Abstract
Background:
Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, cataplexy, and REM sleep abnormalities. A genome-wide association study identified a novel narcolepsy-related single nucleotide polymorphism (SNP), rs5770917, which is located adjacent to CPT1B (carnitine palmitoyltransferase 1B). In this study, we analyzed the CPT1B expression level and measured the carnitine fractions in blood samples obtained from narcolepsy patients and control subjects to test the hypothesis that fatty acid β-oxidation is altered in narcolepsy.
Methodology and Results:
We measured CPT1B mRNA expression in white blood cells of 38 narcolepsy patients and 56 healthy control subjects. The serum carnitine fractions (total carnitine, free carnitine, and acylcarnitine) were measured in the 38 narcolepsy patients and in 30 of 56 control subjects. Stepwise multiple regression analysis revealed that the risk allele (C) for SNP rs5770917 was significantly associated with decreased CPT1B mRNA expression (P = 1.0 × 10−9), and the CPT1B expression was higher in the narcolepsy patients than in the controls (P = 0.005). The acylcarnitine levels were abnormally low in 21% of the narcolepsy patients while those of all the controls were within the normal range. Stepwise multiple regression analysis using the dichotomous variable for acylcarnitine (normal or abnormal) as an objective variable revealed that the diagnosis of narcolepsy but not CPT1B expression level and BMI was associated with abnormally low acylcarnitine levels (P = 0.006).
Conclusions:
Our results indicate that multiple factors are involved in the regulation of serum acylcarnitine levels. Abnormally low levels of acylcarnitine observed in narcolepsy suggest dysfunctional fatty acid β-oxidation pathway.
Citation:
Miyagawa T; Miyadera H; Tanaka S; Kawashima M; Shimada M; Honda Y; Tokunaga K; Honda M. Abnormally low serum acylcarnitine levels in narcolepsy patients. SLEEP 2011;34(3):349-353.
Keywords: Narcolepsy, acylcarnitine, CPT1B mRNA expression level and fatty acid β-oxidation
INTRODUCTION
Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, cataplexy (sudden loss of muscle tone in RESPONSE TO strong emotions such as laughter), sleep paralysis, and hypnagogic hallucinations. Narcolepsy with cataplexy affects 0.16% to 0.18% of the general population in Japan and 0.02% to 0.06% of the population in the United States and Europe.1 It has been reported that both genetic and environmental factors contribute to its development.1 Narcolepsy is closely associated with human leukocyte antigen (HLA)-DQB1*0602.2,3 A genome-wide association study has identified a significant association between narcolepsy and a single nucleotide polymorphism (SNP) located in T-cell receptor α-locus.4 Recently, we also conducted a genome-wide association analysis and identified a novel narcolepsy-related SNP, rs5770917, located between the genes encoding carnitine palmitoyltransferase 1B and choline kinase β (CPT1B and CHKB, respectively).5 The mRNA expression levels of both CPT1B and CHKB were associated with this SNP; these levels were lower in heterozygotes (TC) than in non-risk homozygotes (TT).5
CPT1B is a rate-limiting enzyme in β-oxidation of long-chain fatty acids, which is mainly localized in the muscle mitochondrial outer membrane.6 Conjugation of carnitine to long-chain fatty acyl coenzyme A (CoA) by CPT1B allows the transport of long-chain fatty acids into the mitochondrial matrix for subsequent β-oxidation. The CPT1B expression levels in the blood cells and muscle tissue are known to be correlated7; therefore, they could be used as a marker for systemic CPT1B function. Carnitine fractions (total carnitine, free carnitine, and acylcarnitine) in the blood are commonly used as indicators of the total carnitine status in the body.8 These fractions have also been used to determine dysfunctional fatty acid metabolism in patients with renal dysfunction9,10 and those undergoing pharmacotherapy,11,12 and in neonatal screening for inherited metabolic disorders, including CPT1 deficiency.13–15
Narcolepsy patients are at a higher risk of non–insulin-dependent diabetes mellitus16 and obesity,16–19 suggesting the abnormality in energy homeostasis. The dysfunction in hypocretin/orexin neurotransmission observed as the major pathophysiology of narcolepsy20,21 is suggested to be related to obesity.22 However, animal studies have indicated that several factors other than hypocretin also contribute to obesity in patients with narcolepsy.23,24 Furthermore, mice with systemic carnitine deficiency showed decreased hypocretin neuron activity and fragmented sleep.25
In this study, we analyzed the CPT1B expression level and measured the carnitine fractions in blood samples obtained from narcolepsy patients and control subjects to test the hypothesis that fatty acid β-oxidation is altered in narcolepsy.
MATERIALS AND METHODS
Subjects
This study was approved by the local institutional review boards of the collaborative organizations. A total of 38 patients with narcolepsy (SNP rs5770917; TT: n = 16, TC: n = 14, CC: n = 8) and 56 age-, sex-, and SNP rs5770917 genotype-matched healthy control subjects (SNP rs5770917; TT: n = 29, TC: n = 22, CC: n = 5) were used to compare the relative expression level of the CPT1B gene. Written informed consents were obtained from all the study participants. We measured the serum carnitine fractions (total carnitine, free carnitine, and acylcarnitine) in the narcolepsy patients and in 30 age-, sex-, and body mass index (BMI)-matched control subjects (SNP rs5770917 TT: n = 14, TC: n = 11, CC: n = 5) selected from the 56 controls. All the subjects were unrelated Japanese individuals living in Tokyo or in neighboring areas. All the narcolepsy patients carried the HLA-DRB1*1501-DQB1*0602 haplotype and exhibited unambiguous cataplexy. The patients were diagnosed according to the International Classification of Sleep Disorders, 2nd edition, at the Neuropsychiatric Research Institute. Demographic data of the subjects are summarized in Supplementary Tables S1 and S2. The age and sex of the subjects did not significantly differ among the groups according to the SNP rs5770917 genotype. The BMI was higher in the narcolepsy patients than in the controls, reflecting a tendency toward obesity in narcolepsy patients. Therefore, we measured the carnitine fractions from 30 BMI-matched controls.
Genotyping and Real-Time Quantitative RT-PCR
HLA typing for the HLA-DRB1 and DQB1 loci was performed at the NPO HLA Laboratory (Kyoto, Japan). Genotyping of SNP rs5770917 was performed using the Taqman Genotyping Assays (Applied Biosystems, CA)5 and the relative CPT1B gene expression was measured using Taqman Gene Expression Assays (Applied Biosystems) as described previously.5 Briefly, 5 mL of whole blood was used for total RNA isolation followed by DNase I treatment with a PAXgene Blood RNA Kit (Qiagen, Germany). Single-stranded cDNA was synthesized using SuperScript III and random hexamers (Invitrogen, CA). The relative levels of target gene expression (CPT1B, β-actin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were measured by quantitative RT-PCR using Taqman Gene Expression Assays (Applied Biosystems). To increase the sensitivity and reproducibility of the assays, we used the geometric mean of β-actin and GAPDH as a normalization factor according to the geNorm analysis.26
Analysis of Carnitine Fractions
We collected 5 mL whole blood at the same time as the sampling for RNA extraction, and the sera were separated and stored at −80 °C. The total and free carnitine fractions in the blood were measured by an enzymatic cycling method by using carnitine dehydrogenase (SRL Inc., Tokyo).27 We then calculated the acylcarnitine level on the basis of the amounts of total and free carnitine. The normal ranges of the healthy control subjects for total carnitine, free carnitine, and acylcarnitine were established at 45–91, 36–74, and 6–23 μmol/L, respectively. These normal ranges were calculated from measurement data of 306 healthy subjects (162 males and 144 females) (SRL Inc., Tokyo).
Statistical Analyses
The relative CPT1B mRNA expression levels among groups according to the SNP rs5770917 genotype were compared using the Mann-Whitney U test. To evaluate whether the CPT1B expression level was independently associated with narcolepsy, we used stepwise multiple regression analysis to assess the effect of the following possible confounding factors on the CPT1B expression level: diagnosis (narcolepsy or control), the SNP rs5770917 genotype, age, sex, and BMI. The levels of carnitine fractions were compared using the Mann-Whitney U test, and the distribution of dichotomous variables of each carnitine fraction was compared by Fisher exact test between groups. Further, we assessed the effect of diagnosis, CPT1B mRNA expression level, SNP genotype, age, sex, and BMI on dichotomous carnitine fraction variables using stepwise multiple regression analysis.
RESULTS
CPT1B Expression Analysis
We found that the number of risk alleles (C) of SNP rs5770917 was significantly associated with low levels of CPT1B mRNA expression in both the narcolepsy and healthy control groups (Figure 1 and Table 1). Interestingly, the CPT1B expression levels were higher in narcolepsy patients than in the control subjects of the same genotype (Figure 1 and Table 1). Stepwise multiple regression analysis revealed that SNP rs5770917 and the diagnosis (narcolepsy or control) were included in the model (Table 1). The genotype showed strong association with CPT1B mRNA expression (P = 1.0 × 10−9), and the diagnosis also showed independent association with CPT1B mRNA expression (P = 0.005). The other variables, namely, age, sex, and BMI, were not associated with the CPT1B expression level.
Figure 1.
CPT1B expression from 94 subjects with SNP rs5770917 genotypes (38 patients: TT n = 16, TC n = 14, CC n = 8; 56 controls: TT n = 29, TC n = 22, CC n = 5). The relative mRNA expression level of CPT1B was measured by real-time quantitative RT-PCR and compared by the Mann-Whitney U test. The horizontal lines indicate the mean expression of each group, and the squares indicate the standard error of each group. N, narcolepsy patients; C, healthy control subjects.
Table 1.
Relative expression levels of CPT1B and multiple regression analysis
Narcolepsy |
Healthy Control |
Pgenotype |
Pdiagnosis |
||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
TT (n = 16) |
TC (n = 14) |
CC (n = 8) |
TT (n = 29) |
TC (n = 22) |
CC (n = 5) |
||||||||
Mean | SE | Mean | SE | mean | SE | mean | SE | Mean | SE | mean | SE | ||
6.47 | 0.79 | 3.90 | 0.45 | 1.60 | 0.15 | 4.78 | 0.41 | 2.92 | 0.27 | 1.49 | 0.09 | 1.0 × 10−9 | 0.005 |
A stepwise method (Fin and Fout: 4) was used in the multiple regression analysis. Diagnosis (narcolepsy 0, healthy control 1), genotype of SNP rs5770917 (TT 0, TC 1, CC 2), age, sex (M 0, F 1), and BMI were used as the explanatory variables. The diagnosis and the genotype were included in the model. P values for genotype- (Pgenotype) and diagnosis-based (Pdiagnosis) comparisons were calculated using multiple regression analysis. The other variables were excluded from the stepwise procedure. SE, standard error.
Analysis of Serum Carnitine Fractions
Quantitative analysis of the serum carnitine fractions did not reveal any significant differences in the distribution between narcolepsy patients and control subjects (Table 2 and Supplementary Figure S1). Further, the SNP genotype was not found to affect the serum carnitine levels (Table 2 and Supplementary Figure S1). However, when we treated the acylcarnitine level as a dichotomous variable (normal or abnormal), the acylcarnitine levels were abnormally low in 21% (8 of 38) of the narcolepsy patients but were within the normal range in all the 30 control subjects (Fisher exact test; P = 0.007) (Table 3 and Supplementary Figure S1). The SNP genotype was not associated with the levels of total carnitine, free carnitine, and acylcarnitine fractions (Supplementary Table S3 and Supplementary Figure S1).
Table 2.
Quantitative values of carnitine fractions
Narcolepsy |
||||||||
---|---|---|---|---|---|---|---|---|
TT (n = 16) |
TC (n = 14) |
CC (n = 8) |
Total (n = 38) |
|||||
(μmol/L) | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Total carnitine | 56.6 | 11.4 | 55.6 | 10.6 | 53.1 | 9.4 | 55.5 | 10.6 |
Free carnitine | 45.5 | 9.2 | 46.4 | 8.9 | 45.0 | 8.0 | 45.7 | 8.6 |
Acylcarnitine | 11.1 | 5.1 | 9.2 | 3.4 | 8.1 | 2.0 | 9.7 | 4.1 |
Healthy Control |
||||||||
TT (n = 14) |
TC (n = 11) |
CC (n = 5) |
Total (n = 30) |
|||||
(μmol/L) | Mean | SD | Mean | SD | Mean | SD | Mean | SD |
Total carnitine | 57.6 | 9.0 | 58.2 | 8.3 | 59.0 | 4.4 | 58.1 | 7.8 |
Free carnitine | 49.2 | 7.1 | 48.2 | 7.0 | 48.8 | 3.7 | 48.8 | 6.4 |
Acylcarnitine | 8.4 | 2.4 | 9.9 | 2.4 | 10.3 | 2.5 | 9.3 | 2.5 |
There were no significant inter- and intra- group differences according to the SNP rs5770917 genotype. SD, standard deviation.
Table 3.
Number of subjects with abnormally low carnitine levels
Narcolepsy (n = 38) | Healthy Control (n = 30) | PFisher | Pregression | |
---|---|---|---|---|
Total carnitine | 7 | 1 | 0.07 | — |
Free carnitine | 7 | 1 | 0.07 | — |
Acylcarnitine | 8 | 0 | 0.007 | 0.006 |
Carnitine fractions were treated as a dichotomous variable (normal and abnormal). P values were calculated by Fisher exact test (PFisher) and multiple regression analysis (Pregression). A stepwise method (Fin and Fout: 4) was used in the multiple regression analysis. Diagnosis (narcolepsy 0, healthy control 1), CPT1B mRNA expression level, the SNP rs5770917 genotype (TT 0, TC 1, CC 2), age, sex (M 0, F 1), and BMI were used as the explanatory variables.
Stepwise multiple regression analysis using the dichotomous variable of acylcarnitine (normal or abnormal) as an objective variable and diagnosis (narcolepsy or control), CPT1B mRNA expression level, the SNP rs5770917 genotype, age, sex, and BMI as explanatory variables revealed that only diagnosis (narcolepsy or control) was included in the model, indicating that narcolepsy is significantly associated with abnormally low levels of acylcarnitine (P = 0.006) (Table 3). The other variables were not associated with abnormally low levels of acylcarnitine. Similar analysis using dichotomous variables revealed that narcolepsy patients had low total and free carnitine levels (Table 3 and Supplementary Figure S1), but the differences did not reach statistical significance. None of the subjects, from either of the groups, showed abnormally high total carnitine, free carnitine, or acylcarnitine levels (Supplementary Figure S1).
DISCUSSION
In this study, we extended our previous study,5 that CPT1B mRNA expression levels decreased depending on the number of risk alleles (Figure 1 and Table 1) and found that the CPT1B expression level was higher in narcolepsy patients than in control subjects (Table 1). CPT1B is a rate-limiting enzyme in the β-oxidation of long-chain fatty acids; therefore, we measured the serum carnitine fractions (total and free carnitine and acylcarnitine) to be used as an indicator of the total body carnitine status8 and dysfunction of fatty acid metabolism.14,15 We found that abnormally low serum acylcarnitine level is associated with narcolepsy; 21% (8 of 38) of the narcolepsy patients showed abnormally low serum acylcarnitine levels, while none of the 30 control subjects showed the abnormal values (Table 3). The serum total carnitine level has been reported to be affected by age and sex, while the acylcarnitine level remains stable in the general population regardless of these variables,28 indicating that the abnormally low acylcarnitine levels observed in our study reflect the metabolic alterations associated with the pathophysiology of narcolepsy. Possible reasons of the abnormally low acylcarnitine levels include total carnitine insufficiency, reduced CPT1 activity, and decreased availability of the substrate (acyl-CoA).
Extremely low total carnitine levels could lead to abnormal acylcarnitine level.14 Of the 7 narcolepsy patients with abnormally low total carnitine levels, 3 had abnormally low acylcarnitine level. Since daily food uptake is known to influence carnitine levels,29 dietary or supplemental carnitine intake might be useful to normalize the acylcarnitine levels and reduce some of the narcolepsy symptoms observed in these cases. However, insufficient carnitine levels alone cannot explain the abnormally low acylcarnitine levels observed in the present study. Despite the significant association of the SNP rs5770917 genotype and CPT1B mRNA expression level with narcolepsy, they do not directly affect the serum acylcarnitine level; control subjects with low CPT1B mRNA expression showed normal acylcarnitine level, suggesting the presence of compensatory factors regulating carnitine fractions.
The low acyl-CoA availability for fatty acid oxidation in narcolepsy patients is a plausible hypothesis that requires further investigation. Narcolepsy patients tend to be obese,16–19 suggesting the possibility of preferential utilization of fatty acyl-CoA for lipid synthesis even under increased energy demand. A combination of multiple genetic factors defining the capacity of fatty acid metabolism and environmental factors might determine the individual variation in acylcarnitine levels. Further studies designed with focus on measuring the serum carnitine fractions under controlled conditions, such as food restriction and extensive exercise, should be performed to detect narcolepsy-specific alterations in fatty acid β-oxidation and the basis of the observed higher risk for obesity in narcolepsy.16,17 One limitation is that we could not measure the individual component of acylcarnitine consisting of various lengths of fatty acid chains by the enzymatic cycling method used in this study. Measurement of these individual components by other methods13 would also be useful to identify the disease-specific dysfunction of fatty acid β-oxidation in narcolepsy patients.
There have been limited reports on the molecular basis linking fatty acid β-oxidation and the sleep-wake regulation in the brain.30,31 In addition, AMP-activated protein kinase (AMPK), which is an energy-sensing molecular signal involved in lipid metabolism and regulates CPT1, contributes to homeostatic sleep regulation.32 Low serum acylcarnitine levels have been observed in patients with chronic fatigue syndrome (CFS),33,34 which is a clinically defined condition characterized by severe disabling fatigue and a combination of symptoms, such as musculoskeletal pain, difficulty in concentration and sleep disturbances.35 There might be a common pathological process underlying narcolepsy and CFS accompanying low serum acylcarnitine levels. Animal studies indicate the link between abnormal β-oxidation and decreased hypocretin neuron activity. Fasted juvenile visceral steatosis (jvs−/−) mice with systemic carnitine deficiency exhibit marked reduction in locomotor activity together with the manifestation of fragmented wakefulness and REM sleep,25 similar to the mouse models of narcolepsy.23,36 In these mice, significant reduction in the number of c-Fos-positive hypocretin neurons, hypothalamic hypocretin mRNA expression, and hypocretin-1 concentration in the cerebrospinal fluid (CSF) was observed,25,37 indicating that the acylcarnitine availability is essential for normal sleep regulation and hypocretin cell functions. On the other hand, mice deficient in short-chain acyl-CoA dehydrogenase (encoded by Acads), an enzyme catalyzing the first step of β-oxidation, showed slower theta oscillation during REM sleep.38 The slowing of theta oscillation in these mutant mice was partially recovered by administration of acetyl-l-carnitine, which is known to restore mitochondrial function of β-oxidation.39,40 Potential therapeutic benefits of acetyl-L-carnitine, L-carnitine, or related compounds improving mitochondrial fatty acid oxidation need to be explored in future studies.
In conclusion, we found that CPT1B mRNA expression in blood cells showed genotype- and narcolepsy-specific alterations and that abnormally low serum acylcarnitine levels were significantly associated with narcolepsy. Although other variables such as BMI and CPT1B mRNA expression levels were not found to be associated with abnormally low acylcarnitine levels, further studies are required to clarify the suggested dysfunction in fatty acid β-oxidation pathway and determine the basis for the observed tendency toward obesity in narcolepsy patients.
DISCLOSURE STATEMENT
This was not an industry supported study. The authors have indicated no financial conflicts of interest.
ACKNOWLEDGMENTS
The authors thank all participants in this study. This study was supported by Grants-in-Aid for Scientific Research on Priority Areas “Comprehensive Genomics,” “Applied Genomics,” Scientific Research 19390310 and 21790336 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for “JSPS” fellows and Grants-in-Aid for “Astellas Foundation for Research on Metabolic Disorders,” “Takeda Science Foundation,” “Mitsubishi Pharma Research Foundation,” and “Kowa Life Science Foundation.”
The normal ranges for total carnitine, free carnitine, and acylcarnitine were 45–91, 36–74, and 6–23 μmol/L, respectively. The area within the blue boxes represents the normal ranges. N and C represent the narcolepsy and control groups, respectively.
Table S1.
Demographic data of subjects in CPT1B expression analysis
SNP rs5770917 Genotype | Number of Subjects |
Age |
BMI |
|||
---|---|---|---|---|---|---|
Female | Male | Average | SD | Average | SD | |
TT (case) | 8 | 8 | 49.6 | 11.0 | 27.8 | 4.4 |
TC (case) | 5 | 9 | 46.3 | 13.4 | 29.2 | 4.5 |
CC (case) | 5 | 3 | 34.0 | 17.5 | 27.4 | 5.8 |
TT (control) | 13 | 16 | 45.2 | 13.1 | 24.2 | 3.2 |
TC (control) | 12 | 10 | 49.2 | 10.7 | 24.6 | 3.4 |
CC (control) | 4 | 1 | 39.0 | 18.0 | 21.7 | 2.3 |
Total (case) | 18 | 20 | 45.1 | 14.3 | 28.2 | 4.7 |
Total (control) | 29 | 27 | 46.2 | 12.8 | 24.1 | 3.3 |
There were significant differences in the BMI data between cases and controls in each SNP rs5770917 genotype (TT, P < 0.01; TC, P < 0.01; Student's t-test). SD, standard deviation.
Table S2.
Demographic data of subjects used for the measurement of carnitine fractions
SNP rs5770917 Genotype | Number of Subjects |
Age |
BMI |
|||
---|---|---|---|---|---|---|
Female | Male | Average | SD | Average | SD | |
TT (case) | 8 | 8 | 49.6 | 11.0 | 27.8 | 4.4 |
TC (case) | 5 | 9 | 46.3 | 13.4 | 29.2 | 4.5 |
CC (case) | 5 | 3 | 34.0 | 17.5 | 27.4 | 5.8 |
TT (control) | 7 | 7 | 48.9 | 13.0 | 26.0 | 2.8 |
TC (control) | 6 | 5 | 52.3 | 9.5 | 26.6 | 2.7 |
CC (control) | 4 | 1 | 39.0 | 18.0 | 21.7 | 2.3 |
Total (case) | 18 | 20 | 45.1 | 14.3 | 28.2 | 4.7 |
Total (control) | 17 | 13 | 48.5 | 13.1 | 25.5 | 3.1 |
SD, standard deviation.
Table S3.
Number of subjects in each SNP rs5770917 genotype with abnormally low serum carnitine levels
Narcolepsy |
Healthy Control |
|||||||
---|---|---|---|---|---|---|---|---|
TT (n = 16) | TC (n = 14) | CC (n = 8) | total (n = 38) | TT (n = 14) | TC (n = 11) | CC (n = 5) | total (n = 30) | |
Total carnitine | 3 | 3 | 1 | 7 | 0 | 1 | 0 | 1 |
Free carnitine | 3 | 3 | 1 | 7 | 0 | 1 | 0 | 1 |
Acylcarnitine | 3 | 4 | 1 | 8 | 0 | 0 | 0 | 0 |
There were no significant differences between the groups regardless of their SNP rs5770917genotypes
REFERENCES
- 1.Mignot E. Genetic and familial aspects of narcolepsy. Neurology. 1998;50:S16–22. doi: 10.1212/wnl.50.2_suppl_1.s16. [DOI] [PubMed] [Google Scholar]
- 2.Juji T, Satake M, Honda Y, Doi Y. HLA antigens in Japanese patients with narcolepsy. All the patients were DR2 positive. Tissue Antigens. 1984;24:316–9. doi: 10.1111/j.1399-0039.1984.tb02144.x. [DOI] [PubMed] [Google Scholar]
- 3.Mignot E, Lin L, Rogers W, et al. Complex HLA-DR and -DQ interactions confer risk of narcolepsy-cataplexy in three ethnic groups. Am J Hum Genet. 2001;68:686–99. doi: 10.1086/318799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Hallmayer J, Faraco J, Lin L, et al. Narcolepsy is strongly associated with the T-cell receptor alpha locus. Nat Genet. 2009;41:708–11. doi: 10.1038/ng.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Miyagawa T, Kawashima M, Nishida N, et al. Variant between CPT1B and CHKB associated with susceptibility to narcolepsy. Nat Genet. 2008;40:1324–8. doi: 10.1038/ng.231. [DOI] [PubMed] [Google Scholar]
- 6.McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem. 1997;244:1–14. doi: 10.1111/j.1432-1033.1997.00001.x. [DOI] [PubMed] [Google Scholar]
- 7.Zeibig J, Karlic H, Lohninger A, Damsgaard R, Smekal G. Do blood cells mimic gene expression profile alterations known to occur in muscular adaptation to endurance training? Eur J Appl Physiol. 2005;95:96–104. doi: 10.1007/s00421-005-1334-3. [DOI] [PubMed] [Google Scholar]
- 8.Reuter SE, Evans AM, Chace DH, Fornasini G. Determination of the reference range of endogenous plasma carnitines in healthy adults. Ann Clin Biochem. 2008;45:585–92. doi: 10.1258/acb.2008.008045. [DOI] [PubMed] [Google Scholar]
- 9.Savica V, Bellinghieri G, Di Stefano C, et al. Plasma and muscle carnitine levels in haemodialysis patients with morphological-ultrastructural examination of muscle samples. Nephron. 1983;35:232–6. doi: 10.1159/000183087. [DOI] [PubMed] [Google Scholar]
- 10.Arduini A, Bonomini M, Savica V, Amato A, Zammit V. Carnitine in metabolic disease: potential for pharmacological intervention. Pharmacol Ther. 2008;120:149–56. doi: 10.1016/j.pharmthera.2008.08.008. [DOI] [PubMed] [Google Scholar]
- 11.Coppola G, Epifanio G, Auricchio G, Federico RR, Resicato G, Pascotto A. Plasma free carnitine in epilepsy children, adolescents and young adults treated with old and new antiepileptic drugs with or without ketogenic diet. Brain Dev. 2006;28:358–65. doi: 10.1016/j.braindev.2005.11.005. [DOI] [PubMed] [Google Scholar]
- 12.Reuter SE, Faull RJ, Ranieri E, Evans AM. Endogenous plasma carnitine pool composition and response to erythropoietin treatment in chronic haemodialysis patients. Nephrol Dial Transplant. 2009;24:990–6. doi: 10.1093/ndt/gfn588. [DOI] [PubMed] [Google Scholar]
- 13.Shigematsu Y, Hirano S, Hata I, et al. Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;792:63–72. doi: 10.1016/s1570-0232(03)00281-2. [DOI] [PubMed] [Google Scholar]
- 14.Stanley CA. Carnitine deficiency disorders in children. Ann N Y Acad Sci. 2004;1033:42–51. doi: 10.1196/annals.1320.004. [DOI] [PubMed] [Google Scholar]
- 15.Longo N, Amat di San Filippo C, Pasquali M. Disorders of carnitine transport and the carnitine cycle. Am J Med Genet C Semin Med Genet. 2006;142C:77–85. doi: 10.1002/ajmg.c.30087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Honda Y, Doi Y, Ninomiya R, Ninomiya C. Increased frequency of non-insulin-dependent diabetes mellitus among narcoleptic patients. Sleep. 1986;9:254–9. doi: 10.1093/sleep/9.1.254. [DOI] [PubMed] [Google Scholar]
- 17.Schuld A, Hebebrand J, Geller F, Pollmacher T. Increased body-mass index in patients with narcolepsy. Lancet. 2000;355:1274–5. doi: 10.1016/S0140-6736(05)74704-8. [DOI] [PubMed] [Google Scholar]
- 18.Dahmen N, Bierbrauer J, Kasten M. Increased prevalence of obesity in narcoleptic patients and relatives. Eur Arch Psychiatry Clin Neurosci. 2001;251:85–9. doi: 10.1007/s004060170057. [DOI] [PubMed] [Google Scholar]
- 19.Kok SW, Overeem S, Visscher TL, et al. Hypocretin deficiency in narcoleptic humans is associated with abdominal obesity. Obes Res. 2003;11:1147–54. doi: 10.1038/oby.2003.156. [DOI] [PubMed] [Google Scholar]
- 20.Peyron C, Faraco J, Rogers W, et al. A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nat Med. 2000;6:991–7. doi: 10.1038/79690. [DOI] [PubMed] [Google Scholar]
- 21.Thannickal TC, Moore RY, Nienhuis R, et al. Reduced number of hypocretin neurons in human narcolepsy. Neuron. 2000;27:469–74. doi: 10.1016/s0896-6273(00)00058-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Nishino S, Ripley B, Overeem S, et al. Low cerebrospinal fluid hypocretin (Orexin) and altered energy homeostasis in human narcolepsy. Ann Neurol. 2001;50:381–8. doi: 10.1002/ana.1130. [DOI] [PubMed] [Google Scholar]
- 23.Hara J, Beuckmann CT, Nambu T, et al. Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron. 2001;30:345–54. doi: 10.1016/s0896-6273(01)00293-8. [DOI] [PubMed] [Google Scholar]
- 24.Hara J, Yanagisawa M, Sakurai T. Difference in obesity phenotype between orexin-knockout mice and orexin neuron-deficient mice with same genetic background and environmental conditions. Neurosci Lett. 2005;380:239–42. doi: 10.1016/j.neulet.2005.01.046. [DOI] [PubMed] [Google Scholar]
- 25.Yoshida G, Li MX, Horiuchi M, et al. Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice. Neurosci Res. 2006;55:78–86. doi: 10.1016/j.neures.2006.02.003. [DOI] [PubMed] [Google Scholar]
- 26.Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034. doi: 10.1186/gb-2002-3-7-research0034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Takahashi M, Ueda S, Misaki H, et al. Carnitine determination by an enzymatic cycling method with carnitine dehydrogenase. Clin Chem. 1994;40:817–21. [PubMed] [Google Scholar]
- 28.Takiyama N, Matsumoto K. Age-and sex-related differences of serum carnitine in a Japanese population. J Am Coll Nutr. 1998;17:71–4. doi: 10.1080/07315724.1998.10720458. [DOI] [PubMed] [Google Scholar]
- 29.Bach AC. Carnitine in human nutrition. Z Ernahrungswiss. 1982;21:257–65. doi: 10.1007/BF02020743. [DOI] [PubMed] [Google Scholar]
- 30.Obici S, Feng Z, Arduini A, Conti R, Rossetti L. Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production. Nat Med. 2003;9:756–61. doi: 10.1038/nm873. [DOI] [PubMed] [Google Scholar]
- 31.Cruciani-Guglielmacci C, Hervalet A, Douared L, et al. Beta oxidation in the brain is required for the effects of non-esterified fatty acids on glucose-induced insulin secretion in rats. Diabetologia. 2004;47:2032–8. doi: 10.1007/s00125-004-1569-2. [DOI] [PubMed] [Google Scholar]
- 32.Chikahisa S, Fujiki N, Kitaoka K, Shimizu N, Sei H. Central AMPK contributes to sleep homeostasis in mice. Neuropharmacology. 2009;57:369–74. doi: 10.1016/j.neuropharm.2009.07.015. [DOI] [PubMed] [Google Scholar]
- 33.Kuratsune H, Yamaguti K, Lindh G, et al. Low levels of serum acylcarnitine in chronic fatigue syndrome and chronic hepatitis type C, but not seen in other diseases. Int J Mol Med. 1998;2:51–6. doi: 10.3892/ijmm.2.1.51. [DOI] [PubMed] [Google Scholar]
- 34.Kuratsune H, Yamaguti K, Takahashi M, Misaki H, Tagawa S, Kitani T. Acylcarnitine deficiency in chronic fatigue syndrome. Clin Infect Dis. 1994;18(Suppl 1):S62–7. doi: 10.1093/clinids/18.supplement_1.s62. [DOI] [PubMed] [Google Scholar]
- 35.Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med. 1994;121:953–9. doi: 10.7326/0003-4819-121-12-199412150-00009. [DOI] [PubMed] [Google Scholar]
- 36.Chemelli RM, Willie JT, Sinton CM, et al. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell. 1999;98:437–51. doi: 10.1016/s0092-8674(00)81973-x. [DOI] [PubMed] [Google Scholar]
- 37.Kuwajima M, Fujihara H, Sei H, et al. Reduced carnitine level causes death from hypoglycemia: possible involvement of suppression of hypothalamic orexin expression during weaning period. Endocr J. 2007;54:911–25. doi: 10.1507/endocrj.k07-044. [DOI] [PubMed] [Google Scholar]
- 38.Tafti M, Petit B, Chollet D, et al. Deficiency in short-chain fatty acid beta-oxidation affects theta oscillations during sleep. Nat Genet. 2003;34:320–5. doi: 10.1038/ng1174. [DOI] [PubMed] [Google Scholar]
- 39.Liu J, Killilea DW, Ames BN. Age-associated mitochondrial oxidative decay: improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-L- carnitine and/or R-alpha -lipoic acid. Proc Natl Acad Sci U S A. 2002;99:1876–81. doi: 10.1073/pnas.261709098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Rao KV, Qureshi IA. Decompensation of hepatic and cerebral acyl-CoA metabolism in BALB/cByJ mice by chronic riboflavin deficiency: restoration by acetyl-L-carnitine. Can J Physiol Pharmacol. 1997;75:423–30. [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
The normal ranges for total carnitine, free carnitine, and acylcarnitine were 45–91, 36–74, and 6–23 μmol/L, respectively. The area within the blue boxes represents the normal ranges. N and C represent the narcolepsy and control groups, respectively.
Table S1.
Demographic data of subjects in CPT1B expression analysis
SNP rs5770917 Genotype | Number of Subjects |
Age |
BMI |
|||
---|---|---|---|---|---|---|
Female | Male | Average | SD | Average | SD | |
TT (case) | 8 | 8 | 49.6 | 11.0 | 27.8 | 4.4 |
TC (case) | 5 | 9 | 46.3 | 13.4 | 29.2 | 4.5 |
CC (case) | 5 | 3 | 34.0 | 17.5 | 27.4 | 5.8 |
TT (control) | 13 | 16 | 45.2 | 13.1 | 24.2 | 3.2 |
TC (control) | 12 | 10 | 49.2 | 10.7 | 24.6 | 3.4 |
CC (control) | 4 | 1 | 39.0 | 18.0 | 21.7 | 2.3 |
Total (case) | 18 | 20 | 45.1 | 14.3 | 28.2 | 4.7 |
Total (control) | 29 | 27 | 46.2 | 12.8 | 24.1 | 3.3 |
There were significant differences in the BMI data between cases and controls in each SNP rs5770917 genotype (TT, P < 0.01; TC, P < 0.01; Student's t-test). SD, standard deviation.
Table S2.
Demographic data of subjects used for the measurement of carnitine fractions
SNP rs5770917 Genotype | Number of Subjects |
Age |
BMI |
|||
---|---|---|---|---|---|---|
Female | Male | Average | SD | Average | SD | |
TT (case) | 8 | 8 | 49.6 | 11.0 | 27.8 | 4.4 |
TC (case) | 5 | 9 | 46.3 | 13.4 | 29.2 | 4.5 |
CC (case) | 5 | 3 | 34.0 | 17.5 | 27.4 | 5.8 |
TT (control) | 7 | 7 | 48.9 | 13.0 | 26.0 | 2.8 |
TC (control) | 6 | 5 | 52.3 | 9.5 | 26.6 | 2.7 |
CC (control) | 4 | 1 | 39.0 | 18.0 | 21.7 | 2.3 |
Total (case) | 18 | 20 | 45.1 | 14.3 | 28.2 | 4.7 |
Total (control) | 17 | 13 | 48.5 | 13.1 | 25.5 | 3.1 |
SD, standard deviation.
Table S3.
Number of subjects in each SNP rs5770917 genotype with abnormally low serum carnitine levels
Narcolepsy |
Healthy Control |
|||||||
---|---|---|---|---|---|---|---|---|
TT (n = 16) | TC (n = 14) | CC (n = 8) | total (n = 38) | TT (n = 14) | TC (n = 11) | CC (n = 5) | total (n = 30) | |
Total carnitine | 3 | 3 | 1 | 7 | 0 | 1 | 0 | 1 |
Free carnitine | 3 | 3 | 1 | 7 | 0 | 1 | 0 | 1 |
Acylcarnitine | 3 | 4 | 1 | 8 | 0 | 0 | 0 | 0 |
There were no significant differences between the groups regardless of their SNP rs5770917genotypes