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IntroductionRecent studies identify a mechanistic link between a Western diet, gut microbiota-dependent metabolism, and development of both cardiovascular disease (CVD) and metabolic diseases (1-3). For example, the ingestion of trimethylamine-containing [...], BACKGROUND. L-Carnitine, an abundant nutrient in red meat, accelerates atherosclerosis in mice via gut microbiota- dependent formation of trimethylamine (TMA) and trimethylamine N-oxide (TMAO) via a multistep pathway involving an atherogenic intermediate, [gamma]-butyrobetaine ([gamma]BB). The contribution of [gamma]BB in gut microbiota- dependent L-carnitine metabolism in humans is unknown.METHODS. Omnivores and vegans/vegetarians ingested deuterium-labeled L-carnitine ([d.sub.3]-L-carnitine) or [gamma]BB ([d.sub.9]-[gamma]BB), and both plasma metabolites and fecal polymicrobial transformations were examined at baseline, following oral antibiotics, or following chronic ([greater than or equal to] 2 months) L-carnitine supplementation. Human fecal commensals capable of performing each step of the L-carnitine [right arrow] [gamma]BB [right arrow] TMA transformation were identified.RESULTS. Studies with oral [d.sub.3]-L-carnitine or [d.sub.9]-[gamma]BB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal L-carnitine [right arrow] [gamma]BB [right arrow] TMA [right arrow] TMAO pathway in subjects. Moreover, a striking increase in [d.sub.3]-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral [d.sub.3]-L-carnitine ingestion, whereas fasting endogenous plasma L-carnitine and [gamma]BB levels were similar in vegans/ vegetarians (n = 32) versus omnivores (n = 40). Fecal metabolic transformation studies, and oral isotope tracer studies before versus after chronic L-carnitine supplementation, revealed that omnivores and vegans/vegetarians alike rapidly converted carnitine to [gamma]BB, whereas the second gut microbial transformation, [gamma]BB [right arrow] TMA, was diet inducible (L-carnitine, omnivorous). Extensive anaerobic subculturing of human feces identified no single commensal capable of L-carnitine [right arrow] TMA transformation, multiple community members that converted L-carnitine to [gamma]BB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted [gamma]BB to TMA. In coculture, E. timonensis promoted the complete L-carnitine [right arrow] TMA transformation.CONCLUSION. In humans, dietary L-carnitine is converted into the atherosclerosis- and thrombosis-promoting metabolite TMAO via 2 sequential gut microbiota-dependent transformations: (a) initial rapid generation of the atherogenic intermediate [gamma]BB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota [gamma]BB [right arrow] TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic L-carnitine exposure.TRIAL REGISTRATION. ClinicalTrials.gov NCT01731236.FUNDING. NIH and Office of Dietary Supplements grants HL103866, HL126827, and DK106000, and the Leducq Foundation.

Carnitine is a quaternary amine compound prevalent in animal tissues, and a potential carbon, nitrogen and energy source for pathogens during infection. Characterization of activities in Pseudomonas aeruginosa cell lysates has previously shown that carnitine is converted to 3-dehydrocarnitine (3-dhc) which is in turn metabolized to glycine betaine (GB), an intermediate metabolite in the catabolism of carnitine to glycine. However, the identities of the enzymes required for carnitine catabolism were not known. We used a genetic screen of the P. aeruginosa PAl4 transposon mutant library to identify genes required for growth on carnitine. We identified two genomic regions and their adjacent transcriptional regulators that are required for carnitine catabolism. The PA5388-PA5384 region contains the predicted P. aeruginosa carnitine dehydrogenase homologue along with other genes required for growth on carnitine. The second region identified, PA 1999-PA2000, encodes the [alpha] and [beta] subunits of a predicted 3-ketoacid CoA-transferase, an enzymic activity hypothesized to be involved in the first step of deacetylation of 3-dhc. Furthermore, we confirmed that an intact GB catabolic pathway is required for growth on carnitine. The PA5389 and PA1998 transcription factors are required for growth on carnitine. PA5389 is required for induction of the PA5388-PA5384 transcripts in response to carnitine, and the PA 1999-PA2000 transcripts are induced in a PA1998-dependent manner and induction appears to depend on a carnitine catabolite, possibly 3-dhc. These results provide important insight into elements required for carnitine catabolism in P. aeruginosa and probably in other bacteria.

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by camitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P < 0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P < 0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P < 0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (-17%, P < 0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (-25%, P < 0.05) and the EDL (-45%, P < 0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism. mitochondria; muscle lipids; substrate metabolism

The human isoform 2 of pantothenate kinase (PanK2) is localized to the mitochondria, and mutations in this protein are associated with a progressive neurodegenerative disorder. PanK2 inhibition by acetyl-CoA is so stringent (IC50 < 1 [micro]M) that it is unclear how the enzyme functions in the presence of intracellular CoA concentrations. Palmitoylcarnitine was discovered to be a potent activator of PanK2 that functions to competitively antagonize acetyl-CoA inhibition. Acetyl-CoA was a competitive inhibitor of purified PanK2 with respect to ATP. The interaction between PanK2 and acetylCoA was stable enough that a significant proportion of the purified protein was isolated as the PanK2*acetyl-CoA complex. The longchain acylcarnitine activation of PanK2 explains how PanK2 functions in vivo, by providing a positive regulatory mechanism to counteract the negative regulation of PanK2 activity by acetyl-CoA. Our results suggest that PanK2 is located in the mitochondria to sense the levels of palmitoylcarnitine and up-regulate CoA biosynthesis in response to an increased mitochondrial demand for the cofactor to support [beta]-oxidation. carnitine | coenzyme A | [beta]-oxidation | pantothenate kinase-associated neurodegeneration

A second patient with 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, who was identified through blood spot acylcarnitine analysis showing persistently increased levels of hydroxyl-[C.sub.4]-camitine, is presented. It is concluded that a carefully interpreted acylcarnitine profile would allow more patients with 3-hydroyisobutyryl-CoA hydrolase deficiency to be diagnosed.

A hypothesis that C75 could constitute an acyl-CoA synthetase substrate, with the resulting of carnitine palmitoyltransferase I (CPT I) activity is presented taking into account the CoA derivatives, rather than their free acid forms, are the inhibitory forms of these compounds. It is found that C75-CoA is produced in vitro and inhibits CPT I with competitive inhibition kinetics.

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a [Na.sup.+]-dependent carnitine carrier with [Na.sup.+]-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of AT[B.sup.0+] (an amino acid transporter with a [Na.sup.+]/[Cl.sup.-]-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific [Na.sup.+]-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on [Na.sup.+], and partly on [Cl.sup.-], compatible with hOCTN2 and [ATB.sup.0+] function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by [K.sub.m] of 5.1 [micro]M for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/ nutrient transporters in human mammary gland drug transfer. organic cation transporter; milk; human; novel organic cation transporter 2; amino acid transporter [B.sup.0+]