Oral administration of interferon tau enhances oxidation of energy substrates and reduces adiposity in Zucker diabetic fatty rats.

Male Zucker diabetic fatty (ZDF) rats were used to study effects of oral administration of interferon tau (IFNT) in reducing obesity. Eighteen ZDF rats (28 days of age) were assigned randomly to receive 0, 4, or 8 µg IFNT/kg body weight (BW) per day (n = 6/group) for 8 weeks. Water consumption was measured every two days. Food intake and BW were recorded weekly. Energy expenditure in 4-, 6-, 8-, and 10-week-old rats was determined using indirect calorimetry. Starting at 7 weeks of age, urinary glucose, and ketone bodies were tested daily. Rates of glucose and oleate oxidation in liver, brown adipose tissue, and abdominal adipose tissue, as well as leucine catabolism in skeletal muscle, and lipolysis in white and brown adipose tissues were greater for rats treated with 8 µg IFNT/kg BW/day in comparison with control rats. Treatment with 8 µg IFNT/kg BW/day increased heat production, reduced BW gain and adiposity, ameliorated fatty liver syndrome, delayed the onset of diabetes, and decreased concentrations of glucose, free fatty acids, triacylglycerol, cholesterol, and branched-chain amino acids in plasma, compared with control rats. Oral administration of 8 µg IFNT/kg BW/day ameliorated oxidative stress in skeletal muscle, liver, and adipose tissue, as indicated by decreased ratios of oxidized glutathione to reduced glutathione and increased concentrations of tetrahydrobiopterin. These results indicate that IFNT stimulates oxidation of energy substrates and reduces obesity in ZDF rats and may have broad important implications for preventing and treating obesity-related diseases in mammals.

Efficient mechanical disruption of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity.

Microbiological studies often involve bacterial cell fractionation, which is known to be difficult for Gram-positive as compared to Gram-negative bacteria. Our purpose was to test the breaking efficiency of a new high-pressure pilot homogenizer for three Gram-positive species involved in dairy technology and to assess the activity of an intracellular aminotransferase. Varied pressures (50, 100 and 200 MPa) were applied to concentrated bacterial suspensions (1.2 mg dry weight/ml) of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii. Breaking efficiency was estimated by decreases in optical density at 650 nm, cellular dry weight and viability. The proteins released were quantified and the residual intracellular aminotransferase activity was estimated using leucine as substrate. One run at 50 MPa was sufficient to break 80% of lactobacilli cells whereas 200 MPa were required for the same efficiency for L. lactis and P. freudenreichii. Whatever the pressure, leucine aminotransferase activity was recovered in the supernatant after cell breaking. This new high-pressure pilot homogenizer can allow rapid (20 s/run), easy, continuous and highly efficient cell breaking for intracellular enzyme recovery or other purposes. As the species tested were not phylogenetically related, and had different morphologies and cell wall compositions, we conclude that most Gram-positive bacteria may be broken efficiently by this new device.

Detection of aminotransferase activity of Propionibacterium freudenreichii after SDS-PAGE.

Aminotransferases (ATs) had previously been detected after native electrophoresis. We show now that aminotransferase(s) of Propionibacterium freudenreichii can be detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, it retained a high activity (84%) in the presence of 0.23% SDS, contrary to what was observed for aminotransferase(s) of Bifidobacterium bifidum (54%) and of six other cheese-related species (0-20%).

Decreased pancreatic islet response to L-leucine in the spontaneously diabetic GK rat: enzymatic, metabolic and secretory data.

AIMS/HYPOTHESIS: Pancreatic islets from hereditarily non-insulin-dependent diabetic Goto-Kakizaki (GK) rats have a deficient insulin response not only to D-glucose but also to L-leucine. Our aim was to explain the cellular mechanism(s) underlying the beta-cell unresponsiveness to this amino acid. METHODS: Freshly collagenase isolated islets from GK rats and healthy Wistar control rats matched with them for sex and age were compared. Leucine uptake, metabolic fluxes and insulin secretory capacity were investigated on batch incubated-islets. Enzymatic activities were measured on sonicated islets. RESULTS: In GK rat islets, neither leucine transport nor leucine transaminase activity was disturbed. By contrast, 14CO2 production from either L-[U-14C]leucine or L-[1-14C]leucine was decreased. The L-[U-14C]leucine oxidation: L-[1-14C]leucine decarboxylation ratio was unaffected, indicating that the acetyl-CoA generated from leucine undergoes normal oxidation in the Krebs cycle. The leucine non-metabolizable analogue 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid induced insulin release and enhanced the secretory response to leucine as in controls, whereas leucine failed to amplify the response to the leucine analogue. Moreover, the potentiating action of L-glutamine on leucine-mediated insulin release was preserved. This coincided with normal glutamate dehydrogenase activity and L-[U-14C]glutamine oxidation. Finally, the secretory response to the leucine deamination product 2-ketoisocaproate was decreased, as was the 2-keto[1-14C]isocaproate oxidation. CONCLUSION/INTERPRETATION: In islet beta cells from GK rats, the defective secretory response to leucine cannot be ascribed to a deteriorated leucine-stimulated glutamate metabolism but rather to an impaired leucine catabolism. A reduced generation of acetyl-CoA from 2-ketoisocaproate, due to the defective oxidative decarboxylation of this keto-acid by the mitochondrial branched-chain 2-ketoacid dehydrogenase, is incriminated.

Adaptation of the medullary thick ascending limb to dietary protein intake.

Since the renal growth response to a high-protein diet is characterized by prominent hypertrophy of the medullary thick ascending limb of Henle's loop (MTAL), the functional and metabolic adaptations of this nephron segment to dietary protein were investigated. MTAL suspensions were obtained from rats fed equal amounts of isocaloric food containing either 10% (LP) or 32% (HP) casein for 4-6 weeks. The results show that intact MTAL of HP rats exhibit a blunted respiration rate, sodium pump activity, hormone-sensitive cAMP production and leucine oxidation rate in comparison with those of LP rats. On the other hand, adenylate cyclase and leucine transaminase activities, measured on permeabilized or homogenized MTAL, are enhanced by a HP diet. We conclude that the MTAL adapts to high dietary protein by increasing its maximal enzyme activities, but certain factors, present in intact cells, limit transport and metabolism in HP- more than in LP-fed rats. This reduced function per unit MTAL protein in HP rats is more than compensated for by hypertrophy of the MTAL tissue mass.

[Transferase contents in lymph and blood in fever reaction]. / Soderzhanie transferaz v limfe i krovi pri likhoradochnoi reaktsii.

The aim of the present investigation was the study of the content of alanine, asparagine-transaminases, gamma-glutamyl transferase and their isoenzymes, as well as leucine aminotransferase in the lymph of thoracic duct, hepatic and intestinal lymph and peripheral blood in dynamics of fever reaction of various duration in the experiments on rabbits. Irrespective of its duration, the fever was followed by a significant activation of the enzymes in the body fluids. However, in many-day fever reaction, a rise of enzymes level in the lymph was more prolonged than that in the blood. The above studies make it possible to assume that the released enzymes in fever reaction are primarily resorbed by lymphatic capillaries and their activity indices in the blood serum are largely evidenced by the transport function of the lymphatic system.

Acute testicular toxicity of 1,3-dinitrobenzene and ethylene glycol monomethyl ether in the rat: evaluation of biochemical effect markers and hormonal responses.

The studies described in this paper were undertaken to evaluate the use of plasma enzymes of testicular origin and plasma hormones as markers of acute testicular toxicity. Rats were dosed by gavage with a single dose of either 1,3-dinitrobenzene (1,3-DNB) or ethylene glycol monomethyl ether (EGME). Two experimental designs were used: a dose response and a time-dose response course. Lactate dehydrogenase isozyme C4 (LDH-C4) and sorbitol dehydrogenase (SDH) were used as germ cell markers and leucine aminotransferase (LAT) and androgen binding protein (ABP) were used as Sertoli cell markers. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were also monitored. Histopathology confirmed the known testicular toxicity of 1,3-DNB and EGME. 1,3-DNB induced Sertoli cell damage with associated degenerative changes in late pachytene spermatocytes. The effects of EGME were mainly on early and late pachytene and dividing spermatocytes. No changes in either testicular or plasma SDH or LAT were found. Similarly no effects were observed for plasma LH or testosterone. However testicular LDH-C4 and testosterone, plasma LDH-C4, ABP, and FSH did show compound related effects. LDH-C4 was reduced in testis and increased in plasma with both compounds and plasma LDH-C4 remained elevated up to 14 days after dosing. ABP levels in plasma were increased with 1,3-DNB and EGME. A reduction in testicular testosterone levels was recorded and plasma FSH concentrations were elevated after EGME treatment. It is concluded that plasma LDH-C4 activity and ABP may be of diagnostic value in acute testicular toxicity. Increases in plasma LDH-C4 precede noticeable histological findings.

cpc-2, a new locus involved in general control of amino acid synthetic enzymes in Neurospora crassa.

In Neurospora crassa starvation for single amino acids leads to derepression of enzymes in many amino acid synthetic pathways. Regulation occurs at the level of transcription via "general amino acid (or cross-pathway) control". In this paper a new regulatory gene, cpc-2, is described that specifies a positive, trans-acting effector involved in this control. This gene, located on linkage group VII, was identified by a recessive mutation, U142, which results in sensitivity for two amino acid analogues and a lack of enzyme derepression in response to amino acid limitation. It was shown that cpc-2 (U142) impairs the activation of transcription of amino acid structural genes in several biosyntheses. The only other known regulatory gene involved in general amino acid control of Neurospora is cpc-1. Transcription of the cpc-1 gene, however, is increased in response to amino acid starvation irrespective of the presence of the mutation U142.

Inactivation of leucine aminotransferase with diethylpyrocarbonate and rose bengal: evidence for an active site histidine residue.

Modification of leucine aminotransferase by diethylpyrocarbonate or rose bengal-sensitized photo-oxidation caused rapid inactivation of the enzyme. The inactivation of leucine aminotransferase depended on the concentration of the reagent, the time of incubation and exhibited pseudo-first order kinetics. Rose bengal-sensitized photo-oxidation was maximum at pH 6.5 and 9. Substrates leucine and alpha-ketoglutarate protected the enzyme against inactivation by these reagents, thus suggesting participation of histidine residue at the substrate binding site.

Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria.

Regulation of leucine catabolism by caloric sources. Role of glucose and lipid in nitrogen sparing during nitrogen deprivation.

Previously we showed that hypocaloric amounts of glucose reduce leucine catabolism while an isocaloric amount of fat does not (1985. J. Clin. Invest. 76:737.). This study was designed to investigate whether the same difference exists when the entire caloric need is provided either as glucose or lipid. Rats were maintained for 3 d on total parenteral nutrition (350 cal/kg per d), after which the infusion of amino acids was discontinued and rats received the same amount of calories entirely as glucose or lipid for three more days. A third group of rats was infused with saline for 3 d. In comparison to glucose, lipid infusion resulted in higher urinary nitrogen excretion (55 +/- 3 vs. 37 +/- 2 mg N/24 h, P less than 0.05), muscle concentrations of tyrosine (95 +/- 8 vs. 42 +/- 8 microM, P less than 0.01), and leucine (168 +/- 19 vs. 84 +/- 16 microM, P less than 0.01), activity of BCKA dehydrogenase in muscle (2.2 +/- 0.2 vs. 1.4 +/- 0.04 nmol/mg protein per 30 min, P less than 0.05), and whole body rate of leucine oxidation (3.3 +/- 0.5 vs. 1.4 +/- 0.2 mumol/100 g per h, P less than 0.05). However, all these parameters were significantly lower in lipid-infused than starved rats. There was no significant difference between leucine incorporation into liver and muscle proteins of lipid and glucose-infused rats. On the other hand, starved rats showed a lower leucine incorporation into liver proteins. The data show that under conditions of adequate caloric intake lipid has an inhibitory effect on leucine catabolism but not as great as that of glucose. The mechanism of this difference may be related to a lesser inhibition of muscle protein degradation by lipid than glucose, thereby increasing the leucine pool, which in turn stimulates leucine oxidation.

Branched chain amino acid transaminases in brain in methionine sulphoximine (MSI) toxicity.

The effect of intraperitoneal administration of L-methionine-DL-sulphoximine (MSI) was studied on branched-chain amino acid transaminases (BCAA-T) in different regions of rat brain and in liver. Administration of an acute dose of MSI (300 mg/kg body weight) resulted in a significant decrease in leucine aminotransferase activity in cerebral cortex, cerebellum, and brain-stem, while the activity of isoleucine aminotransferase was enhanced in hippocampus, corpus striatum, brain stem, and midbrain. Activities of both these enzymes changed marginally or remained unaltered in other regions of the brain. Valine aminotransferase showed a significant decrease in all the regions of the brain except in cerebellum. Following the administration of a sub-acute dose of MSI (150 mg/kg body wt.), the activities of the three BCAA aminotransferases were found to be enhanced in all regions of the brain. The results are discussed in relation to the utilization of BCAA for the production of glutamate and glutamine in hyperammonemia.

Oxidation of leucine in human lymphocytes.

Appropriate conditions for analysis of leucine oxidation in peripheral human lymphocytes were determined. Lymphocytes, isolated by Ficoll centrifugation, were washed in phosphate-buffered saline without additions, with 5 mmol/l ADP or with 5 mmol/l ADP plus 25 mmol/l NaF for determination of transaminase activity, and total and basal activity of branched chain keto acid dehydrogenase (BCKA-D), respectively. Cells were incubated for 20-60 min with [1-14C]-leucine, or [1-14C]-alpha-ketoisocaproic acid (KIC) in the presence of various concentrations of the respective unlabelled substrates. Pyridoxal phosphate (0.1 mmol/l) augmented transaminase activity, coenzyme A, NAD and thiaminepyrophosphate (0.5 mmol/l each) enhanced BCKA-D activity. Apparent Km values for transamination and for BCKA-D were 40 and 17 microM, respectively. Total capacity for leucine transamination was about five times greater than for oxidation of KIC. The mean activity state of BCKA-D was 30%. Oxidation of KIC declined when 5.6 mmol/l glucose was added to the medium. It is concluded that: (1) BCKA-D is rate-limiting for leucine catabolism in peripheral human lymphocytes, (2) the BCKA-D complex is normally only partially active, and (3) flux of leucine through BCKA-D is inhibited by physiological glucose concentrations.

[Diagnostic importance of determining leucine aminotransferase activity in acute pancreatitis]. / Diagnosticheskoe znachenie opredeleniia aktivnosti leitsin-aminotransferazy pri ostrom pankreatite.

It has been experimentally and clinically established that the determination of leucine-aminotransferase activity in the blood serum and abdominal exudate may serve as a marker for the early determination of pancreonecrosis and edematous (serous) pancreatitis.

Subcellular localization of leucine aminotransferase and alpha-hydroxyacid dehydrogenase in Trypanosoma cruzi.

Homogenates of Trypanosoma cruzi epimastigotes (Tulahuén strain) show L-leucine aminotransferase activity (EC 2.6.1.6). Subcellular distribution of this enzyme and of alpha-hydroxyacid dehydrogenase, enzymes which share a common substrate/product (alpha-ketoisocaproate), has been studied by means of differential centrifugation, digitonin treatment of entire parasites, isopycnic centrifugation and determination of latency of enzymes in the large granule fraction. The results indicate that both enzymes have a dual localization, in the cytosol and in the mitochondrion, probably in the matrix. On the basis of this location, it is proposed that they operate in a shuttle system transferring reducing equivalents between the cytosol and the mitochondrion.

Ethanol and leucine oxidation--III. Leucine oxidation by rat heart in vitro.

The activities of leucine aminotransferase (BCAT) and 2-oxoisocaproate dehydrogenase (OADH) were measured in rat heart in vitro. The effect upon these enzyme activities of both ethanol and acetaldehyde, administered either acutely or chronically, was determined. Enzyme activities were not significantly altered by either acetaldehyde or ethanol when given chronically. Ethanol administered acutely to rats decreased OADH activity but BCAT was unaffected. Acetaldehyde administered acutely did not alter significantly BCAT activity but significantly increased OADH activity.

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids.

Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of L-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.

Effects of carbon tetrachloride and azathioprine on diethylnitrosamine and N-2-fluorenylacetamide-induced hyperplastic liver nodule and hepatocellular carcinoma.

Effects of carbon tetrachloride (CCl4) and azathioprine (AZP) on the evolution of hyperplastic liver nodules and foci and hepatocellular carcinoma (HCC) were tested in short- and long-term in vivo experiments. In diethylnitrosamine (DEN)-treated rats, which were fed a N-2-fluorenylacetamide (FAA)-containing diet and additionally treated with repeated CCl4 injections, gamma-glutamyl transpeptidase (gamma-GTP)-positive hyperplastic nodules were markedly developed in the 8th week of the experiment. However, their number and area in liver sections were remarkably small in DEN-treated rats fed a diet containing both FAA and AZP. Increased area of gamma-GTP-positive foci was also observed in the 12th week in DEN-injected rats fed a choline-devoid died alone or treated with repeated doses of CCl4 alone. Hepatocellular carcinoma in DEN-injected rats treated with both FAA and CCl4 was first detected in the 21st week, and the incidence up to the 36th week was very high. However, no hepatocellular carcinoma developed in DEN-injected rats treated with both FAA and AZP. The increased activity of liver aniline hydroxylase observed 12 h after the administration of FAA, AZP or DEN alone was not observed when AZP was administered simultaneously with FAA to DEN-injected rats. The mechanisms of the effects of CCl4 and AZP on hepatocarcinogenesis are discussed with special reference to drug interaction.