Bohdan Luhovyy

Response of brain amino acid metabolism to ketosis.

Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.

Ifosfamide-Induced Nephrotoxicity: Mechanism and Prevention

The efficacy of ifosfamide (IFO), an antineoplastic drug, is severely limited by a high incidence of nephrotoxicity of unknown etiology. We hypothesized that inhibition of complex I (C-I) by chloroacetaldehyde (CAA), a metabolite of IFO, is the chief cause of nephrotoxicity, and that agmatine (AGM), which we found to augment mitochondrial oxidative phosphorylation and beta-oxidation, would prevent nephrotoxicity. Our model system was isolated mitochondria obtained from the kidney cortex of rats treated with IFO or IFO + AGM. Oxidative phosphorylation was determined with electron donors specific to complexes I, II, III, or IV (C-I, C-II, C-III, or C-IV, respectively). A parallel study was done with (13)C-labeled pyruvate to assess metabolic dysfunction. Ifosfamide treatment significantly inhibited oxidative phosphorylation with only C-I substrates. Inhibition of C-I was associated with a significant elevation of [NADH], depletion of [NAD], and decreased flux through pyruvate dehydrogenase and the TCA cycle. However, administration of AGM with IFO increased [cyclic AMP (cAMP)] and prevented IFO-induced inhibition of C-I. In vitro studies with various metabolites of IFO showed that only CAA inhibited C-I, even with supplementation with 2-mercaptoethane sulfonic acid. Following IFO treatment daily for 5 days with 50 mg/kg, the level of CAA in the renal cortex was approximately 15 micromol/L. Taken together, these observations support the hypothesis that CAA is accumulated in renal cortex and is responsible for nephrotoxicity. AGM may be protective by increasing tissue [cAMP], which phosphorylates NADH:oxidoreductase. The current findings may have an important implication for the prevention of IFO-induced nephrotoxicity and/or mitochondrial diseases secondary to defective C-I.

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine.

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.

Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N

The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.

Agmatine Stimulates Hepatic Fatty Acid Oxidation A POSSIBLE MECHANISM FOR UP-REGULATION OF UREAGENESIS

We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled β-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled β-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial β-oxidation and suggest a coupling between the stimulation of hepatic β-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.

Acute sodium ingestion has no effect on short-term food and water intake, subjective appetite, thirst, or glycemic response in healthy young men

The high intake of dietary sodium (Na(+)) has been associated with obesity and insulin resistance, sparking the hypothesis that the consumption of salty foods affects food intake (FI) and postprandial blood glucose (BG) response. Therefore, we conducted 2 randomized repeated-measures experiments to examine the acute effects of the Na(+) content of solid food and beverage on FI, water intake (WI), subjective appetite, thirst, and BG. FI and WI were measured at ad libitum pizza test meals; appetite, thirst, and BG were measured at baseline and at regular intervals before and after meals. In the first experiment, 16 males (mean body mass index (BMI), 22.2 kg·m(-2)) consumed a low-Na(+) (71 mg) bean preload (300 kcal) with or without 740 mg or 1480 mg of added Na(+) 120 min prior to the pizza meal. Participants ate 116 kcal more at the test meal after consuming beans with 740 mg of added Na(+) than after beans with 1480 mg of added Na(+). In the second experiment, 19 males (mean BMI, 23.2 kg·m(-2)) consumed a low-Na(+) (62 mg) tomato beverage (73 kcal) with or without 500, 1000, 1500, or 2000 mg of added Na(+) 30 min prior to a pizza meal. The beverage with 2000 mg of added Na(+) led to higher WI during the pizza meal than the beverage with 500 mg of added Na(+). However, compared with the control conditions (no added Na(+)), added Na(+) treatments had no effect on dependent measures in either experiment. In conclusion, the acute intake of Na(+), in a solid or liquid form, did not affect short-term subjective ratings of appetite or thirst, ad libitum FI or WI, or BG in healthy young men.

Canned Navy Bean Consumption Reduces Metabolic Risk Factors Associated with Obesity

The high prevalence of obesity and its metabolic co-morbidities require dietitians to promote lifestyle modifications that can be effectively implemented into practice and are feasible for customers to adhere to. The objective of this study was to determine the effect of commercially available ready-to-eat canned navy beans added to the habitual diet on risk factors associated with obesity. Fourteen overweight and obese adults consumed 5 cups of canned navy beans per week for 4 weeks. The study results demonstrated that bean consumption results in reduced waist circumference in females by 2.5 cm and males by 2.1 cm (P < 0.001). The effect of beans on pulse rate, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL) were sex dependent (P < 0.05). In males, pulse rate, TC, and LDL were decreased by 6.5%, 11.5%, and 18%, respectively. In females, pulse rate increased by 9.6%, and TC and LDL were relatively unchanged. There was a trend for a decreased glucose AUC (P = 0.06) in response to a glucose load. This study demonstrates that consuming 5 cups per week of ready-to-eat canned navy beans for 4 weeks reduces metabolic risk factors associated with obesity and therefore can be used as a tool in dietetic practice.

The effect of dairy products consumed with high glycemic carbohydrate on subjective appetite, food intake and post-prandial glycemia in older adults

The objective was to compare the effect of liquid, semi-solid, and solid dairy products and a nondairy beverage when consumed with glycemic carbohydrate on subjective appetite, food intake (FI), and post-prandial glycemia (PPG) in healthy older adults. Thirty healthy men and women (14 males and 16 females; age: 64.6 ± 2.4 y; BMI: 25.6 ± 2.5 kg/m2) participated in a randomized crossover study. Treatments were one of 250 mL of 2% fat milk and soy beverage, 175 g of 2% Greek yogurt, and 30 g of Cheddar cheese consumed as part of an isocaloric (380 kcal) meal with bread and jam. Water alone served as the energy-free control for subjective appetite. At 180 min after consumption, the participants were fed an ad libitum meal to measure FI. Subjective appetite, blood glucose, and insulin were measured at baseline and at intervals both before (post-treatment) and after the meal (postmeal). Cheese and yogurt resulted in lower post-treatment blood glucose than milk and soy beverage when consumed with carbohydrate (p < 0.0001), but no differences among any treatments were observed postmeal. Treatments led to similar insulin concentrations. Post-treatment appetite was lower than after the water control for all treatments but suppressed more by cheese and yogurt compared with milk (p < 0.0001). There were no differences in FI among treatments. Cheese and yogurt increase satiety and lower PPG more than milk or a soy beverage when consumed with carbohydrate.

The effect of dairy and non-dairy beverages consumed with high glycemic cereal on subjective appetite, food intake and post-prandial glycemia in young adults

The objective was to compare the effect of dairy and nondairy beverages when consumed with carbohydrate at breakfast on subjective appetite, food intake (FI), and postprandial glycemia (PPG) in healthy young adults. Twenty-six healthy males and females (13 males and 13 females; 23.0 ± 2.6 years; BMI: 22.3 ± 1.5 kg/m2) participated in a randomized crossover study. They consumed nonisocaloric amounts (250 mL) of almond beverage, soy beverage, 1% fat milk, yogurt beverage, and water (control) with cereal and 120 min later, an ad libitum meal. Subjective appetite, PPG, and insulin were measured at baseline and at intervals before and after the meal at which FI was measured. Post-treatment blood glucose was lowest following soy beverage compared with all treatments but was not different from milk (p = 0.0002). There were no differences between any other treatments. However, over the first hour, PPG for all treatments was 27% lower compared with water (p < 0.0001). Milk and yogurt beverage led to the highest insulin concentrations post-treatment (p < 0.0001) but there were no differences between treatments postmeal. All treatments reduced appetite and led to lower FI at the meal compared with water, but FI was lower after milk compared with all treatments except yogurt beverage (p < 0.0001). Both dairy and nondairy beverages consumed with a high glycemic cereal at breakfast increased satiety and decreased FI compared with water with cereal. Despite higher carbohydrate content, all beverages led to similar or lower PPG than the water breakfast, but dairy beverages increased insulin more than nondairy beverages.