Sirt5 improves cardiomyocytes fatty acid metabolism and ameliorates cardiac lipotoxicity in diabetic cardiomyopathy via CPT2 de-succinylation.

RATIONALE: The disruption of the balance between fatty acid (FA) uptake and oxidation (FAO) leads to cardiac lipotoxicity, serving as the driving force behind diabetic cardiomyopathy (DbCM). Sirtuin 5 (Sirt5), a lysine de-succinylase, could impact diverse metabolic pathways, including FA metabolism. Nevertheless, the precise roles of Sirt5 in cardiac lipotoxicity and DbCM remain unknown. OBJECTIVE: This study aims to elucidate the role and underlying mechanism of Sirt5 in the context of cardiac lipotoxicity and DbCM. METHODS AND RESULTS: The expression of myocardial Sirt5 was found to be modestly elevated in diabetic heart failure patients and mice. Cardiac dysfunction, hypertrophy and lipotoxicity were exacerbated by ablation of Sirt5 but improved by forced expression of Sirt5 in diabetic mice. Notably, Sirt5 deficiency impaired FAO without affecting the capacity of FA uptake in the diabetic heart, leading to accumulation of FA intermediate metabolites, which mainly included medium- and long-chain fatty acyl-carnitines. Mechanistically, succinylomics analyses identified carnitine palmitoyltransferase 2 (CPT2), a crucial enzyme involved in the reconversion of fatty acyl-carnitines to fatty acyl-CoA and facilitating FAO, as the functional succinylated substrate mediator of Sirt5. Succinylation of Lys424 in CPT2 was significantly increased by Sirt5 deficiency, leading to the inactivation of its enzymatic activity and the subsequent accumulation of fatty acyl-carnitines. CPT2 K424R mutation, which mitigated succinylation modification, counteracted the reduction of enzymatic activity in CPT2 mediated by Sirt5 deficiency, thereby attenuating Sirt5 knockout-induced FAO impairment and lipid deposition. CONCLUSIONS: Sirt5 deficiency impairs FAO, leading to cardiac lipotoxicity in the diabetic heart through the succinylation of Lys424 in CPT2. This underscores the potential roles of Sirt5 and CPT2 as therapeutic targets for addressing DbCM.

Clinical characteristics and genetic analysis of six children with carnitine palmitoyltransferase 2 deficiency. / å ­ä¾‹è‚‰ç¢±æ£•æ¦ˆé °åŸºè½¬ç§»é ¶2ç¼ºä¹ç—‡æ‚£å„¿ä¸´åºŠç‰¹å¾åŠåŸºå› å˜å¼‚åˆ†æž.

OBJECTIVES: To investigate the clinical characteristic and genetic variants of children with carnitine palmitoyltransferase 2 (CPT2) deficiency. METHODS: The clinical and genetic data of 6 children with CPT2 deficiency were retrospectively analyzed. The blood acylcarnitines and genetic variants were detected with tandem mass spectrometry and whole-exon gene sequencing, respectively. RESULTS: There were 4 males and 2 females with a mean age of 32 months (15 d-9 years) at diagnosis. One case was asymptomatic and with normal laboratory test results, 2 had delayed onset, and 3 were of infantile type. Three cases were diagnosed at neonatal screening, and 3 cases presented with clinical manifestations of fever, muscle weakness, and increased muscle enzymes. Five children presented with decreased free carnitine and elevated levels of palmitoyl and octadecenoyl carnitines. CPT2 gene variants were detected at 8 loci in 6 children (4 harboring biallelic mutations and 2 harboring single locus mutations), including 3 known variants (p.R631C, p.T589M, and p.D255G) and 5 newly reported variants (p.F352L, p.R498L, p.F434S, p.A515P, and c.153-2A>G). It was predicted by PolyPhen2 and SIFT software that c.153-2A>G and p.F352L were suspected pathogenic variants, while p.R498L, p.F434S and p.A515P were variants of unknown clinical significance. CONCLUSIONS: The clinical phenotypes of CPT2 deficiency are diverse. An early diagnosis can be facilitated by neonatal blood tandem mass spectrometry screening and genetic testing, and most patients have good prognosis after a timely diagnosis and treatment.

Low C0 and normal C16 and C18:1 masking the diagnosis of carnitine palmitoyltransferase II deficiency including a novel CPT2 variant: A case report.

The cases were a pair of siblings with a carnitine palmitoyltransferase (CPT2) deficiency detected by tandem mass spectrometry. Their C16 and C18:1 levels were both within the normal range, while C0 was low, and the (C16+C18:1)/C2 ratio was high. Following genetic testing, a novel CPT2 gene mutation was identified in both patients. The male patient had a normal growth rate during 5 years of follow-up after treatment. By contrast, the female patient did not take l-carnitine supplements and died after an infectious disease-associated illness when she was 1 year old. These data emphasize the need to raise awareness about CPT2 deficiency so as to correctly diagnose and accurately manage the disease.

Newborn Screening for Mitochondrial Carnitine-Acylcarnitine Cycle Disorders in Zhejiang Province, China.

Background: Disorders of mitochondrial carnitine-acylcarnitine cycle is a heterogeneous group of hereditary diseases of mitochondrial ß-oxidation of fatty acids tested in NBS program in Zhejiang province, China. Large-scale studies reporting disorders of mitochondrial carnitine-acylcarnitine cycle among Chinese population in NBS are limited. The aim of this study was to explain the incidence and biochemical, clinical, and genetic characteristics of disorders of mitochondrial carnitine-acylcarnitine cycle in NBS. Methods: From January 2009 to June 2021, 4,070,375 newborns were screened by tandem mass spectrometry. Newborns with elevated C0 levels and/or C0/(C16 + C18) ratios were identified as having CPT1D, whereas those with decreased C0 levels and/or C0/(C16 + C18) ratios and/or elevated C12-C18:1 level were identified as having CPT2D or CACTD. Suspected positive patients were further subjected to genetic analysis. All confirmed patients received biochemical and nutritional treatment, as well as follow-up sessions. Results: Overall, 20 patients (12 with CPT1D, 4 with CPT2D, and 4 with CACTD) with disorders of mitochondrial carnitine-acylcarnitine cycle were diagnosed by NBS. The overall incidence of these disorders was one in 203,518 newborns. In toal, 11 patients with CPT1D exhibited increased C0 levels and C0/(C16 + C18) ratios. In all patients of CPT2D, all long chain acyl-carnitines levels were elevated except for case 14 having normal C12 levels. In all patients with CACTD, all long chain acyl-carnitines levels were elevated except for case 17 having normal C12, C18, and C18:1 levels. Most patients with CPT1D were asymptomatic. Overall, two of 4 patients with CPT2D did not present any clinical symptom, but other two patients died. In 4 cases with CACTD, the disease was onset after birth, and 75% patients died. In total, 14 distinct mutations were identified in CPT1A gene, of which 11 were novel and c.1910C > A (p.S637T), c.740C > T (p.P247L), and c.1328T > C (p.L443P) were the most common mutations. Overall, 3 novel mutations were identified in CPT2 gene, and the most frequent mutation was c.1711C > A (p.P571T). The most common variant in SLC25A20 gene was c.199-10T > G. Conclusion: Disorders of mitochondrial carnitine-acylcarnitine cycle can be detected by NBS, and the combined incidence of these disorders in newborns was rare in Zhejiang province, China. Most patients presented typical acylcarnitine profiles. Most patients with CPT1D presented normal growth and development, whereas those with CPT2D/CACTD exhibited a high mortality rate. Several novel CPT1A and CPT2 variants were identified, which expanded the variant spectrum.

Skeletal muscle undergoes fiber type metabolic switch without myosin heavy chain switch in response to defective fatty acid oxidation.

OBJECTIVE: Skeletal muscle is a heterogeneous and dynamic tissue that adapts to functional demands and substrate availability by modulating muscle fiber size and type. The concept of muscle fiber type relates to its contractile (slow or fast) and metabolic (glycolytic or oxidative) properties. Here, we tested whether disruptions in muscle oxidative catabolism are sufficient to prompt parallel adaptations in energetics and contractile protein composition. METHODS: Mice with defective mitochondrial long-chain fatty acid oxidation (mLCFAO) in the skeletal muscle due to loss of carnitine palmitoyltransferase 2 (Cpt2Sk-/-) were used to model a shift in muscle macronutrient catabolism. Glycolytic and oxidative muscles of Cpt2Sk-/- mice and control littermates were compared for the expression of energy metabolism-related proteins, mitochondrial respiratory capacity, and myosin heavy chain isoform composition. RESULTS: Differences in bioenergetics and macronutrient utilization in response to energy demands between control muscles were intrinsic to the mitochondria, allowing for a clear distinction of muscle types. Loss of CPT2 ablated mLCFAO and resulted in mitochondrial biogenesis occurring most predominantly in oxidative muscle fibers. The metabolism-related proteomic signature of Cpt2Sk-/- oxidative muscle more closely resembled that of glycolytic muscle than of control oxidative muscle. Respectively, intrinsic substrate-supported mitochondrial respiration of CPT2 deficient oxidative muscles shifted to closely match that of glycolytic muscles. Despite this shift in mitochondrial metabolism, CPT2 deletion did not result in contractile-based fiber type switching according to myosin heavy chain composition analysis. CONCLUSION: The loss of mitochondrial long-chain fatty acid oxidation elicits an adaptive response involving conversion of oxidative muscle toward a metabolic profile that resembles a glycolytic muscle, but this is not accompanied by changes in myosin heavy chain isoforms. These data suggest that shifts in muscle catabolism are not sufficient to drive shifts in the contractile apparatus but are sufficient to drive adaptive changes in metabolic properties.

mTORC1 activation is not sufficient to suppress hepatic PPARα signaling or ketogenesis.

The mechanistic target of rapamycin (mTOR) is often referred to as a master regulator of the cellular metabolism that can integrate the growth factor and nutrient signaling. Fasting suppresses hepatic mTORC1 activity via the activity of the tuberous sclerosis complex (TSC), a negative regulator of mTORC1, to suppress anabolic metabolism. The loss of TSC1 in the liver locks the liver in a constitutively anabolic state even during fasting, which was suggested to regulate peroxisome proliferator-activated receptor alpha (PPARα) signaling and ketogenesis, but the molecular determinants of this regulation are unknown. Here, we examined if the activation of the mTORC1 complex in mice by the liver-specific deletion of TSC1 (TSC1L-/-) is sufficient to suppress PPARα signaling and therefore ketogenesis in the fasted state. We found that the activation of mTORC1 in the fasted state is not sufficient to repress PPARα-responsive genes or ketogenesis. Furthermore, we examined whether the activation of the anabolic program mediated by mTORC1 complex activation in the fasted state could suppress the robust catabolic programming and enhanced PPARα transcriptional response of mice with a liver-specific defect in mitochondrial long-chain fatty acid oxidation using carnitine palmitoyltransferase 2 (Cpt2L-/-) mice. We generated Cpt2L-/-; Tsc1L-/- double-KO mice and showed that the activation of mTORC1 by deletion of TSC1 could not suppress the catabolic PPARα-mediated phenotype of Cpt2L-/- mice. These data demonstrate that the activation of mTORC1 by the deletion of TSC1 is not sufficient to suppress a PPARα transcriptional program or ketogenesis after fasting.

Recurrent acute necrotizing encephalopathy in a boy with RANBP2 mutation and thermolabile CPT2 variant: The first case of ANE1 in Japan.

BACKGROUND: Acute necrotizing encephalopathy (ANE) is a severe encephalopathy associated with acute viral infection. While most ANE cases are sporadic, pathogenic variants in the gene RAN binding protein 2 (RANBP2) have been identified as a major cause of familial or recurrent ANE (ANE1). Although sporadic ANE predominantly affects Asian children, ANE1 is very rare in east Asia. CASE REPORT: A 1-year-7-month-old boy, born to unrelated Japanese parents, presented with a seizure and impaired consciousness after 3 days of fever. Brain magnetic resonance imaging (MRI) showed a characteristic involvement of the bilateral thalami, external capsules, insular cortices, and brainstem, suggesting ANE. He received intravenous steroids. Two months later, he had another episode of acute encephalopathy during respiratory syncytial virus infection, from which he recovered relatively well. The recurrent encephalopathic episodes and the characteristic MRI suggested ANE1. Genetic analyses revealed two variants: a rare heterozygous missense variant of RANBP2 [c.1754C>T; p.Thr585Met], and a thermolabile polymorphism in carnitine palmitoyltransferase 2 (CPT2) [c. 1055T>G; p.Phe352Cys]. CONCLUSION: This is the first case of recurrent ANE with an RANBP2 mutation in Japan. The patient also harbored a CPT2 polymorphism that is linked to acute encephalopathy in Japanese patients. Thus, he had a genetic background with two susceptibility variants for acute encephalopathy, RANBP2 (frequent in the Caucasians), and CPT2 (frequent in the Japanese). Further studies are needed to fully discover the genetic predisposition to familial or recurrent ANE in the Asian population.

CPT2 down-regulation promotes tumor growth and metastasis through inducing ROS/NFκB pathway in ovarian cancer.

BACKGROUND: Carnitine palmitoyltransferase 2 (CPT2) is a rate-limiting enzyme involved in fatty acid ß-oxidation (FAO) regulation. Recently, it has been increasingly recognized that lipid metabolism dysregulation is closely implicated in tumorigenesis. However, the involvement of CPT2 in the progression of cancer is still largely unclear, especially in ovarian cancer (OC). METHODS: In the present study, CPT2 expression and its clinical significance were determined in OC tissues and cells. The biological functions and molecular mechanisms of CPT2 in OC growth and metastasis were determined by in vitro and in vivo assays. FINDINGS: We found that CPT2 was frequently down-regulated in primary ovarian serous carcinomas, which is significantly correlated with poor survival of ovarian cancer patients. Functional experiments revealed that CPT2 inhibited OC cell growth and metastasis via suppression of G1/S cell cycle transition and epithelial to mesenchymal transition (EMT), as well as induction of cell apoptosis. Mechanistically, suppression of ROS/NFκB signaling pathway by increasing fatty acid oxidation-derived NADPH production was involved in the anti-tumorigenic functions of CPT2 in OC cells. INTERPRETATION: Altogether, our findings demonstrate that CPT2 functions as a potential tumor suppressor in OC progression. CPT2 may serve as a novel prognostic marker and therapeutic target in OC.

Loss of Muscle Carnitine Palmitoyltransferase 2 Prevents Diet-Induced Obesity and Insulin Resistance despite Long-Chain Acylcarnitine Accumulation.

To assess the effects of acylcarnitine accumulation on muscle insulin sensitivity, a model of muscle acylcarnitine accumulation was generated by deleting carnitine palmitoyltransferase 2 (CPT2) specifically from skeletal muscle (Cpt2Sk-/- mice). CPT2 is an irreplaceable enzyme for mitochondrial long-chain fatty acid oxidation, converting matrix acylcarnitines to acyl-CoAs. Compared with controls, Cpt2Sk-/- muscles do not accumulate anabolic lipids but do accumulate ∼22-fold more long-chain acylcarnitines. High-fat-fed Cpt2Sk-/- mice resist weight gain, adiposity, glucose intolerance, insulin resistance, and impairments in insulin-induced Akt phosphorylation. Obesity resistance of Cpt2Sk-/- mice could be attributed to increases in lipid excretion via feces, GFD15 production, and energy expenditure. L-carnitine supplement intervention lowers acylcarnitines and improves insulin sensitivity independent of muscle mitochondrial fatty acid oxidative capacity. The loss of muscle CPT2 results in a high degree of long-chain acylcarnitine accumulation, simultaneously protecting against diet-induced obesity and insulin resistance.

Mitochondrial carnitine palmitoyltransferase 2 is involved in Nε-(carboxymethyl)-lysine-mediated diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in the world. Advanced glycation end products (AGEs) are thought to be involved in the pathogenesis of DN via multifactorial mechanisms including the generation of oxidative stress and overproduction of various growth factors and cytokines. AGEs are heterogeneous cross-linked sugar-derived proteins, and Nε-(carboxymethyl)-lysine (CML)-conjugated BSA is a major component of AGEs. However, the proteins involved in DN induction by CML have never been reported. Herein, we investigated specific protein regulators of AGE-mediated DN via proteomic analysis of streptozotocin (STZ)-induced diabetic mice kidneys. We identified 937, 976, and 870 proteins in control, STZ, and STZ + CML-BSA samples, respectively. Bioinformatics analysis identified several CML-mediated proteins potentially involved in kidney damage, activation of fatty acid oxidation (FAO), and mitochondrial dysfunction. Furthermore, we identified the CML-specific differential protein carnitine palmitoyltransferase 2 (CPT2), related to FAO. To confirm the effect of CPT2 and the CML-mediated mechanism, human renal tubular HK-2 cells were treated with CML-BSA and cpt2 siRNA, and examined for FAO-mediated fibrosis and mitochondrial dysfunction. CML-BSA and CPT2 knockdown induced fibrosis-related gene expression and damage to mitochondrial membrane potential. Moreover, CPT2 overexpression recovered CML-induced fibrosis-related gene expression. Based on these results, a decrease in CML-induced CPT2 expression causes mitochondrial FAO damage, leading to renal fibrosis and DN.

Maternal Lipid Metabolism Directs Fetal Liver Programming following Nutrient Stress.

The extreme metabolic demands of pregnancy require coordinated metabolic adaptations between mother and fetus to balance fetal growth and maternal health with nutrient availability. To determine maternal and fetal contributions to metabolic flexibility during gestation, pregnant mice with genetic impairments in mitochondrial carbohydrate and/or lipid metabolism were subjected to nutrient deprivation. The maternal fasting response initiates a fetal liver transcriptional program marked by upregulation of lipid- and peroxisome proliferator-activated receptor alpha (Pparα)-regulated genes. Impaired maternal lipid metabolism alters circulating lipid metabolite concentrations and enhances the fetal response to fasting, which is largely dependent on fetal Pparα. Maternal fasting also improves metabolic deficits in fetal carbohydrate metabolism by increasing the availability of alternative substrates. Impairment of both carbohydrate and lipid metabolism in pregnant dams further exacerbates the fetal liver transcriptional response to nutrient deprivation. Together, these data demonstrate a regulatory role for mitochondrial macronutrient metabolism in mediating maternal-fetal metabolic communication, particularly when nutrients are limited.

Thermolabile polymorphism of carnitine palmitoyltransferase 2: A genetic risk factor of overall acute encephalopathy.

OBJECTIVES: Acute encephalopathy is an acute brain dysfunction after preceding infection, consisting of multiple syndromes. Some syndromes, such as acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), are severe with poor outcome, whereas others, such as clinically mild encephalitis/encephalopathy with reversible splenial lesion (MERS), are mild with favorable outcome. Previous study reported the association of the thermolabile polymorphism in Carnitine Palmitoyltransferase 2 (CPT2) gene and severe syndromes of acute encephalopathy. To further explore the pathogenetic role of CPT2 in acute encephalopathy, we conducted a case-control association study of a typical thermolabile CPT2 polymorphism, rs2229291, in 416 patients of acute encephalopathy, including both severe and mild syndromes. METHODS: The case cohort consisted of 416 patients, including AESD, MERS, and other syndromes. The control subjects were 100 healthy Japanese. rs2229291 was genotyped by Sanger sequencing. Genetic distribution was compared between the patients and controls using Cochran-Armitage trend test. RESULTS: Minor allele frequency of rs2229291 was significantly higher in AESD (p = 0.044), MERS (p = 0.015) and entire acute encephalopathy (p = 0.044) compared to the controls. The polymorphism showed no significant association with influenza virus, or with outcome. CONCLUSIONS: This study provided evidence that CPT2 is a susceptibility gene for overall acute encephalopathy, including both severe and mild syndromes, and suggested that impairment of mitochondrial metabolism is common to various syndromes of acute encephalopathy.

A novel mutation leading to the lethal form of carnitine palmitoyltransferase type-2 deficiency.

Background The clinical phenotypes of carnitine palmitoyltransferase type-2 deficiency (CPT2D) are classified into lethal neonatal, severe infantile and muscle forms. The rarest form is the lethal neonatal form. Case presentation The patient was hypotonic and bradycardic at admission. Blood urea nitrogen and creatinine were high. He had polycystic kidneys, patent foramen ovale and aortic valve insufficiency. Cranial magnetic resonance imaging (MRI) revealed increased signal intensities in the periventricular white matter. Tandem mass spectrometry (MS) analysis was compatible with CPT2D. We found a homozygous in-frame deletion in the CPT2 gene using next-generation sequencing. Conclusions We identified a novel mutation leading to the lethal form of CPT2D with polycystic kidney, cardiac malformation and cranial MRI findings. Our findings expand the spectrum of causative mutations and clinical findings in CPT2D.

Characterisation of the Urinary Metabolic Profile of Liver Fluke-Associated Cholangiocarcinoma.

BACKGROUND: Human infection with Opisthorchis viverrini, a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis. METHODS: Global molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand. RESULTS: Multivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury. CONCLUSIONS: Several metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA.

Acute necrotizing encephalopathy and a carnitine palmitoyltransferase 2 variant in an adult.

A 54-year-old Japanese man had a fever of over 40 °C for 7 days and developed unconsciousness, seizure and respiratory arrest. T2-weighted imaging magnetic resonance imaging revealed high-intensity signals on bilateral thalamus and it gradually extended to the brain white matter. Moreover, the lesion progressed to the spinal gray matter. The patient was diagnosed with acute necrotizing encephalopathy. CPT2 variants have been reported to be associated with acute necrotizing encephalopathy particularly in children and spinal cord lesions are extremely rare. We report a case of ANE in an adult with a CPT2 variant who developed spinal cord lesions.

Lipid Metabolic Reprogramming in Hepatocellular Carcinoma.

Metabolic reprogramming for adaptation to the local environment has been recognized as a hallmark of cancer. Although alterations in fatty acid (FA) metabolism in cancer cells have received less attention compared to other metabolic alterations such as glucose or glutamine metabolism, recent studies have uncovered the importance of lipid metabolic reprogramming in carcinogenesis. Obesity and nonalcoholic steatohepatitis (NASH) are well-known risk factors of hepatocellular carcinoma (HCC), and individuals with these conditions exhibit an increased intake of dietary FAs accompanied by enhanced lipolysis of visceral adipose tissue due to insulin resistance, resulting in enormous exogenous FA supplies to hepatocytes via the portal vein and lymph vessels. This "lipid-rich condition" is highly characteristic of obesity- and NASH-driven HCC. Although the way in which HCC cells adapt to such a condition and exploit it to aid their progression is not understood, we recently obtained new insights into this mechanism through lipid metabolic reprogramming. In addition, accumulating evidence supports the importance of lipid metabolic reprogramming in various situations of hepatocarcinogenesis. Thus, in this review, we discuss the latest findings regarding the role of FA metabolism pathways in hepatocarcinogenesis, focusing on obesity- and NASH-driven lipid metabolic reprogramming.

Adiponectin activates the AMPK signaling pathway to regulate lipid metabolism in bovine hepatocytes.

Adiponectin (Ad) plays a crucial role in hepatic lipid metabolism. However, the regulating mechanism of hepatic lipid metabolism by Ad in dairy cows is unclear. Hepatocytes from a newborn female calf were cultured in vitro and treated with different concentrations of Ad and BML-275 (an AMPKα inhibitor). The results showed that Ad significantly increased the expression of two Ad receptors. Furthermore, the phosphorylation and activity of AMPKα, as well as the expression levels and transcriptional activity of peroxisome proliferator activated receptor-α (PPARα) and its target genes involved in lipid oxidation, showed a corresponding trend of upregulation. However, the expression levels and transcriptional activity of sterol regulatory element binding protein 1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP) decreased in a similar manner. When BML-275 was added, the p-AMPKα level as well as the expression and activity of PPARα and its target genes were significantly decreased. However, the expression levels of SREBP-1c, ChREBP and their target genes showed a trend of upregulation. Furthermore, the triglyceride (TG) content was significantly decreased in the Ad-treated groups. These results indicate that Ad activates the AMPK signaling pathway and mediates lipid metabolism in bovine hepatocytes cultured in vitro by promoting lipid oxidation, suppressing lipid synthesis and reducing hepatic lipid accumulation.

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient.

Fatty acid ß-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal ß-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid ß-oxidation deficiency or overload.

Differences between acylcarnitine profiles in plasma and bloodspots.

UNLABELLED: Quantification of acylcarnitines is used for screening and diagnosis of inborn error of metabolism (IEM). While newborn screening is performed in dried blood spots (DBSs), general metabolic investigation is often performed in plasma. Information on the correlation between plasma and DBS acylcarnitine profiles is scarce. In this study, we directly compared acylcarnitine concentrations measured in DBS with those in the corresponding plasma sample. Additionally, we tested whether ratios of acylcarnitines in both matrices are helpful for diagnostic purpose when primary markers fail. STUDY DESIGN: DBS and plasma were obtained from controls and patients with a known IEM. (Acyl)carnitines were converted to their corresponding butyl esters and analyzed using HPLC/MS/MS. RESULTS: Free carnitine concentrations were 36% higher in plasma compared to DBS. In contrast, in patients with carnitine palmitoyltransferase 1 (CPT-1) deficiency free carnitine concentration in DBS was 4 times the concentration measured in plasma. In carnitine palmitoyltransferase 2 (CPT-2) deficiency, primary diagnostic markers were abnormal in plasma but could also be normal in DBS. The calculated ratios for CPT-1 (C0/(C16+C18)) and CPT-2 ((C16+C18:1)/C2) revealed abnormal values in plasma. However, normal ratios were found in DBS of two (out of five) samples obtained from patients diagnosed with CPT-2. CONCLUSIONS: Relying on primary acylcarnitine markers, CPT-1 deficiency can be missed when analysis is performed in plasma, whereas CPT-2 deficiency can be missed when analysis is performed in DBS. Ratios of the primary markers to other acylcarnitines restore diagnostic recognition completely for CPT-1 and CPT-2 in plasma, while CPT-2 can still be missed in DBS.