A new LC–ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells

Jáuregui, Olga; Sierra, Adriana Y.; Carrasco, P.; Gratacós, Esther; Hegardt, Fausto G.; Casals, Núria

doi:10.1016/j.aca.2007.07.066

Cited by 20 publications

(12 citation statements)

References 16 publications

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“…Accordingly, the sensitivity of this method for carnitine, ␥-butyrobetaine and trimethyllysine was slightly higher (about three times) than in the method of Vaz et al [12]. However, sensitivities of about 1 nM as limit of quantitation as described by Jáuregui et al [7] for long-chain acyl carnitines were not achieved in our method. However, these authors used an API 3000 which is in the multi-reaction mode about 10 times sensitiver as the API 2000 used in our study.…”

Section: Resultscontrasting

confidence: 72%

“…In order to get more insight into carnitine metabolism and synthesis, a sensitive analysis for the determination of the concentrations of free carnitine, carnitine esters and the carnitine precursors trimethyllysine and ␥-butyrobetaine is required. Currently, carnitine is mostly analyzed by LC-MS/MS methods [3][4][5][6][7][8]. The use of multi-reaction mode (MRM) transitions for detection allows the determination of underivatized carnitine and acylcarnitines with sufficient sensitivity [6].…”

Section: Introductionmentioning

confidence: 99%

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Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

Hirche

Fischer

Keller

et al. 2009

Journal of Chromatography B

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Section: Resultscontrasting

confidence: 72%

Section: Introductionmentioning

confidence: 99%

Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

Hirche

Fischer

Keller

et al. 2009

Journal of Chromatography B

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“…1A). The levels of long-chain acylcarnitines, products and a reliable indicator of CPT-1 acyltransferase activity (24,26), were not changed following the overexpression (Fig. 1A), which is consistent with the prediction that CPT-1c has a very low CPT-1 activity in vivo (21).…”

Section: Cpt-1csupporting

confidence: 75%

Important roles of brain-specific carnitine palmitoyltransferase and ceramide metabolism in leptin hypothalamic control of feeding

Gao

Zhu

Gao

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Brain-specific carnitine palmitoyltransferase-1 (CPT-1c) is implicated in CNS control of food intake. In this article, we explore the role of hypothalamic CPT-1c in leptin's anorexigenic actions. We first show that adenoviral overexpression of CPT-1c in hypothalamic arcuate nucleus of rats increases food intake and concomitantly up-regulates orexigenic neuropeptide Y (NPY) and Bsx (a transcription factor of NPY). Then, we demonstrate that this overexpression antagonizes the anorectic actions induced by central leptin or compound cerulenin (an inhibitor of fatty acid synthase). The overexpression of CPT-1c also blocks leptin-induced downregulations of NPY and Bsx. Furthermore, the anorectic actions of central leptin or cerulenin are impaired in mice with brain CPT-1c deleted. Both anorectic effects require elevated levels of hypothalamic arcuate nucleus (Arc) malonyl-CoA, a fatty acidmetabolism intermediate that has emerged as a mediator in hypothalamic control of food intake. Thus, these data suggest that CPT-1c is implicated in malonyl-CoA action in leptin's hypothalamic anorectic signaling pathways. Moreover, ceramide metabolism appears to play a role in leptin's central control of feeding. Leptin treatment decreases Arc ceramide levels, with the decrease being important in leptin-induced anorectic actions and downregulations of NPY and Bsx. Of interest, our data indicate that leptin impacts ceramide metabolism through malonyl-CoA and CPT-1c, and ceramide de novo biosynthesis acts downstream of both malonyl-CoA and CPT-1c in mediating their effects on feeding and expressions of NPY and Bsx. In summary, we provide insights into the important roles of malonyl-CoA, CPT-1c, and ceramide metabolism in leptin's hypothalamic signaling pathways. O besity is a major cause of insulin-resistance and diabetes. An imbalance between food intake and energy expenditure contributes to the development of obesity. The CNS regulates energy homeostasis with the hypothalamus playing a critical role (1, 2). Mounting evidence now shows that fatty acid metabolism in the hypothalamus plays important roles in the central regulation of energy homeostasis (3-17). In this regard, malonyl-CoA, an intermediate in fatty acid de novo biosynthesis, is emerging as a player in the hypothalamus (3,5,6,(8)(9)(10)(11)(12)(13).Hypothalamic malonyl-CoA metabolism has been implicated in leptin-induced anorectic actions (9, 12, 13). Leptin, an important physiological regulator of food intake and body weight, exerts its anorectic effects partly by inducing an increase in malonyl-CoA level in the hypothalamic arcuate nucleus (Arc) (12). In addition to fatty acid biosynthesis, recent data also suggest a role for carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid β-oxidation (7, 18), in hypothalamic regulation of energy balance. Fatty acid biosynthesis and β-oxidation are linked by malonyl-CoA-mediated inhibition of CPT-1 acyltransferase activity (12). In the hypothalamus, the CPT-1 liver isoform (CPT-1a) is the predominant t...

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“…Malonyl-CoA levels were quantified by the CoA recycling assay (21). The levels of long-chain acyl-CoAs (LCFA-CoAs; consisting of palmitoyl-CoA, oleoyl-CoA, and stearoyl-CoA) and acylcarnitines (consisting of palmitoylcarnitine, oleoylcarnitine, and stearoylcarnitine) were measured by HPLC and mass spectrometry (15,16,24).…”

Section: Methodsmentioning

confidence: 99%

“…Levels are presented as %null. *, **wt or mt vs. null, P Ͻ 0.05. a surrogate assay of CPT-1a activity (15,24). The typical acyltransferase activity of CPT-1 converts palmitoyl-, oleoyl-, and stearoyl-CoA, the major LCFA-CoA species, to palmitoyl-, oleoyl-, and stearoylcarnitine, the major LC-AC species, respectively (15,24).…”

Section: Activation Of Cpt-1a Induces Orexigenic Effectsmentioning

confidence: 99%

Important role of ventromedial hypothalamic carnitine palmitoyltransferase-1a in the control of food intake

Gao

Serra

Keung

et al. 2013

American Journal of Physiology-Endocrinology and Metabolism

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Gao S, Serra D, Keung W, Hegardt FG, Lopaschuk GD. Important role of ventromedial hypothalamic carnitine palmitoyltransferase-1a in the control of food intake. Am J Physiol Endocrinol Metab 305: E336 -E347, 2013. First published June 4, 2013 doi:10.1152/ajpendo.00168.2013.-Carnitine palmitoyltransferase-1 (CPT-1) liver isoform, or CPT-1a, is implicated in CNS control of food intake. However, the exact brain nucleus site(s) in mediating this action of CPT-1a has not been identified. In this report, we assess the role of CPT-1a in hypothalamic ventromedial nucleus (VMN). We stereotaxically injected an adenoviral vector containing CPT-1a coding sequence into the VMN of rats to induce overexpression and activation of CPT-1a. The VMN-selective activation of CPT-1a induced an orexigenic effect, suggesting CPT-1a in the VMN is involved in the central control of feeding. Intracerebroventricular administration of etomoxir, a CPT-1 inhibitor, decreases food intake. Importantly, in the animals with VMN overexpression of a CPT-1a mutant that antagonizes the CPT-1 inhibition by etomoxir, the anorectic response to etomoxir was attenuated. This suggests that VMN is involved in mediating the anorectic effect of central inhibition of CPT-1a. In contrast, arcuate nucleus (Arc) overexpression of the mutant did not alter etomoxir-induced inhibition of food intake, suggesting that Arc CPT-1a does not play significant roles in this anorectic action. Furthermore, in the VMN, CPT-1a appears to act downstream of hypothalamic malonyl-CoA action of feeding. Finally, we show that in the VMN CPT-1 activity was altered in concert with fasting and refeeding states, supporting a physiological role of CPT-1a in mediating the control of feeding. All together, CPT-1a in the hypothalamic VMN appears to play an important role in central control of food intake. VMN-selective modulation of CPT-1a activity may therefore be a promising strategy in controlling food intake and maintaining normal body weight.THE CENTRAL NERVOUS SYSTEM (CNS) regulates energy homeostasis by tightly balancing energy (food) intake, energy expenditure, and energy deposition (6,50,54). The hypothalamus in the CNS plays a crucial role in this process. Diverse neuronal, humoral, and nutrient signals indicating energy status converge at the hypothalamus, and the hypothalamus processes and integrates these signals to exert the controls of feeding and energy balance (18,47,50,51,54). It has been shown that fatty acid metabolism plays important roles in the intracellular signaling mechanisms underlying hypothalamic control of energy balance (12,32,34). Carnitine palmitoyltransferase-1 (CPT-1) is a key enzyme in shuttling fatty acid into mitochondria for ␤-oxidation by converting fatty acyl-CoA to acylcarnitine (38, 48). Of relevance, it has been proposed that hypothalamic CPT-1 plays a key role in the central control of food intake (42). In the hypothalamus, the predominant CPT-1 isoform possessing acyltransferase activity is the CPT-1 liver isoform (CPT-1a). Previous studies have s...

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A new LC–ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells

Cited by 20 publications

References 16 publications

Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

Determination of carnitine, its short chain acyl esters and metabolic precursors trimethyllysine and γ-butyrobetaine by quasi-solid phase extraction and MS/MS detection

Important roles of brain-specific carnitine palmitoyltransferase and ceramide metabolism in leptin hypothalamic control of feeding

Important role of ventromedial hypothalamic carnitine palmitoyltransferase-1a in the control of food intake

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