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,1
* Gastroenterology Unit, Department of Clinical and Experimental Medicine, University Federico II, Naples, Italy;
Scientific Department, Sigma-Tau, Pomezia, Roma, Italy;
Institute of Genetics and Biophysics "A. Buzzati-Traverso," IGB-CNR and
Institute of Protein Biochemistry, IBP-CNR, Naples, Italy;
|| Telethon Institute of Genetics and Medicine, Naples, Italy
1Correspondence: Institute of Protein Biochemistry, IBP-CNR, Via P.Castellino 111, Naples, Italy. E-mail: g.peluso{at}ibp.cnr.it
SPECIFIC AIMS
The aims of this study were to determine whether: a) there is decreased expression of plasma-membrane carnitine transporters and impaired carnitine uptake in colonocytes in a rat model of experimental colitis; b) a decreased carnitine cell content perturbs the bioenergetic metabolism of colonocytes; and c) carnitine-loaded liposomes reverse carnitine depletion and restore normal metabolism thereby alleviating experimental colitis.
PRINCIPAL FINDINGS
1. Carnitine uptake in isolated colonocytes
Carnitine transport has been shown to be sodium-dependent in many tissues. This characteristic is ascribed mainly to carnitine transport by Octn2 and Atb0+. We examined Na-dependence of [3H]-carnitine uptake in colonocytes from control and trinitrobenzene sulfonic acid (TNBS)-treated rats.
Control colonocytes efficiently accumulated carnitine, but carnitine uptake was significantly decreased when sodium was replaced by N-methylglucamine. We tested the specificity of carnitine transport using Octn2 and Atb0+ substrates. All Octn2 substrates tested significantly inhibited [3H]-carnitine uptake in colonocytes. Glycine, which affects the Atb0+ transporter, also caused decreased carnitine accumulation in colonocytes, although to a much lesser extent. Sodium-dependent carnitine uptake was significantly reduced in colonocytes from TNBS-treated rats vs. controls. Differently, in the absence of sodium no difference was found in carnitine uptake between TNBS-treated and untreated rats.
2. Octn2 and Atb0+ expression
We investigated the mechanism by which colon inflammation prevents carnitine accumulation in colonocytes by measuring Octn2 (Slc22a5) and Atb0+ (Slc6a14) expression in rat colon samples by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Fourteen days after colitis induction, Octn2 mRNA in the colonic tissue was decreased nearly 5-fold, whereas Atb0+ expression was undetectable. Real-time PCR analysis with the same RNAs confirmed high Octn2 expression in the colon of control rats and showed that Octn2 expression was significantly reduced in TNBS-treated rats. Real-time RT-PCR analysis showed that Atb0+ was expressed on control colonocytes, whereas it was barely discernable from background activity in colonocytes from TNBS-treated rats. To verify that the effect of TNBS on carnitine transporters of colonocytes is specific and not a generalized defect in membrane transporters due to mucosal injury and inflammation, we measured the expression of the Abcd1 gene on the same samples. Abcd1 gene expression in colonocytes did not differ between control and TNBS-treated animals. An in situ hybridization experiment confirmed the pronounced decrease of Atb0+ and Octn2 gene expression in the epithelial cell that survived TNBS treatment (Fig. 1
).
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3. Carnitine and carnitine derivatives in isolated colonocytes
The presence of Octn2 and the ability to accumulate carnitine by isolated colonocytes prompted us to undertake a more detailed analysis of carnitine content in colonocytes. Carnitine was present in control colonocytes mainly in its free form (
60%), with only a minor contribution from acetyl- and propionyl-carnitine (19% and 11%, respectively). Long-chain acyl-carnitines were present in traces. Carnitine and its derivatives were dramatically decreased in colonocytes of TNBS-treated rats. The most evident change occurred in the case of acetyl-carnitine. Although the free-form and the short-chain acyl-carnitines remained predominant, there was a slight increase in carnitine acylated in the form of long-chain acyl derivatives.
4. CO2 production from butyrate in colonocytes
To investigate the effect of carnitine on butyrate metabolism, we measured the rates of 14CO2 production by 14C-labeled butyrate in colonocytes from control and TNBS-treated rats.
14CO2 by 14C-labeled butyrate in isolated colonocytes decreased significantly in samples from TNBS-treated animals vs. controls. The addition of carnitine-loaded liposomes restored butyrate oxidation in colonocytes from TNBS-treated rats. Empty liposomes, with or without carnitine admixed, did not affect metabolism in control and TNBS-treated cells. The addition of free or carnitine-loaded liposomes to control cells did not significantly increase butyrate metabolism.
5. Effect of carnitine on acetyl-coenzyme A distribution in butyrate-treated colonocytes
To examine the role of butyrate ± carnitine in the distribution of acetyl-coenzyme A in the different cell compartments, we examined acetyl-coenzyme A cell content and acetyl-coenzyme A intracellular distribution in cells incubated with butyrate in the presence or absence of carnitine-loaded liposomes. Butyrate did not significantly affect mitochondrial acetyl-coenzyme A level in control colonocytes, whereas it caused a 3-fold increase of cytosolic acetyl-coenzyme A. Carnitine supplementation of the cell suspension did not change either total cellular acetyl-coenzyme A content or its cell distribution. On the contrary, mitochondrial acetyl-coenzyme A content was significantly higher in colonocytes from TNBS-treated rats vs. control colonocytes. The addition of butyrate did not affect either mitochondrial or cytosolic acetyl-coenzyme A level. The addition of carnitine-loaded liposomes to these cells dramatically increased cytosolic acetyl-coenzyme A level and decreased mitochondrial acetyl-coenzyme A level.
6. Effect of carnitine-loaded liposome treatment on TNBS-induced colitis
To evaluate the in vivo effects of carnitine on colitis, we instilled carnitine-loaded liposomes in the colon of rats twice a day for two weeks beginning the day after TNBS administration. On completion of treatment, total carnitine content in colon mucosa was higher in carnitine-encapsulated liposome treated-rats than in untreated and empty liposomes-treated animals. Treatment with empty liposomes plus carnitine admixed gave results similar to those obtained with empty liposomes. Anatomic and histological evaluations demonstrated that the treatment with carnitine-loaded liposomes ameliorated the macroscopic appearance of the colon and significantly improved colitis.
CONCLUSIONS AND SIGNIFICANCE
This study demonstrates that normal colonocytes express both Octn2 and Atb0+ carnitine transporters. Because absorption of carnitine by the colon does not appear to be relevant for the maintenance of systemic carnitine homeostasis in physiological conditions, we speculate that both carriers are needed to supply colonocytes with carnitine. It is tempting to speculate that carnitine transporters may be abundantly expressed because carnitine might be required for the metabolism of butyrate, which is the preferential respiratory fuel of colonocytes.
A novel finding of this study is that experimental colitis simultaneously decreased both the expression of carnitine transporters (confirmed by in situ hybridization studies) and the colonocyte carnitine content. This condition parallels the impaired ability of colonocytes to oxidize butyrate, whereas butyrate uptake is not modified.
It is generally agreed that butyrate does not require a supply of carnitine for its metabolism, since it crosses the double mitochondrial membrane very rapidly and, unlike long chain fatty acids, does not require the presence of carnitine. Although apparently not controlled by carnitine, butyrate metabolism can be subjected to several intramitochondrial controls, which may involve carnitine. For example, in the case of free carnitine deficiency, an impairment of transformation of acetyl-coenzyme A esters in their carnitine esters would result in the breakdown of the mitochondrial oxidative metabolism of butyrate (Fig. 2
). These assumptions are supported by our observation that the increase in total acetyl-coenzyme A level in butyrate-treated colonocytes may be attributable to an increased metabolic flow of butyrate into mitochondria and a consequent export of acetate groups from mitochondria to the cytosol. Indeed, in control colonocytes, where acetate is efficiently transported from mitochondria to the cytosol through the carnitine acetyl-transferase pathway, the increase of acetyl-coenzyme A after butyrate addition is mainly in the cytoplasm. The finding that carnitine supplementation did not affect cellular acetyl-coenzyme A distribution in control cells indicates that carnitine-dependent acetate transport is normally well supported by endogenous carnitine under physiological conditions.
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In the case of carnitine depletion, the finding that the increase of acetyl-coenzyme A was confined to the mitochondrial compartment of colonocytes shows that carnitine plays an important role in the regulation of cell metabolic fluxes. The dramatic increase in cytosolic acetyl-coenzyme A content induced by carnitine supplementation in colonocytes from TNBS-treated rats indicates that substrate flow at mitochondrial level was markedly decreased by Octn2/Atb0+ down-regulation through carnitine uptake decrease and intracellular carnitine depletion. This down-regulation of Octn2/Atb0+ genes seems to be a specific event induced by inflammation because it was not associated with decreased expression of another gene encoding cell membrane transporters, namely, Abcd1.
The results of this study are compatible with a causal relationship between depletion of colonocyte carnitine and the inability of mitochondria to maintain normal butyrate ßbeta;-oxidation. Indeed, ßbeta;-oxidation flux may at least partially depend on the [acetyl-coenzyme A]/[coenzyme A] ratio. In the case of a high ratio, several intramitochondrial enzymes dependent on CoASH (namely, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase) would be inhibited. Hence, complete acylation of the mitochondrial coenzyme A (CoA) pool would result in the breakdown of mitochondrial oxidative metabolism. We also demonstrate that supplementation with carnitine-loaded liposomes restores butyrate metabolism in colonocytes from rats with colitis by decreasing the level of acetyl-coenzyme A. These conclusions are supported by a significant inverse correlation between in vivo carnitine treatment and histological indicators of mucosal damage induced by TNBS.
In conclusion, our data support the concept that carnitine is a rate-limiting factor for the maintenance of physiological butyrate oxidation in colonocytes.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5950fje
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  S. R. Srinivas, P. D. Prasad, N. S. Umapathy, V. Ganapathy, and P. S. Shekhawat Transport of butyryl-L-carnitine, a potential prodrug, via the carnitine transporter OCTN2 and the amino acid transporter ATB0,+ Am J Physiol Gastrointest Liver Physiol, November 1, 2007; 293(5): G1046 - G1053. [Abstract] [Full Text] [PDF]
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