Linda J. Brady

Regulation of the long-chain carnitine acyltransferases.

Long‐chain carnitine acyltransferases are a family of enzymes found in mitochondria, peroxisomes, and endoplasmic reticulum that catalyze the exchange of carnitine for coenzyme A in the fatty acyl‐CoA. Conversion of the fatty acyl‐CoA to fatty acylcarnitine renders the fatty acid more permeable to the various cellular membranes. The mitochondrial carnitine palmitoyltransferases are considered important in the regulation of mitochondrial β‐oxidation of long‐chain fatty acids, However, palmitoylcarnitine produced by peroxisomal carnitine octanoyltransferase or by microsomal carnitine palmitoyltransferase is not different from that produced by the mitochondrial enzyme. Therefore, for there to be control of fatty acid oxidation by the long‐chain carnitine acyltransferases, there would have to be some mechanism to coordinately regulate these varied enzymes. The first system of regulation involves inhibition by malonyl‐CoA, an intermediate in the synthesis of fatty acids. Malonyl‐CoA inhibits long‐chain carnitine acyltransferase activity by all three enzymes at similar concentrations in the physiological range. In addition, the mitochondrial and peroxisomal enzymes are known to be regulated at the level of mRNA transcription by a number of shared factors. Although the microsomal enzyme is less well studied, there does, indeed, appear to be a pattern of coordinate regulation for this system.—Brady, P. L., Ramsay, R. R., Brady, L. J. Regulation of the long‐chain carnitine acyltransferases, FASEB J. 7: 1039‐1044; 1993.

Carbohydrate source and bifidobacteria influence the growth of Clostridium perfringens in vivo and in vitro

Abstract The purpose of this study was to test the effects of administering bifidobacteria with either 2% fructooligosaccharide (FOS), 2% wheat bran oligosaccharide (WBOS), 2% soybean oligosaccharide (SBOS) or no added oligosaccharide (control) on cecal populations of bifidobacteria and C. perfringens , a potential pathogen and normal inhabitant of the mammalian colon, in the rat. Rats were randomly assigned to one of four treatment groups: basal diet + 1 ml daily gavage of skim milk containing 10 8 bifidobacteria, 2% FOS + bifidobacteria, 2% SBOS + bifidobacteria or 2% WBOS + bifidobacteria. The rats were fed the diets for 4 wk, after which cecal concentrations of bifidobacteria and C. perfringens were assessed. WBOS and SBOS feeding resulted in higher concentrations of bifidobacteria and C. perfringens relative to FOS-feeding or control. However, no differences in either bifidobacteria or C. perfringens were detected between control and FOS groups. Parallel to this animal study, in vitro competition experiments with bifidobacteria and C. perfringens were performed in growth media containing either glucose, FOS or WBOS as the primary carbon source. The concentrations of bifidobacteria and C. perfringens were determined at each hour for 10 h and specific growth rates (μ) were calculated. The μ for C. perfringens , co-cultured with bifidobacteria in glucose-based or WBOS-based media was significantly decreased relative to C. perfringens alone. However, no significant difference was found between the μ for C. perfringens co-cultured in FOS-based media with bifidobacteria and the C. perfringens grown alone in FOS-based media. These findings indicate that different oligosaccharides have differential effects on the populations of bifidobacteria and C. perfringens in vivo and that certain oligosaccharides may potentiate an inhibitory action of bifidobacteria against C. perfringens .

Prophylactic feeding of Lactobacillus acidophilus NCFM to mice attenuates overt colonic hyperplasia.

The objective of this project was to determine if the probiotic Lactobacillus acidophilus NCFM would protect mice from developing transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium. Our hypothesis was that the oral administration of L. acidophilus NCFM to mice would mitigate colonic hyperplasia and modulate the host immune response. A concurrent administration (CA) study was performed by feeding mice phosphate-buffered saline (PBS), C. rodentium only, L. acidophilus NCFM only, or C. rodentium and NCFM concurrently on the same day. The mice in the CA study were not protected by the probiotic, since their mean colon sample weights (0.109 g) were significantly higher than those of the PBS controls (0.0774 g), and the hematoxylin and eosin-stained samples showed histological changes typically associated with TMCH. A prophylactic feeding (PF) study was performed by orally feeding mice PBS or NCFM once daily for 20 consecutive days; in addition, on day 7, mice were challenged with either PBS or C. rodentium. Mice in the PF study were protected when they consumed the probiotic prior to the pathogen challenge, since their mean colon sample weights (0.0812 g) were not significantly higher than those of the controls (0.0753 g). The hematoxylin and eosin-stained samples appeared similar to the control samples, and the intestinal interleukin (IL)-15 and gamma interferon (IFN-gamma) mRNA levels were reduced. L. acidophilus NCFM did attenuate overt colonic hyperplasia when fed to mice prior to challenge with C. rodentium. The mouse model used in this study enabled us to investigate the efficacy of the L. acidophilus NCFM in preventing gastrointestinal disease and is a valid model for future probiotic research.

Pharmacologic action of l-carnitine on hypertriglyceridemia in obese Zucker rats

Administration of pharmacologic amounts of L-carnitine was studied in the hypertriglyceridemic Zucker rat. When administered subcutaneously, doses from 250 to 2,000 mg/kg/d significantly decreased plasma triglycerides in obese rats over eight to 12 weeks, with no effect on plasma triglycerides in lean rats. Oral doses at the same high levels were not effective in decreasing plasma triglycerides. Triglyceride secretion rate was reduced from 367 micrograms/min to 168 micrograms/min in treated obese rats. Concurrently, liver lipid was increased twofold in obese treated rats, and the livers of these rats showed significant fatty infiltration. The mechanism of action of carnitine in decreasing plasma triglycerides appeared to be via decreased secretion of triglycerides by the liver of obese rats. There was no effect of L-carnitine in lean or obese rats on the following variables: carnitine palmitoyltransferase-A kinetics or malonyl CoA inhibition, mitochondrial or peroxisomal oxidative capacity, lipoprotein lipase in heart, muscle, and adipose, or fecal lipids. The effect of pharmacologic L-carnitine thus appears to be an inhibition of triglyceride synthesis and/or secretion by the liver.

Immunoquantitation of carnitine palmitoyl transferase in skeletal muscle of 31 patients

We studied 31 patients suspected of having muscle carnitine palmitoyl transferase 2 (CPT2) deficiency. The catalytic activity of CPT2 was measured in muscle biopsies by the isotope exchange method and CPT2 immunoreactivity was quantitated by an enzyme-linked immunosorbent assay. Nine patients had normal enzyme activity and immunoreactivity. Eight patients had significant deficiencies in catalytic activity (> 3 S.D. below reference mean) of which six were also deficient in immunoreactivity. An additional nine patients were significantly deficient in immunoreactivity with normal catalytic activity and five patients had partial deficiencies in both. At least two categories of alterations in CPT may exist which lead to a deficiency based on the data presented: (1) a regulatory defect in CPT which only alters the enzyme active site; and (2) a structural defect due to altered synthesis, increased degradation, or changes in the immunoreactive site. It may prove to be of diagnostic importance to combine the analysis of enzyme activity and immunoreactivity in patients suspected of having a CPT deficiency and to further investigate the condition of partial CPT deficiency.

Effects of clofibrate and acetylsalicylic acid on hepatic carnitine palmitoyltransferase synthesis

Clofibrate and acetylsalicylic and have both been reported to increase carnitine palmitoyltransferase (CPT) activity when administered to rats. The purpose of the present study was to determine the mechanism of the increase in CPT activity. Rats (150-200 g) were fed one of the following: chow, chow with 0.5% clofibrate, or chow with 1% acetylsalicylic acid for 2 weeks. At the end of this time, hepatic CPT activity was increased 4-fold over control in the clofibrate group and 3.6-fold over control in the acetylsalicylic acid group. Immunoreactive protein increased 4.0- and 3.6-fold, respectively, over control. Transcription rates of hepatic nuclei were increased 2.8- and 1.9-fold over control in the clofibrate and acetylsalicylic acid groups, and hepatic mRNA levels increased 2.8- and 2.0-fold respectively. These data indicate that increases in CPT activity caused by clofibrate and acetylsalicylic acid administration are due, at least in part, to increased CPT protein, resulting from increased transcription rate and levels of mRNA specific for CPT.

Regulation of Carnitine Palmitoyltransferase Synthesis in Spontaneously Diabetic BB Wistar Rats

The long-term regulation of hepatic mitochondrial carnitine palmitoyltransferase (CPT) was studied in control, insulin-treated, and untreated spontaneously diabetic BB Wistar rats. The activity of CPT was elevated approximately twofold in the untreated diabetic rats. This corresponded to an approximately equivalent elevation in the immunoreactive CPT activity. mRNAcpT was assayed by reticulocyte lysate translation and by dot blot to a CPT oligonucleotide probe. The level of mRNACPT was approximately proportional to the observed CPT activity. A cDNA probe to CPT was developed, and transcriptional activity for CPT was assessed in isolated hepatic nuclei. Again, transcription of CPT mRNA was approximately proportional to the observed activity. We therefore conclude that at least part of the long-term regulation of hepatic CPT in spontaneously diabetic BB Wistar rats is the product of increased de novo synthesis of CPT protein brought about by regulation at the transcriptional level. Additional control of the amount of CPT may be via the regulation of RNA processing and turnover and enzyme insertion into the mitochondrial membrane.

Hemipalmitoylcarnitinium a strong competitive inhibitor of purified hepatic carnitine palmitoyltransferase

We have synthesized (2S,6R:2R,6S)-6-carboxymethyl-2-hydroxy-2-pentadecyl-4,4-dimethylmorp holinium bromide (hemipalmitoylcarnitinium, HPC) which is a conformationally restricted analog inhibitor of carnitine palmitoyltransferase (CPT; EC 2.3.1.21). rac-HPC inhibits catalytic activity in purified rat liver CPT. In the forward reaction, HPC competes with both (R)-carnitine (Ki(app) = 5.1 +/- 0.7 microM) and palmitoyl-CoA (Ki(app) = 21.5 +/- 4.9 microM). In the reverse reaction, inhibition by HPC is competitive with palmitoyl-(R)-carnitine (Ki(app) = 1.6 +/- 0.6 microM), but inhibition is uncompetitive with CoA. The forward reaction is also competitively inhibited by its product, palmitoyl-(R)-carnitine, Ki(app)s 14.2 +/- 2.1 microM relative to (R)-carnitine and 8.7 +/- 2.6 microM relative to palmitoyl-CoA. rac-HPC is the most potent synthetic reversible inhibitor of purified CPT. HPC fails to inhibit carnitine acetyltransferase (CAT; EC 2.3.1.7). Palmitoylcholine also inhibits CPT in the forward reaction, competing with (R)-carnitine (Ki(app) = 18.6 +/- 4.5 microM) and with palmitoyl CoA (Ki(app) = 10.4 +/- 2.5 microM). Choline is not an effective CPT inhibitor. We have shown [R.D. Gandour et al. (1986) Biochem. Biophys. Res. Commun. 138, 735-741] that hemiacetylcarnitinium inhibits CAT but not CPT. The combined data demonstrate further differences between the carnitine recognition sites in CPT and CAT.

Trimethyllysine metabolism in lean and obese zucker rats during fasting

Abstract The effect of starvation upon the metabolism of trimethyllysine in lean and obese female Zucker rats was studied. All rats were fed a carnitine and trimethyllysine limiting diet for one to two weeks before starvation was initiated. Fed rats and rats starved for three, six, and nine days were used. Liver free and peptide-linked trimethyllysine, and urine total trimethyllysine were measured. Lean and obese Zucker rats had similar hepatic-free and peptide-linked trimethyllysine content when expressed per g protein or per mg DNA. Obese Zucker rats excreted more total trimethyllysine during starvation relative to lean rats. Starvation did not affect trimethyllysine excretion over time, although there were significant decreases in total carnitine excretion in both lean and obese starved rats. Both phenotypes demonstrated a high efficiency of entry of trimethyllysine into the carnitine biosynthetic pathway. We conclude that lean and obese female Zucker rats, similar to male Sprague-Dawley rats, are extremely efficient in the conversion of trimethyllysine into carnitine.