Development and characterization of an animal model of carnitine deficiency

Abstract

Mammals cover their carnitine needs by diet and biosynthesis. The last step of carnitine biosynthesis is the conversion of butyrobetaine to carnitine by butyrobetaine hydroxylase. We investigated the effect of N-trimethylhydrazine-3-propionate (THP), a butyrobetaine analogue, on butyrobetaine hydroxylase kinetics, and carnitine biosynthesis and body homeostasis in rats fed a casein-based or a vegetarian diet. The K m of butyrobetaine hydroxylase purified from rat liver was 41^9 mmol´L 21 for butyrobetaine and 37^5 mmol´L 21 for THP, and THP was a competitive inhibitor of butyrobetaine hydroxylase (K i 16^2 mmol´L 21 ). In rats fed a vegetarian diet, renal excretion of total carnitine was increased by THP (20 mg´100 g 21´d ay 21for three weeks), averaging 96^36 and 5.3^1.2 mmol´day 21 in THP-treated and control rats, respectively. After three weeks of treatment, the total carnitine plasma concentration (8.8^2.1 versus 52.8^11.4 mmol´L 21 ) and tissue levels were decreased in THP-treated rats (liver 0.19^0.03 versus 0.59^0.08 and muscle 0.24^0.04 versus 1.07^0.13 mmol´g 21 ).Carnitine biosynthesis was blocked in THP-treated rats (20.22^0.13 versus 0.57^0.21 mmol´100 g 21´d ay 21 ). Similar results were obtained in rats treated with the caseinbased diet. THP inhibited carnitine transport by rat renal brush-border membrane vesicles competitively (K i 41^3 mmol´L 21 ). Palmitate metabolism in vivo was impaired in THP-treated rats and the livers showed mixed steatosis. Steady-state mRNA levels of the carnitine transporter rat OCTN2 were increased in THP-treated rats in skeletal muscle and small intestine. In conclusion, THP inhibits butyrobetaine hydroxylase competitively, blocks carnitine biosynthesis in vivo and interacts competitively with renal carnitine reabsorption. THP-treated rats develop systemic carnitine deficiency over three weeks and can therefore serve as an animal model for human carnitine deficiency.

The plasma carnitine concentration regulates renal OCTN2 expression and carnitine transport in rats

“…Rats treated with THP (THP rats; n=6) were treated with 20 mg per 100 g body weight THP per day via the ground-up food for 2 weeks (Spaniol et al, 2001).…”

“…The real-time PCR was carried out in a total reaction volume of 10 µl containing the TaqMan mastermix, cDNA corresponding to 10 ng total RNA as described previously (Spaniol et al, 2001). The primers and probe were: forward primer 5'-C(A/C)TATGTGTTGGCCTGGCTG-3'; reverse primer 5'-AACTTGCCCACCATCACCAG-3'; probe: 5'-FAM-CTCTTCCTGGGTGGCAGTGTCCTTCTCT-TAMRA-3'.…”

“…Quantification of OCTN2 tissue and cellular mRNA levels was performed with GAPDH or 18S rRNA as an endogenous control as described previously (Spaniol et al, 2001). …”

Pharmacological manipulation of L-carnitine transport into L6 cells with stable overexpression of human OCTN2

“…L-[ 3 H]-carnitine hydrochloride (81 Ci/mmol) was obtained from Amersham-Pharmacia Biotech (Little Chalfont, Buckinghamshire, England). Trimethylhydraziniumpropionate was synthesized as described previously [28].…”

Mitochondria as the target for mildronate's protective effects in azidothymidine (AZT)‐induced toxicity of isolated rat liver mitochondria

“…10 Furthermore, it was also shown that mildronate decreases the concentration of lactic acid and increases ATP level in cardiac tissue after coronary artery occlusion; 11 as well as preventing the drop of ATP content during hypoxia of isolated rat heart . 12 A plausible mechanism to explain mildronate's protective action is related to its ability to regulate carnitine levels by acting as a competitive inhibitor of gammabutyrobetaine (GBB) hydroxylase, 13,14 thus inhibiting GBB transformation to carnitine. In fact, in rat liver mitochondria, mildronate was found to inhibit carnitine-palmitoyltransferase-1, thus protecting against long-chain fatty acid accumulation and subsequent damage of mitochondrial membranes.…”