLDL, which has been previously shown to be immunologically and chemically indistinguishable from the major component of apoLDL1–3. In VLDL isolated from blood drawn within two hours following 75Se-SM injection, the Sf-I polypeptide fraction of apoVLDL 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° 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 apoVLDL and apoLDL. These findings suggest that a component of the Sf-I polypeptide fraction of apoVLDL 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 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.

A new and highly sensitive fluorimetric assay for hexokinase (HK) isoenzymes which requires only 5 μg. of tissue protein is described. This assay has been used to examine the alterations in the HK isoenzymes of rat skeletal muscle and adipose tissue under a variety of physiologic conditions. In both tissues, HK-II declined progressively with increasing age, reaching plateau levels in animals larger than 300 gm., whereas HK-I remained constant. HK-II activity decreased markedly in both tissues of young rats after a twenty-four to forty-eight hour fast and was restored after two days of refeeding. HK-I was not affected by the fasting-refeeding regimen. In older rats (300 gm.), adipose tissue HK-II did not fall during fasting but exhibited a significant rise when refeeding was instituted. “Pulsing” the fasted rat with periodic episodes of hyperglycemia (0.5 gm. glucose intraperitoneally every eight hours) did not prevent the fall in HK-II during fasting or restore HK-II activity in the two day fasted animal. Approximately 50 per cent of HK-I, but little or none of HK-II, is bound to subcellular organelles in skeletal muscle. In adipose tissue, however, about one-third of the HK-II and insignificant amounts of HK-I are in the bound form. Ratios of bound to free HK are not altered during aging, but HK-II binding in adipose tissue is decreased during refeeding. Normal mice exhibited a decrease in both adipose tissue HK isoenzymes with aging. Adipose tissue of sixteen week old obese hyperglycemic mice (obob) has lower HK-II and higher HK-I activity than that of normal litter mates, but no differences were detected in obob and normal mice at five weeks of age. The changes in muscle and adipose tissue HK-II activity associated with aging and fasting-refeeding parallel the known changes in insulin sensitivity of these tissues under comparable conditions.

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 effects of dexamethasone and growth hormone on hexokinase (HK) isoenzymes and glucose utilization were studied in adipose tissue from young rats. Dexamethasone (0.1 mg./kg. twice a day) caused hyperinsulinemia with normal blood glucose and marked diminution of adipose tissue HK-II after twenty-four and forty-eight hours. HK-I activity was reduced after twenty-four hours but normal after forty-eight hours. There was no change in HK four hours after a single injection of 0.2 mg./kg. Bovine growth hormone (1 mg. daily) had no effect after twenty-four hours, but increased HK-II after forty-eight hours. Plasma glucose and insulin remained unaltered by GH, and HK-I was also unchanged. Isolated adipocytes showed the same changes as whole fat pads with dexamethasone, aging, fasting and refeeding; GH had no significant effect. HK-I activity under all circumstances was correlated with in vitro lipogenesis from glucose in the absence of insulin, but not with glucose oxidation. Both lipogenesis and oxidation in the presence of insulin (100 μU./1 ml.) werestrongly correlated with HK-II and glucose-6-phosphate dehydrogenase activįties. These results suggest that control of phosphorylation by HK-II may be a mechanism for mediating hormonal actions on insulin-stimulated glucose utilization.

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.

Tubulin represents a major protein in rat pancreatic islets, which averages 0.5% of the total protein content and 6% of the noninsulin protein synthesized under basal metabolic conditions. Glucose increases the synthesis of tubulin twofold to threefold. Tubulin synthesis is also stimulated by adenosine 3':5'-cyclic monophosphate in both the absence and presence of glucose; this effect of adenosine 3':5'-cyclic monophosphate occurs preferential to noninsulin protein synthesis at physiological glucose concentrations. Tubulin synthesis was decreased more than 75% by fasting, an effect prevented by maintaining animals on glucose exclusively. The fasting-induced reduction in tubulin synthesis is corrected in vitro by increasing adenosine 3':5'-cyclic monophosphate levels. These findings parallel changes previously reported in insulin release and suggest that physiological agents can exert their insulin secretory action through an effect upon the rate of tubulin synthesis.

Data obtained in the dog during thyroid perfusion support the hypothesis that glucagon and epinephrine stimulate thyrocalcitonin release via adenyl cyclase activation and consequently by the intracellular level of cyclic adenosine monophosphate. The studies also suggest that agonists acting at alpha adrenergic loci may lead to a reduction in cyclic adenosine monophosphate in the parafollicular thyroid cells. (Endocrinology 88: 1337, 1971)

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.

The involvement of cyclic 3′,5′ AMP has been examined in the regulation of protein synthesis by ACTH, epinephrine and insulin in fat cells isolated from rat epididymal adipose tissue. Cyclic AMP was measured in fat cell suspensions by radioimmunoassay and found to be 7.9 ± 0.63 pmoles/mg protein. The cyclic 3′,5′ GMP level was an order of magnitude lower at 0.46 ± 0.07 pmoles/mg protein and was unaffected by ACTH, epinephrine or insulin. Increasing doses of theophylline, ACTH and epinephrine produced a progressive increase in cyclic AMP levels and glycerol release coupled with a reciprocal inhibition of protein synthesis. For the same elevation of cyclic AMP levels, ACTH inhibited protein synthesis to a greater degree than epinephrine while theophylline produced an inhibition much greater than either of the lipolytic hormones. Dibutyryl cyclic AMP, 0.05–5.0 mM, produced an increasing rate of lipolysis with a concomitant decrease in the rate of protein synthesis. Time course studies revealed that cyclic AM...

Highly purified human placental lactogen in physiological concentrations exhibits significant lipolytic activity on the rat isolated fat cell preparation. The onset of lipolysis is preceded by a 60–90-min lag period and is inhibited by the addition of actinomycin or puromycin to the incubation medium. During the first 2 h of incubation, human placental lactogen (HPL) does not significantly affect glucose or acetate oxidation or incorporation into fatty acids. Following the onset of activated lipolysis, HPL decreases [1-14C]glucose oxidation 30–35%, increases [6-14C]glucose oxidation 30–35% and increases [1-14C]acetate oxidation 50–55%. The incorporation of all precursors into fatty acids is decreased to 25–45% of control levels. These effects on glucose and acetate metabolism are abolished by incubation in the presence of high glucose concentration in order to accelerate free fatty acid re-esterification and prevent free fatty acid accumulation. Furthermore, they can be reproduced in the absence of HPL by the addition of palmitate to the medium, suggesting the changes in glucose and acetate metabolism are secondary to the adipokinetic effect of the hormone and the associated accumulation of free fatty acid.

Immunization of rabbits and guinea pigs with glucagon conjugated to Limulus hemocyanin using the bifunctional imidoester diethylmalonimidate as the coupling agent resulted in the production of high titered glucagon antiserum. The production of acute glucagon deficiency in the fasted rat by the intravenous injection of glucagon antiserum resulted in significant hypoglycemia. This is the first demonstration of a biologic effect of acute glucagon deficiency and supports the contention that glucagon plays an important regulatory role in the maintenance of euglycemia in the starved rat. Similar effects were not noted in fed rats, suggesting that the hormone does not have a comparable gluco-regulatory function in the fed rat. Glucagon antibodies also inhibited the lipolytic effects of the hormone on the isolated rat epididymal fat cell. The ability to induce a specific and isolated deficiency of glucagon in the intact animal should permit elucidation of the physiological role of the hormone in the maintenance o...

Presentation or Case A 72-year-old man was admitted to the hospital because of fever. One month previously he was evaluated on the Neurology Service because of ataxia. Physical examination revealed bilateral cerebellar deficits. The urine gave a trace-positive test for protein; the sediment contained 3 red cells, 2 white cells and occasional finely granular and hyaline casts per high-power field. The hematocrit was 38.5 per cent; the white-cell count was 5600, with 83 per cent neutrophils. The urea nitrogen was 18 mg per deciliter. The folic acid was 3.8 ng (normal, 6 to 15 ng); the vitamin B12 was . . .

Guanosine 3′, 5′ cyclic monophosphate (cGMP) and adenosine 3′,5′ cyclic monophosphate (cAMP) were measured in the cerebellum and cerebral cortex of mice after treatment with oxotremorine and atropine. Oxotremorine treatment results within 3–5 minutes in a 70% increase in cGMP in cerebral cortex and cerebellum. Pretreatment with atropine prevents the oxotremorine induced rise in cGMP. Following treatment with atropine alone, cGMP levels rise in cerebral cortex and fall in cerebellum. Atropine has no effect on cAMP levels, but oxotremorine decreases cAMP levels in both cerebral cortex and cerebellum. These results are interpreted as indicating that 1) cGMP is involved in cholinergic neurotransmission, and 2) cGMP and cAMP have idependent functional roles in the central nervous system.

1. 1. The kinetics of tracer amino acid appearance in the intracellular amino acid pool and in cellular protein was examined under steady-state conditions in the intact rat-diaphragm preparation and in isolated guinea pig and rabbit-lymph node cells. 2. 2. The concentration ratio of tracer amino acid between the extracellular and intracellular amino acid pools approached equilibrium exponentially whereas incorporation into cellular protein proceeded linearly from the earliest time period studied. 3. 3. A theoretical formulation is presented predicting the kinetics of amino acid incorporation into cellular protein if the total intracellular amino acid pool were an obligatory intermediate in protein synthesis. 4. 4. The marked deviation of experimental observations from those theoretically predicted has been interpreted to indicate functional heterogeneity (compartmentation) of the intracellular amino acid pool.

During two successive three-week periods, seven obese subjects were fed isocaloric diets, the first low, the second high in carbohydrate. In all, basal plasma insulin levels decreased 50 per cent on the low-carbohydrate diet and increased on the high-carbohydrate diet. Three obese subjects were fed, during three successive four-week periods, 1500-calorie diets with high, then low, and then high-carbohydrate content. Basal plasma insulin levels were significantly reduced on the low-carbohydrate diet. Refeeding of the high-carbohydrate diet, despite continued weight loss, resulted in markedly increased basal plasma insulin. In both protocols, most patients also exhibited a decreased insulin secretory response to oral glucose when on a low intake of carbohydrate and an increased response on a high intake. Thus the hyperinsulinemia characteristic of obesity may be a result, in part, of dietary factors rather than exclusively a consequence of the insulin antagonism associated with obesity.

Antiserum specific to porcine ACTH was used to study nonpituitary tumors from patients with hyperadrenocorticism by the fluorescent antibody technique in order to characterize further a substance in the tumors which is biologically similar to ACTH. These tumors stained specifically for ACTH by this technique. The pituitaries of 2 of the patients contained lower than normal amounts of ACTH and the plasma of the patients contained increased levels of ACTH-like activity by bio-assay. One of the tumors with biological activity did not stain. The histopathology of these tumors and its relation to the ACTH content of the tumors is discussed. From the data presented, it would seem that certain tumors are capable of producing a substance biologically and immunologically similar to ACTH which suppresses the normal ACTH-producing mechanism in the pituitary.

(1) Acute insulin deficiency was produced in rats by the intravenous administration of potent anti-insulin guinea pig serum. Plasma glucose and nonesterified fatty acid (NEFA) levels rapidly increased to an excess of 350 mg. per cent and 1,200 μEq/L. respectively, and were kept elevated by repeated injections of anti-insulin serum. (2) Hepatic fatty acid synthesis was determined by measuring the incorporation of acetate-1-C-14, acetyl-1-C-14 CoA, and malonyl-1,3-C-14 CoA into fatty acid by cell-free liver homogenates. Within 90 min. after the induction of acute insulin deficiency, acetate and acetyl CoA incorporation were markedly decreased, but malonyl CoA conversion to fatty acids was unaffected, indicating a block in lipogenesis at the acetyl CoA carboxylase step. (3) The acute inhibition of carboxylase appeared to be a feedback mechanism since significant increases in hepatic tissue NEFA were observed when fatty acid synthesis decreased, and similar patterns of inhibition could be reproduced in normal rat liver homogenates by addition of either palmitate or palmityl CoA. (4) Hepatic protein synthesis, measured by the incorporation of lysine-U-C-14 did not become impaired until three hours after the induction of acute insulin deficiency. The delayed onset of inhibition and the failure to observe impairment of protein synthesis following the in vitro addition of palmitate, suggest that the factor(s) responsible for impaired hepatic protein synthesis following acute insulin deprivation differ from those causing impaired lipogenesis under similar conditions. (5) In contrast to previous reports using chronically insulin deprived animals (alloxan diabetes), the protein synthesizing activity of both soluble and microsomal fractions was impaired following acute insulin deprivation.

Sensitive and specific radioimmunoassays have been developed for cyclic adenosine 3',5'-monophosphate (cAMP), cyclic guanosine 3',5'-monophosphate (cGMP), cyclic inosine 3',5'-monophosphate (cIMP), and cyclic uridine 3',5'-monophosphate (cUMP). These assays are based upon competition of the cyclic nucleotide with isotopically labeled cyclic nucleotide derivatives for binding sites on specific antibody. Antibodies to the cyclic nucleotides were obtained by immunizing rabbits with an antigen prepared by conjugating the 2'-O-succinyl derivative of the cyclic nucleotide with protein (human serum albumin or keyhole limpet hemocyanin). High specific activity derivatives of the cyclic nucleotides (g150 Ci per mmole) were prepared by iodinating (125I) the tyrosine methyl ester derivative of the succinylated cyclic nucleotide. Free and antibody bound 125I-labeled cyclic nucleotides were separated by either the "second antibody" precipitation technique or (NH4)2SO4 fractionation. Binding equilibrium was reached in 24 hours, but sensitive and reproducible assays were obtained after 4 to 6 hours of incubation. The sensitivity and assay range for the various radioimmunoassays are (picomoles per tube): cAMP, 0.01 to 2; cGMP, 0.01 to 1.0; cIMP, 0.12 to 10.0; and cUMP, 0.1 to 10.0. Structurally related purine and pyrimidine nucleotides and nucleosides exhibited ≤0.005% competitive binding in all immunoassays except cIMP which showed 0.35% competitive binding in the cGMP assay, cGMP 1% in the cIMP assay, and cIMP 1% in the cUMP assay.

The utilization of glucose by striated muscle of the diabetic animal is markedly impaired. Whether this is a consequence of decreased cellular permeability to glucose or a diminution in the ability of muscle to phosphorylate glucose has not been resolved, since previous investigations have been concerned with measurements of utilization rates, a parameter which does not enable one to distinguish between transport and subsequent phosphorylation. Recent observations on intact animals (1) suggested that both processes are impaired in the diabetic state. A further exploration of this problem has been carried out in vitro with an intact rat diaphragm preparation with the glucose analogue 2-deosyglucose. This sugar is phosphorylated by hexokinase as rapidly as is glucose,’ but the phosphorylated product, 2-deoxyglucose g-phosphate, is not acted upon appreciably by other muscle enzymes and hence accumulates within the cell. These properties permitted the investigation of cellular penetration and phosphorylation as separate events. In the present paper it will be shown that there is a disturbance of both penetration and phosphorylation in diabetic muscle. The former is caused by a lack of insulin, whereas the latter can be attributed, at least in part, to the action of adrenal cortical secretions. Additional observations are recorded which indicate that the cut hemidiaphragm preparation is much more permeable to sugars than the intact preparation.*

Six cases of ACTH-secreting tumors—which are shown to secrete additional hormonal substances—are reported. The high incidence of this phenomenon among our 15 observed cases with ACTH-secreting tumor suggests that poly-humoral secretion in ACTH-secreting tumors is more common than previous reports would indicate. Although several of our cases are unique in that the particular combination of endocrine substances produced has not been previously reported, the tumor types (islet-cell carcinoma, carcinoid, pheochromocytoma, oat-cell carcinoma) are in general those which may be endocrine active in a variety of ways. Ectopic corticotrophin production arises principally in tumors of endocrine tissue, neuroendocrine tissue, endocrine dependent tissue and in oat-cell carcinoma of the thorax. The possibility that oat-cell carcinoma of the lung and mediastinum may be a more malignant variety of carcinoid tumor is not proven but should not yet be discounted.

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.

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.

Studies were designed to determine the effect of glucose on RNA metabolism in isolated Islets of Langerhans. [3H]Uridine incorporation into RNA by isolated islets was linear for 2 hours and was increased 2-fold by raising glucose in the incubation media from 2.8 to 15.3 mm. Previously incubating islets with [3H]uridine at 2.8 mm glucose and then transferring them into 15.3 mm glucose in the absence of [3H]uridine showed that [3H]uridine incorporation into RNA was stimulated by high glucose independent of effects on the intracellular transport of uridine. Glucose also stimulated the uptake and phosphorylation of uridine and increased the islet UTP pool size, but under the conditions of these studies the relative specific activity of the UTP pool was the same at 2.8 or 16.6 mm glucose. Since high glucose did not affect the rate of degradation of islet RNA, it was concluded that increasing the glucose concentration in the incubation medium increased the over-all rate of islet RNA synthesis. Base composition analysis of islet RNA labeled for 1 hour in 32PO4 revealed a guanosine + cytosine content of 29.6% for islets incubated in 2.8 mm glucose and a guanosine + cytosine content of 45.6% for islet RNA synthesized in 16.6 mm glucose. Sucrose gradient analysis indicated that the [3H]RNA synthesized after 1 hour of incubation was heterodisperse (i.e. from 4 S to g45 S), and that glucose stimulated the greater production of large molecular weight RNA. Islets pulsed with [3H]uridine for 1 hour in 16.6 mm glucose and then "chased" with cold uridine for 3 hours showed that much of the large molecular weight RNA was ribosomal precursor. Fractionation of islets into crude nuclear, soluble, and polysomal fractions revealed that glucose increased RNA synthesis in each fraction. After 1 hour of incubation, 65% of the labeled RNA was still in the nucleus. Glucose had no effect on distribution of RNA within these fractions. Sucrose gradient analysis of the RNA extracted from these subcellular fractions revealed a predominant 4 S peak for the soluble fraction, partially degraded RNA in the nuclear fraction, and a prominent 8 to 10 S peak for the polysomal fraction. DNA-RNA hybridization studies showed that RNA synthesized in 16.6 mm glucose hybridized to DNA in greater amounts than RNA labeled in 2.8 mm glucose. Thermal elution profiles showed that the RNA labeled in 16.6 mm glucose dissociated from the DNA at lower temperatures. These results indicate that glucose stimulates the over-all rate of islet RNA synthesis and alters the nature of the RNA species synthesized.

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) were measured in mammalian tissues and fluids by radioimmunoassay in a variety of physiological and pharmacological conditions. Basal concentrations and responses to hormonal stimuli were in close agreement with results from previous studies using other assay techniques. Cyclic nucleotide phosphodiesterase treatment of urine and trichloroacetic acid extracts of various tissues completely destroyed the immunoreactive material in each assay except for a tissue blank in kidney, liver, and skeletal muscle of 24%, 40%, and 15%, respectively, in the cGMP assay. The nature of this cross-reacting substance has not been identified. Hormonal stimuli causing marked increases in cAMP in vivo or in vitro did not affect the level of cGMP, indicating that the concentration of each of these nucleotides is controlled independently. Adrenalectomy caused an increase in cGMP in skeletal muscle, but did not affect cAMP in this or other tissues. The concentration of cGMP in skeletal muscle from adrenalectomized rats reverted toward normal after treatment with dexamethasone. Adenylate cyclase and guanylate cyclase activities in subcellular fractions were estimated by immunoassay of the amount of cAMP and cGMP formed from ATP and GTP, respectively. The high specificity and sensitivity of the cAMP and cGMP immunoassays allowed detection of cyclic nucleotide in the supernatant of the reaction mixture without prior concentration or separation procedures. No difference in adenylate cyclase activity in liver plasma membranes was observed between this technique and that of measuring conversion of [α-32P]ATP to [32P]cAMP. Guanylate cyclase activity was detectable in soluble fractions from liver and renal cortex and was not affected by glucagon, dexamethasone, thyroxine, or sodium fluoride added in vitro.

Plasma insulin responses to oral glucose (100 gm.) and intravenous tolbutamide (1.0 gm.) before and after dexamethasone were determined in normal, obese nondiabetic, normal-weight diabetic and obese diabetic subjects. Fasting plasma insulin levels were significantly higher in obese nondiabetic and obese diabetic subjects than in normalweight nondiabetic and diabetic individuals. Total plasma insulin response to oral glucose in obese nondiabetics was threefold greater, in obese diabetic 2.2-fold greater, and in normal-weight diabetics 1.7-fold greater than that observed in normal individuals. When the plasma insulin responses of obese diabetic and nondiabetic subjects were measured at comparable blood sugar levellN insulin secretion was demonstrated to be significantly impaired in the diabetic group. The initial plasma insulin response (i.e., thirty-minute level) to glucose of diabetic subjects is markedly impaired when compared to normal-weight and obese nondiabetic individuals. Total plasma insulin response to intravenous tolbutamide was fourfold greater in obese nondiabetic and obese diabetic subjects than in normal-weight nondiabetic and diabetic individuals. Following dexamethasone (2 mg. q.i.d. × 2), plasma insulin response to intravenous tolbutamide was increased fivefold in normal subjects, threefold in obese nondiabetics, 1.6-fold in obese diabetics and twofold in nonobese diabetics. The results of these studies indicate that obesity is associated with a hypersecretory insulin response to both tolbutamide and glucose. The presence of overt diabetes melitus indicates an impairment in insulin response in both obese and nonobese individuals when compared to their appropriate nondiabetic controls. Dexamethasone provokes a marked compensatory insulin response in nondiabetics and makes more apparent the impaired secretory capacity of diabetic subjects. Maturity-onset diabetes in nonobaese individuals appears to be a consequence primarily of insulin deficiency. In obese diabetic individuals, however, even though secretory capacity may be significantly greater than observed in nonobese diabetics, impaired carbohydrate tolerance develops because insulin antagonism associated with obesity per se.

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.

A 53 year old woman presented with diabetes mellitus, hyperglucagonemia (600 to 1,500 pg/ml), clinical hyperparathyroidism and an abdominal mass diagnosed on biopsy as an islet cell carcinoma. Glucagon content of the tumor was 0.78 μg/g wet weight. Hourly blood samples during a 24 hour period revealed a direct correlation between plasma glucose and glucagon. The oral administration of glucose paradoxically increased whereas the intravenous administration decreased plasma glucagon. Circulating glucagon levels were markedly increased with arginine and epinephrine infusion. Both short- and long-term administration of alpha adrenergic blockade depressed the glucagon response to epinephrine infusion. In contrast, long-term alpha adrenergic blockade increased glucagon secretion despite improved glucose tolerance during a second 24 hour study. Although the patient demonstrated overt clinical and chemical findings of hyperparathyroidism, parathyroid hormone (PTH) was not detected in her plasma. The pattern of tumor growth was consistent with an origin from pancreatic islets. We conclude that (1) the tumor was responsive to physiologic stimuli known to affect glucagon secretion; (2) elevations of plasma glucagon levels with oral and dietary glucose suggest regulation of secretion by intestinal factors; and (3) improvement of glucose tolerance with alpha adrenergic blockade may be related to increased insulin secretion.