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Diets containing high quantities of individual branched-chain alpha-keto acids (BCKAs) or a combination of BCKAs as used for treatment of renal disease were fed to rats. When the diet contained a single BCKA, its concentration was high in plasma and the concentration of its corresponding amino acid was high in plasma and brain. Liver BCKA dehydrogenase (BCKD) was 42% active in control rats. Consumption of diets containing 0.38 mol/kg diet of alpha-ketoisocaproate (KIC), alpha-keto-beta-methylvalerate (KMV), or alpha-ketoisovalerate (KIV) resulted in complete activation of liver BCKD. Consumption of the diet containing the combination of BCKAs increased basal BCKD activity of liver twofold. Muscle BCKD was activated after feeding the KIV diet (2-fold), the KIC diet (3-fold), and the KMV diet (15-fold). Total BCKD activity of liver and muscle was unaffected by dietary treatments. Activation of liver and muscle BCKD by dietary BCKA is consistent with their ability to inhibit BCKD kinase in vitro.

Effects of feeding frequency on liver branched-chain alpha-keto acid dehydrogenase (BCKAD) activity are unknown. In the present study, rats were trained to consume their daily allotment of food in 6 h (meal-feeding). Rats were fed diets containing 0, 9, 25 or 50% casein and after 10 d were killed before or 3 h after the meal. The enzyme in rats fed diets containing 0, 9 and 25% casein was activated three- to sixfold after meal consumption. Previous studies showed that the liver enzyme is essentially fully activated in post-absorptive rats fed an adequate protein diet ad libitum. Meal-feeding an adequate protein (25% casein) diet resulted in a marked decrease in the postabsorptive percentage of active complex compared to ad libitum feeding of the same diet (29 +/- 6% vs. 93 +/- 6% active). Administration of alpha-ketoisocaproate (200 mumol/100 g body weight, an inhibitor of BCKAD kinase) reversed the meal-feeding-induced inactivation of the complex within 10 min. We conclude that the frequency of food intake, in addition to the level of dietary protein, influences the proportion of liver BCKAD in the active state. Inactivation of hepatic BCKAD in rats trained to feed once a day may be an adaptive mechanism that results in increased efficiency of branched-chain amino acid utilization between meals.

In a patient with third trimester abdominal pregnancy with fetal demise, technetium-99m-labeled erythrocytes (99mTc-RBCs) localized the placenta preoperatively, after nonvisualization by ultrasonography and arteriography. Extrauterine placental localization by blood-pool imaging may be useful when ultrasound fails.

Branched-chain alpha-keto acid dehydrogenase (BCKAD) is a multisubunit complex regulated by phosphorylation and is considered to be rate-limiting for branched-chain amino acid (BCAA) metabolism in skeletal muscle. Glucocorticoids increase net protein degradation in muscle; associated with this increased breakdown of muscle protein is an elevated rate of BCAA oxidation. The effects of glucocorticoids on skeletal muscle BCKAD were investigated in different rat models. BCKAD was activated after glucocorticoid treatment (both acutely, within 2 h, and chronically). The amount of enzyme per muscle cell increased after 5 d of cortisone acetate treatment. Insulin administration partially blocked the acute effects of glucocorticoids on muscle BCKAD. Activation was also observed during metabolic acidosis, insulinopenic diabetes mellitus, and endotoxic shock, three conditions characterized by elevated circulating glucocorticoids, increased BCAA oxidation, and increased net protein breakdown. Activation of BCKAD may account for the increased oxidation of BCAA observed during hypercortisolemia. The sequelae of this accelerated catabolism may include increased glutamine and alanine production for gluconeogenesis and provision of ATP for muscle work.

Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones. To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT). HPT cells showed specific binding of IGF-1, insulin, and EGF. IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (alpha-IR3). Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking. IGF-1, insulin, and EGF stimulated [3H]thymidine incorporation by 77, 73, and 87%, respectively. Half maximal stimulation by IGF-1, insulin, and EGF were produced with 4 X 10(-9) M, 2.5 X 10(-8) M, and 8 X 10(-10) M concentrations of these hormones. Alpha-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation. EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively. These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor.

The free amino acid content of diaphragm muscles of control and diabetic rats was studied 5 days after the injection of streptozotocin. Muscles were prepared for analysis either immediately after sacrifice or following incubation in balanced salt solution containing 5.5 mM glucose, with or without an electron acceptor, 0.02 mM methylene blue. Diaphragms of diabetic rats contained significantly more free taurine, glutamate, and branched chain amino acids than the controls at sacrifice, and significantly less glutamine, serine, asparagine, lysine, arginine, histidine, threonine, citrulline, and carnosine. Alanine decreased in plasma of diabetic rats but not in diaphragms before incubation. Hemidiaphragms of diabetic rats produced less alanine and more glutamate during incubation than controls. After incubation they contained less than half as much alanine and glutamine and twice as much glutamate than the controls, having released approximately 40% less alanine and 25% more glutamate into the medium than the controls. Glutamine release was not significantly different between the two groups. Methylene blue increased the free alanine content in the tissue water as well as alanine release by control and by diabetic muscles; the glutamate content of muscles decreased concomitantly. The effects of methylene blue were greater in the diabetic group. Branched chain amino acid release by diabetic muscles decreased during incubation with methylene blue. Muscles of diabetic rats contained more alpha-ketoglutarate than the controls after incubation with or without methylene blue. Methylene blue increased the alpha-ketoglutarate content of muscles and its release into the medium, the effect being greater in diabetics than in controls. Hemidiaphragms from diabetic rats released less pyruvate during incubation than controls, while lactate release by the two groups was not significantly different. Incubation with methylene blue caused a marked increase in pyruvate release by diabetic muscles, and a lesser stimulation in controls; lactate release increased in both groups. After incubation the lactate/pyruvate ratio in muscles was lower in the methylene blue treated group. The in vitro effect of 0.02 mM phenazine methosulfate on alanine production was similar to that of methylene blue. The data is compatible with the hypothesis that the NADH/NAD ratio may exert a restraining effect on alanine production and release by muscle. The progressive increase in this ratio may play a role in the eventual deceleration of gluconeogenesis during a prolonged fast and may restrain this process in uncompensated diabetes.

A patient with clinical and biochemical evidence of Cushing's disease and severe hyperlipidemia underwent an adrenal imaging procedure with NP-59 (6 beta-[131I]iodomethyl-19-norcholesterol), without visualization of either gland. Correction of the hyperlipidemia followed by repeated adrenal imaging resulted in bilateral visualization. A pituitary tumor was removed at surgery, confirming the diagnosis of Cushing's disease.

Radionuclide bone imaging can be of value in assessing the osseous changes around porous-coated femoral prostheses. Scintimetry appears to be a promising method for determining if radiodensities seen in radiographs of long-term prostheses are indicative of accelerated bone turnover or represent stable osseous structures. In dogs, the periosteal reaction which often occurs within two months of joint surgery masks endosteal-intramedullary osseous changes, including bone ingrowth into the porous coating.

Parenteral administration of iron as the complex with nitrilotriacetate resulted in moderate iron deposition in the renal cortex along with heavy deposition of iron in liver, heart, and exocrine pancreas. Iron treated rats exhibited slight but significant changes in serum chemistries consistent with mild renal insufficiency: increased chloride and creatinine and decreased sodium, calcium and CO2 content as a group with isolated increases in urea nitrogen (2/9) and phosphorus (3/9). Serum albumin was decreased and the normal correlation of serum calcium with serum albumin was abolished by iron overload. Although food intake/g body weight was similar in the two groups, both water intake and urine output relative to solute load were increased in iron-overloaded rats. We conclude that the renal insufficiency occasionally reported in human hemochromatosis may reflect a direct nephrotoxic action of iron.

The oxidation of 4-methyl-2-oxopentanoate (alpha-ketoisocaproate) by rat liver mitochondria was shown to be independent of exogenous coenzyme A and exogenous NAD+. Carnitine stimulated the oxidation of 4-methyl-2-oxopentanoate in rat liver homogenates and mitochondria; octanoate, DL-octanoylcarnitine, 4-pentenoate, and 3-methylbutyrate (isovalerate) were inhibitory, and 2-bromopalmitate had no effect. Addition of carnitine was found to increase the export from the mitochondria of acylcarnitines derived from 4-methyl-2-oxopentanoate in the presence of absence of 4-pentenoate but not in the presence of octanoylcarnitine. It is concluded that the branched chain oxoacid dehydrogenase is localized on the inner surface of the inner mitochondrial membrane and that it is regulated in part by the intramitochondrial levels of branched chain fatty acyl-CoA esters and/or free CoA.

This is a correlative study of ultrastructural changes in the myocardium and the blood lead levels (BLLs) in acute lead toxicity in mice. No substantial ultrastructural changes were observed with BLLs of less than 20 mug/100ml. Blood lead levels of 20 mug/100ml and above were associated with nuclear chromatin clumping and nucleolar disorganization. Sarcotubular dilation and mitochondrial changes characterized by mitochondrial enlargement, cristal disorientation, and an increase in intramitochondrial matrix were observed with BLLs of 40 mug/100ml and over. Blood lead levels of 60 mug/100 ml and above were associated with local myofibrillar degeneration, focal intercalated disk disjunction, and interstitial cell changes characterized by prominent endoplasmic reticulum and the appearance of cytoplasmic lysosome-like dense bodies. Our findings indicate a differential sensitivity of various myocardial cell organelles to lead and suggest that the changes observed are directly related to the BLLs.

Histone-H1 purified from rat skeletal muscle is a relatively potent inhibitor of peptide chain initiation in a cell free system, the rabbit reticulocyte lysate (50% inhibition at approximately 0.4 microM). H1 does not inhibit formation of the ternary complex nor its attachment to 40S ribosomes; the data are compatible with H1 binding to mRNA. The inhibition shows mRNA selectivity: translation of beta-globin mRNA is more affected than that of alpha-globin mRNA and hepatic albumin mRNA more than total hepatic mRNA. Whether or not histone-H1 plays a role in translational regulation in intact cells is conjectural, it may serve as a useful model for protein-mRNA interactions.

Amino acid availability rapidly regulates protein synthesis and degradation. Increasing amino acid concentrations above the levels found in post-absorptive plasma stimulates protein synthesis in a dose-dependent manner at the level of mRNA translation-initiation and inhibits protein degradation by inhibiting lysosomal autophagy. The anabolic effects of insulin on protein synthesis and protein degradation are exerted at the same sites (i.e., peptide chain initiation and lysosomal stabilization) allowing for a rapid synergistic response when both amino acids and insulin increase after a protein-containing meal. In perfused liver preparations, protein anabolic effects are exerted by a group of amino acids acting in concert. The BCAA are among the amino acids required for stimulation of hepatic protein synthesis, but there is no evidence that BCAA or leucine alone are effective. Leucine alone is an important inhibitor of hepatic protein degradation, but maximal inhibition requires in addition several other regulatory amino acids. In heart and skeletal muscle in vitro, increasing the concentration of the three BCAA or of leucine alone reproduces the effects of increasing the supply of all amino acids in stimulating protein synthesis and inhibiting protein degradation. Skeletal muscle is the largest repository of metabolically active protein and a major contributor to total body nitrogen balance. Supplying energy alone (i.e., carbohydrate and lipids) cannot prevent negative nitrogen balance (net protein catabolism) in animals or humans; only provision of amino acids allows the attainment of nitrogen balance. In rats and in humans nourished parenterally, provision of balanced amino acid solutions or of only the three BCAA cause similar improvements in nitrogen balance for several days. There is some evidence that infusions of leucine alone can stimulate muscle protein synthesis in vivo; the effect may be transitory and was not observed by all investigators; provisions of excess leucine alone does not seem to affect total body or muscle protein degradation in vivo. In postabsorptive rats, in vivo, infusion of the three BCAA together stimulates muscle protein synthesis as much as the infusion of a complete amino acid mixture or of a mixture of essential amino acids; the in vivo effect requires coinfusion of glucose or of small (physiological) doses of insulin, suggesting synergism between insulin and amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Rats (80-100 g) were fasted for 48 hours prior to receiving i.v. infusions of [(14)C]tyrosine (0.5 ml/hour)in 0.9% saline or 2.8 M glucose or 2.5 M glucose with 0.1 M each of leucine, isoleucine and valine. The incorporation of [(14)C]tyrosine into proteins of different muscles was measured at the end of six hour infusions. Heart, soleus and diaphragm muscles incorporated more tyrosine into proteins than psoas and gastrocnemius under all conditions. Infusions of glucose alone did not accelerate tyrosine incorporation significantly in the muscles tested. The addition of branched chain amino acids to glucose markedly increased tyrosine incorporation into proteins in the diaphragm, and to a lesser extent in soleus and psoas. No effect was detectable in heart and gastrocnemius. Previous reports of differences in protein synthetic rates between different types of muscles are confirmed. Additionally, the data suggest that branched chain amino acids may be rate limiting for muscle protein synthesis in fasted rats in vivo, and that the responsiveness to branched chain amino acids differs between muscles.

The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, increases after a protein meal. The effect of amino acid mixtures on hepatic and renal ODC activity and polyamine content was studied in postabsorptive and 72-hour fasted rats. Fasting decreased ODC activity in liver and in kidney by approximately 50%. Hepatic ODC activity increased tenfold 4 hours after intraperitoneal injection of either 1 g/kg of a synthetic mixture of 17 amino acids or of casein hydrolysate to fed rats and about 20-fold in fasted rats. Renal ODC activity increased four- and tenfold respectively. A mixture of glutamate, aspartate, and alanine at concentrations given in the hydrolysate reproduced the full amino acid effect. No amino acid was effective when given alone, nor were mixtures of the other amino acid constituents of the hydrolysate. Glutamate + alanine was ineffective as were glucose or various combinations of arginine, ornithine, aspartate and NH3. Ornithine + glutamate or aspartate + glutamate were active but stimulated less than aspartate + glutamate + alanine. Hepatic and renal putrescine content increased in parallel with ODC activity. The data suggest that specific amino acids possess the full ODC-stimulating capability of a high quality protein and that polyamine synthesis is linked to urea cycle activity.

Insulin receptors have been demonstrated on activated but not resting T-lymphocytes. We compared insulin binding to several T- and B-cell tissue culture lines transformed by either radiation or lymphotrophic Herpes virus. Two T-cell lines derived from mouse lymphomas, one T-cell derived from the cotton top marmoset and one mouse B-cell line bound, at most, 3% of a tracer dose of [125I]insulin. In contrast, positive B-cell line (1605 L) derived from a leukemic cotton top marmoset, bound 40% of tracer [125I]insulin. The binding was of high affinity (1.9 nM-1), displayed analogue specificity characteristic of the insulin receptor and receptor number per cell was 16,600. The data show that a transformed B-lymphocyte binds substantial levels of insulin. The magnitude of such expression might be related to the fact that this B-cell line also expresses Epstein-Barr virus.

An improved method for the evaluation of aldosterone antagonists in adrenalectomized, glucocorticoid-treated rats is described. The method involved assessing the pharmacological effects of spironolactone and potassium canrenoate and comparing them with the action of prorenone and potassium prorenoate, respectively. Adrenalectomized rats were pretreated with fluocortolone caproate (10 mg/kg s.c.), a long-acting glucocorticoid, immediately after surgery. Fluocortolone (1.25 mg/kg s.c.), a short acting preparation, was administered 4 days after this treatment. On the 5th day after adrenalectomy, the actual diuresis experiment was performed. The rats received a continuous i.v. infusion of aldosterone [1 microgram/(kg x h)] for 10, 15 or 20 h. Spirnolactone or prorenone (6.7, 13.4 or 26.8 mg/kg of each steroid) were administered in single oral doses 1 h before or 4 h after the start of the i.v. infusion. Potassium canrenoate and potassium prorenoate (1.9, 3.8, or 6.7 mg/(kg x h) of each compound) were infused intravenously over 10 or 15 h. Urine was collected in 1 h fractions and the anti-aldosterone activity was assessed by the ability of the compounds to reverse the aldosterone effect on the Na/K ratio. The anti-aldosterone activity of the steroids studies was clearly detectable with the method described. Prorenone was as potent as spironolactone and potassium prorenoate was on the average 3.9 times as potent as potassium canrenoate. The method appears suitable for the characterization of the time course and duration of anti-aldosterone activity and for the calculation of relative potencies in comparison to standard compounds.

The inhibitory effect of epinephrine on basal and tolbutamide-stimulated insulin release was studied in 5 patients with hyperinsulinemic hypoglycemia. Epinephrine inhibited both basal and tolbutamide-induced insulin release in patients with beta-cell adenoma and hyperplasia, but failed to inhibit insulin release in a patient with beta-cell carcinoma. The inhibition of basal insulin with epinephrine was maximum in patients with beta-cell hyperplasia. This differential inhibitory effect of epinephrine on insulin release may prove to be a useful screening test in the preoperative diagnosis of the nature of the lesion producing hyperinsulinemia.

In vitro catabolism of branched-chain amino acids, leucine and valine, was investigated using diaphragm muscles from normal, streptozotocin-diabetic and overnight fasted rats. Oxidation and transamination of [1-14C] branched-chain amino acids were both stimulated to a similar extent by diabetes or fasting, when diaphragms were incubated with glucose. Transamination of leucine and valine was increased when diaphragms were incubated with pyruvate; stimulation of transamination was greatest in diaphragms from diabetic rats. Leucine and valine oxidation by control diaphragms was inhibited by pyruvate while it was unchanged or slightly stimulated in diaphragms from fasted or diabetic rats. Thus diaphragms from diabetic rats oxidized two to threefold more branched-chain amino acids than controls when they were incubated with pyruvate. The specific radioactivity of extracellular alpha-ketoisocaproate (KIC; the product of leucine transamination) produced by diaphragms incubated with [14C]leucine was similar for all groups (fed, fasted, or diabetic) in the presence or absence of pyruvate. Oxidation of [1-14C]KIC by diaphragms from fasted or diabetic rats, incubated with glucose, was the same or less than KIC oxidation by control diaphragms. Incubation with pyruvate inhibited KIC oxidation by control diaphragms to a significantly greater degree than that by diaphragms from diabetic or fasted rats. These data suggest the following Flux through branched-chain amino acid transaminase is limited by the availability of amino group acceptors in diaphragms from normal and overnight fasted rats, and to a greater extent in diaphragms from diabetic rats. Flux through the transaminase may be a major determinant of accelerated branched-chain amino acid oxidation by diaphragms in fasting and diabetes. In diaphragms of fasted and diabetic rats, flux through the branched-chain alpha-ketoacid dehydrogenase complex is resistant to inhibition by pyruvate, which is normally observed in controls.

The role of the type I insulinlike growth factor (IGF) receptor in regulating growth of Wilms' tumor (WT) was evaluated by examining the effect of antibody-mediated inhibition of this receptor on tumor growth in cell cultures and as heterotransplants in athymic mice. An antibody to the human type I IGF receptor (alpha IR-3) inhibited 125I-IGF-1 binding and prevented stimulation of thymidine incorporation by IGF-1 in vitro. Intraperitoneal administration of alpha IR-3 to nude mice bearing WT heterotransplants prevented tumor growth for 4 weeks and resulted in partial regression of established tumors. These data indicate the importance of IGF action in control of WT growth in vivo, and suggest potential therapeutic application using antigrowth factor receptor antibodies to block growth factor action.

The protein anabolic effect of branched chain amino acids was studied in isolated quarter diaphragms of rats. Protein synthesis was estimated by measuring tyrosine incorporation into muscle proteins in vitro. Tyrosine release during incubation with cycloheximide served as an index of protein degradation. In muscles from normal rats the addition of 0.5 mM leucine stimulated protein synthesis 36--38% (P less than 0.01), while equimolar isoleucine or valine, singly or in combination were ineffective. The three branched chain amino acids together stimulated no more than leucine alone. The product of leucine transamination, alpha-keto-isocaproate, did not stmino norborane-2-carboxylic acid (a leucine analogue) were ineffective. Leucine and isoleucine stimulated protein synthesis in muscles from diabetic rats.Leucine, isoleucine, valine and the norbornane amino acid but not alpha-ketoisocaproate or beta-hydroxybutyrate decreased the concentration of free tyrosine in tissues during incubation with cycloheximide; tyrosine release into the medium did not decrease significantly. Leucine caused a small decrease in total tyrosine release, (measured as the sum of free tyrosine in tissues and media), suggesting inhibition of protein degradation. The data suggest that leucine may be rate limiting for protein synthesis in muscles. The branched chain amino acids may exert a restraining effect on muscle protein catabolism during prolonged fasting and diabetes.

The effects of streptozotocin(SZ)-induced diabetes on renal ornithine decarboxylase (ODC) activity, the rate-limiting enzyme in polyamine biosynthesis, were studied. Sixteen hours after the injection of SZ, renal ODC activity increased 50% above that of the vehicle-injected controls. The maximum increase in activity--600%--was observed from 24 to 72 h after SZ. Within a week, ODC activity fell below control levels and remained suppressed during a 3 wk follow-up period. Insulin treatment within 10 h of the SZ injection prevented the increase of ODC activity; however, insulin given after enzyme activity had increased did not restore ODC activity to control levels. The early increase of ODC activity occurred in the presence of mild hyperglycemia without ketosis or hyperglucagonemia, but the levels were much higher in severely diabetic animals. Adrenalectomy, performed before the initial increase in enzyme activity, prevented the subsequent increase in diabetic animals; however, when adrenalectomy was performed after the enzyme had increased, enzyme activity did not normalize.

Skeletal muscle rapidly develops severe insulin resistance following denervation, although insulin binding is unimpaired. Insulin-stimulated receptor tyrosyl kinase activity was studied in intact and 24-h denervated rat hind limb muscles using three preparations: (a) solubilized insulin receptors incubated +/- insulin with gamma-[32P]ATP and histone H2b; (b) soleus muscles prelabeled in vitro with [32P]phosphate with subsequent insulin-stimulated phosphorylation of the receptor in situ; (c) assessment of in vivo activation of muscle receptor tyrosyl kinase by insulin. The latter was achieved by solubilizing muscle insulin receptors in the presence of phosphoprotein phosphatase and kinase inhibitors and measuring receptor-catalyzed histone H2b phosphorylation in the presence of limiting (5 microM) gamma-[32P]ATP. Receptors isolated 5 and 30 min after intravenous insulin injection catalyzed 32P incorporation into histone H2b twice as fast as those from saline-treated controls; insulin stimulated histone H2b labeling exclusively on tyrosine. In vivo activation was demonstrated using solubilized and insulin-agarose-bound receptors. Autophosphorylation of the beta-subunit and receptor tyrosyl kinase activity toward histone H2b was stimulated by insulin in denervated muscles as in controls, although the biological response to insulin, in vitro and in vivo, was markedly impaired after denervation, suggesting a postreceptor defect. The method developed to assess insulin-stimulated receptor activation in vivo seems useful in characterizing mechanisms of insulin resistance.

Isoelectric focusing (IEF) of human erythrocyte hemolystaes on polyacrylamide slab gels over a pH gradient of 6 to 8 provides sufficient resolution of hemoglobin AIc (HbAIc) from other hemoglobin components (AIa, AIb, AII, S, and F) to allow quantification by high-resolution microdensitometry. Twice-chromatographed HbAIc alone and in admixtures with normal and diabetic hemolysates were employed to verify the identification and quantification of the AIc component. Relative concentration was determined as a per cent of the total hemoglobin absorption at 556 nm on acid-fixed, unstained gels. A total of 60 patient samples were examined by IEF, and, for 35 samples, both column chromatography and IEF determination were obtained, revealing excellent correlation between these two methods.

The in vitro incorporation of lucine, isoleucine and pyruvate into lipids was compared and the possibility that leucine might serve as an in situ precursor to the corresponding iso fatty acids in the rat sciatic nerve was studied. The relative incorporation of 14C from leucine into lipids vs. nonlipids was 20%, and the incorporation of label into total lipids from leucine was one-halp that from pyruvate. The incorporation of label from leucine and pyruvate into sterols was nearly equivalent, but the incorporation of label into all other lipid classes from leucine was less than that from pyruvate, and the incorporation of label from isoleucine into lipids was much less in all cases. No detectable label from leucine was incorporated into brached chain fatty acids. It is concluded that leucine may be a substantial in vitro precursor to all major lipids in peripheral nerve, espeically sterols. The possibility and significance of a leucine catabolic pathway in the cytosol in relation to availability of 3-hydroxy-3-methylglutaryl CoA for sterol biosynthesis is discussed.

The aldosterone antagonistic activity in vivo and the affinity for mineralocorticoid receptors (MCR) in vitro of 3-(17 beta-hydroxy-6 beta,7 beta; 15 beta,16 beta-dimethylene-3-oxo-1, 4-androstadiene-17 alpha-yl) propionic acid gamma-lactone (spirorenone), a new aldosterone antagonist, and four of its derivatives were studied in comparison with spironolactone in rats. Spirorenone was 8.6 times as potent as spironolactone, but showed a lower affinity for the MCR than the standard. The C1/C2 saturated derivative (compound I) had 7.6 times the antialdosterone potency and 2 times the binding activity of spironolactone. The 17-spiroether derivative of spirorenone (compound II) showed a lower aldosterone antagonistic activity (1.7 the potency of spironolactone) as well as a lower affinity for the MCR (0.5 the affinity of spironolactone). The analogue with a 17 beta-hydroxyl group and a 17 alpha-hydroxypropyl side chain (compound III) possessed a very low binding capacity for MCR (1/10 of that of spironolactone) but still a 1.3 times higher antialdosterone activity than spironolactone. This result is probably due to a transformation of compound III to an active metabolite in vivo. Finally, the derivative with a reversed configuration of the 17-spirolactone ring (compound IV) had no biological activity in vivo and no affinity for the MCR. These results show that spirorenone or one of its active derivatives might lead to a new series of potent aldosterone antagonists.

Copper-zinc superoxide dismutase (SOD) is present in relatively high concentrations in the beta-cells of human islets. The activity of the extracted enzyme is partially inhibited upon incubation with the diabetogenic drugs alloxan, streptozotocin, or Vacor. The role of this enzyme in protecting beta-cells against chemically induced diabetes was further investigated. Incubation of intact canine islets with alloxan (0.2 mg/ml) and 4 mM glucose decreased the insulin secretory response by 87% during subsequent exposure to 28 mM glucose. Concomitantly the SOD-specific activity (units of enzyme activity per milligram immunoreactive SOD) decreased 50% in alloxan-exposed islets. When islets were protected from alloxan toxicity by including 28 mM glucose with alloxan, the insulin secretory response and SOD specific activity remained identical to controls. Thus, SOD specific activity correlates with maintenance of beta-cell function. To test the effectiveness of SOD against streptozotocin in vitro, canine islets were incubated 10 min with or without streptozotocin (0.1 mg/ml) with 4 mM glucose; their functional integrity was tested subsequently as the insulin secretory response to 28 mM glucose. Exposure to streptozotocin alone decreased the response by 70%; inclusion of SOD (1.5 mg/ml) before and during exposure to streptozotocin completely prevented this effect. Cyanide-inactivated SOD was not effective. The potential of SOD to prevent streptozotocin-induced diabetes was tested in rats in vivo. SOD injected 10 s or 50 min before streptozotocin prevented or significantly attenuated diabetes. Injection of SOD and streptozotocin simultaneously was much less effective, and cyanide-inactivated SOD was ineffective. No protection was afforded by injection of SOD 12 or 24 h before streptozotocin. Our results support hypotheses that (a) oxygen radicals mediate the beta-cell toxicity of both alloxan and streptozotocin, and (b) beta-cells may be particularly vulnerable to oxygen radical damage.

Branched-chain amino acid (BCAA) metabolism is frequently abnormal in pathological conditions accompanied by chronic metabolic acidosis. To study how metabolic acidosis affects BCAA metabolism in muscle, rats were gavage fed a 14% protein diet with or without 4 mmol NH4Cl X 100 g body wt-1 X day-1. Epitrochlearis muscles were incubated with L-[1-14C]-valine and L-[1-14C]leucine, and rates of decarboxylation, net transamination, and incorporation into muscle protein were measured. Plasma and muscle BCAA levels were lower (P less than 0.05) in acidotic rats. Rates of valine and leucine decarboxylation and net transamination were higher (P less than 0.05) in muscles from acidotic rats; these differences were associated with a 79% increase in the total activity of branched-chain alpha-keto acid dehydrogenase and a 146% increase in the activated form of the enzyme. We conclude that acidosis affects the regulation of BCAA metabolism by enhancing flux through the transaminase and by directly stimulating oxidative catabolism through activation of branched-chain alpha-keto acid dehydrogenase.

Catabolism in denervated and diabetic rat skeletal muscle undergoing accelerated protein degradation has been investigated with methods for demonstrating acid phosphatase ultrastructurally. Control muscles displayed strong acid phosphatase activity in lateral sacs and in sparse secondary hysosomes distributed mainly near nuclear poles. Muscles from diabetic rats and, to a lesser extent, 2-day denervated rats, revealed increased secondary lysosomes apparently derived from fusion of mitochondria with acid-phosphatase-reactive vesicles and cisternae. The latter were interpreted as possibly originating from T tubules. Reaction product was also noted in the junctional folds of the motor end plate of a denervated muscle. At the longer post denervation intervals studied, deposits indicative of acid phosphatase were dispersed throughout the sarcoplasm with greater concentration in the I band and appeared more abundant in denervated than in contralateral control muscles. The enzymatic basis for the sarcoplasmic deposits and other deposits was confirmed by their absence from cytochemical controls, which included incubation in substrate-free medium, heat or NaF inactivation of enzyme, and exposure sequentially to PbNO3 and NaH2PO or PbNO3 and beta-glycerophosphate.

Unlabelled: We have recently described structural differences between insulin receptors isolated from rat skeletal muscle and liver. In this report we compared their intrinsic kinase activity and their precipitability with antiphosphotyrosine and polyclonal anti-beta-subunit antibodies before and after stimulation by insulin in vitro or in vivo. Liver derived receptors incubated with insulin showed 40-50% less autophosphorylation and exogenous substrate tyrosine kinase activity per insulin binding site than those from muscle, as well as a 50% reduction in the proportion of antiphosphotyrosine precipitable receptors. After tyrosine kinase was maximally activated by iv insulin injection (in vivo), antiphosphotyrosine antibodies precipitated 60% and 38% of insulin receptors from muscle and liver, respectively. Addition of insulin in vitro to in vivo activated receptors increased the proportion of antiphosphotyrosine antibody-precipitable receptors in both tissues but increased the tyrosine kinase activity only in liver-derived receptors, indicating that most activatable insulin receptors reside on the plasmalemma in muscle whereas a significant proportion (35%) of liver receptors are not accessible to insulin in vivo. No tissue specific differences were apparent in the interaction of three site specific anti-beta-subunit antibodies with the receptors., Conclusions: 1) The intrinsic kinase activity per insulin binding site is less in liver than in muscle. This reflects in part the greater proportion of insulin receptors from liver which do not autophosphorylate on tyrosine upon insulin binding. 2) Some insulin receptors can be phosphorylated on tyrosine in vitro without concomitant exogenous tyrosine kinase activation.

A one-dimensional model of human thermo-regulation is used to solve a variety of basic problems in determining an adequate structure of the controller for metabolic heat production, skin blood flow and sweat production. Assuming one integrated central and one integrated peripheral afferent signal the controller parameters are evaluated by analysis of the control performance. Based on a validation by experimental results this allows the determination of a first optimized set of values for controller gains and weight of skin temperature feedback. Furthermore we analyse the effect of inhomogeneous distribution of heat production and blood flow, the influence of body fat content, of controller gains, of weight of skin temperature feedback and of depth of peripheral receptors on the dynamic performance. Increase of peripheral blood flow in particular evokes essentially both an increase of energy requirement in the cold and a quicker system response. Differing rates of increase of metabolic heat production are the consequence of differing body fat content. The weight of skin temperature feedback can be limited to 5...20%, because values outside this range evoke dynamic responses incompatible with the experiments. The actual value can only be determined if there is a correct assumption for the depth of the skin receptors. The use of measured superficial skin temperatures brings about an underestimation of the peripheral afferent signal. Of the controller gains it is primarily the gain of the metabolic controller which affects the dynamic response of the system. The experimental fact of a delayed onset of sweat production after a transition from cold to heat is the consequence of a high gain of the vasomotor system.

Seven patients showing a localized area of increased uptake (hot spot) on 99mTc sulfur colloid liver scans are described. Four also had dynamic studies. In five patients the hot spot was associated with superior vena caval obstruction, in one it was associated with inferior vena caval obstruction, and in one with a hepatoma. Although a hot spot on static liver images usually indicates superior vena caval obstruction, the addition of radionuclide venacavography is recommended to confirm the diagnosis. A hot spot in the liver area in a radionuclide venogram of the lower limbs suggests inferior vena caval obstruction with collaterals via the liver.

Diabetes is known to produce increased levels of the branched chain amino acids in plasma, heart and muscle as well as increased oxidation of [14C]-leucine by nerves and muscles from rats. Plasma and retinas from streptozotocin diabetic rats had significant elevations in branched chain amino acid levels compared to control. Retinas from diabetic rats have been found to oxidize significantly more of the branched chain amino acids, leucine, isoleucine and valine than did control retinas when incubated in media containing 16.5 mmol/l glucose. Neither the extracellular space nor the tissue pool of leucine was significantly different in the two groups. The addition of 19 amino acids, at normal plasma concentrations, to the incubation media resulted in 80 percent suppression of leucine oxidation without significant change in incorporation of [14C] into protein. These results suggest that the major role for the branched chain amino acids in the rat retina is in protein synthesis which is not affected by short-term diabetes.

The ability of 11 steroids with varying degrees of progestogenic potency to inhibit the renal actions of aldosterone was tested in adrenalectomized, glucocorticoid-treated rats. The rats were continuously infused with an isotonic solution of low sodium content (0.05% NaCl + 5.2% glucose, 3 ml/rat/h) supplemented with d-aldosterone [1 microgram/(kg X h)] resulting in a long-lasting reduction in renal sodium excretion, increase in renal potassium excretion and hence decrease in the urinary Na/K-ratio. The test drugs were administered either subcutaneously or orally 1 h before start of infusion. Their antimineralocorticoid activity was judged by the increase in the aldosterone-lowered Na/K-ratio in urine which was collected at hourly intervals for 15 or 21 h, respectively. Adrenalectomized, glucocorticoid-treated rats receiving an i.v. infusion without aldosterone were used for assessing possible mineralocorticoid effects of the steroids tested. D-Norgestrel, norethisterone acetate, 3-keto-desogestrel (the active metabolite of desogestrel) and cyproterone acetate, respectively, did neither show antimineralocorticoid nor mineralocorticoid activity when injected subcutaneously at a dose of 53.3 mg/kg. In contrast, gestodene (26.7 or 53.3 mg/kg s.c., respectively), progesterone (53.3 mg/kg s.c.), spironolactone (26.7 mg/kg s.c.), spirorenone (13.3 mg/kg s.c.), 1,2-dihydro-spirorenone (13.3 mg/kg s.c.), or 1,2 alpha-methylene-spirorenone (13.3 mg/kg s.c.) exhibited significant anti-mineralocorticoid activity. Canrenone (53.3 mg/kg s.c.) slightly increased the urinary Na/K-ratio. This effect, however, was not significant. Gestodene, like spironolactone or progesterone, was devoid of aldosterone-like mineralocorticoid activity. With the exception of progesterone, the antimineralocorticoid activity of the steroids tested could also be demonstrated after oral administration. Based on the Na/K-ratio or the log (Na X 100)/K-ratio, the potency of each test compound relative to spironolactone was evaluated using regression analysis. The following results (average relative potency, spironolactone = 1.0) were obtained: gestodene: approximately 0.2; canrenone: approximately 0.3-0.35; spirorenone: approximately 7-8; 1,2-dihydro-spirorenone: approximately 8; 1,2 alpha-methylene-spirorenone: approximately 3.5. Progesterone which was evaluated after s.c. injection exhibited an average relative potency of approximately 0.35. Due to these results, gestodene may be regarded as the first progestogen of the nortestosterone series exhibiting a more natural, progesterone-like profile of activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.

Human dialyzable leukocyte extract (10(7) - 10(8) leukocyte equivalents, containing transfer factor) was administered intraperitoneally to CFW mice the day of and 2 days after intravenous infection with Candida albicans. Tissue Candida populations were determined immediately after and 2, 4, 7 and 14 days after infection. Kidney populations were significantly reduced on 27% of the days studied. Similar reduction in C. albicans census was obtained after injection of leukocyte extracts from donors skin test-positive to Candida antigens or donors negative to Candida antigens by skin test and migration inhibition analyses. There was no evidence of a dose-response relationship for leukocyte extract in the range 10(5) - 10(9) leukocyte equivalents. When mice were primed with C. albicans antigen 4 weeks prior to challenge the efficacy of leukocyte extracts was not augmented. There was no evidence that the infection-reducing effects were related to augmented polymorphonuclear leukocyte mobilization, increased mononuclear clearance of C. albicans, or to a direct toxic effect on C. albicans blastospores. These studies suggest that the reduction in Candida populations was non-specific and give further impetus to the use of the dialyzable leukocyte extracts as non-specific supplements to antibiotics in overwhelming or recalcitrant infections in man.

Copper-zinc superoxide dismutase is known to protect pancreatic beta-cell function from the damage of alloxan. A morphological investigation of copper-zinc superoxide dismutase prophylaxis against alloxan was undertaken in rats to investigate the mechanism of this protective action. Exogenous copper-zinc superoxide dismutase reproducibly protected the morphological features of pancreatic beta cells against damage by alloxan as determined by light microscopic immunostaining for insulin and by ultrastructural examination. By the same criteria, alpha and delta cells appeared unaffected by administration of a combination of alloxan and copper-zinc superoxide dismutase or either agent alone. Autoradiography after injection of 125I labelled copper-zinc superoxide dismutase into normal rats showed no evidence that the enzyme enters viable islet cells, suggesting an extracellular site of protection against alloxan.

The thyroid traps the pertechnetate ion and iodine in a similar manner. Thus, 99mTc-pertechnetate may be used for thyroid uptake studies (including stimulation and suppression tests)8 as well as imaging. Images are obtained after an intravenous administration of 2--4 mCi of 99mTc-pertechnetate, and the scan is preceded by a trapping index at 20 minutes, measured as the neck to thigh ratio. In 1973, we compared 99mTc-pertechnetate with 131I in diagnostic uptake and imaging studies of the thyroid gland in 100 patients. We have had experience with an additional 100 patients, and modified our procedure so that the Anger camera with pinhole collimator will adequately replace the standard scintillation probe. Using the camera alone, with the 99mTc-pertechnetate makes the study less expensive and briefer, with no loss of information. Routine use of the scan aids in distinguishing diffuse from localized diseases.

Erythroblastic leukemic (EBL) cells incubated in media containing essential amino acids, glutamine and serine incorporate more [3H]-leucine into protein than those incubated without serine. Cells incubated with serine contain higher intracellular serine concentrations and display increased rates of peptide chain initiation on polyribosomal profile analysis. Deficiency of serine inhibited protein synthesis more than deficiencies of most other single essential amino acids, but no further inhibition was seen when single essential amino acids were removed from serine deficient media. Serine also enhanced the uptake of [3H]-uridine and its transfer to RNA while several essential amino acids had no effect. We conclude that in EBL cells, serine is an essential amino acid and that exogenous repletion of intracellular concentrations induces a positive pleiotypic response. We have previously shown that after incubation with serine for 15 min. EBL cells have greater numbers of plasmalemma insulin receptors. Regulation of cell surface receptors may therefore comprise another limb of the pleiotypic response.

The structure of insulin receptors, solubilized from rat skeletal muscle and liver, was studied. The alpha subunit was identified by specific cross-linking to A14 125I-insulin with disuccinimidyl suberate. Muscle- and liver-derived alpha subunits migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a Mr of 131,000 and 135,000, respectively. There was no significant difference in insulin binding affinity. Treatment of cross-linked, immunoprecipitated receptors with either neuraminidase or endoglycosidase H decreased the Mr of muscle- and liver-derived alpha subunits but did not affect the difference in Mr. Autophosphorylated beta subunits migrated with a Mr of 98,000 for muscle and 101,000 for liver. After partial V8 digestion of autophosphorylated, immunoprecipitated receptors the major phosphopeptide fragment migrated on SDS-PAGE at Mr 57,000 from muscle and 60,000 from liver. Glycosidase digestion of autophosphorylated receptors suggested that Mr heterogeneity was due in part to differences in the sialic acid content of beta subunits. Muscle and liver are the major target organs of insulin; the apparent heterogeneity of insulin receptor structure may be relevant to tissue-specific differences in insulin action.

The etiology of diabetes in some conditions of iron overload is not known. We studied growth, glucose tolerance, and pancreatic islet cell morphology and cytochemistry in rats administered parenteral FeNTA. These rats developed glucosuria, slowing of growth with eventual weight loss, polyuria, polydipsia, and death. They had normal fasting plasma glucose levels but decreased glucose tolerance and insulin response to glucose. Although no Prussian blue staining of iron was observed in pancreatic islets by light microscopy, at the ultrastructural level insulin-secreting beta-cells showed ferrin iron deposits localized to the plasmalemma and the cytoplasmic surface of secretory granule membranes. Prussian blue staining was also observed in parenchymal cells of the liver, heart, and kidney, in order of decreasing intensity. Animals treated with an equivalent dose of NTA, saline, or iron-dextran in saline had normal growth and response to glucose and did not exhibit pancreatic iron deposits at the light or ultrastructural level. These results support the hypothesis that iron affects pancreatic islet cell function and may be an etiologic agent of diabetes mellitus in hemochromatosis.