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The binding affinity of monoiodoinsulin analogues to receptors purified from rat skeletal muscle and liver were compared. Insulin iodinated at tyrosine B26 bound to both muscle and liver derived insulin receptors with higher affinity than the A14-iodoisomer or native insulin. The affinity of the B26-iodoanalogue was greater for muscle than for liver derived receptors; by Scatchard analysis the affinity ratio B26/A14 was 2.8 for muscle and 1.3 for liver. The affinity of muscle and liver derived receptors for A14-iodoinsulin was not different. Dose response curves of autophosphorylation and exogenous tyrosine kinase activation showed significantly increased sensitivity to the B26-iodoanalogue (compared to the A14-iodoisomer or native insulin) in muscle derived receptors, but not in liver. The difference in affinity between muscle and liver derived insulin receptors towards B26-monoiodotyrosyl-insulin likely reflects the observed structural difference between the insulin receptor alpha-subunits from muscle and liver.

Quarter rat diaphragms were incubated for 150 min. with or without stimulators of protein synthesis and/or inhibitors of protein glycosylation. Tyrosine incorporation into protein was measured during the last 120 min. Branched chain amino acids stimulated protein synthesis by 40-60%. Tunicamycin or 2-deoxy-D-glucose did not affect baseline protein synthesis, but inhibited or abolished stimulation of protein synthesis by branched chain amino acids. Tunicamycin did not prevent stimulation of protein synthesis by insulin or affect amino acid transport under these conditions. The data suggest that a) glycoprotein(s) may be involved in the post-transcriptional stimulation of protein synthesis by leucine, b) leucine and insulin may stimulate peptide chain initiation in muscle through different mechanisms.

Tobaccos from countries with high and low incidences of lung cancer were analyzed. Tobacco concentrations of polonium-210 were similar in cigarettes from high- and low-incidence countries, as were levels of cigarette smoke tar and nicotine. Tobaccos from low-incidence countries had significantly lower Alternaria spore counts. Mean selenium concentrations of tobaccos from the high-incidence countries (0.16 +/- 0.05 micrograms/g) were significantly lower than those of tobaccos from the low-incidence countries (0.49 +/- 0.22 micrograms/g).

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.

Immunoelectron microscopy and cellular fractionation on sucrose density gradients have been used to examine the intracellular distribution of copper-zinc superoxide dismutase (SOD) in the canine and rat endocrine pancreas. Using rabbit anti-canine copper-zinc SOD as the primary antiserum, immunostaining in canine beta and non-beta islet cells was significantly greater than that in serial sections of the same islet incubated with preimmune serum from the same rabbit. Within the cells, immunostaining was associated with spherical and crystalloid granules as well as with the cytoplasm. Radioimmune assays of cellular fractions or rat islet-cell preparations showed that the granule-rich fraction containing large amounts of insulin was also rich in SOD. This interesting observation of association of SOD with hormone-containing granules suggests that SOD may play a role in protecting these oxidation-sensitive proteins.

The ability of a series of 15,16-methylene-spirolactones in comparison to known antimineralocorticoids to inhibit the renal actions of aldosterone was tested in adrenalectomized, glucocorticoid-treated rats. The standard procedure involved continuous i.v. infusion with an isotonic solution of low sodium content (0.05% NaCl + 5.2% glucose, 3 ml/rat/h) supplemented with d-aldosterone [1 micrograms/(kg X h)] resulting in a long-lasting reduction of renal sodium excretion, increase of renal potassium excretion and hence decrease of the urinary Na/K-ratio. In some experiments sodium input was increased (0.2% NaCl + 4.3% glucose or 0.9% NaCl, respectively). The test drugs either were administered orally 1 h before start of the infusion or were added to the infused solution. With the exception of two steroids which could only be tested at single doses, all compounds were administered at three doses ranging from 2.2 to 40 mg/kg (p.o.) or from 0.83 to 6.7 mg/kg/h (i.v.). Spironolactone or spirorenone (oral administration) and potassium canrenoate (i.v. infusion) served as reference compounds. The antimineralocorticoid activity of the steroids 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 as controls. To obtain preliminary information on potential antiandrogenic and progestogenic (side-)effects, binding of the test-compounds to androgen receptors (rat prostate cytosol) and progestogen receptors (rabbit uterus cytosol) was measured in vitro using 3H-dihydrotestosterone (DHT) and 3H-progesterone (prog.) as tracer and unlabelled DHT and prog. as references. All steroids tested exhibited antimineralocorticoid activity. For compounds tested at three doses levels the potency relative to the standard used was evaluated using regression analysis based on the Na/K-ratio or the log (Na X 100)/K-ratio. The relative potency of the other compounds was estimated by comparing the biological effect of single doses of test drug and standard drug, respectively, using nonparametric statistical tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.

The response of rat skeletal muscle branched-chain alpha-keto acid dehydrogenase to administration of branched-chain amino acids in vivo was determined using a soluble preparation of the enzyme. After detergent extraction of the complex in the presence of kinase and phosphatase inhibitors, initial in vivo activity was typically 1 nmol X min-1 X g muscle-1, with 0.1 mM alpha-[1-14C]ketoisocaproate as substrate. Total activity of the dephosphorylated complex, measured after preincubation with 15 mM Mg2+, typically reached a maximum of 29 nmol X min-1 X g-1. Thus in overnight-fasted rats the complex was 2-3% active. Initial activity increased three- to fivefold after leucine or isoleucine (at higher concentrations) but not valine administration in vivo. After intravenous leucine injection (0.25 mmol/kg) initial muscle enzyme activity increased rapidly and subsequently decreased, paralleling plasma leucine concentrations, while plasma valine and isoleucine decreased. In conclusion, muscle branched-chain alpha-keto acid dehydrogenase complex is rapidly activated when circulating leucine is increased to concentrations that may occur after meals. During hyperleucinemia accelerated valine and isoleucine degradation by muscle may account for the observed "antagonism" among the branched-chain amino acids.

1. Blood plasma obtained from the hypophysial portal vessels of rats has been tested in assay animals for effects on the content of growth hormone in the anterior pituitary.2. Growth hormone was estimated by radioimmuno-assay at 15 min, 30 min, 1 hr and 2 hr after intracarotid or intravenous infusion of portal plasma. There was a significant increase in growth hormone content at 15 min after infusion, but not at 30 min, 1 hr and 2 hr.3. Insulin, cold stress, haemorrhage and treatment with growth hormone had no effect on growth hormone content of the pituitary under the conditions of application of these stimuli.4. Growth hormone was measured in plasma obtained from the pituitary of control and insulin-treated animals. In control animals the amount of growth hormone in this plasma was higher than in base line samples and in the insulin-treated animals it was lower than in base line samples. A calculation based on the growth hormone content of blood obtained directly from the pituitary indicates a daily rate of synthesis and release of about 10 mug.5. The hypothesis that portal plasma stimulated the synthesis of immuno-assayable growth hormone by the anterior pituitary is considered.6. Discrepancies between our results using immuno-assay and those of others using bio-assay are discussed.

Immunoreactive growth hormone (GH) released into an incubation medium and (14)C-leucine incorporation into GH by the anterior pituitary were determined after incubation for 15 min and one hr with hypophysial portal plasma and with peripheral plasma from the same animals as a control substance. Twenty-three hemipituitaries in which (14)C-leucine incorporation was measured at IS min showed a mean difference of 2,980 +/- 1,030 SEM cpm/mg of GH (t = 2.89, p < .01). After one hr of incubation no significant difference could be determined. After one hr of incubation the amount of GH released into the medium containing the portal plasma was significantly greater than the amount released into the medium containing peripheral plasma. The mean difference was 409 +/- 179 SEM ng/mg pituitary (* = 2.87, p < .05). After 15 min of incubation no differences in GH released into the medium could be detected. Differences in GH content of the pituitary could not be detected at either time period. The study supports the hypothesis that hypophysial portal plasma contains a factor which stimulates GH synthesis by the pituitary. In addition, the earlier finding of Wilber and Porter that the hypophysial portal plasma contains a factor which will release GH from the pituitary is confirmed.