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The stimulus-secretion coupling of amino acid-induced insulin release: metabolism and cationic effects of leucine.

When isolated rat pancreatic islets are exposed to L-leucine (20 mM), the rate of NH4 production is close to the summed rates of L-[1-14C] leucine decarboxylation and alpha-ketoisocarproate production, whereas the rates of acetoacetate production and L-[U-14C]-leucine oxidation are compatible with conversion of each mole of the amino acid to one mole of acetoacetate and three moles of CO2. ATP content, ATP/ADP ratio, and adenylate charge are maintained at normal values by L-leucine, whereas the NADH/NAD+ ratio (but not the NADPH/NADP+ ratio) is significantly increased. The release of insulin evoked by L-leucine is potentiated by 2-ketoisovalerate, unaffected by L-valine, and inhibited by menadione. L-leucine mimicks the effect of D-glucose on 86Rb+ and 45Ca2+ handling by the islets. However, relative to its rate of oxidation, the insulinotropic effect of L-leucine is less marked than that of D-glucose. This may be due, in part at least, to a decrease in the oxidation of endogenous nutrients. It is concluded that the metabolic, cationic, and secretory effects of L-leucine in isolated islets are not incompatible with the fuel hypothesis for insulin release.

Acetoacetates↗

Slow [Ca2+]i oscillations induced by ketoisocaproate in single mouse pancreatic islets.

The effect of alpha-ketoisocaproate (KIC), the first catabolic metabolite of the amino acid leucine, on [Ca2+]i, insulin release, and membrane potential was measured in mouse pancreatic islets of Langerhans. Stimulatory concentrations of KIC (2.5-10 mmol/l) caused slow oscillations of [Ca2+]i and cyclic variations of the membrane potential. Slow [Ca2+]i oscillations depended on extracellular calcium. Simultaneous measurements of [Ca2+]i and insulin release resolved pulsatile insulin secretion that paralleled slow [Ca2+]i oscillations. Whereas 11 mmol/l glucose induced a significant increase in cAMP, KIC was unable to modify it. Glucagon (10 nmol/l), which significantly increased cAMP in mouse islets, also increased the frequency of glucose-induced fast [Ca2+]i oscillations. However, neither glucagon (10 nmol/l) nor dibutyryl cAMP (1 mmol/l) was able to change the slow oscillation pattern into a fast pattern. Imaging of Ca2+ showed that KIC-induced slow oscillations were synchronic throughout the whole islet. It is suggested that beta-cell electrical activity plays a role in the origin of slow [Ca2+]i oscillations.

Animals↗

Skeletal muscle myosin heavy chain synthesis in type 1 diabetes.

Although insulin's anticatabolic effect on protein metabolism in type 1 diabetes has been clearly shown to be related to the inhibition of protein breakdown, insulin's effect on muscle protein synthesis remains controversial. Cross-limb studies and measurements of synthesis rates of mixed muscle protein have yielded conflicting results. These measurements represent the mean synthesis of several muscle proteins and may miss changes in the synthesis rates of individual muscle proteins. We measured the fractional synthesis rates of myosin heavy chain (MHC), the principal muscle contractile protein, and mixed muscle protein (MMP) in six type 1 diabetic patients during insulin deprivation and insulin treatment. Comparisons were made with six healthy control subjects. Muscle biopsies were taken at 2 h and 8 h during a primed continuous infusion of L-[1-13C]leucine. MHC was purified by a preparative continuous elution gel electrophoresis, and fractional synthesis rates were calculated. We found that in type 1 diabetic subjects, the fractional synthesis rates of MHC and MMP during insulin treatment are similar to those of control subjects. Acute insulin deprivation did not affect either the synthesis rate or the ratio of MHC to MMP in type 1 diabetic subjects. In the postabsorptive state, acute insulin deprivation has no effect on MHC or MMP synthesis in type 1 diabetic patients.

Adult↗

Insulin's effect on synthesis rates of liver proteins. A swine model comparing various precursors of protein synthesis.

Insulin's effect on the synthesis of liver proteins remains to be fully defined. Previous studies using various surrogate measures of amino acyl-tRNA have reported variable results of insulin's effect on liver protein synthesis. We determined the effect of insulin with or without amino acid supplementation on the synthesis rates of liver proteins (tissue, albumin, and fibrinogen) using L-[1-13C]Leu as a tracer in 24 male miniature swine. In addition, we compared the isotopic enrichment of different precursors of liver proteins with that of amino acyl-tRNA using L-[1-13C]Leu and L-[15N]Phe as tracers. Although liver tissue fluid enrichment of [13C]Leu and [15N]Phe and that of plasma [13C]ketoisocaproatic acid (KIC) were very similar to that of tRNA, plasma isotopic enrichment of both Leu and Phe were substantially higher (P < 0.01) and VLDL apolipoprotein-B100 enrichment was lower (P < 0.01) than the respective amino acyl-tRNA enrichment. Plasma KIC enrichment most accurately predicted leucyl-tRNA enrichment, whereas plasma Leu enrichment was best correlated with that of tRNA. Neither insulin alone nor insulin plus amino acid infusion had an effect on liver tissue protein synthesis. In contrast, insulin alone decreased the albumin synthesis rate, and insulin with amino acids maintained the albumin synthesis rate. Insulin with or without amino acids inhibited the fibrinogen synthesis rate. These results, based on synthetic rates using amino acyl-tRNA, were consistent with those obtained using KIC or tissue fluid Leu or Phe as precursor pools. These studies demonstrated that plasma KIC enrichment is a convenient and reliable surrogate measure of leucyl-tRNA in liver. We also concluded that insulin has differential effects on the synthesis rates of liver proteins. Whereas insulin with or without amino acid supplement has no acute effect on the synthesis of liver tissue protein, insulin has a substantial inhibitory effect on fibrinogen synthesis. In contrast, insulin administration along with amino supplement is necessary to maintain albumin synthesis rate.

Amino Acids↗

Influence of alpha-ketoisocaproate on lamb growth, feed conversion, and carcass composition.

Four experiments were conducted to determine whether leucine's alpha-ketoacid, alpha-ketoisocaproate (KIC), would influence lamb growth, feed conversion, and carcass composition. In the first experiment, lambs were injected intraperitoneally with 3.5 g of Na-KIC per day. In the second experiment, KIC unprotected from rumen degradation was fed at a rate of 15 g per animal daily. In a third experiment, KIC, leucine, and isovalerate (IVA), protected from rumen degradation, were fed to growing lambs at a rate of 1 g per animal per day. Finally, a fourth experiment was conducted in which ruminally protected KIC was fed to growing lambs at a rate of 1 g per animal per day. Ketoisocaproate tended to increase ADG and decrease fat deposition in all four experiments. Ketoisocaproate increased ADG by 11 (P less than .09), 10 (P less than .05), 9, and 13% in 1 through 4, respectively, and feed efficiency improved 5, 9 (P less than .02), 5, and 5%, respectively. Fat thickness over the 12th rib decreased 28 (P less than .06), 11, 17 (P less than .04), and 5% in Exp. 1 through 4, and the perirenal fat depot also decreased 13, 5, 18, and 3%, respectively. In contrast, neither ruminally protected leucine nor IVA affected the growth of young lambs. Together these studies indicate that administration of KIC to growing lambs can increase weight gain and muscle growth while decreasing fat deposition.

Adipose Tissue↗

The importance of gestagen, tamoxifen and steroid receptors in the therapy of endometrial cancer. Report of an experiment.

Six human endometrial cancers in different clinical stages were xenotransplanted into thymusaplastic nude mice. Estrogen and progesterone receptors in the tumor tissue were determined in each case. Two carcinomas with both estrogen and progesterone receptors showed a better clinical course and grew more slowly in nude mice than the four carcinomas with no receptors at all. Treatment with the antiestrogen Tamoxifen in the carcinoma with positive estrogen receptors showed a significant growth retardation compared with the control group. Estrogen treatment gave accelerated growth, whereas gestagen treatment showed no significant effect. The progesterone receptor content of the receptor-positive tumor tissue might have been too small. None of the four receptor-negative carcinomas showed any influence of tumor growth during endocrine therapy. The results demonstrate the importance of receptor determination as a prognostic sign and, further, allow the endocrine therapy of endometrial carcinomas to be complemented with Tamoxifen in estrogen-receptor-positive tumor tissue.

Aged↗

Propionyl-CoA carboxylase deficiency: case report, effect of low-protein diet and identification of 3-oxo-2-methylvaleric acid 3-hydroxy-2-methylvaleric acid, and maleic acid in urine.

Vomiting, lethargy and metabolic acidosis were the main initial symptoms of metabolic disease in a 1 month old girl. Her older sister had died from a similar disease, considered to be Reye's syndrome, at an age of 15 months. The urine of the present case contained 2-methylcitric acid, 3-hydroxypropionic acid, N-propionylglycine, 2-hydroxy-3-methylbutyric acid, N-tiglylglycine, 3-hydroxyvaleric acid and glutaric acid. These metabolites are all known to be associated with propionyl-CoA accumulation. Free propionic acid was not detected in the urine. In addition, the urine contained 3-oxo-2-methylvaleric acid and 3-hydroxy-2-methylvaleric acid, probably formed by condensation of two molecules of propionyl-CoA. The identity of these metabolites was confirmed by synthesis. An elevated urinary concentration of maleic acid and fumaric acid was another constant abnormality. The activity of propionyl-CoA carboxylase in leucocytes was about 20% of the normal activity. The girl was teated with a low-protein diet since the diagnosis was made at an age of 1 month, and her psychomotor development was satisfactory at an age of 2 1/2 years. She had a few episodes of acidosis during infections.

Amino Acid Metabolism, Inborn Errors↗

On gestagen treatment of advanced prostatic carcinoma.

Depostat treatment was found to reduce plasma testosterone and to have a gonadotrophin suppressive effect in males. In our study the clinical effect on prostatic cancer was, however, disappointing when compared to the well established effect of orchiectomy. The therapeutic failure of Depostat might be related to the incomplete suppression of plasma testosterone compared to that observed after orchiectomy.

Adenocarcinoma↗

The metabolism of 2-ethylhexanol in rats.

1. 2-Ethylhexanol was efficiently absorbed following oral administration to rats. 14C associated with 2-ethyl[1-14C]hexanol was rapidly excreted in respiratory CO2 (6-7%), faeces (8-9%) and urine (80-82%), with essentially complete elimination by 28 h after administration. 2. The amount of label recovered in 14CO2 matched the amount of unlabelled 2-heptanone plus 4-heptanone recovered from urine, suggesting that both types of metabolite may have been derived form the major urinary metabolite, 2-ethylhexanoic acid, by decarboxylation following partial beta-oxidation. The 14CO2 appeared not to be derived from acetate (urinary acetic acid and liver and brain cholesterol were not labelled) or by reductive decarboxylation (heptane was not present.) 3. Other identified metabolites were 2-ethyl-5-hydroxyhexanoic acid, 2-ethyl-5-ketohexanoic acid, and 2-ethyl-1,6-hexanedioic acid. Only about 3% of the ethylhexanol was excreted unchanged. 4. Ethylhexanol was a competitive inhibitor of yeast alcohol dehydrogenase, but a good substrate for horse alcohol dehydrogenase. 5. Other relationships between metabolism and toxicity of 2-ethylhexanol are discussed.

Administration, Oral↗

Metabolism of 2-ethylhexanol administered orally and dermally to the female Fischer 344 rat.

1. Excretion balance studies were conducted with 2-ethylhexanol (2-EH) in female Fischer 344 rats following single high (500 mg/kg) and low (50 mg/kg) oral doses of [14C]2-EH, following repeated oral dosing with unlabelled 2-EH at the low level, following dermal exposure for 6 h with a 1 g/kg applied dose of [14C]2-EH, and following a 1 mg/kg i.v. dose of [14C]2-EH. 2. The high, low and repeated low oral dose studies with 2-EH showed similar excretion balance profiles of [14C], with some evidence of metabolic saturation at the high dose. 3. No evidence of metabolic induction was seen following the repeated low oral dosing. 4. All of the oral doses were eliminated rapidly, predominantly in the urine during the first 24 h following dosing. 5. The dermal dosing resulted in only about 5% absorption of the 1 g/kg dose, with the major portion of the dose recovered unabsorbed from the dermal exposure cell at 6 h. 6. Urinary metabolites eliminated following the oral and dermal doses were predominantly glucuronides of oxidized metabolites of 2-EH, including glucuronides of 2-ethyladipic acid, 2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid and 6-hydroxy-2-ethylhexanoic acid.

Adipates↗

Inhibition studies on LDH isoenzyme purified from Uromastix testes.

An LDH isoenzyme was purified to homogeneity from uromastix testes and its inhibition spectrum towards known LDH isoenzyme inhibitors studied. Platinum compounds inhibited the enzyme in the forward reaction (pyruvate-->lactate) only, n-hexanediol and colchicine showed no inhibition and gossypol acetic acid (GAA) strongly inhibited both the forward and reverse reactions and the reactions were time-dependent. Oxalate caused non-competitive inhibition (Ki app = IC50 = 0.15 mM) of the forward reaction, NADH was more effective in blocking inhibition by GAA than pyruvate. This enzyme was also unable to use ketocaproic acid as a substrate.

Animals↗

Accumulation of short n-chain ethyl esters by esterases of lactic acid bacteria under conditions simulating ripening Parmesan cheese.

EstA from Lactobacillus helveticus CNRZ32 (Lbh-EstA), EstB, and EstC from Lactobacillus casei LILA, and EstA from Lactococcus lactis MG1363 (Lcl-EstA) were evaluated for their ability to accumulate esters in a model system simulating Parmesan cheese ripening conditions (10 degrees C, 2 to 3% NaCl, pH 5.4 to 5.5, aw = 0.850 to 0.925) using Capalase K from kid goat as a positive control. All of the LAB esterases and Capalase K mediated the accumulation of esters in the model system in an enzyme specific manner, which was influenced by a, and selectivity for fatty acid chain-length. In general, enzyme mediated accumulation of ethyl esters was higher at aw values of 0.850 and 0.900 than at aw of 0.925, demonstrating that aw is a critical parameter influencing ester accumulation. The substrate selectivity of esterases, aw, and enzyme type may be important factors in the development of fruity flavors, as evidenced by results in this model system simulating Parmesan cheese ripening conditions.

Butyrates↗