Kazunori Mawatari

Branched-Chain Amino Acid Catabolism in Exercise and Liver Disease

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, the enzyme catalyst for the second step of the BCAA catabolic pathway, plays a central role in the regulation of BCAA catabolism. The activity of the complex is regulated by a covalent modification cycle in which phosphorylation by BCKDH kinase inactivates and dephosphorylation by BCKDH phosphatase activates the complex. Many studies suggest that control of the activity of the kinase is a primary determinant of the activity of the complex. The kinase exists at all times in the mitochondrial matrix space in two forms, with a large amount being free and a smaller amount bound rather tightly to the BCKDH complex. Only the bound form of the kinase appears to be catalytically active and, therefore, responsible for phosphorylation and inactivation of the complex. alpha-Ketoisocaproate, the transamination product of leucine and the most important known physiological inhibitor of BCKDH kinase, promotes release of the kinase from the complex. alpha-Chloroisocaproate, the analogue of leucine and the most potent known inhibitor of the kinase, is more effective than alpha-ketoisocaproate in promoting release of BCKDH kinase from the complex. Exercise and chronic liver disease (liver cirrhosis) likewise decrease the amount of the kinase bound to the complex in rat liver. The resulting activation of the BCKDH complex appears responsible for the increase in BCAA catabolism caused by exercise and liver cirrhosis. Our findings support the use of BCAA supplements for patients with liver cirrhosis.

Thirteen-week oral toxicity study of branched-chain amino acids in rats.

Branched-chain amino acids (l-isoleucine, l-valine, and l-leucine) are being increasingly used in sport supplements. This study evaluated toxicological and behavioral effects of l-isoleucine (Ile), l-valine (Val), and l-leucine (Leu) during a dosing study with male and female Sprague-Dawley rats. The amino acids were incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. No significant, dose-related effects on body weight were found in rats fed a Leu-and Ile-supplemented diet. Val mixed into a diet at 5.0% (w/w) decreased slightly, but significantly body weight gain in females, but not males. Ile (5.0% w/w) affected the urine electrolytes, protein, ketone bodies, urine glucose, and urobilinogen in both genders, yet the observed changes remained mostly within the range observed in controls. The random findings in hepatology and ophthalmology at the 13-week sacrifice were not considered toxicologically relevant to effects of the tested amino acids. No significant changes in organ weights were recorded. We estimate the no-observed-adverse-effect level (NOAEL) for Ile at 2.5% for both genders (male, 1.565 ± 0.060 g/kg/day; females, 1.646 ± 0.095 g/kg/day), Val at 5.0% for males (3.225 ± 0.135 g/kg/day) and 2.5% for females (1.853 ± 0.060 g/kg/day), and Leu at 5.0% for both genders (males, 3.333 ± 0.101 g/kg/day: females, 3.835 ± 0.257 g/kg/day).

Thirteen-week oral toxicity study of L-arginine in rats.

The amino acid l-arginine (Arg) has been used extensively in dietary and pharmacological products. This study evaluated toxicological and behavioral effects of Arg produced by Ajinomoto Co. (Tokyo, Japan) during a dosing study with male and female Sprague-Dawley rats. The amino acid was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received only a standard diet. All diets were administered ad libitum for 13 continuous weeks. To examine recoverability of any potential effects, the administration period was followed by a 5-week-long recovery, during which only a standard diet was provided. In male and female rats in each concentration group, treatment-related changes were not observed for clinical signs, body weights, diet consumption, ophthalmology, gross pathology, organ weight, or histopathology. An elevated level of plasma glucose was detected in some male rats (5.0%, w/w) during the analysis conducted in the fifth week of administration; however, the degree of the change was within the physiological range, and no changes were observed at the end of the administration period. In the same group, an increase in hemoglobin, together with a tendency toward an increase in the red blood cell counts, was found, but the change was considered toxicologically insignificant. The no-observed-adverse-effect level (NOAEL) for Arg was estimated at 5.0% (w/w) for both genders (males, 3.3 ±0.1 g/kg/day; females, 3.9 ±0.2 g/kg/day).

Alterations in cognitive function in prepubertal mice with protein malnutrition : Relationship to changes in choline acetyltransferase

We have found that protein malnutrition (PM) causes a significant impairment of memory-related behavior on the 15th and 20th day after the start of PM (5% casein) feeding in prepubertal mice but not in postpubertal mice, as measured by a passive-avoidance task. This impairment was almost completely reversed by merely switching to a standard protein (20% casein) diet on the 10th day after the start of PM. However, the reversal was not observed when the switching to a standard protein regimen was done on the 15th day of the PM diet. Interestingly, the impairment of memory-related behavior on the 20th day was improved by the chronic administration of physostigmine (0.1 mg/kg/day x last 10 days, i.p.), a cholinesterase inhibitor. To correlate brain cholinergic neuron function with the memory-related behavior impairment induced by PM, microphotometry was used to determine the histological distribution of the imunofluorescence intensity for choline acetyltransferase (ChAT), a functional marker of presynapse in cholinergic neurons. The change in the intensity of fluorescence indicated that ChAT protein was decreased in the hippocampus (CA1, CA3 and dentate gyrus) on the 20th day after PM feeding in comparison with controls. These results suggest the possibility that the memory-related behavior deficits observed in prepubertal mice with PM are caused by a dysfunction of the cholinergic neurons in the hippocampus.

Thirteen-week oral toxicity study of L-lysine hydrochloride in rats.

l-Lysine hydrochloride (Lys) is an essential amino acid in humans and animals, and it is used in animal feeds, in prevention of herpes simplex recurrence, and cereal fortification in some developing countries. This study evaluated toxicological and behavioral effects of Lys during a dosing study with male and female Sprague-Dawley rats. The amino acid was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine stability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. In male and female rats in each concentration group, treatment-related changes were not observed in the clinical signs, body weights, diet consumption, water intake, ophthalmology, gross pathology, organ weights, or histology. A Lys-related drop in serum concentration and an increase in urine excretion of chlorides was a compensatory reaction to the ingested hydrochloride. No functional, biochemical, or histological changes in renal function were found. The no-observed-adverse-effect level (NOAEL) for Lys was estimated at 5.0% for both genders (male, 3.36 ± 0.12 g/kg/day; female, 3.99 ± 0.28 g/kg/day).

Thirteen-Week Oral Toxicity Study of l-Glutamine in Rats

l-Glutamine (Gln) is a semiessential amino acid used in enteral feeding in critically ill patients, and is contained in numerous dietary supplements available to the general public. This study evaluated toxicological effects of Gln in male and female Sprague-Dawley rats. Gln produced by Ajinomoto Co. (Tokyo, Japan) was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w), respectivelly. A control group of rats received only a standard diet. All diets were administered ad libitum for 13 consecutive weeks. To examine recoverability of any potential effects, the administration period was followed by a 5-week recovery period, during which only the standard diet was provided to all animals. Throughout the administration and recovery periods, no deaths were observed, and no changes in diet consumption, ophthalmologic findings, gross pathology, and histopathology were detected. Several changes in urine parameters (total protein, urine pH, and a positive incidence (±) of ketone bodies) were observed in the 2.5% and 5.0% groups at the end of the administration period. Minor increases were found in hematology parameters for the 5.0% group (platelet count, γ-globulin, lactate dehydrogenase [LDH]), but all changes were within physiological range. No effects of administration were observed in the 1.25% group. The no-observed-adverse-effect level (NOAEL) for Gln was estimated at 1.25% for both genders (males 0.83 ± 0.01 g/kg/day; females, 0.96 ± 0.06 g/kg/day).

Restoration by dietary glutamine of reduced tumor necrosis factor production in a low-protein-diet-fed rat model

Tumor necrosis factor-α (TNF) production by peritoneal macrophages and its dietary modification were investigated by using rats fed on a low-protein diet. The rats were given a 20% casein (control) diet or a 3% casein diet for 21 days, and TNF production was measured in activated macrophages of these animals. TNF production was significantly lower in macrophages from rats fed on the low-protein diet than that in macrophages from rats fed on the control diet. Oral administration of a cabbage extract, a known modulator of TNF production, to the low-protein-diet-fed rats significantly enhanced TNF production by macrophages. Glutamine supplementation to the low-protein diet significantly enhanced TNF production as well as TNF mRNA expression. These results indicate that the 3%-casein-diet-fed rat would be useful as a model for reduced TNF production in protein malnutrition. These results also suggest that glutamine administration restored the reduced TNF production associated with protein malnutrition.