During postnatal development, a number of nutrient homeostasis pathways undergo ontogenesis to satisfy the nutrient requirements for growth and development. Although many of the developmental changes in these nutrient homeostasis pathways are genetically programmed, exogenous factors such as dietary components, environmental compounds and therapeutic drugs might affect their maturation. Our laboratory is interested in the developmental outcomes of interactions between drugs and nutrients that share the same absorption and/or disposition (i.e. distribution, elimination mechanisms) mechanisms. Our investigations began with L-carnitine homeostasis pathways and we systemically evaluated the ontogeny of key L-carnitine homeostasis pathways in the rat. Such information can help us to further understand the impact of exogenous factors on the maturation of nutrient homeostasis processes and the possible long-term consequences of drug-nutrient interactions during ontogeny.
In mammals, fatty acid oxidation becomes the main source of energy for many tissues with transition to extrauterine life . Mitochondrial fatty acid utilization, though, requires a sufficient supply of L-carnitine to shuttle long-chain fatty acids across the mitochondrial membrane making them available for β-oxidation . The maternal circulation supplies L-carnitine to the developing fetus. During late gestation, L-carnitine concentrations significantly increase in fetal tissues and this storage of L-carnitine assures adequate levels in the immediate postpartum period [9, 26]. These tissue stores quickly become depleted due to the immaturity of many of the L-carnitine homeostasis mechanisms and maintenance of L-carnitine concentrations requires an exogenous source (i.e. milk) of L-carnitine . In our study, postnatal increases in serum free L-carnitine were consistent with levels reported in the literature  and these increases correlated with maturation of a number of enzymes and transporter systems that critically determine L-carnitine levels in the body.
Lower serum free L-carnitine levels in the early postnatal period is, in part, due to the limited capacity for endogenous biosynthesis by the young neonate [5, 19]. As noted in other studies, TLMH mRNA expression remained constant with postnatal development  and in agreement with other studies we found that hepatic γ-Bbh mRNA expression and activity was significantly lower at early postnatal development in rat pups [19, 28]. Young neonates are highly dependent on exogenous sources of L-carnitine, which is usually supplied in sufficient amounts by the breast milk during nursing . Interestingly, L-carnitine levels in the milk of nursing rat dams decrease significantly by mid-lactation [30, 31]. Despite the reduced exogenous L-carnitine, serum free L-carnitine levels in rat pups increase with advancing age . The maturation of hepatic γ-Bbh contributes to the postnatal increase in L-carnitine levels in the body. However, our study also suggests that maturation of other processes, namely gastrointestinal absorption and renal reabsorption of L-carnitine additionally contributed to the postnatal rise in serum L-carnitine.
Absorption of dietary sources of L-carnitine requires the function of several transporter systems expressed at the gastrointestinal epithelial barrier . In our study expression of small intestinal Octn2 and Octn3 did not change with postnatal development, while Octn1 expression increased significantly between PD4 to PD8 remaining relatively constant after PD8. A previous study, which examined the postnatal maturation of the intestinal uptake L-carnitine, noted that Na+-dependent and Na+-independent intestinal uptake of L-carnitine was high in late gestation and in the newborn and significantly decreased between PD1 and PD15 . Furthermore, mRNA expression of Octn2 demonstrated only a 20 % decrease between PD1 and PD15 in the jejunum, while ileal expression demonstrated a 100 % decrease between these two postnatal age groups . In our study Octn2 mRNA expression was evaluated in the jejunum, and the constant expression of Octn2 and Octn3 is consistent with these findings. Octn2 and Octn3 mediates the Na+-dependent uptake of L-carnitine at the small intestine [11, 33], and although not statistically significant, our data demonstrates that Octn2 expression did decrease at PD11 and PD20 and Octn3 at PD20, which might suggest a reduced ability to absorb L-carnitine with advancing postnatal age as previously reported .
Renal reabsorption of L-carnitine from the urinary filtrate plays a significant role in maintenance of L-carnitine levels in the body. Almost 95 % of the excreted L-carnitine is reabsorbed by transporters expressed in the proximal tubules of the kidney with Octn2 as the principal transporter involved in this process . In our study renal Octn2 expression increased during postnatal development in the rat, which is consistent with the literature [12, 14, 34]. The increase in renal Octn2 expression correlated strongly with increases in serum L-carnitine levels suggesting that renal Octn2 plays a significant role in the postnatal pattern of serum L-carnitine development. Overall our data suggest the developmental changes in hepatic γ-Bbh expression, intestinal Octn1 expression, and renal Octn2 expression might systemically contribute to the postnatal increase in serum L-carnitine levels. However, the precise interconnections of these pathways and their overall contribution to L-carnitine homeostasis during development is not known and further studies are required to clarify their contributions.
The distribution of L-carnitine in the body is organ dependent with the highest concentration of L-carnitine in the heart . In our study, heart L-carnitine levels increased during postnatal development and these increases were correlated with increased expression of Octn2 in the heart. L-Carnitine has a significant function in energy production in neonatal cardiac tissue due to its role in fatty oxidation and the reliance of neonatal hearts on fatty acids as the primary energy substrate . The dramatic increase in fatty acid oxidation rates in early heart development after birth has been attributed to an increase in L-carnitine levels . Although we observed a significant increase in L-carnitine levels in the heart with advancing age of the neonate, cardiac ATP levels remained constant through postnatal development. Interestingly, we found that creatine and ADP levels were ontogenically regulated during postnatal development. The significance of such developmental changes is not clear and requires investigation.
We also evaluated heart Cpt enzyme expression and activity due to the pivotal role of these enzymes in heart energy production. The postnatal increase in both Cpt1b and Cpt2 mRNA expression at postnatal day 20 are paralleled by the increases in heart L-carnitine concentrations. Indeed, Cpt1a and Cpt2 mRNA levels were increased by carnitine administration in cell culture systems . Thus, the significant increase in heart L-carnitine levels at postnatal day 20 might account for the transcriptional enhancement of both Cpt1b and Cpt2. Despite these transcriptional increases in Cpt enzymes, we observed no significant changes in heart Cpt enzyme activity. Cpt enzyme activities have been reported to increase with increasing mitochondrial L-carnitine levels [38–40]. Unfortunately, L-carnitine levels in the whole heart tissue rather than in the mitochondria were measured in our study.
In conclusion, several L-carnitine homeostasis pathways underwent significant ontogenesis during postnatal development in the rat. However, the exact relationship between these pathways and their contribution to L-carnitine homeostasis during development is not completely known and further studies are required to clarify their contributions. Such a clarification is necessary to understand the impact of exogenous and endogenous factors on L-carnitine status during development. Nonetheless, this systematic evaluation of key pathways in the L-carnitine homeostasis pathway provides a basis from which we can conduct further evaluations regarding the effects of exogenous (i.e. drug) and endogenous factors (i.e. disease) on L-carnitine status during postnatal development and possible long-term consequences of any disturbance in the normal ontogeny of these pathways.