Nutrient Metabolism Carnitine Deficiency and Supplementation Do Not Affect the Gene Expression of Carnitine Biosynthetic Enzymes in Rats1,2 Alan T. Davis3 and Thomas J. Monroe* Departments of Surgery, Michigan State University and Spectrum Health, Grand Rapids, MI and *Department of Molecular Biology, Spectrum Health, Grand Rapids, MI ABSTRACT Starved male weanling rats supplemented with 20 mmol/L pivalate in their drinking water exhibit Downloaded from https://academic.oup.com/jn/article/135/4/761/4663769 by guest on 30 September 2021 significantly depressed concentrations of carnitine in tissues and plasma. In addition, pivalate supplementation has been linked with increased renal and hepatic trimethyllysine hydroxylase (TMLH) activity, whereas carnitine supplementation has been associated with significantly decreased hepatic ␥-butyrobetaine hydroxylase (BBH) activity. The purpose of this study was to determine whether pivalate or carnitine supplementation affects the activity and genetic expression of 2 enzymes of carnitine (Cn) biosynthesis, TMLH and BBH, expressed as mRNA abundance, relative to the abundance of ␤-actin mRNA. Male weanling rats were administered the control treatment (C; n ϭ 6), the pivalate treatment (P; n ϭ 7), or the pivalate treatment plus supplemental dietary carnitine (PϩCn; n ϭ 7). Rats in group P had elevated renal TMLH activity, relative to the other groups (P Ͻ 0.05). The groups did not differ in the abundance of renal or hepatic TMLH or BBH mRNA. A previously unreported finding was the quantifiable level of renal BBH mRNA, which was verified by direct sequencing of the BBH cDNA product amplified from kidney RNA. The groups did not differ in renal BBH mRNA abundance and renal BBH enzyme activity was not detected. Thus, the alterations in enzyme activities in the pivalate-treated rats are not regulated at the transcrip- tional level, and are apparently related to post-transcriptional effects on the enzymes themselves. J. Nutr. 135: 761–764, 2005. KEY WORDS: ● carnitine ● carnitine biosynthesis ● trimethyllysine ● ␥-butyrobetaine Carnitine is a naturally occurring compound in mammalian ciency of ␥-butyrobetaine metabolized to carnitine. In addi- energy metabolism. Its functions include the facilitation of tion, thyroxine was reported to significantly increase liver long-chain fatty acid oxidation, elimination of toxic metabo- carnitine concentration, as well as hepatic BBH activity lites of acyl CoA excess, modulation of the free CoA to acyl (11,12). Whether these alterations in carnitine concentration CoA ratio, storage of energy as acetylcarnitine, and the inter- are directly related to or enhanced by altered TMLH or BBH compartmental shuttling of energy substrates (1). The first activities is unknown. enzyme in the carnitine biosynthetic pathway, trimethyllysine A previous study from this laboratory, using the pivalate 4 hydroxylase (TMLH), hydroxylates trimethyllysine to 3-hy- model of secondary carnitine deficiency in rats (13), showed droxy-trimethyllysine, whereas the final enzyme in the path- that TMLH activity was greater in kidney, liver, and heart of ␥ ␥ way, -butyrobetaine hydroxylase (BBH), hydroxylates -bu- pivalate-treated rats compared with controls (14). In addition, tyrobetaine to carnitine. The biosynthesis of carnitine is BBH activity was depressed in rats fed a carnitine-supple- thought to be regulated by the availability of trimethyllysine mented diet relative to controls. It was unclear from these (2,3). In their review of previous studies (2–6), Vaz et al. (1) results, however, whether these effects were related to a direct noted that the capacity of the carnitine biosynthetic pathway ␥ effect upon the enzymes themselves, or to an alteration in the to generate carnitine from trimethyllysine and -butyrobe- expression of the enzymes. taine far exceeds the amount of carnitine utilized. The purpose of the current study was to determine whether It has been noted, however, that during starvation (7,8), pivalate alone or in combination with supplemental carnitine clofibrate administration (9), and lactation (10), there are marked effects upon carnitine distribution and/or the effi- alters the metabolism of trimethyllysine, via alterations in TMLH activity, expression of the TMLH mRNA, tissue con- centration, and/or urinary excretion. In addition, a goal was to 1 Presented at Experimental Biology 03, April, 2003, San Diego, CA [Davis, determine whether pivalate alone or in combination with A. T. & Monroe, T. J. (2003) Expression of carnitine biosynthetic enzymes is supplemental carnitine alters the biosynthesis of carnitine, via unaltered in carnitine deficient and carnitine supplemented rats (Program adden- dum, abstract LB403)]. alterations in BBH activity and expression of the BBH 2 Supported by grants from the Blodgett Butterworth Health Care Foundation. mRNA, as well as tissue concentration and/or urinary excre- 3 To whom correspondence should be addressed. E-mail: [email protected]. tion of ␥-butyrobetaine and carnitine. Specifically, the work- 4 Abbreviations used: BBH, ␥-butyrobetaine hydroxylase; C, control rats; P, rats receiving pivalate; PϩCn, rats receiving pivalate and supplemental carnitine; ing hypothesis was that the activity and mRNA expression of TMLH, trimethyllysine hydroxylase. both enzymes would be increased in the pivalate-treated rats 0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences. Manuscript received 3 November 2004. Initial review completed 23 December 2004. Revision accepted 11 January 2005. 761 762 DAVIS AND MONROE and decreased in the carnitine-supplemented rats relative to 100% Eluent C. From 40.9 to 41 min after injection, a linear gradient controls. between Eluents C and A resulted in a concentration of 100% Eluent A. The column was then reequilibrated for 15 min before the next injection. MATERIALS AND METHODS RNA isolation and PCR amplification. Expression of TMLH mRNA was determined using RT-PCR with fluorescence quantita- Animals and diets. Male weanling Sprague-Dawley rats (n ϭ 20; tion, using the set of primers described by Vaz et al. (23). Expression Charles River) were housed individually in polycarbonate cages in a of BBH mRNA was determined using RT-PCR with fluorescence room maintained at 21 Ϯ 2°C and 50 Ϯ 10% humidity with a 12-h quantitation, using a set of primers described by Galland et al. (24). light:dark cycle. The rats were housed at the West Michigan Regional The expression of ␤-actin was used as a control (25). TMLH, BBH, Laboratory, whose Animal Care and Use Committee approved the and ␤-actin cDNA were amplified from 600 ng total RNA extracted study. Rats were maintained in accordance with the NIH guidelines from rat kidney and liver using the Superscript 1-step RT-PCR system for the care and use of laboratory animals. The rats were randomly (Invitrogen). Amplification linearity of the 3 RNA species using the assigned to 1 of 3 groups. Control rats (group C, n ϭ 6) were freely same thermocycling profile and cycle number was determined empir- fed a nutritionally complete purified diet, AIN-76A [(15); Harlan ically before quantitative experiments. The RT-PCR amplification Teklad]. Analysis in this laboratory determined the carnitine con- product quantification was determined using PicoGreen (Molecular centration of this diet to be 1.2 nmol/g. Rats in Group C were Probes) and a fluorescence microplate reader (Cytofluor series 4000, Downloaded from https://academic.oup.com/jn/article/135/4/761/4663769 by guest on 30 September 2021 administered 20 mmol/L sodium bicarbonate in their drinking water, Perspective Biosystems). as described previously (13). Rats in the pivalate group (Group P, n Statistics. The values shown in the text and tables are means ϭ 7) were fed the same diet as the rats in Group C, and their water Ϯ SEM, except where indicated otherwise. Due to the wide range of contained 20 mmol/L sodium pivalate. Rats in the carnitine-supple- variability for the total carnitine concentrations and for the urine mented group (Group PϩCn, n ϭ 7) were fed the AIN-76A diet ␥-butyrobetaine excretion, all of these values were transformed by supplemented with 0.067 mmol carnitine/g diet. This concentration taking the natural logarithm of the original data before analysis. The of carnitine in the diet was used previously and produced 400 and data were analyzed using 1-way ANOVA and the Fisher’s Protected 200% increases in plasma and skeletal muscle total carnitine concen- Least Significant Difference (FPLSD) test. Differences were consid- tration, respectively (16). Rats in Group PϩCn were administered 20 ered significant at P Ͻ 0.05. All analyses were conducted with NCSS mmol/L sodium pivalate in their drinking water. The rats in all 3 2004 (Number Cruncher Statistical Systems). groups remained in their cages and were administered these treat- ments for 14 d, at which time the rats were transferred to individual metabolism cages for an additional 48-h period. During this time, RESULTS 24-h urinary excretions were collected (in 6 mol/L HCl), and 24-h food and fluid intake were recorded. Carnitine and carnitine precursor concentrations in tissue At the end of the study period, the rats were anesthetized with and urine. Provision of pivalate in the drinking water of the isoflurane and killed by decapitation. Blood was collected into hep- Group P rats significantly decreased total carnitine in plasma arinized tubes and plasma separated by centrifugation at 1500 ϫ g for and urine compared with controls (Table 1). Addition of 10 min. Plasma samples were frozen at Ϫ80°C until they were carnitine to the diet of Group PϩCn rats significantly and analyzed. Samples of liver, skeletal muscle, heart, and kidney were markedly increased plasma and urine total carnitine relative to obtained from each rat, and freeze-clamped in aluminum tongs cooled rats in the other 2 groups. Group PϩCn rats excreted signif- in liquid nitrogen. A smaller sample of liver and kidney from each rat icantly less trimethyllysine than either of the other 2 groups. was first submerged in RNAlater (Ambien) before freeze-clamping ϩ Ϫ Group P Cn rats had significantly higher concentrations of the tissue.