![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
4 Departments of Animal Sciences and Nutritional Sciences, University of Wisconsin, Madison, WI 53706 and 5 Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, WV 26506
* To whom correspondence should be addressed. E-mail: njbeneve{at}ansci.wisc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT LITERATURE CITED |
|---|
-ketoglutarate reductase (LKR), is found only in the mitochondrial matrix. For Lys catabolism to occur, Lys must first enter the matrix of the mitochondrion. LKR, saccharopine dehydrogenase, mitochondrial lysine uptake, and lysine oxidation (LOX) all increased >3-fold in rats fed high levels of dietary protein (up to 60%). The activities of mitochondrial Lys uptake and LOX were similar when expressed as mmol/(d · 100 g body weight). Thus, LOX can be a proxy for mitochondrial Lys uptake. Piglet liver LKR and LOX increase 5- to 10-fold when piglets are fed high-protein (50 or 75%) diets. In both the rat and piglet, after adapting to the high protein diet, the activity of LKR is 400–500 times that of LOX, suggesting that Lys uptake by a transporter(s) is rate limiting. Quantitative 24-h dietary infusion studies in piglets revealed that >80% of the Lys infused (4% of the diet) could not be recovered in the urine or body or accounted for by calculated Lys oxidation based on liver activity of LOX. Other pathways and tissues may account for the Lys oxidation in piglets.
In his review on the determination of the nutritive value of proteins, Bender (1) commented on the early work of Block and Mitchell. They were trying to develop a means of estimating the relative nutritive value of proteins while avoiding the then current demanding biological tests used to assess amino acid content of protein. As the availability of methods for determining the amino acid content of proteins by column chromatography became more widespread, a relation between the amino acid concentration in a protein or proteins and animal response resulting from the use of that protein could be contemplated. An initial assessment of the nutritive value of a protein could be estimated from a calculated chemical score based on the ratio of the concentration of each indispensable amino acid in the protein under consideration to the concentration of that same amino acid in a reference standard. The chemical score could be calculated from the following formula:
 |
The amino acid with the lowest ratio was considered "first limiting." As more data became available, it became possible to construct a plot of animal response (Y) to the chemical score (X) of the dietary protein. One of the measurements of animal response is net protein utilization (NPU),6 which can be determined in an animal trial, usually with laboratory rats. NPU could be estimated from the following equation:
 |
If all first-limiting essential amino acids in dietary proteins behaved similarly in support of animal maintenance and growth, then one would expect a linear relation between NPU and chemical score. If the appropriate scale is selected, a slope of 45° and intercept of zero is expected. Although a linear relation was expected, it was not realized as more data became available. Figure 1 taken from Bender (1,2) shows the dependence of NPU on chemical score. This figure shows that the deviation from a linear relation occurs only at chemical scores <50. Note also that the expected linear relation was not achieved for all amino acids but that the greatest deviation was with Lys. Rats fed diets with proteins limiting in Lys and having chemical scores from 0 to 40 act as if they are consuming a protein with a chemical score of 40 or above. It seems "as if" rats could save (maybe protect against loss) and reuse Lys, whereas that was not as evident with other amino acids. However, in exploring other potential sources of dietary Lys, Bender found Lys in rat feces. He determined that total consumption of daily fecal output could meet 10–25% of the Lys requirement of the rat. This source of Lys would not likely be responsible for the unique response of the rat shown in Figure 1. Bender comments on Lys, "It seems likely that when the animal breaks down tissue protein in the normal turnover processes, part, at least, of the Lys can be reused for new protein synthesis. We have been able to keep rats alive for periods longer than 6 mo on an amino acid diet from which Lys was omitted." This unique response to diets limiting in Lys is not confined to rats; as Ousterhout (3) reported, the survival time of chicks fed a Lys-free diet was double that when other amino acids were eliminated from the diet.
|
|
At a whole-animal level, work by Gahl et al. (6) showed that factors having an effect on the rate of protein synthesis impacted total body Lys catabolism in rats. Along with increasing growth of mature female rats from 0.5 g/d to 5 g/d, use of bovine somatotropin decreased the rate of whole-body Lys catabolism (P < 0.05) from 293 ± 15 µmol/(d · 100 g BW) to 163 ± 15 µmol/(d · 100 g BW). The same treatment (7) was shown to decrease the activity of liver LKR, the initial enzyme in the saccharopine-dependent pathway of Lys catabolism, to 65% of control and the oxidation of Lys, methionine, and valine to 
65% of control as well. These studies demonstrated that an enzyme involved in Lys catabolism is hormonally sensitive. The response of LKR to dietary factors became more interesting, especially in light of observations on Lys conservation mentioned above. More recent work (8–11) has shown that diet-induced alterations in LKR activity are not well correlated with LKR mRNA or protein abundance consistent with posttranslational mechanisms of regulation as in plants (12).
The work of Higashino et al. (13) is the basis for considering the mitochondrion as the initial site of cellular Lys catabolism in rat liver. When U-14C-L-Lys was incubated with rat liver subcellular fractions, whole homogenate and mitochondria were able to produce 14CO2. Fractions that produced essentially no 14CO2 did so when mitochondria were added to them. As the saccharopine-dependent pathway of Lys catabolism shows in Figure 3, 14CO2 production from labeled Lys is dependent on Lys being metabolized by LKR and saccharopine dehydrogenase before labeled CO2 is released. Although Lys catabolism has been shown to be associated with mitochondria, the location of LKR in the mitochondrion was not known. By use of a mitochondrial marker enzyme, ornithine aminotransferase (OT), known to be exclusively housed in the mitochondrial matrix (14), Blemings et al. (15) showed that LKR and OT were found in the same subcellular location, the mitochondrion (Table 1). Interestingly, the patterns of LKR and OT recovery were similar in the cytosol, nuclear, microsomal, and mitochondrial fractions. The total recovery of homogenate LKR and OT activities (97–98%) across the subcellular fractions lent confidence to the methods used to prepare these subcellular fractions. To determine their submitochondrial location, marker enzymes are required for reference to assess recoveries across submitochondrial fractions. Monoamine oxidase is used as a marker of the outer membrane, cytochrome oxidase is the marker enzyme for the inner membrane, and OT is used as a marker for the mitochondrial matrix. Because the LKR distribution is the same as OT, it too is exclusively housed in the mitochondrial matrix of rat liver mitochondria (Table 2). The exclusive mitochondrial matrix location of LKR in rat liver is also seen for LKR in the neonatal piglet liver (Fig. 4). When the recovery for LKR is corrected for OT recovery in the various fractions, 100% of the LKR activity is recovered in the mitochondrial matrix of the piglet liver mitochondrion. This subcellular and submitochondrial location of LKR in the rat and piglet is consistent with the mRNA sequence data for LKR, which suggest a matrix leader sequence required of proteins synthesized in the cytosol but destined for the mitochondrial matrix (16). This matrix location of LKR forces one to consider amino acid transport in addition to enzyme activity when assessing metabolic factors that may affect Lys catabolism.
|
|
|
|
4-fold increase in all 4 activities. Two important observations were made. First, the values for Lys uptake and oxidation (mmol/(d · 100 g BW) are not different (P < 0.05). Thus, Lys oxidation can be used as an approximation of mitochondrial Lys uptake, which is a very challenging measurement to make because individual measurements must be made at <20-s intervals. LOX on the other hand involves incubation of a homogenate or isolated mitochondria with L-[1-14C]Lys and collection and measurement of the rate of 14CO2 production. Second, the activity of Lys -ketoglutarate reductase and that of saccharopine dehydrogenase are 5 to 10 times that of Lys uptake or oxidation. This observation suggests that Lys transport into the mitochondrial matrix is rate limiting for Lys oxidation. Little is known about Lys uptake in mitochondria. Hommes et al. (18) reported on the competitive uptake of Lys and ornithine in rat liver mitochondria. At least 2 different proteins transport ornithine across the mitochondrial inner membrane (19). Whether these transporters are responsible for Lys uptake into the matrix is still unknown.
|
-lactalbumin) allowed for a gain of 110 ± 75 g/d, whereas 33% was required to maximize growth at 278 ± 69 g/d. Figure 5 shows the activity of liver LOX and LKR determined 6, 8, and 12 d after the initiation of feeding piglets diets containing 10, 50, or 75% protein. When compared with 10% protein, consumption of diets containing 50 or 75% protein resulted in a 5-fold increase in LOX, a proxy for mitochondrial Lys uptake, and a 10-fold increase of LKR. Both were expressed as µmol/(h · kg pig). As with the rat, LKR activity in the piglet liver, after adapting to the high-protein diet, was 400–500 times that of LOX when expressed as µmol/(h · kg pig).
|
A liquid formula diet containing 10% of a high-quality whey protein isolate (BiPro) was used in these studies. This 10% protein diet provides sufficient protein to maintain weight but does not allow for weight gain. This means that the added Lys must be catabolized because it cannot be stored in newly synthesized body protein. Liver LOX and LKR were determined on the 8 piglets fed the diet containing 10% protein with no added Lys, and 2 pigs each received the 10% protein diet supplemented with 2, 4, or 6% of L-Lys. Surprisingly, piglets seemed unaffected by the additional dietary Lys, as no decrease in piglet weight over the 12-d experiment was noted even when the diet contained 6% added Lys. By the end of the 12-d experiment, liver LOX and LKR (Fig. 6) had not increased relative to the activities in livers of piglets receiving no added Lys [units, mmol/(d · piglet)]. Based on total liver LOX activity of 0.9 mm/d, then, on average, <10% of the Lys consumed could be oxidized.
|
0.9 mmol of Lys was recovered in urine of piglets infused with the 10% protein diet with 4% of Lys added. On average the percentage of infused Lys recovered in urine over the 4 6-h intervals was 1, 4, 6, and 11%, respectively. This accounted for <6% of the Lys infused. Gastric infusion of the diet with added Lys resulted in an average 4-fold increase in blood Lys (Fig. 8). Note that even though the rate of diet infusion did not change over the 24 h, blood Lys concentration increased and then came to a plateau at 12 h, indicating that the rate of Lys destruction was equal to the rate of Lys infusion. Some type of metabolic adaptation must have occurred because the plateau could not be caused by an increase in the rate of urinary excretion of Lys, as it accounted for 6% of that infused from 12 to 18 h and 11% from 18 to 24 h.
|
|
90% of the Lys infused is no longer recoverable as Lys.
|
12 mmol/24 h. The average increment in piglet total body free Lys was 1.22 mmol/24 h. The average increase in urinary Lys excretion (Fig. 7) caused by the infusion was 0.9 mmol/24 h. Of the 12 mmol of Lys infused, 9.9 mmol cannot be accounted for [12 – 1.2 (body free Lys) – 0.9 (urine) = 9.9 mmol]. If the results for total piglet liver LOX activity from Figure 6, where piglets consumed a diet with 2, 4, or 6% added L-Lys can be used, then the potential for Lys catabolism can be estimated. Using the data for the 6 piglets yields an average value of 0.7 mmol/d. Thus, 9.2 mmol [12 – (1.2 + 0.9 + 0.7)] of the 12 mmol increment infused cannot be accounted for. It is not in the body or in the urine, nor can it be accounted for by liver LOX activity, which is 0.2% of LKR activity. LOX was selected because it is a proxy for Lys uptake into the mitochondrial matrix where LKR is housed.
From the material presented, the nutrition of Lys is unique relative to the other indispensable amino acids in that it can be conserved and can be fed 12 h out of phase with the other indispensable amino acids. Rats fed Lys 12 h out of phase are able grow at about two-thirds to three-fourths of the rate of rats receiving Lys with the other amino acids. Similar studies with rats fed Trp 12 h out of phase revealed that they lost weight at the same rate as they did when fed a protein-free diet. Lys may also be unique in that it appears not to be toxic; it can be added at levels from 2 to 6% to diets containing 10% protein without substantial decrements in growth. One wonders if the mitochondrial matrix location of Lys -ketogluterate reductase, the first enzyme in the saccharopine-dependent pathway of Lys degradation, can account for the unique nutritional behavior of Lys. If the Lys is prevented from interacting with a major pathway for its destruction by a transport barrier, then it may be incorporated and turned over from protein to protein without being destroyed. This would appear nutritionally "as if" Lys were more metabolically available than would be expected based on the Lys content of the diet. In both the rat and the piglet, consumption of high dietary levels of protein results in increased LKR activity and, more importantly, increased transport capacity (mitochondrial Lys uptake) and LOX, which is suggested as representative of transport activity. Consumption of diets with excessive levels of Lys (2–4%) results in increased LKR activity in the rat but not the piglet. Although both liver LOX and LKR respond to high dietary protein (50 or 75%) in the piglet, neither LOX nor LKR responded to high dietary Lys levels (2–6%) in piglets. Quantitative 24-h dietary infusion studies in piglets revealed that >80% of the Lys infused (4% of the diet) could not be recovered in the urine or body or be accounted for by calculated Lys oxidation based on liver activity of LOX. It appears, at least in piglets, that other pathways and tissues are necessary to account for the Lys oxidation. Other tissues have been reported to contain LKR in pigs (23), rats (24), chickens (25), and humans (26,27). Other known pathways that contribute to Lys degradation include those depending on lysyl oxidase (28), L-amino acid oxidase (29), and carnitine biosynthesis. However, relative to LKR, the activities of lysyl oxidase and L-amino acid oxidase are orders of magnitude lower (11). Perhaps other as yet undefined pathways make substantial contributions to Lys degradation. It seems that there is much more work to be done before a comprehensive understanding of Lys degradation and its regulation is realized.
|
FOOTNOTES |
|---|
2 Author disclosures: N. J. Benevenga, the International Council on Amino Acid Science provided for travel and lodging for this meeting and K. P. Blemings, no conflicts of interest.
3 Supported by Hatch WISO 4241.
6 Abbreviations used: BW, body weight; LKR, lysine -ketoglutarate reductase; LOX, lysine oxidation; NPU, net protein utilization; OT, ornithine aminotransferase.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTLITERATURE CITED |
|---|
1. Bender AE. 1961. Determination of the nutritive value of proteins by chemical analysis. In Progress in meeting protein needs of infants and preschool children, Publ. 843. Washington, DC: National Academy of Sciences. p. 407–24.
2. Bender AE. Evaluation of protein quality. methodological considerations. Proc Nutr Soc. 1982;41:267–76.[Medline]
3. Ousterhout LE. Survival time and biochemical changes in chicks fed diets lacking different essential amino acids. J Nutr. 1960;70:226–34.
4. Yang SP, Tilton KS, Ryland LL. Utilization of a delayed Lys or Trp supplement for protein repletion of rats. J Nutr. 1968;94:178–84.
5. Baker DH, Izquierdo OA. Effect of meal frequency and spaced crystalline Lys ingestion on the utilization of dietary Lys by chickens. Nutr Res. 1985;5:1103–12.
6. Gahl MJ, Benevenga NJ, Crenshaw TD. Rates of Lys catabolism are inversely related to rates of protein synthesis when measured concurrently in adult female rats induced to grow at different rates. J Nutr. 1998;128:1503–11.
7. Blemings KP, Gahl MJ, Crenshaw TD, Benevenga NJ. Recombinant bovine somatotropin decreases hepatic amino acid catabolism in female rats. J Nutr. 1996;126:1657–61.
8. Kiess AS, Stinefelt BM, Cantrell CM, Wilson ME, Klandorf H, Blemings KP. Lys -ketoglutarate reductase activity appears to be post-transnationally regulated in mice fed high protein containing diets [abstract]. FASEB J. 2005;19:A438.
9. Stinefelt BM, Cantrell CM, Kiess AS, Blemings KP. RNA interference mediated decrease of -aminoadipate 
-semialdehyde synthase mRNA in a murine hepatic cell line. FASEB J. 2005;19:A438–439.
10. Higgins AD, Silverstein JT, Wilson ME, Rexroad, III CE, Blemings KP. Starvation-induced alterations in hepatic Lys catabolism in different families of rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem. 2006;31:33–44.
11. Kiess AS, Stinefelt BM, Gentilin AJ, Wilson ME, Klandorf H, Blemings KP. Lys catabolism in chickens fed at or below their Lys requirement [abstract]. FASEB J. 2006;20:A1042.
12. Karchi H, Miron D, Ben-Yaacov S, Galili G. The Lys-dependent stimulation of Lys catabolism in tobacco seed required calcium and protein phosphorylation. Plant Cell. 1995;7:1963–70.[Abstract]
13. Higashino K, Tsukada K, Lieberman I. Saccharopine, a product of Lys breakdown by mammalian liver. Biochem Biophys Res Commun. 1965;20:285–90.[Medline]
14. Ip MM, Chee PY, Swick RW. Turnover of hepatic mitochondrial ornithine amiontransferase and cytochrome oxidase using [14C] carbonate as tracer. Biochim Biophys Acta. 1974;354:29–38.[Medline]
15. Blemings KP, Crenshaw TD, Swick RW, Benevenga NJ. Lys--ketoglutarate reductase and saccharopine dehydrogenase are located only in the mitochondrial matrix in rat liver. J Nutr. 1994;124:1215–21.
16. Chua N-H, Schmidt GW. Transport of proteins into mitochondria and chloroplasts. J Cell Biol. 1979;81:461–83.
17. Blemings KP, Crenshaw TD, Benevenga NJ. Mitochondrial Lys uptake limits Lys oxidation in rats fed diets containing 5, 20, or 60% casein. J Nutr. 1998;128:2427–34.
18. Hommes FA, Kitchings L, Eller AG. The uptake of ornithine and Lys by rat liver mitochondria. Biochem Med. 1983;30:313–21.[Medline]
19. Fiermonte G, Dolce V, David L, Santorelli FM, Dionisi-Vici C, Palmieri F, Walker JE. The mitochondrial ornithine transporter. Bacterial expression, reconstitution, functional characterization and tissue distribution of two human isoforms. J Biol Chem. 2003;278:32778–83.
20. Chu S-HW, Hegsted DM. Adaptive response of Lys and threonine degrading enzymes in adult rats. J Nutr. 1976;106:1089–96.
21. Foster AR, Scislowski PWD, Harris CI, Fuller MF. Metabolic response of liver Lys -ketoglutarate reductase activity in rats fed Lys limiting or Lys excessive diets. Nutr Res. 1993;13:1433–43.
22. Wang S-H, Crosby LO, Nesheim MC. Effect of dietary excesses of Lys and arginine on the degradation of Lys by chicks. J Nutr. 1973;103:384–91.
23. Pink D, Elango R, Dixon WT, Ball RO. Regulation of Lys -ketoglutarate reductase varies during postnatal stages of growth and development in the pig [abstract]. FASEB J. 2004;18:A539.
24. Rao VV, Pan X, Chang Y. Developmental changes of L–Lys -ketoglutarate reductase in rat brain and liver. Comp Biochem Physiol. 1992;103B:221–4.[Medline]
25. Manangi MK, Hoewing SFA, Engles JG, Higgins AD, Killefer J, Wilson ME, Blemings KP. Lys -ketoglutarate reductase and Lys oxidation are distributed in the extrahepatic tissues of chickens. J Nutr. 2005;135:81–5.
26. Hutzler J, Dancis J. Conversion of Lys to saccharopine by human tissue. Biochim Biophys Acta. 1968;158:62–9.[Medline]
27. Hutzler J, Dancis J. Lys -ketoglutarate reductase in human tissues. Biochim Biophys Acta. 1975;377:42–51.[Medline]
28. Trackman PC, Kagan HM. Nonpeptidyl amino inhibitors are substrates for lysyl oxidase. J Biol Chem. 1979;254:7831–6.
29. Murthy SN, Janardanasarma MK. Identification of L-amino acid/L-Lys a-amino acid oxidase in mouse brain. Mol Cell Biochem. 1999;197:13–23.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. H. Baker Lysine, Arginine, and Related Amino Acids: An Introduction to the 6th Amino Acid Assessment Workshop J. Nutr., June 1, 2007; 137(6): 1599S - 1601S. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
