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Nutrient Metabolism

Lysine -Ketoglutarate Reductase and Lysine Oxidation Are Distributed in the Extrahepatic Tissues of Chickens^1,2

Division of Animal and Veterinary Sciences, West Virginia University-Morgantown, WV

⁶To whom correspondence should be addressed. E-mail: kbleming{at}wvu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In animals, lysine oxidation is thought to occur primarily via the activity of lysine -ketoglutarate reductase (LKR). This activity was reported previously in chicken liver, but no work on the tissue distribution of the enzyme in chickens has been reported. Therefore, LKR activity was assayed in liver, kidney, pancreas, heart, brain, lung, spleen, muscle, and intestinal tissues in chickens as was the in vitro ability of tissue homogenates to oxidize lysine. Additionally, the expression of LKR mRNA was assessed by RT-PCR. We found LKR to be present in all tissues studied by both enzymatic analysis and mRNA abundance. Additionally, all tissues assayed oxidized lysine. The extent of lysine oxidation differed among the tissues, consistent with the different pathways of lysine oxidation in the different tissues. These studies demonstrate that LKR is widely distributed in chicken tissues and that tissues other than liver can contribute to whole-body lysine oxidation.

KEY WORDS: • chickens • lysine -ketoglutarate reductase • lysine • amino acid metabolism

The essential amino acid lysine is often the limiting amino acid in diets of farm animals and human populations consuming predominantly cereal grain–based diets (1). In some agriculture production systems, this necessitates the addition of relatively expensive dietary components rich in lysine, thus increasing production costs. From the standpoint of human health, relative shortages of lysine impair well-being. Given the importance of dietary lysine, improving the efficiency of lysine retention would be valuable from both a human health and animal agriculture perspective. To improve the efficiency of lysine retention, a detailed understanding of how and where lysine is degraded in the body would clearly be beneficial.

The "primary" or "major" route of lysine oxidation is via the lysine -ketoglutarate reductase (LKR)⁷ pathway (2). In this pathway, lysine and -ketoglutarate are converted to saccharopine by LKR; then saccharopine is converted to -aminoadipate semialdehyde and glutamate by saccharopine dehydrogenase. The assertion that the LKR pathway is the primary or major route rests on descriptions of familial hyperlysinemias that are the result of LKR (EC 1.5.1.8) deficiency with or without saccharopine dehydrogenase (EC 1.5.1.9) deficiency (2). Lysine -ketoglutarate reductase was initially identified in rat liver as the enzyme that carries out the reverse of a step in yeast lysine biosynthesis (3–5). Later it was determined that in rat liver, this enzyme is present only in the mitochondrial matrix (6). The unique location in liver raised the possibility that transport into the mitochondrion could limit lysine oxidation, as was subsequently demonstrated (7).

Less is known about the oxidative disposal of L-lysine in chickens. Both the saccharopine-dependent and the pipecolic acid–dependent pathways appear to function in chickens (8). Lysine -ketoglutarate reductase activity was identified in chicken liver (9). Given the paucity of knowledge regarding lysine metabolism in chickens, we investigated the tissue distribution of LKR and of lysine oxidation measured as the conversion of lysine carbon to CO₂. We expected to find LKR activity in several but not all tissues. Additionally, on the basis of Miller’s work (10), we expected that perhaps only liver and kidney would oxidize lysine to CO₂. Instead, LKR and lysine oxidation activity were found in all tissues studied.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Mixed-sex chicks (n = 10; 1 d old; Ross x Ross, a generous gift of Wampler Foods) were housed in dirt floor pens with fresh bedding and given free access to a commercial starter diet from Carl S. Akey beginning when they were 2 d old. The diet contained corn (611.8 g/kg), soybean meal (329.7 g/kg), soybean oil (22.0 g/kg), dicalcium phosphate (18.5 g/kg), calcium carbonate (9.5 g/kg), NaCl (3.5 g/kg), a vitamin/mineral premix (2.5 g/kg), methionine (1.5 g/kg as the DL-hydroxy analog), and Coban 45 (1 g/kg, a coccidiostat). The diet was calculated to contain 21% crude protein, 0.84% methionine and cystine, 1.17% lysine, and 3076 kcal/kg (12.87 MJ/kg) of metabolizable energy. Water was always available. The chickens were electrically stunned and then killed by exsanguination at 3 wk of age. The experimental protocol was approved by the West Virginia University Animal Care and Use Committee.

    Tissue preparation. Organs were harvested immediately after death, and placed in ice-cold H-medium [mannitol, 220 mmol/L; sucrose, 70 mmol/L; HEPES, 5 mmol/L, EGTA, 1 mmol/L, ß-mercaptoethanol, 5 mmol/L; and bovine serum albumin (BSA), 0.05% wt/v], pH = 7.4. All organs except breast muscle were weighed to calculate the whole organ contribution. The breast muscle was assumed to be 21% of live weight given a 70% dressing weight and a data set (unpublished) showing that breast muscle is 30% of dressing weight. Organs were minced with scissors and homogenized with a polytron (Kinematica AG, PT 2100). A 25% (w/v) homogenate was prepared. Liver, kidney, pancreas, heart, brain, lung, and spleen were removed whole and homogenized. For the intestine, a section 20 cm in length beginning at the pyloric sphincter was removed and flushed with ice-cold H-medium and used to represent the entire intestine. For muscle, a superficial portion of the breast muscle, pectoralis major, was removed as representative of breast muscle.

    Assays. LKR activity was measured spectrophotometrically as the lysine-dependent oxidation of NADPH at 340 nm in a Beckman-Coulter DU-640 spectrophotometer as previously described (6), except that the temperature was 41°C to reflect the temperature of chickens. All LKR reactions were assayed in duplicate. Reaction mixtures contained 150 mmol/L HEPES, 135 mmol/L mannitol, 45 mmol/L sucrose, 5 mmol/L 2-mercaptoethanol, 0.05% (wt/v) BSA, 0.25 mmol/L NADPH, 15 mmol/L -ketoglutarate, 0.05% (v:v) Triton X-100 ± 40 mmol/L L-lysine HCl in a final volume of 2 mL. Reactions were started by the addition of lysine. In preliminary studies, a K_m of LKR for lysine of 6.96 ± 1.87 mmol/L was determined using liver homogenate as an enzyme source and a Lineweaver-Burke approach (n = 4).

Lysine oxidation was measured as described previously (11) except that 0.125 g of tissue was used, the L-lysine concentration was raised to 10 mmol/L, and the specific activity to 8.4 Bq/nmol due to the lower rate of oxidation in chickens relative to rodents. The final concentration of reagents in a 2 mL volume was 10 mmol/L L-lysine · HCl, 10 mmol/L HEPES, 3 mmol/L MgCl₂, 0.2 mmol/L EDTA, 182 mmol/L mannitol, and 61 mmol/L sucrose. Incubations were performed in 25-mL Erlenmeyer flasks and lasted 30 min in a shaking water bath set at 50 oscillations/min at 41°C. Incubations were initiated by the addition of 1 mL of 25% (wt/v) homogenate and terminated by the injection of 0.5 mL of 35% perchloric acid. The hanging well (Eppendorf tube) contained ethanolamine and methylcellosolve 1:2 to trap the CO₂. After acid killing of the incubations, flasks remained in the shaking bath for an additional 180 min. Preliminary studies indicated that no increase in ¹⁴CO₂ could be detected after 180 min. Additionally, preliminary studies also determined the assay to be linear for 45 min. The rate of lysine oxidation was determined in triplicate less a heat-inactivated blank for each sample. The radiotracer lysine was either uniformly labeled {NEN, L-[¹⁴C(U)]} or labeled only at the 1-carbon (American Radiolabeled Chemicals, L-[1-¹⁴C]).

    RT-PCR. RT-PCR was performed on RNA isolated from the various tissues using TRI Reagent (MRC) and the manufacturer’s protocol. Integrity of the RNA was assessed spectrophotometrically by OD 260/OD 280. RNA was treated with RNase-free DNase according to the manufacturer’s protocol (Promega) to eliminate genomic DNA as a source of signal. Reverse transcription was performed with random hexamers and Moloney-murine leukemia virus reverse transcriptase as described by the manufacturer’s protocol (Promega). For LKR mRNA the forward primer was 5'-AAC ACC AGC CAT GAA GGA AC-3' and the reverse primer was 5'-TGA ACG GTG TTC AGC AAG AC-3' with an expected product size of 251 bp. The primers were designed to span 2 intron/exon boundaries to eliminate concerns of a signal from genomic DNA. For LKR mRNA the following "touchdown" program was used: Cycle 1 (1X) 95°C, 4 min; Cycle 2 (10X) step 1: 95°C, 1 min, step 2: 75°C, 1 min (decrease temp 1°C every cycle), step 3: 72°C, 1.5 min; Cycle 3 (35X) step 1: 95°C, 1 min, step 2: 65°C, 1 min, step 3: 72°C, 1.5 min; Cycle 4: (1X) 72°C, 10 min; Cycle 5: 4°C. The identity of the product was confirmed by sequencing of the PCR products. For the "control gene," ß-actin, we used primer pairs supplied by Promega (catalog G5740). The ß-actin has an expected product size of 285 bp in chickens. For ß-actin, the following PCR program was used: Cycle 1 (1X) 95°C, 5 min; Cycle 2 (1X) 55°C, 1 min; Cycle 3 (30X) step 1: 72°C, 1.5 min, step 2: 94°C, 1 min, step 3: 55°C, 1 min; Cycle 4: (1X) 72°C, 10 min; Cycle 5: 4°C. The PCR products were visualized on 1.5% agarose gels in Tris-acetate-EDTA buffer using ethidium bromide. All samples were assayed at least in duplicate. The 100-bp ladder used was from Promega (catalog #G3161).

    Statistics. Data were analyzed using the general linear model procedure in PC SAS, version 8.1. The data were analyzed for the effect of tissue. In the event of a significant F, multiple comparisons were made using Tukey’s test. Linear regression was used to assess the relation between LKR and lysine oxidation. Data are presented as means ± SEM. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Tissue distribution of LKR activity and lysine oxidation. Lysine oxidation and LKR activity were detected in every tissue surveyed and there were significant differences among the 9 tissues (Table 1). Several tissues had very low and variable LKR activity, and it was desirable to use another method to confirm the presence of LKR.

View this table: [in this window] [in a new window]   TABLE 1 Lysine -ketoglutarate reductase activity and lysine oxidation in 9 tissues of 3-wk-old chicks1

View larger version (125K): [in this window] [in a new window]   FIGURE 1 Representative gels showing the tissue distribution of LKR and ß-actin in 3-wk-old chicks. The prominent band in the bp ladder is at 500 bp. (A) Negative image of an ethidium bromide–stained gel of RT-PCR products using chicken LKR primers. Lane 1, base pair ladder; Lane 2, liver; Lane 3, kidney; Lane 4, heart; Lane 5, brain; Lane 6, lung; Lane 7, spleen; Lane 8, muscle; Lane 9, intestine; Lane 10, base pair ladder; Lane 11, blank; Lane 12, no cDNA control. (B) Negative image of an ethidium bromide–stained gel of RT-PCR products using chicken ß-actin primers. Lane 1, base pair ladder; Lane 2, liver; Lane 3, kidney; Lane 4, heart; Lane 5, brain; Lane 6, lung; Lane 7, spleen; Lane 8, muscle; Lane 9, intestine; Lane 10, base pair ladder. For both LKR and ß-actin, tissues for all 10 chickens were analyzed.

View larger version (17K): [in this window] [in a new window]   FIGURE 2 Regression of lysine oxidation with LKR activity for 9 tissues of 3-wk-old chicks. The regression equation was: y = 0.31x + 0.34, R2 = 0.08, P > 0.10.

An investigation using 5 tissues from 8 chickens was performed to investigate the differential oxidation of these 2 lysine tracers. The oxidation ratios of U-labeled to 1-labeled tracer (U:1) for muscle, heart, liver, lung, and intestine were 0.93, 0.80, 0.78, 0.46, and 0.45, respectively, with a SEM of 0.15. Lung and intestine tended (P = 0.10) to have less complete oxidation of lysine. The addition of an alternative energy source (glucose at 5 mmol/L) to the oxidation medium did not affect the U:1 ratio (data not shown), suggesting that lysine oxidation was not increased to use lysine as a fuel source in the homogenate.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In the current study, a K_m of LKR for lysine of nearly 7 mmol/L was determined in crude homogenates of chicken liver. The K_m agrees well with values obtained in crude homogenates of mouse (Manangi, unpublished results) and rat liver (Blemings, unpublished results) of 6.2 and 5.0 mmol/L, respectively. The K_m is also similar to that determined in rainbow trout of 7.3 mmol/L (12). Purified preparations of rat (13) and human (14) liver enzymes reported K_m values of 2.2 and 1.5 mmol/L, respectively. Given that the lysine concentration in blood is in the range of 100 to 200 µmol/L and perhaps 3 to 4 mmol/L in liver mitochondria (7), it would seem that LKR activity is substrate limited.

Miller (10) found that, in rats, lysine oxidation based on the release of ¹⁴CO₂ from labeled lysine was largely a hepatic event. Therefore, finding LKR in tissues other than liver (15,16) was somewhat surprising. However, LKR is present in several different human tissues (14,17). The extrahepatic presence of LKR raised the possibility that lysine could be converted to saccharopine and eventually to -aminoadipate in tissues other than liver and later oxidized by the liver, consistent with the findings of Edmonds and Baker (18).

The data of Table 1 and Figure 1 clearly demonstrate that LKR is widely distributed in chicken tissues. The wide distribution of the enzyme is most similar to work in humans (14,17,19). Work in rats showed the enzyme to be located in liver (3,6,20) as well as kidney (15,21) and brain (22), similar to chickens as reported here. This makes the findings concerning LKR distribution in mice somewhat unclear because the enzyme activity was measured in liver (23) and the transcript was detected by Northern blot analysis in liver and kidney but not heart, brain, spleen, lung, skeletal muscle, or testis (23). However, other work suggests that the enzyme is present in brain (24). In pigs, LKR was detected in liver (25–29), kidney (27–29), and intestine (27–29). Additionally, the enzyme was reported in pig heart, muscle, and brain (28) with evidence that the heart and muscle forms were developmentally regulated (28,29). Given the above, it seems clear that the extrahepatic tissues of most animals contribute to lysine catabolism via LKR.

In examining the oxidation of lysine in various tissues, the current data set do not allow us to determine the extent to which the oxidation depends on LKR activity. Certainly in the avian liver, lysine oxidation is not totally dependent on LKR. The pipecolate acid–dependent pathway, which depends on L-amino acid oxidase (EC 1.4.3.2), functions in avian liver (8,9,30–32). Additionally, L-amino acid oxidase/L-lysine -amino oxidase activity was determined in mouse brain (33). The participation of other enzyme systems that may degrade lysine and their quantitative importance is open to question. The best quantitative data to date concerning lysine oxidation and LKR activity suggest that LKR cannot account for whole-body lysine oxidation (26).

What is different about lysine oxidation in lung and intestine (Fig. 2)? Two possibilities that are not mutually exclusive would be alternative pathways of lysine catabolism and decreased mitochondrial uptake of lysine. One pathway that likely contributes to whole-body lysine oxidation, particularly in extrahepatic tissues, is lysyl oxidase (EC 1.4.3.13). Although lysyl oxidase was originally thought to use only peptide-bound lysine residues as a substrate, later work demonstrated that free lysine could serve as a substrate with the purified bovine aorta enzyme (34). It is intriguing that lung, which would be expected to have a relatively high lysyl oxidase activity for elastin formation, at least in mammalian species, is 1 of the 2 tissues that does not fit the regression line of Figure 2. The other tissue that does not fit well is intestine. It is of interest that a high activity of ornithine decarboxylase (EC 4.1.1.17) for polyamine synthesis in the rapidly proliferating mucosa is expected, and lysine was shown to be a substrate for ornithine decarboxylase (35,36). Consistent with this is the low ratio for the oxidation of uniformly labeled lysine to 1-labeled lysine found in the present study. However, the quantitative importance of the ornithine decarboxylase pathway is questionable in vivo because the K_m of ornithine decarboxylase for lysine is 100 times greater than for ornithine (36). However, whole-animal data suggest the quantitative importance of intestinal lysine catabolism (37).

Uptake of lysine by mitochondria is another possibility for the displacement of lung and intestine from the regression line in Figure 2. Previous evidence showed that the LKR is a mitochondrial enzyme in rat liver (6). The chicken enzyme, like the mouse enzyme, seems to contain a mitochondrial localization signal, suggesting that LKR is mitochondrial in chickens as well. Perhaps the decreased lysine oxidation relative to LKR activity in lung and intestine is a function of decreased mitochondrial lysine uptake by these tissues.

The activity of LKR was determined to be a part of a larger bifunctional -aminoadipic semialdehyde synthase (AASS) in bovine liver (38,39). The bifunctional AASS has both LKR and saccharopine dehydrogenase activity. Saccharopine dehydrogenase is the second enzyme in the saccharopine-dependent pathway. The AASS gene was then identified in humans and determined to be the locus of some cases of hyperlysinemia (19). The chicken gene sequence data would support the presence of a bifunctional AASS in chickens as well.

It is clear that alterations in dietary protein (7,40) or lysine (40,41) affect the hepatic activity of LKR in rats. How these alterations occur mechanistically is unclear. Similarly, in chickens, alterations in dietary lysine affect LKR activity (9,42), but no mechanisms have been proposed to date. The human data (19) certainly raise the possibility that splicing could be a control point for LKR activity. Additionally, as suggested by Papes et al. (23), a post-translational modification, specifically phosphorylation, may regulate LKR activity as is the case in plants (43,44). That regulation of LKR activity may be effected by a post-translational modification fits well with our own recent reports (45,46).

	ACKNOWLEDGMENTS

 
The technical expertise of Hakan Kocamis and Scott Gahr is greatly appreciated.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 2000, April 2000, Orlando, FL [Manangi, M. K., Hoewing, S.F.A., Engels, J. G. & Blemings, K. P. (2000) Extra-hepatic tissues contribute to lysine oxidation in chicks. FASEB J. 15: A268 (abs.)].

² Supported by the West Virginia Agriculture and Forestry Experiment Station (H413), a WVU Senate Research Grant, British United Turkey of America (Lewisburg, WV), the WVU Research Corporation, and USDA-NRI (2002–35206-12859). This is paper 2884 of the West Virginia Agriculture and Forestry Experiment Station.

³ Present address: O-220, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701.

⁴ Present address: VA-MD Regional College of Veterinary Medicine, Virginia Tech, Duck Pond Drive (0442), Blacksburg, VA 24061.

⁵ Present address: Department of Animal Sciences, University of Illinois, 210 Meat Science Lab MC 010, Urbana, IL 61801.

⁷ Abbreviations used: AASS, -aminoadipic semialdehyde synthase; BSA, bovine serum albumin; LKR, lysine -ketoglutarate reductase.

Manuscript received 9 July 2004. Initial review completed 4 August 2004. Revision accepted 6 October 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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