Effect of dietary lysine on hepatic lysine catabolism in broilers.

Lysine is frequently a first- or second-limiting amino acid in poultry diets. Improving the efficiency of lysine use for protein synthesis would effectively lower the lysine requirement and decrease feed costs. Understanding how lysine is degraded and how the degradation is regulated would identify potential molecular targets for interventions to decrease lysine degradation. To better understand lysine degradation in poultry, 3 experiments were conducted. In experiment 1, one-day-old chicks were fed 1.07, 1.25, 1.73, or 3.28% dietary lysine for 2 wk. In experiments 2 and 3, fourteen-day-old chicks were fed 1.07 or 1.25% dietary lysine for 2 wk. Measures of liver lysine catabolism including lysine α-ketoglutarate reductase (LKR) and lysine oxidation (LOX) were assessed. The α-aminoadipate δ-semialdehyde synthase (AASS) is a bifunctional enzyme composed of both LKR and saccharopine dehydrogenase activities, and the relative abundance of this protein and mRNA were likewise assessed. Moreover, potential alternative pathways of lysine catabolism that depend on l-amino acid oxidase (AAOX) and on lysyl oxidase (LYLOX) were considered. In experiment 1, chicks fed lysine-deficient diets had decreased (P < 0.05) LKR activities compared with chicks fed at or above the requirement. However, the lowered LKR activities were not associated with a decreased (P > 0.05) LOX as measured in vitro. In experiments 2 and 3, chicks 28 d of age did not decrease LKR activity (P > 0.05) in response to a lysine-deficient diet. No changes in AASS protein abundance or mRNA were detected. Likewise, no differences in the mRNA abundances of AAOX or LYLOX were detected. The activity of AAOX did increase (P < 0.05) in birds fed a lysine-adequate diets compared with those fed a lysine-deficient diet. Based on kinetic parameters and assumed concentrations, AAOX could account for about 20% of liver lysine oxidation in avians.

Tissue distribution of indices of lysine catabolism in growing swine.

The primary pathway of lysine degradation in pigs presumably depends on the bifunctional protein α-aminoadipate δ-semialdehyde synthase (AASS), which contains lysine α-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) activities. In liver, AASS is restricted to the mitochondrial matrix and lysine is presumptively transported through the plasma membrane by a cationic AA transporter (CAT1/2) and through the inner mitochondrial membrane by 1 or both mitochondrial ornithine transporters (ORC-1/ORC-2). Lysyl oxidase (LO) may represent an alternative pathway of lysine oxidation. The objective of this experiment was to analyze the distribution of indices of lysine catabolism in various pig tissues. We assessed LKR, SDH, and LO activities, lysine oxidation, mRNA abundance of LKR, CAT1/2, and ORC1/2, and AASS protein abundance (via SDH antibody) in liver, heart, kidney medulla and cortex, triceps, longissimus, whole intestine, enterocytes, and intestine stripped of enterocytes in 10 growing pigs, weighing ∼25 kg. The LKR activity differed across tissues (P<0.001) and was greatest in liver, intestine, and kidney samples, and LKR mRNA abundance (P<0.001) was greatest in liver; although, LKR activity and mRNA abundance were detected in all other tissues. Activity of SDH (P<0.001) and SDH mRNA abundance (P<0.001) were affected by tissue and were greatest in liver compared with all other tissues analyzed. The AASS protein abundance (P<0.001) was greatest in whole intestine and liver. Activity of LO (P<0.0001) was greatest in muscle samples. The abundance of ORC-1 (P<0.001) and ORC-2 mRNA (P<0.001) differed among tissues, and ORC-1 was greatest in liver, kidney, and intestinal preparations, and ORC-2 mRNA abundance was greatest in liver and intestine. Interestingly, LKR activity was correlated with ORC-1 (r=0.32, P<0.05) and ORC-2 (r=0.41, P<0.05) expression. The expression of CAT-1 was uniform in all tissues, whereas CAT-2 (P<0.01) was greatest in liver. In conclusion, these data indicate that extra-hepatic tissues contribute to lysine catabolism as do enzymes other than LKR.

Determination of xanthine oxidoreductase activity in broilers: effect of pH and temperature of the assay and distribution in tissues.

Xanthine oxidoreductase (XOR) is the enzyme responsible for the synthesis of uric acid, which exists primarily in the dehydrogenase form in birds. Uric acid is the major end product of the metabolism of nitrogen-containing compounds in birds and it functions as an antioxidant to reduce oxidative stress. Despite the importance of this enzyme, the tissue distribution of XOR in physiologically normal chickens is not well known. In this study, we analyzed XOR activity in extracts of 8 tissues from broilers at 7 and 10 wk of age. No differences in XOR activity due to the age were found in any tissue. Liver and kidney showed the greatest activity, that in the kidney being about 89% of the activity in the liver. Enzyme activity in intestine and pancreas was about 60 and 37% of that in the liver. All breast muscle, heart, and lung samples showed enzyme activity, but values were only 3.0, 1.2, and 0.6% of those found in the liver. Traces of enzyme activity were also detected in 3 out of 10 brain samples, and no activity was found in the plasma. Our results show that XOR distribution in chickens differs from that in mammals, in which the highest levels have been found in liver and intestine. An additional objective was the evaluation of the effect of pH (7.2, 7.7, 8.2, and 8.7) and temperature (25 and 41 degrees C) on the enzyme activity in liver and kidney samples. Temperature had a similar effect on both tissues, with the activity at 25 degrees C being about 30% of that measured at 41 degrees C. At 41 degrees C, the enzyme activity in liver and kidney decreased quadratically as pH decreased from 8.7 to 7.2. The highest activity in kidney was measured at pH 8.2, although there were no differences between enzyme activities at pH 8.7 or 8.2 in the liver. Our results indicate that the optimum pH of the enzyme in chicken liver and kidney is around 8.2.

Concomitant changes in progesterone catabolic enzymes, cytochrome P450 2C and 3A, with plasma insulin concentrations in ewes supplemented with sodium acetate or sodium propionate.

Progesterone is essential for maintaining pregnancy, and several authors have suggested that low peripheral concentrations of progesterone may be responsible for high rates of embryonic loss. The primary organ involved in the catabolism of progesterone is the liver, and cytochrome P450 2C and 3A sub-families account for a large proportion of this catabolism. Elucidating a mechanism to decrease progesterone catabolism, thereby increasing embryonic and uterine exposure to progesterone, seems a logical approach to ameliorate high rates of embryonic loss. The objectives of the current experiment were to determine the pattern of insulin secretion after supplementing feed with either sodium acetate or sodium propionate and to determine any association between the differential patterns of insulin secretion with the hepatic activity of cytochrome P450 2C and 3A and progesterone clearance. Sixteen ovariectomized ewes were fed 3 kg/day for 10 days of a diet consisting of 50% corn silage, 38% triticale haylage, 12% soybean meal and 600 ml of 3.5 M sodium acetate (energy control; n = 8) or 2.0 M sodium propionate (gluconeogenic substrate; n = 8). Equal portions of the ration (1 kg as-fed basis along with 200 ml of 3.5 M sodium acetate or 2.0 M sodium propionate) were offered three times daily at 0600, 1400 and 2200 h. Concentrations of insulin in plasma were determined immediately before feeding and at 15, 30, 60, 90, 120, 180, 240 and 300 min after feeding. Progesterone clearance from peripheral circulation (ng/ml per min) was measured by giving a 5 mg injection of progesterone into the left jugular vein and collecting blood via the right jugular vein at 0, 2, 4, 6, 8, 10, 15, 20 and 30 min afterwards. Liver biopsies were taken 1 h after feeding to determine cytochrome P450 2C and 3A activities. Insulin concentrations in ewes supplemented with sodium propionate were elevated at 15, 30 and 60 min after feeding compared to the sodium acetate group. Cytochrome P450 2C and 3A activities were decreased 1 h after feeding in the sodium propionate-treated ewes relative to sodium acetate. Insulin appears to down-regulate cytochrome P450 activity, which could be used to decrease the catabolism of progesterone during early gestation, thereby increasing peripheral concentrations of progesterone and, consequently, embryonic exposure to progesterone.

Diet-induced alterations in progesterone clearance appear to be mediated by insulin signaling in hepatocytes.

Factors that affect progesterone clearance from plasma and by hepatocytes in culture were examined in a series of experiments. In Exp. 1, the objective was to determine whether an increase in hepatic portal blood acetate or propionate could alter progesterone metabolism by the liver. For ewe lambs gavaged orally with sodium propionate compared with those gavaged orally with sodium acetate, serum progesterone concentrations began to diverge as early as 0.5 h after administration and were greater (P < 0.05) at 3 and 4 h after administration. The objective of Exp. 2 was to determine the effect of a single oral gavage of either sodium acetate or sodium propionate on peripheral insulin and glucagon concentrations. Ewes gavaged orally with sodium propionate had greater (P < 0.05) insulin concentrations at 0.5 and 1 h after gavage than ewes gavaged with sodium acetate. Furthermore, glucagon concentrations were greater (P < 0.05) at 0.5, 1, and 2 h for ewe lambs gavaged orally with sodium propionate compared with those receiving sodium acetate. The third experiment investigated the rate of in vitro progesterone clearance by cultured hepatocytes in response to treatment with different concentrations of insulin and glucagon. Progesterone clearance was reduced (P < 0.05) with the addition of 0.1 nM insulin compared with the control. Furthermore, there was a greater reduction (P < 0.05) in progesterone clearance in response to 1.0 and 10 nM insulin compared with the control and 0.1 nM insulin. No change was observed in progesterone clearance in hepatocytes treated with either physiological (0.01 and 0.1 nM) or supraphysiological (1.0 nM) glucagon. Supraphysiological concentrations of glucagon (1.0 nM) negated the effects of either 0.1 or 1.0 nM insulin on progesterone clearance by hepatocytes. However, with physiological concentrations of glucagon (0.1 nM) and 1.0 nM insulin, glucagon was not able to negate the reduction in progesterone clearance caused by insulin. These data are consistent with a paradigm in which elevated hepatic portal vein propionate increases plasma insulin in ruminants, which decreases progesterone clearance, thereby increasing serum progesterone concentrations.

Increased dietary protein elevates plasma uric acid and is associated with decreased oxidative stress in rapidly-growing broilers.

Uric acid is an important antioxidant and methods to elevate its plasma concentration may be important in animal health. In a first study, the effect of dietary protein on plasma uric acid (PUA) and glucose concentrations were determined in 3-week-old chicks. Twenty-four broiler chicks were randomly assigned to four diets: a commercial control diet (C, 20% crude protein), low protein (LP) containing 10% casein, medium protein (MP) containing 20% casein or high protein (HP) containing 45% casein for a 3-week experiment. PUA concentration increased (P<0.05) in chicks fed HP diet and declined (P<0.05) in chicks fed LP while plasma glucose concentrations were lower (P<0.05) in chicks fed the LP diet at the end of the study. In a second study, PUA and leukocyte oxidative activity (LOA) were determined in broilers fed C, LP, MP or HP diets for 4 weeks. As in the first study, dietary protein directly affected PUA concentrations. In birds consuming HP diets, PUA was negatively correlated (P=0.06) with lowered LOA. These data support the view that increases in dietary protein can increase PUA concentrations, which can ameliorate oxidative stress.

Iron regulatory proteins, iron responsive elements and iron homeostasis.

The discovery of iron regulatory proteins (IRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of vertebrate iron metabolism. IRPs bind to iron responsive elements (IREs) in specific mRNAs and regulate their utilization. The targets of IRP action now appear to extend beyond proteins that function in the storage (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic acid cycle. To date, it appears that IRPs modulate the utilization of six mammalian mRNAs. Current studies are aimed at defining the mechanisms responsible for the hierarchical regulation of these mRNAs by IRPs. In addition, much interest continues to focus on the signaling pathways through which IRP function is regulated. Multiple factors modulate the RNA binding activity of IRP1 and/or IRP2 including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress and hypoxia/reoxygenation. Because IRPs are key modulators of the uptake and metabolic fate of iron in cells, they are focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances.

Mitochondrial lysine uptake limits hepatic lysine oxidation in rats fed diets containing 5, 20 or 60% casein.

Sixty male Sprague-Dawley rats were randomly allotted to receive diets containing 5, 20 or 60% casein. Rats had access to the diet only during the initial 8 h of the daily 12-h dark period. Hepatic mitochondrial lysine uptake, lysine alpha-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SacD) activities, and in vitro lysine oxidation (LOX) were measured 0, 6, 12, 18 and 24 h after the start of the dark period. Diurnal variation of mitochondrial lysine uptake was not detected (P > 0.10) although uptake varied 3-fold over 24 h. Mitochondrial lysine uptake was greater (P < 0.05) for rats fed diets containing 60% casein than for rats fed diets containing 5% casein. Diurnal variation of LKR was detected (P < 0. 05) in rats fed diets containing 20 and 60% casein. Diurnal variation of SacD was detected (P < 0.05) in rats fed diets containing 60% casein. Increased casein consumption resulted in increased LKR and SacD activities (4- to 5-fold; P < 0.05). Diurnal variation of LOX was detected in rats fed diets containing 20 and 60% casein (P < 0.05). Increasing the casein concentration in the diet from 5 to 60% resulted in a 7-fold increase in LOX (P < 0.05). To make rate comparisons, LKR and SacD activities and LOX were predicted from a range of substrate concentrations (0.1 to 5.0 mmol/L). Overall, LKR and SacD were 6-107 times that of LOX, suggesting that, in liver, mitochondrial lysine uptake limits LOX.

Dietary iron intake rapidly influences iron regulatory proteins, ferritin subunits and mitochondrial aconitase in rat liver.

Iron regulatory protein 1 (IRP1) and IRP2 are cytoplasmic RNA binding proteins that are central regulators of mammalian iron homeostasis. We investigated the time-dependent effect of dietary iron deficiency on liver IRP activity in relation to the abundance of ferritin and the iron-sulfur protein mitochondrial aconitase (m-acon), which are targets of IRP action. Rats were fed a diet containing 2 or 34 mg iron/kg diet for 1-28 d. Liver IRP activity increased rapidly in rats fed the iron-deficient diet with IRP1 stimulated by d 1 and IRP2 by d 2. The maximal activation of IRP2 was five-fold (d 7) and three-fold (d 4) for IRP1. By d 4, liver ferritin subunits were undetectable and m-acon abundance eventually fell by 50% (P < 0.05) in iron-deficient rats. m-Acon abundance declined most rapidly from d 1 to 11 and in a manner that was suggestive of a cause and effect type of relationship between IRP activity and m-acon abundance. In liver, iron deficiency did not decrease the activity of cytosolic aconitase, catalase or complex I of the electron transport chain nor was there an effect on the maximal rate of mitochondrial oxygen consumption with the use of malate and pyruvate as substrates. Thus, the decline in m-acon abundance in iron deficiency is not reflective of a global decrease in liver iron-sulfur proteins nor does it appear to limit ATP production. Our results suggest a novel role for m-acon in cellular iron metabolism. We conclude that, in liver, iron deficiency preferentially affects the activities of IRPs and the targets of IRP action.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line.

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5' or 3' untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Recombinant bovine somatotropin decreases hepatic amino acid catabolism in female rats.

The effect of recombinant bovine somatotropin (rbST) on hepatic amino acid catabolism in female rats was investigated. Daily injections of rbST for 5 d decreased liver homogenate lysine alpha-ketoglutarate reductase (EC 1.5.1.8) activity (P < 0.05) and liver homogenate lysine oxidation (P < 0.05) approximately 35%. Liver homogenate methionine and valine oxidation were depressed approximately 20 (P = 0.13) and 35% (P < 0.05), respectively. These data show a decrease in hepatic capacity to oxidize amino acids in rats administered rbST. Whether depressed liver amino acid degrading enzyme activity plays a role in amino acid oxidation in vivo remains to be evaluated.

Lysine-alpha-ketoglutarate reductase and saccharopine dehydrogenase are located only in the mitochondrial matrix in rat liver.

In rat liver, comparisons of marker enzyme activities (beta-hexosaminidase, lysosomes; catalase, peroxisomes; cytochrome oxidase, mitochondrial-inner membrane; monoamine oxidase, mitochondrial outer membrane; ornithine aminotransferase, mitochondrial matrix) show that lysine-alpha-ketoglutarate reductase and saccharopine dehydrogenase, the initial enzymes of saccharopine-dependent lysine degradation, are found only in the mitochondrial matrix. These results are consistent with obligatory uptake of lysine into the matrix for lysine catabolism and raise the possibility that lysine transport into the mitochondrion may control lysine degradation.

Role of protein synthesis in amino acid catabolism.

Except for branched chain amino acids, the site of indispensable amino acid degradation is the liver. Location of amino acid degradation capacity in a single organ may play an important role in the reutilization of amino acids derived from protein turnover. The importance of preferential utilization of amino acids for protein synthesis on catabolism of amino acids is demonstrated in two ways. First, by minimal oxidation of an amino acid at dietary concentrations below that required for maximum gain followed by a near proportionate oxidation with increased dietary level, and second, by increased oxidation of an indispensable amino acid when another amino acid limits protein synthesis. A direct effect of protein synthesis on amino acid catabolism can be shown by a marked increase in amino acid catabolism when protein synthesis is inhibited.