Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on plasma amino- and keto-acid concentrations and branched-chain keto-acid dehydrogenase activity.

The present experiment was designed to elucidate the mechanism of the methionine-sparing effect of excess branched-chain amino acids (BCAA) reported in the previous paper (Langer & Fuller, 2000). Twelve growing gilts (30-35 kg) were prepared with arterial catheters. After recovery, they received for 7 d a semipurified diet with a balanced amino acid pattern. On the 7th day blood samples were taken before (16 h postabsorptive) and after the morning meal (4 h postprandial). The animals were then divided into three groups and received for a further 7 d a methionine-limiting diet (80% of requirement) (1) without any amino acid excess; (2) with excess leucine (50% over requirement); or (3) with excesses of all three BCAA (leucine, isoleucine, valine, each 50% over the requirement). On the 7th day blood samples were taken as in the first period, after which the animals were killed and liver and muscle samples taken. Plasma amino acid and branched-chain keto acid (BCKA) concentrations in the blood and branched-chain keto-acid dehydrogenase (BCKDH; EC 1.2.4.4) activity in liver and muscle homogenates were determined. Compared with those on the balanced diet, pigs fed on methionine-limiting diets had significantly lower (P < 0.05) plasma methionine concentrations in the postprandial but not in the postabsorptive state. There was no effect of either leucine or a mixture of all three BCAA fed in excess on plasma methionine concentrations. Excess dietary leucine reduced (P < 0.05) the plasma concentrations of isoleucine and valine in both the postprandial and postabsorptive states. Plasma concentrations of the BCKA reflected the changes in the corresponding amino acids. Basal BCKDH activity in the liver and total BCKDH activity in the biceps femoris muscle were significantly (P < 0.05) increased by excesses of leucine or all BCAA.

Chronic infusion of norepinephrine into the VMH of normal rats induces the obese glucose-intolerant state.

Increases in ventromedial hypothalamic (VMH) norepinephrine (NE) levels and/or activities have been observed in a variety of animal models of the obese insulin-resistant condition. This study examined the metabolic effects of chronic NE infusion (25 nmol/h) into the unilateral VMH of normal rats. Within 4 days, VMH NE infusion significantly increased plasma insulin (140%), glucagon (45%), leptin (300%), triglyceride (100%), abdominal fat pad weight (50%), and white adipocyte lipogenic (100%) and lipolytic (100%) activities relative to vehicle-infused rats. Furthermore, isolated islet insulin secretory response to glucose (15 mM) within 4 days of such treatment was increased over twofold (P < 0.05). Among treated animals, fat stores continued to increase over time and plateaued at approximately 2 wk (3-fold increase), remaining elevated to the end of the study (5 wk). By week 4 of treatment, NE infusion induced glucose intolerance as evidenced by a 32% increase in plasma glucose total area under the glucose tolerance test curve (P < 0.01). Whole body fat oxidation rate measured after 5 wk of infusion was significantly increased among treated animals as evidenced by a reduced respiratory quotient (0.87 +/- 0.01) relative to controls (0. 90 +/- 0.01). VMH NE infusion induced hyperphagia (30%) only during the first week and did not affect body weight over the 5-wk period. Increases in VMH NE activity that are common among obese insulin-resistant animal models can cause the development of this obese glucose-intolerant (metabolic) syndrome.

Bromocriptine/SKF38393 treatment ameliorates dyslipidemia in ob/ob mice.

Our previous studies have shown that the dopaminergic D1 receptor agonist SKF38393 (SKF) plus the D2 receptor agonist bromocriptine (BC) act synergistically to reduce obesity in obese C57BL/6J (ob/ob) mice. The present study investigated the effects of this combination on dyslipidemia in ob/ob mice. Female ob/ob mice were treated daily with vehicle or SKF (20 mg/kg body weight [BW]) plus BC (16 mg/kg BW [BC/SKF]) for 14 days. The animals were then used for the characterization of plasma lipoprotein profiles, hepatic triacylglycerol synthesis and secretion, adipocyte lipolysis, adipose and muscle lipoprotein lipase (LPL) activity, and muscle triglyceride (TG) content. The treatment significantly reduced plasma glucose 54%, TG 41%, cholesterol 21%, phospholipid 20%, and free fatty acid (FFA) 36% (P < .01). Hepatic triacylglycerol synthesis was 55% lower in treated mice versus control mice (P < .01). The cell size of isolated adipocytes was significantly reduced (41%) by treatment. LPL activity was increased in soleus skeletal muscle (25%, P < .05) but was sharply reduced in adipose tissue (91%, P < .01) in treated versus control mice. The TG content of hindlimb muscle was about 49% lower in treated versus control mice (P < .05). The basal and isoproterenol-stimulated lipolytic rate was decreased (approximately 53%) in adipocytes from treated animals compared with the control (P < .01). In conclusion, BC/SKF normalized the hypertriglyceridemia likely via its simultaneous antilipogenic action in liver tissue and antilipolytic action in adipose tissue. Decreased plasma flux of FFA partially contributed to the reduced hepatic lipogenesis, plasma very-low-density lipoprotein (VLDL)-TG, and TG in skeletal muscle. The above-described effects of BC/SKF treatment are largely independent of its effect to normalize hyperphagia in ob/ob mice.

Biochemical mechanisms responsible for the attenuation of diabetic and obese conditions in ob/ob mice treated with dopaminergic agonists.

OBJECTIVE: We previously reported that a two week treatment with SKF 38393 (SKF, a dopamine D1 receptor agonist), plus bromocriptine (BC, a dopamine D2 receptor agonist) acted synergistically to normalize hyperphagia, body fat, hyperglycaemia and hyperlipidaemia in ob/ob mice. The present study further investigates the biochemical mechanisms triggered by this drug treatment. DESIGN: Six week old female C57BL/6J ob/ob mice were divided into three groups and treated for two weeks with either BC and SKF, vehicle (control), or vehicle and pair fed to match the drug-treated group's daily food intake. RESULTS: BC/SKF treatment reduced food consumption by 55%, and treated mice weighed less than either pair fed or ad libitum fed controls after two weeks of treatment. Moreover, oxygen consumption was increased by 2.4-fold and the respiratory quotient (RQ) decreased from 1.23 to 0.96 (indicating a reduction in de novo lipogenesis) by drug treatment relative to ad libitum fed controls, but these parameters were unaffected by pair feeding control mice. The treatment also reduced blood glucose and free fatty acids (FFA) relative to pair fed and ad libitum fed controls. BC/SKF treatment (but not pair feeding) concurrently reduced lipolysis, lipogenic enzyme activities and hepatic gluconeogenic enzyme activities. Treatment also increased hepatic concentrations of glycogen and xylulose-5-phosphate (X-5-P), a key stimulator of glycolysis. Finally, BC/SKF, but not pair feeding, reduced the circulating concentrations of thyroxine and corticosterone, two hormones known to increase lipolysis, lipogenesis and hyperglycaemia. Drug treatment also increased serum dehydroepiandrosterone (DHEA) sulfate concentrations, an inhibitor of body fat store accumulation. CONCLUSION: These findings demonstrate that BC/SKF treatment not only normalizes hyperphagia of ob/ob mice, but also redirects several metabolic and endocrine activities, independent of its effects on feeding to improve the obese-diabetic syndrome in ob/ob mice.

Dopamine agonist treatment ameliorates hyperglycemia, hyperlipidemia, and the elevated basal insulin release from islets of ob/ob mice.

One of the characteristics of obesity-associated diabetes is an elevated fasting plasma insulin concentration with a weak insulin secretory response to subsequent glucose stimulation. Evidence suggests that hyperglycemia and hyperlipidemia may contribute to the initiation and progression of this disordered islet glucose sensing. It has been proposed that reducing hyperglycemia and hyperlipidemia per se may improve islet glucose sensing. Here we studied glucose-dependent insulin release in islets isolated from ob/ob mice treated with dopamine agonists (bromocriptine and SKF38393, BC/SKF) which significantly reduced circulating glucose and lipid levels of ob/ob mice. Islets from BC/SKF-treated mice showed a marked decrease of the elevated basal insulin release to levels similar to lean mice. Such treatment also induced a higher secretory response to glucose stimulation compared with that in ob/ob mice with sustained hyperglycemia and hyperlipidemia. Similarly, when islets from untreated ob/ob mice were cultured for 7 days in 11 mM glucose in the absence of free fatty acid, the basal insulin release was significantly decreased and high glucose stimulated insulin release increased compared with that from islets cultured in medium containing 30 mM glucose and 2 mM oleate. The BC/SKF-induced reduction of elevated basal insulin release was associated with decreased hexokinase activity and basal cyclic AMP content in islet tissue. Our results demonstrate that dopamine agonist treatment improves basal insulin release in ob/ob mice and this effect may be mediated, in part, by a reduction of hyperglycemia and hyperlipidemia.

Bromocriptine/SKF38393 treatment ameliorates obesity and associated metabolic dysfunctions in obese (ob/ob) mice.

It has been postulated that dopaminergic activities comprise a major functional component of a central regulatory system for metabolism which can be manipulated by dopamine modulating drugs. The present study is aimed at delineating the role and importance of pharmacological dopaminergic activation in the regulation of metabolism during obesity and diabetes. We treated C57BL/6J ob/ob mice for 2 weeks with bromocriptine (dopamine D2 agonist), SKF38393 (dopamine D1 agonist), both drugs combined or vehicle and monitored the effects of such treatment on body composition, food consumption, and serum metabolites. Bromocriptine and SKF38393 individually produced moderate improvements in obesity, hyperglycemia, and hyperinsulinemia. However, a combination of bromocriptine plus SKF38393 resulted in major reductions in body weight (7.5 g), body fat (40%), food consumption (42%), and serum concentrations of glucose (59%), triglyceride (37%), free fatty acid (45%) and insulin (49%) while increasing protein mass (8%). These results indicate that regulatory components of metabolism in the ob/ob mouse are modulated by and/or are comprised of dopaminergic activities. Importantly, dopaminergic D1/D2 receptor coactivation maximizes this dopaminergic response (i.e., improvement of metabolic abnormalities) in these mice.

The utilization of lupin (Lupinus angustifolius) and faba bean globulins by rats is poorer than of soybean globulins or lactalbumin but the nutritional value of lupin seed meal is lower only than that of lactalbumin.

The effects of dietary sweet lupin (Lupinus angustifolius, Unicrop) seed meal or its insoluble fiber (nonstarch polysaccharides + lignin) on performance, digestibility and nitrogen utilization in growing rats were studied in four experiments. Globulin proteins isolated from lupin, faba bean (Vicia faba L. minor) or soybean (Glycine max) were also incorporated into purified diets as replacements for lactalbumin (control) and the nutritional effects were evaluated. Isocaloric, legume-based diets supplemented with amino acids were used. Final weight gain, gain:feed ratios, nitrogen retention and net protein utilization of the animals fed whole lupin meal-based diets for 10 d were inferior to those of controls. In contrast, adding lupin insoluble fiber to a control diet produced no adverse effects. Ileal starch and apparent nitrogen digestibilities, and fecal digestibility of starch in lupin-fed rats were higher than those of controls, but fecal true nitrogen digestibility was lower. Replacement of lactalbumin with globulin proteins from lupin or faba bean depressed food intake and protein utilization, but only performance was affected by consumption of soybean globulins. Rats consuming lupin or faba bean globulins excreted significantly more nitrogen, particularly as urea through the urine. This did not occur in rats fed soybean globulins. Urea concentration in plasma was higher in rats fed diets containing lupin meal or legume globulins. The concentrations of urea, arginine and ornithine in plasma increased significantly compared with control values after 3 to 9 h of a lupin diet. After 9 h, plasma lysine was also decreased. We concluded that the main reasons for the low nutritional value of sweet lupin seed meal are likely to be related to the chemical structure of the globulin proteins and their adverse effects on growth and nitrogen metabolism, and not to any known antinutritional factor or poor digestibility.

The regulation of transaminative flux of methionine in rat liver mitochondria.

We have demonstrated methanethiol production from methionine in isolated rat liver mitochondria and shown how it is affected by other metabolites. The enzymes involved include several transaminases, branched chain 2-oxoacid dehydrogenase, acyl-CoA dehydrogenase, and crotonase. Methanethiol production from methionine in mitochondria isolated from rat liver was increased by 50% after the rats had been given a single injection of glucagon, but was reduced by 25% when the rats had been starved for 24 h. These results indicate the physiological importance of the transaminative pathway of methionine metabolism.

Regulation of oxidative degradation of L-lysine in rat liver mitochondria.

The generation of 14CO2 from [1-14C]lysine by hepatic mitochondria through the saccharopine pathway is controlled by intramitochondrial concentrations of lysine, 2-oxoglutarate and NADPH. Mitochondria, isolated from rats pre-treated with glucagon, exhibited higher activities of L-lysine: 2-oxoglutarate reductase, saccharopine dehydrogenase and 2-aminoadipate aminotransferase. The flux through this pathway is stimulated in liver mitochondria after glucagon treatment. Multiple regulation of lysine oxidation in liver mitochondria confirms a complex mechanism of 'mitochondrial activation' by glucagon.

Methionine transamination--metabolic function and subcellular compartmentation.

Enzymatic activities catalysing the inter-conversion of L-methionine and its oxy analogue 4-methylthio-2-oxobutyric acid (2,4-KMB) were detected in the liver, skeletal muscle and heart of the laboratory rat and of sheep. In both species the highest activity of methionine transamination was found in the liver and was located in the cytoplasm and mitochondria. We propose that physiological and nutritional role of the cytoplasmic methionine transamination is amination of 2,4 KMB and formation of L-methionine while in mitochondria the activity is responsible for disposal of excess methionine is oxidised through oxidative decarboxylation of 2,4 KMB.

Determination of L-methionine-dl-sulphoxide in tissue extracts.

The amino acid fraction from rat liver, heart and skeletal muscle was prepared by the separation of sulphosalicylic acid extract on Dowex 50 H+ form. The presence of L-methionine-dl-sulphoxide in these extracts was identified and compared by three independent chromatographic methods: ion-exchange, Pico-Tag and reversed-phase high-performance liquid chromatography after precolumn derivatisation with diethylethoxymethylenemalonate. Quantitative data indicate that L-methionine-dl-sulphoxide is present in the intracellular pool at the levels of free methionine.

Characterization of sheep hepatocytes in primary culture.

1. Primary cultures of isolated sheep hepatocytes were used to characterize metabolic functions of liver: gluconeogenesis, ureagenesis and protein synthesis. The rates of all three metabolic activities were linear over a 20 hr culture period. 2. Hepatocytes in the presence of glucagon increased the synthesis of urea by approx 30% (P < 0.05) and increased release of glucose into the medium by 60% (P < 0.05). 3. In the absence of insulin, significantly more (35%; P < 0.05) glucose was released in the medium than in the presence of insulin. 4. Results help evaluate the primary culture of sheep hepatocytes as an appropriate experimental model to study nutritional and hormonal regulation of liver in the ruminant species.

Urea synthesis in rats fed diet containing kidney beans.

When rats were fed a diet containing kidney bean (Phaesolus vulgaris) urea excretion was increased 3-5 fold. Isolated liver mitochondria from rats fed the kidney bean diet produced 40% more citrulline in the presence of arginine than mitochondria isolated from control rats. Mitochondrial activities of urea cycle enzymes and N-acetylglutamate synthetase were similar in animals fed diets containing kidney bean or lactalbumin. The possible mechanisms causing acute urea production in rats fed with kidney bean are discussed.

Metabolic effects of proglycosyn.

Proglycosyn, a phenylacyl imidazolium compound that lowers blood glucose levels, was demonstrated previously to promote hepatic glycogen synthesis, stabilize hepatic glycogen stores, activate glycogen synthase, inactivate glycogen phosphorylase, and inhibit glycolysis. In the present study proglycosyn was found to inhibit fatty acid synthesis, stimulate fatty acid oxidation, and lower fructose 2,6-bisphosphate levels, but to have no significant effects on cell swelling and the levels of cAMP in hepatocytes prepared from fed rats. Verapamil and atropine blocked the effects of proglycosyn on glycogen metabolism, but these compounds inhibit proglycosyn accumulation by hepatocytes. Proglycosyn stimulated phosphoprotein phosphatase activity in postmitochondrial extracts, as measured by dephosphorylation of phosphorylase a and glycogen synthase D, but this action required a very high concentration of the compound, making it unlikely to be the actual mechanism involved. It is proposed that a metabolite of proglycosyn is responsible for its metabolic effects.

Heart mitochondria metabolize 3-methylthiopropionate to CO2 and methanethiol.

3-Methylthiopropionate (MTP) stimulates respiration of substrate-depleted heart mitochondria. This is blocked by uncouplers and by malonate. With the use of methyl-14C- and uniformly 14C-labeled MTP, it was found that methanethiol and CO2 are reaction products. The methyl carbon was not significantly oxidized. This study, together with a recent report [P. W. D. Scislowski et al. (1987) Biochem. J. 247, 35-40], demonstrates the existence of a transsulfuration independent pathway of methionine metabolism by muscle, and that the complete pathway following the initial transamination is a mitochondrial process. The data suggest that MTP is oxidized via acetyl-CoA.

Sulfur oxidation of free methionine by oxygen free radicals.

The oxidation of free methionine to methionine sulfoxide by chemically or enzymatically generated oxygen free radicals is presented. The physiological significance of this process in living cells is suggested.

Methionine metabolism by rat muscle and other tissues. Occurrence of a new carnitine intermediate.

Perfused rat hindquarter preparations were shown to incorporate radioactivity from [U-14C]methionine into citrate-cycle intermediates, lactate, alanine, glutamate, glutamine and CO2. During perfusion, large amounts of methionine were also oxidized to methionine sulphoxide. The capacity for transamination of methionine or its oxo analogue, 4-methylthio-2-oxobutyrate, by muscle extracts was demonstrated. Rat skeletal muscle, heart, liver and kidney mitochondria, when incubated with the latter plus radiolabelled carnitine, formed a newly identified carnitine derivative, 3-methylthiopropionylcarnitine. It is concluded that the capacity for oxidation of methionine by a trans-sulphuration-independent pathway occurs in several mammalian tissues. The extent of inter-organ handling of intermediates in this pathway(s) is discussed.

Amino acid catabolism by perfused rat hindquarters: degradation of threonine and isoleucine.

Rat hindquarters were perfused without added substrate other than trace amounts of [U-14C]threonine or [U-14C]isoleucine. Comparison of incorporation of radiolabel into some nonessential amino acids, citrate cycle intermediates, and lactate is presented. Activities of three enzymes for the initial reactions in threonine degradation are reported. It is concluded that skeletal muscle catabolizes threonine, and that the latter is a potential source of carbon for glucogenic precursors for the liver. In contrast, label from isoleucine was incorporated into glutamate, glutamine, and alanine much more than was that from threonine. Large amounts of organic acids accumulated, and more than 60% of total radioactivity was lost as CO2 during a 2-h perfusion period.

A sensitive spectrophotometric assay of pyruvate dehydrogenase activity.

The recently reported highly sensitive method for assay of acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) [H. H. Andres, A. J. Klein, S. M. Szabo, and W. W. Weber (1985) 145, 367-375] has been adapted for determination of pyruvate dehydrogenase activity. This method provides an improvement in sensitivity over extant spectrophotometric methods and circumvents limitations of assays using radioactive pyruvate. In addition, the assay is simple and inexpensive and can be readily adapted for measurement of enzyme activity in crude tissue extracts or homogenates.

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.