Responses of circulating urea cycle and branched-chain amino acids to feeding in adult and aged Fischer-344 rats.

Amino acid concentrations in plasma and blood were compared between adult (6 months) and aged (22 months) male rats in fed and food-deprived (fasted) states. Since feeding is known to elevate plasma amino acids, rats were sampled after food had been freely available to them and after food was withheld for 20 hours. Fourteen amino acids increased (8 to 74%) in blood and plasma in fed rats compared to food-deprived rats. Blood concentrations of four amino acids (cystine, aspartate, threonine, and phosphoserine) were increased in aged compared to adult rats, while tyrosine was 10% lower. The interaction between fed state and age was significant for the branched-chain amino acids (BCAA) as they increased 23% in the blood and plasma of fed compared to food-deprived adult rats; however, BCAA were not affected by feeding state in the aged rats. Plasma arginine concentration was 12% lower (p < 0.001) and citrulline was 20% higher (p < 0.005) in aged compared to adult rats, and consequently the citrulline:arginine ratio was elevated by 36% in aged rats. Our results demonstrate that, although the total arginine in whole blood is not altered or is increased with aging, less of the arginine is transported in the plasma of aged compared to adult rats. Furthermore, these results indicate altered metabolism of the urea cycle amino acids and BCAA with aging in the rat.

Protein metabolism in rheumatoid arthritis and aging. Effects of muscle strength training and tumor necrosis factor alpha.

OBJECTIVE: To determine the effects of rheumatoid arthritis (RA) on whole-body protein metabolism. METHODS: We examined protein metabolism and its hormonal and cytokine mediators before and 12 weeks after progressive resistance muscle strength training in 8 healthy young (mean +/- SD age 25 +/- 2 years) and 8 healthy elderly (70 +/- 5 years) men and women, and in 8 adults with RA (42 +/- 13 years). An additional 6 healthy elderly subjects (69 +/- 3 years) served as a swimming-only control group. RESULTS: Subjects with RA had higher rates of protein breakdown than did young or elderly healthy subjects (79.9 +/- 17.2 versus 60.3 +/- 5.8 and 63.7 +/- 12.4 mumoles/gm total body potassium/hour, respectively, P < 0.05), while there was no effect of age per se. Patients treated with methotrexate had normal rates of protein breakdown (P < 0.01 versus RA without methotrexate; P not significant versus healthy young subjects). Increased protein catabolism in RA was no longer evident after strength training. In multiple regression analysis, levels of tumor necrosis factor alpha (TNF alpha) (r = 0.47, P = 0.01) and growth hormone (r = -0.51, P = 0.006) were associated with protein breakdown, and plasma glucagon levels were inversely correlated with protein synthesis (r = -0.45, P = 0.02). Growth hormone (r = -0.56, P = 0.002) and glucagon (r = 0.45, P = 0.04, levels were associated with protein oxidation. CONCLUSION: Adults with RA have increased whole-body protein breakdown, which correlates with growth hormone, glucagon, and TNF alpha production.

Dietary citrulline but not ornithine counteracts dietary arginine deficiency in rats by increasing splanchnic release of citrulline.

Ornithine and citrulline have different arginine-sparing capabilities when provided in an arginine-deficient diet. Rats were fed arginine-deficient diets containing ornithine or citrulline to see the effect on net fluxes of free arginine, ornithine and citrulline across the portal-drained viscera (PDV) and liver. The diets contained either 10 g arginine/kg diet (+Arg) or no arginine: the -Arg/+Ala diet contained additional alanine and the -Arg/+Orn and -Arg/+Cit diets contained ornithine and citrulline, respectively, in amounts isomolar to the amount of arginine in the control (+Arg) diet. Blood arginine concentrations were 50% lower than control values (P < 0.001) in rats fed the -Arg/+Ala or the -Arg/+Orn diet. However, addition of citrulline to the arginine-deficient diet restored blood arginine concentrations. The source of the circulating arginine in the rats fed the -Arg/+Cit diet was neither the PDV nor the liver; net splanchnic uptake of arginine was 0.3 mumol/min. Net citrulline release was substantial from the PDV (0.65 mumol/min) as well as from the splanchnic bed (0.67 mumol/min) of rats fed the -Arg/+Cit diet, such that blood citrulline concentrations were more than double (P < 0.001) those in rats fed the +Arg or -Arg/+Orn diet. Splanchnic release of citrulline in rats fed the -Arg/+Cit diet but not in rats fed the -Arg/+Orn diet supported the production of arginine in non-splanchnic organs such as, presumably, the kidney.

Supplemental arginine and ornithine do not affect splenocyte proliferation in surgically treated rats.

The present study was designed to determine whether arginine or ornithine supplementation enhanced immune responsiveness in surgically stressed rats. Young rats (130 to 150 g; n = 72) were fed one of three nonpurified diets: control, arginine-supplemented (30 g/kg of diet), or supplemented with ornithine on an equimolar basis to supplemental arginine. Control and ornithine-supplemented diets were made isonitrogenous to the arginine-supplemented diet with alanine. Food intake and body weight were monitored throughout the experimental period. Eight days after initiation of dietary treatments, 36 rats were given dorsal skin wounds. Rats were killed 7 days later. Blood was collected, spleen and thymus were weighed, and splenocytes were isolated to measure proliferation in response to mitogens and interleukin-2 production. Food intake, body weight gain, and thymus weight were lower in rats subjected to surgery than in controls rats (p < .01). Neither supplemental dietary arginine nor ornithine affected food intake, body weight gain, thymus weight, splenocyte proliferation, or splenocyte interleukin-2 production in any treatment group (p < .1). These data suggest that low-level dietary supplementation of arginine and ornithine did not ameliorate detrimental effects of minor surgery in rats.

Oral arginine supplementation does not affect lymphocyte proliferation during endotoxin-induced inflammation in rats.

Earlier studies from this laboratory were unable to confirm reported immunostimulatory effects of supplemental dietary arginine on healthy, unstressed young or aged rats. The present study was undertaken to determine effects of oral arginine supplementation on in vitro measures of immune function using a stressed rat model. The stressor used was intraperitoneal injection of bacterial lipopolysaccharide (1 mg/kg body wt). Four-month-old male Sprague-Dawley rats were placed in either a control or an arginine-supplemented (7.5 g/L arginine-HCl in drinking water) group for 7 d, after which control and supplemented rats received injections of endotoxin or phosphate-buffered saline. Rats were killed 3 d following injections. Endotoxin treatment resulted in lower food intake, less thymic cellularity and greater splenic weight. Endotoxin injections also enhanced proliferative response of rat splenocytes to pokeweed mitogen (1 mg/L) and lipopolysaccharide (25 and 100 mg/L) and enhanced response of thymocytes to concanavalin A (10 mg/L), phytohemagglutinin (25 and 100 mg/L) and pokeweed mitogen (1 mg/L). Supplemental arginine did not reduce thymic weight loss or influence mononuclear cell proliferation or interleukin-2 production in the presence or absence of endotoxin stress. These data indicate no benefit of arginine supplementation during endotoxin stress in rats.

Dietary arginine deficiency alters flux of glutamine and urea cycle intermediates across the portal-drained viscera and liver of rats.

The effect of an arginine-deficient diet on net flux of amino acids across the portal-drained viscera and across the liver was studied in rats. Blood was obtained after food deprivation and 1 and 2 h after a meal of a 1.0% arginine control diet or an arginine-deficient diet containing 3.4% glutamate. The arginine-deficient diet decreased net portal-drained viscera flux of arginine and increased net portal-drained viscera flux of ornithine and proline. However, net portal-drained viscera flux of citrulline (0.35 +/- 0.05 mumol/min) was not influenced by diet; of this rate, 46% (0.16 mumol/min) bypassed the liver and was available for extrahepatic arginine synthesis. However, rats continued to exhibit signs of arginine deficiency such as decreased blood arginine concentrations (by 28%) and increased orotic acid excretion (90-fold). Arterial blood glutamine concentration was 25% higher in rats fed the arginine-deficient diet. In the fed state, net hepatic flux of glutamine was elevated from 0.15 (control) to 1.39 mumol/min, indicating that the liver was a major source of the increased blood glutamine concentrations. Increased production of hepatic glutamine and orotic acid may help rats compensate for dietary arginine deficiency, whereas splanchnic output of citrulline was not increased with dietary arginine deficiency even with a substantial dietary supply of glutamate.

Arginine-deficient diets alter plasma and tissue amino acids in young and aged rats.

Blood and urine metabolites were measured in two experiments for young (2-mo-old) and aged (20-mo-old) male Sprague-Dawley rats fed arginine-devoid diets made isonitrogenous to a control 1.12% arginine diet by adding alanine or glycine. Diet, fed for 7 or 13 d, had little effect on urinary or plasma ammonia and urea. Urinary orotate excretion was more than 40-fold higher in rats fed the arginine-deficient diets (P less than 0.01) in both experiments. Source of nonessential N (alanine or glycine) in the arginine-deficient diets did not alter orotic acid excretion or plasma or urine ammonia or urea. Changes in plasma arginine, alanine and glycine concentrations reflected the levels of these amino acids in the diet. Tissue ornithine levels reflected dietary arginine level, but tissue citrulline was unaffected by dietary arginine. Glutamate and glutamine were greater in the plasma and liver of rats fed arginine-deficient diets. Plasma concentrations of glutamate and glutamine were positively correlated with urinary orotic acid excretion (P less than 0.05) and ornithine and arginine were negatively correlated with orotic acid excretion (P less than 0.01). Increased tissue glutamine may be related to the greater orotate excretion in rats fed arginine-devoid diets. The metabolic responses to dietary arginine deficiency were similar in young and aged rats. In general, concentrations of amino acids in plasma, liver and spleen were higher in aged rats.

Dietary arginine supplementation does not enhance lymphocyte proliferation or interleukin-2 production in young and aged rats.

Recent studies indicate that supplemental arginine may enhance in vitro lymphocyte mitogenesis. To determine whether dietary arginine could reverse age-associated losses in immune functions, we fed purified amino acid diets to young (2-mo-old) and aged (24-mo-old) Fischer 344 rats. Rats receiving control (1.12% arginine) or supplemented (3% arginine) diets were pair fed to intakes of deficient (0% arginine) rats. Another group was fed the supplemented diet ad libitum. On d 15, responses of splenocytes to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) were lower (P less than 0.01), but interleukin-2 (IL-2) production was higher (P less than 0.05) in aged rats than in young rats. At mitogen doses producing maximal stimulation, supplemental arginine did not enhance PHA-, Con A- or PWM-stimulated lymphocyte proliferation; PWM responses at sub-maximal doses were higher in pair-fed supplemented rats than in control or ad libitum supplemented rats (P less than 0.05). Arginine supplements did not increase thymus weights or IL-2 production above controls. In another experiment, weanling rats received control and supplemented diets in amounts equal to the intake of deficient rats for an average of 37 d. Splenocytes were cultured with mitogens at various arginine levels. No diet effect was observed. Mitogenesis was maximal when media arginine approximated normal plasma levels. Our results suggest that supplemental arginine has little effect on lymphocyte proliferation or IL-2 production in healthy young and aged rats.

Dietary self-selection and the Zucker rat.

Impaired protein deposition has been suggested to be a critical factor promoting the hyperphagia of the obese Zucker rat. The selection patterns of adult, male, genetically obese (fa/fa) and lean (Fa/-) Zucker rats were studied to determine if obese rats would select a diet that was higher in protein than the diet selected by lean littermates. Rats were provided ad libitum access to three macronutrient sources and were allowed to compose their own diets for 9 days. The three dietary items were: a vitamin + mineral--supplemented carbohydrate source (cornstarch), a vitamin + mineral--supplemented protein source (casein) and commercially available corn oil. Obese rats ate 43% more calories than lean littermates. Further, obese rats selected a diet that provided 12% of their total caloric intake as protein, 24% as carbohydrate and 64% as fat. Lean rats selected a diet that provided 33% of their total caloric intake as protein, 37% as carbohydrate and 30% as fat. These selection data are not consistent with the hypothesized importance of the role of dietary protein and its incorporation into lean body mass as a stimulus promoting the hyperphagia demonstrated by the genetically obese Zucker rats.

Incidence of increased numbers of Clostridium perfringens in the intestinal tract of rats fed xylitol.

Sprague-Dawley rats were fed diets containing up to 20% xylitol for 49 days. When the rats were fed a xylitol regimen intended to produce adaptation to xylitol, approximately half of the animals adapted to xylitol and remained free from diarrhea during the feeding regimen. The other half did not adapt to xylitol and developed severe and persistent diarrhea accompanied by large volumes of intestinal gas. These non-adapted rats had significantly higher levels of intestinal tract Clostridium perfringens (10(6)--10(11) organisms per gram intestinal contents) than did control rats fed a xylitol-free cornstarch diet (0-10(4) organisms per gram). Rats adapted to dietary xylitol did not have detectable levels of C. perfringens in the gastrointestinal tract.

Effects of dietary xylitol on redox state and gluconeogenesis in the rat liver.

Rats fed a 20% xylitol diet were compared to rats fed for 6 weeks a diet of either 20% glucose, cornstarch or sucrose (plus 45% cornstarch), or the AIN-76TM diet (basal diet). There were no differences between the rats (diarrhea-free) fed xylitol and those fed the other carbohydrate sources in the lactate/pyruvate ratio in freeze clamped livers. Gluconeogenesis was measured from 10 mM of various precursors in isolated hepatocytes. Rats fed xylitol had: a) a lower rate of glucose production from lactate compared to rats fed the sucrose, glucose or basal diet; b) a lower rate of gluconeogenesis from xylitol than in rats fed either the basal or glucose diets, and c) equal capacity to produce glucose from pyruvate, glycerol, dihydroxyacetone or alanine as rats fed the other diets. Ethanol added to isolated hepatocyte incubations did not have a greater inhibitory effect on gluconeogenesis in xylitol-fed animals than in those fed the other carbohydrates. Results of this study indicate that dietary xylitol has little effect on the hepatic cytosolic redox state or on the ability of the rat liver to produce glucose in the presence of ethanol, but may have an effect on the rate of glucose production from some precursors.