Maternofetal transport of l-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that l-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K(m) = 11.09 +/- 1.32 microM; V(max) = 41.75 +/- 0.94 pmol.mg protein(-1).min(-1)), and was unchanged over the pH range from 5.5 to 8.5. l-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of l-carnitine, including d-carnitine, acetyl-d,l-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-l-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na(+)-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for l-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans.

Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of L-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of L-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of L-[ 3 H]carnitine uptake were investigated with or without specific inhibitors. L-Carnitine uptake in mature cells was sodium dependent and linear with time. Km and Vmax values for saturable uptake were 14.07 Ϯ 1.70 M and 26.3 Ϯ 0.80 pmol ⅐ mg protein Ϫ1 ⅐ 6 min Ϫ1 , respectively. L-carnitine uptake was inhibited (P Ͻ 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that L-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM. intestine; brush-border membrane; organic cation transport; anti-OCTN2 antibodies; valproate.

Inflammatory bowel disease (IBD) patients are at increased risk of developing colorectal cancer (CRC). Vitamin D (vD) induces NOD2 gene expression, enhancing immunity, while deficiency impairs intestinal epithelial integrity, increasing inflammation. This study investigated the effect of vD on CRC in colitis, and if preventive benefits are mediated via NOD2. Inflammation-associated CRC was induced by treating C57BL/6J and Nod2 mice with azoxymethane (AOM) and dextran sodium sulfate (DSS) cycles (×3). vD-deficient mice displayed more severe colitis compared to vD-supplemented mice, with greater weight loss, higher colitis activity index, increased colonic weight/length ratios, and lower survival rates. Increased histological inflammation score and increased IL-6 were observed in the mucosa of vD-deficient mice. Overall incidence of colonic tumors was not significantly different between vD-deficient and vD-supplemented mice. Higher tumor multiplicity was observed in vD-deficient vs vD-supplemented groups (both mouse strains). After AOM/DSS treatment, decreased plasma 25(OH)D levels and downregulation of vD target genes Cyp24 and Vdr were observed in both mice strains (vD-deficient or vD-supplemented diet), compared to saline-treated controls on the vD-deficient diet. In conclusion, vD supplementation reduced colitis severity and decreased the number of inflammation-associated colorectal tumors in both C57BL/6J and Nod2 mice, independent of NOD2.

The present data reveal that OCTN protein levels appear to be similar in intestinal tissue from Crohn's Disease patients and controls. Overall, ileal carnitine transport appears to as well equal in Crohn's Disease and control groups. However, there was a trend towards higher carnitine transport in subjects with OCTN1 and OCTN2 mutations.

Accumulating evidence implicates defective innate immunity in the pathogenesis of Crohn's disease (CD). Ineffectual NOD2 (nucleotide-binding oligomerization domain 2) is the most common susceptibility gene contributing to CD. Vitamin D (vD), a potent modulator of innate and adaptive immunity, induces NOD2 gene expression and its downstream function. We hypothesized that the hormonal form of vD (1,25D) could beneficially modulate innate immune function in CD. Using peripheral mononuclear cells and monocyte-derived dendritic cells (Mo-DCs) from CD, it was found that 1,25D decreased Toll-like receptor (TLR)-induced cytokine production and enhanced cytokine levels induced by muramyl dipeptide (MDP), the NOD2 ligand. 1,25D increased the synergistic effect provided by NOD2 and TLR co-activation on interleukin (IL)-10, IL-23, and tumor necrosis factor-alpha (TNF-α). Whereas 1,25D inhibits Mo-DC TLR-induced cytokines, co-stimulation of NOD2 results in increased IL-10 and IL-23. IL-12p70 was completely abrogated by 1,25D. 1,25D similarly modulated cytokine production by immune cells in ulcerative colitis patients and healthy controls. Mo-DCs from CD patients heterozygous for NOD2 mutations had a response similar to those from patients without NOD2 mutations. Immune cells from patients homozygous for the 1007 fs mutation were unresponsive to MDP and 1,25D. Our in vitro data support 1,25D as a potential modulator of immunity. However, these results cannot be extrapolated to CD patients without further controlled studies.