The role of CPT1A as a biomarker of breast cancer progression: a bioinformatic approach.

Breast cancer is the commonest malignancy of women and with its incidence on the rise, the need to identify new targets for treatment is imperative. There is a growing interest in the role of lipid metabolism in cancer. Carnitine palmitoyl-transferase-1 (CPT-1); the rate limiting step in fatty acid oxidation, has been shown to be overexpressed in a range of tumours. There are three isoforms of CPT-1; A, B and C. It is CPT-1A that has been shown to be the predominant isoform which is overexpressed in breast cancer. We performed a bioinformatic analysis using readily available online platforms to establish the prognostic and predictive effects related to CPT-1A expression. These include the KM plotter, the Human Protein Atlas, the cBioPortal, the G2O, the MethSurvand the ROC plotter. A Network analysis was performed using the Oncomine platform and signalling pathways constituting the cancer hallmarks, including immune regulation as utilised by NanoString. The epigenetic pathways were obtained from the EpiFactor website. Spearman correlations (r) to determine the relationship between CPT-1A and the immune response were obtained using the TISIDB portal. Overexpression of CPT-1A largely confers a worse prognosis and CPT-1A progressively recruits a range of pathways as breast cancer progresses. CPT-1A's interactions with cancer pathways is far wider than previously realised and includes associations with epigenetic regulation and immune evasion pathways, as well as wild-type moderate to high penetrant genes involved in hereditary breast cancer. Although CPT-1A genomic alterations are detected in 9% of breast carcinomas, both the alteration and the metagene associated with it, confers a poor prognosis. CPT-1A expression can be utilised as a biomarker of disease progression and as a potential therapeutic target.

Measuring CPT-1-mediated respiration in permeabilized cells and isolated mitochondria.

Carnitine palmitoyltransferase-1 (CPT-1) is a rate-controlling enzyme for long-chain fatty acid oxidation. This manuscript provides protocols for measuring CPT-1-mediated respiration in permeabilized, adherent cell monolayers and mitochondria freshly isolated from tissue, along with examples to assess the potency and specificity of interventions targeting CPT-1. Strengths of the approach include ease, speed, and breadth of analysis, whereas drawbacks include loss of physiological regulation in reductionist systems and indirect assessment of CPT-1 enzymatic activity. For complete details on the use and execution of this protocol, please refer to Divakaruni et al. (2018).

Mass spectrometry imaging-based metabolomics to visualize the spatially resolved reprogramming of carnitine metabolism in breast cancer.

New insights into tumor-associated metabolic reprogramming have provided novel vulnerabilities that can be targeted for cancer therapy. Here, we propose a mass spectrometry imaging (MSI)-based metabolomic strategy to visualize the spatially resolved reprogramming of carnitine metabolism in heterogeneous breast cancer. Methods: A wide carnitine coverage MSI method was developed to investigate the spatial alternations of carnitines in cancer tissues of xenograft mouse models and human samples. Spatial expression of key metabolic enzymes that are closely associated with the altered carnitines was examined in adjacent cancer tissue sections. Results: A total of 17 carnitines, including L-carnitine, 6 short-chain acylcarnitines, 3 middle-chain acylcarnitines, and 7 long-chain acylcarnitines were imaged. L-carnitine and short-chain acylcarnitines are significantly reprogrammed in breast cancer. A classification model based on the carnitine profiles of 170 cancer samples and 128 normal samples enables an accurate identification of breast cancer. CPT 1A, CPT 2, and CRAT, which are extensively involved in carnitine system-mediated fatty acid ß-oxidation pathway were also found to be abnormally expressed in breast cancer. Remarkably, the expressions of CPT 2 and CRAT were found for the first time to be altered in breast cancer. Conclusion: These data not only expand our understanding of the complex tumor metabolic reprogramming, but also provide the first evidence that carnitine metabolism is reprogrammed at both the metabolite and enzyme levels in breast cancer.

Actions of annatto-extracted tocotrienol supplementation on obese postmenopausal women: study protocol for a double-blinded, placebo-controlled, randomised trial.

INTRODUCTION: Obesity is a major health concern in postmenopausal women, and chronic low-grade inflammation contributes to the development of obesity. Cellular studies and high-fat-diet-induced obese mouse model mimicking obesity show the antiobesity effect of annatto-extracted tocotrienols (TT) with antioxidant capability. We aim to assess the safety and efficacy of TT consumption for lipid-related parameters in obese postmenopausal women. METHODS AND ANALYSIS: Eligible obese postmenopausal women will be randomly assigned to placebo group (430 mg olive oil) and TT group (DeltaGold Tocotrienol 70%) for 24 weeks. In the present study, the primary outcome is total/regional fat mass and visceral adipose tissue. The secondary outcomes include lipid profile in serum, mRNA expression of fatty acid synthase and carnitine palmitoyltransferase 1A in fat tissue, oxylipins and endocannabinoids in plasma and adipose tissue, abundance and composition of intestinal microbiome in faeces, high-sensitivity C-reactive protein (hs-CRP) in serum and leptin in serum. Every participant will be evaluated at 0 (prior to starting intervention) and 24 weeks of intervention, except for serum lipid profile and hs-CRP at 0, 12 and 24 weeks. 'Intent-to-treat' principle is employed for data analysis. Hierarchical linear modelling is used to estimate the effects of dietary TT supplementation while properly accounting for dependency of data and identified covariates. To our knowledge, this is the first randomised, placebo-controlled, double-blinded study to determine dietary TT supplementation on an obese population. If successful, this study will guide the future efficacy TT interventions and TT can be implemented as an alternative for obese population in antiobesity management. ETHICS AND DISSEMINATION: This study has been approved by the Bioethics Committee of the Texas Tech University Health Sciences Center, Lubbock. An informed consent form will be signed by a participant before enrolling in the study. The results from this trial will be actively disseminated through academic conference presentation and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03705845.

Molecular characterization and tissue distribution of carnitine palmitoyltransferases in Chinese mitten crab Eriocheir sinensis and the effect of dietary fish oil replacement on their expression in the hepatopancreas.

The carnitine palmitoyltransferase (CPT) family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for ß-oxidation. In this study, three isoforms (CPT 1α, CPT 1ß and CPT 2) of the CPT family were cloned from Chinese mitten crab (Eriocheir sinensis) and their complete coding sequences (CDS) were obtained. Sequence analysis revealed deduced amino acid sequences of 915, 775 and 683 amino acids, respectively. Gene expression analysis revealed a broad tissue distribution for all three isoforms, with high CPT 1α and CPT 2 mRNA levels in the hepatopancreas of males and females. In males, CPT 1ß was highly expressed in gill, heart, brain ganglia and muscle, while in females, CPT 1ß-mRNA levels were relatively high in muscle, hepatopancreas and ovary tissue. The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0, 25, 50, 75 and 100% fish oil by 1:1 rapeseed oil: soybean oil. The results showed that Diets 2# and 5# yielded higher CPT 1α and CPT 2 mRNA expression in males (P < 0.05), while in females, expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement. The observed changes in CPT gene expression varied in different isoforms and gender, suggesting the three CPT genes might play different roles in fatty acid ß-oxidation in E. sinensis.

Dietary soya protein improves intra-myocardial lipid deposition and altered glucose metabolism in a hypertensive, dyslipidaemic, insulin-resistant rat model.

This study investigates the effects of replacing dietary casein by soya protein on the underlying mechanisms involved in the impaired metabolic fate of glucose and lipid metabolisms in the heart of dyslipidaemic rats chronically fed (8 months) a sucrose-rich (62·5 %) diet (SRD). To test this hypothesis, Wistar rats were fed an SRD for 4 months. From months 4 to 8, half the animals continued with the SRD and the other half were fed an SRD in which casein was substituted by soya. The control group received a diet with maize starch as the carbohydrate source. Compared with the SRD-fed group, the following results were obtained. First, soya protein significantly (P<0·001) reduced the plasma NEFA levels and normalised dyslipidaemia and glucose homoeostasis, improving insulin resistance. The protein levels of fatty acid translocase at basal state and under insulin stimulation and the protein levels and activity of muscle-type carnitine palmitoyltransferase 1 were normalised. Second, a significant (P<0·001) reduction of TAG, long-chain acyl CoA and diacylglycerol levels was observed in the heart muscle. Third, soya protein significantly increased (P<0·01) GLUT4 protein level under insulin stimulation and normalised glucose phosphorylation and oxidation. A reduction of phosphorylated AMP protein kinase protein level was recorded without changes in uncoupling protein 2 and PPARα. Fourth, hydroxyproline concentration decreased in the left ventricle and hypertension was normalised. The new information provided shows the beneficial effects of soya protein upon the altered pathways of glucose and lipid metabolism in the heart muscle of this rat model.

Newborn screening for carnitine palmitoyltransferase II deficiency using (C16+C18:1)/C2: Evaluation of additional indices for adequate sensitivity and lower false-positivity.

BACKGROUND: Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder (FAOD). However, newborn screening (NBS) for this potentially fatal disease has not been established partly because reliable indices are not available. METHODS: We diagnosed CPT II deficiency in a 7-month-old boy presenting with hypoglycemic encephalopathy, which apparently had been missed in the NBS using C16 and C18:1 concentrations as indices. By referring to his acylcarnitine profile from the NBS, we adopted the (C16+C18:1)/C2 ratio (cutoff 0.62) and C16 concentration (cutoff 3.0nmol/mL) as alternative indices for CPT II deficiency such that an analysis of a dried blood specimen collected at postnatal day five retroactively yielded the correct diagnosis. Thereafter, positive cases were assessed by measuring (1) the fatty acid oxidation ability of intact lymphocytes and/or (2) CPT II activity in the lysates of lymphocytes. The diagnoses were then further confirmed by genetic analysis. RESULTS: The disease was diagnosed in seven of 21 newborns suspected of having CPT II deficiency based on NBS. We also analyzed the false-negative patient and five symptomatic patients for comparison. Values for the NBS indices of the false-negative, symptomatic patient were lower than those of the seven affected newborns. Although it was difficult to differentiate the false-negative patient from heterozygous carriers and false-positive subjects, the fatty acid oxidation ability of the lymphocytes and CPT II activity clearly confirmed the diagnosis. Among several other indices proposed previously, C14/C3 completely differentiated the seven NBS-positive patients and the false-negative patient from the heterozygous carriers and the false-positive subjects. Genetic analysis revealed 16 kinds of variant alleles. The most prevalent, detected in ten alleles in nine patients from eight families, was c.1148T>A (p.F383Y), a finding in line with those of several previous reports on Japanese patients. CONCLUSIONS: These findings suggested that CPT II deficiency can be screened by using (C16+C18:1)/C2 and C16 as indices. An appropriate cutoff level is required to achieve adequate sensitivity albeit at the cost of a considerable increase in the false-positive rate, which might be reduced by using additional indices such as C14/C3.

Muscle Carnitine Palmitoyltransferase II Deficiency: A Review of Enzymatic Controversy and Clinical Features.

CPT (carnitine palmitoyltransferase) II muscle deficiency is the most common form of muscle fatty acid metabolism disorders. In contrast to carnitine deficiency, it is clinically characterized by attacks of myalgia and rhabdomyolysis without persistent muscle weakness and lipid accumulation in muscle fibers. The biochemical consequences of the disease-causing mutations are still discussed controversially. CPT activity in muscles of patients with CPT II deficiency ranged from not detectable to reduced to normal. Based on the observation that in patients, total CPT is completely inhibited by malony-CoA, a deficiency of malonyl-CoA-insensitive CPT II has been suggested. In contrast, it has also been shown that in muscle CPT II deficiency, CPT II protein is present in normal concentrations with normal enzymatic activity. However, CPT II in patients is abnormally sensitive to inhibition by malonyl-CoA, Triton X-100 and fatty acid metabolites. A recent study on human recombinant CPT II enzymes (His6-N-hCPT2 and His6-N-hCPT2/S113L) revealed that the wild-type and the S113L variants showed the same enzymatic activity. However, the mutated enzyme showed an abnormal thermal destabilization at 40 and 45 °C and an abnormal sensitivity to inhibition by malony-CoA. The thermolability of the mutant enzyme might explain why symptoms in muscle CPT II deficiency mainly occur during prolonged exercise, infections and exposure to cold. In addition, the abnormally regulated enzyme might be mostly inhibited when the fatty acid metabolism is stressed.

Lipid metabolism and inflammation modulated by Vitamin D in liver of diabetic rats.

BACKGROUND: In recent years, much evidence suggested that vitamin D plays an important role in decreasing the risk of type 2 diabetes. The purpose of this study was to investigate whether 1, 25 (OH) 2D3 can modulate inflammation and lipid metabolism in type 2 diabetic rat liver. METHODS: Type 2 diabetes was induced in SD rat with high-fat and high-sugar diets and multiple low-dose streptozotocin. The levels of serum calcium, phosphorus, glucose, TC, TG, AST, ALT and hepatic TG were determined. H & E staining were performed to assess the effects of vitamin D treatment on pathological changes in the liver tissues. Immunohistology, real-time PCR and Western blot were used to evaluate the expressions of NF-κ B, MCP-1, ICAM-1, TGF-ß1, PPAR-α and CPT-1. RESULTS: The administration of 1, 25 (OH) 2D3 reduced liver weight. Compared to DM rats, 1, 25 (OH) 2D3-treated DM rats had lower liver weight. Moreover, compared to healthy or 1, 25 (OH) 2D3-treated DM rats, DM rats had increased hepatic transcription factors (NF-κ B), monocyte chemoattractant protein -1 (MCP-1), intercellular adhesion molecule -1 (ICAM-1), transforming growth factor-ß1 (TGF-ß1) expressions, but had fewer hepatic PPAR- α and CPT-1 expressions. CONCLUSIONS: 1, 25 (OH) 2D3 significantly modulated the liver inflammation and lipid metabolism in diabetic rat models, which may be caused by its regulations on hepatic signaling NF-κ B pathway and PPAR- α.

Apolipoprotein E isoforms disrupt long-chain fatty acid distribution in the plasma, the liver and the adipose tissue of mice.

Evidences suggest that omega-3 fatty acid (n-3 PUFA) metabolism is imbalanced in apolipoprotein E epsilon 4 isoform carriers (APOE4). This study aimed to investigate APOE genotype-dependant modulation of FA profiles, protein and enzyme important to fatty acid (FA) metabolism in the adipose tissue, the liver and the plasma using human APOE-targeted replacement mouse-model (N=37). FA transport (FATP) and binding (FABP) protein levels in tissues and concentrations of liver carnitine palmitoyltransferase 1 (CPT1) were performed. N-3 PUFA concentration was >45% lower in the adipose tissue and liver of APOE4 mice compared to APOE3 mice. In APOE4 mice, there were higher levels of FATP and FABP in the liver and higher FATP in the adipose tissue compared to APOE2 mice. There was a trend towards higher CPT1 concentrations in APOE4 mice compared to APOE3 mice. Therefore, since APOE-isoform differences were not always in line with the unbalanced n-3 PUFA profiles in organs, other proteins may be involved in maintaining n-3 PUFA homeostasis in mice with different APOE-isoforms.

Elevated expression of fatty acid synthase and nuclear localization of carnitine palmitoyltransferase 1C are common among human gliomas.

Fatty acid synthase (FASN) and carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform of the CPT1 family, are upregulated in certain types of cancers, including gliomas. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis, and its phosphorylated form inhibits lipid synthesis. We examined the expression and subcellular localization of these fatty acid metabolism-related molecules in human gliomas. We performed immunostaining of two glioma cell lines (U373MG and U87MG) and 41 surgical specimens of diffuse gliomas with various histological grades (21 with the isocitrate dehydrogenase 1(IDH1) R132H mutation and 20 without the mutation). In the cultured glioma cells, CPT1C and phosphorylated ACC (p-ACC) were mainly localized to the nuclei, whereas FASN localized to the cytoplasm. In the surgical specimens, most glioma tissues showed nuclear staining for CPT1C and p-ACC, and cytoplasmic staining for FASN, regardless of the genetic status of IDH1 and the histological grade. Therefore, elevated cytoplasmic expression of FASN and nuclear localization of CPT1C are common among human diffuse gliomas, which may be regulated by the differential phosphorylation status of ACC in the cellular compartment.

Short communication: high insulin concentrations inhibit fatty acid oxidation-related gene expression in calf hepatocytes cultured in vitro.

In dairy cows, ketosis is an important disease associated with negative energy balance, which leads to low blood glucose levels and high blood nonesterified fatty acid levels. The liver is the most active organ in cows for the metabolism of nonesterified fatty acids. Insulin is an anabolic hormone that plays numerous roles in the metabolism of carbohydrates, lipids, and proteins, as well as being a potent regulator of fatty acid oxidation. In this study, using fluorescent quantitative reverse-transcription PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of insulin (0, 5, 10, 20, 50, and 100 nmol/L) on fatty acid oxidation by monitoring mRNA and protein expression levels of key enzymes: long-chain acyl-coenzyme A synthetase, carnitine palmitoyltransferase I, and long-chain acyl-coenzyme A dehydrogenase. The results showed that the mRNA and protein expression of long-chain acyl-coenzyme A synthetase, carnitine palmitoyltransferase I, and long-chain acyl-coenzyme A dehydrogenase was markedly decreased when the concentration of insulin in the media was increased. These findings indicate that high levels of insulin significantly inhibit the expression of genes related to fatty acid oxidation and consequently results in a decreased level of fatty acid oxidation in calf hepatocytes.

Ellagic acid attenuates high-carbohydrate, high-fat diet-induced metabolic syndrome in rats.

BACKGROUND: Fruits and nuts may prevent or reverse common human health conditions such as obesity, diabetes and hypertension; together, these conditions are referred to as metabolic syndrome, an increasing problem. This study has investigated the responses to ellagic acid, present in many fruits and nuts, in a diet-induced rat model of metabolic syndrome. METHODS: Eight- to nine-week-old male Wistar rats were divided into four groups for 16-week feeding with cornstarch diet (C), cornstarch diet supplemented with ellagic acid (CE), high-carbohydrate, high-fat diet (H) and high-carbohydrate, high-fat diet supplemented with ellagic acid (HE). CE and HE rats were given 0.8 g/kg ellagic acid in food from week 8 to 16 only. At the end of 16 weeks, cardiovascular, hepatic and metabolic parameters along with protein levels of Nrf2, NF-κB and CPT1 in the heart and the liver were characterised. RESULTS: High-carbohydrate, high-fat diet-fed rats developed cardiovascular remodelling, impaired ventricular function, impaired glucose tolerance, non-alcoholic fatty liver disease with increased protein levels of NF-κB and decreased protein levels of Nrf2 and CPT1 in the heart and the liver. Ellagic acid attenuated these diet-induced symptoms of metabolic syndrome with normalisation of protein levels of Nrf2, NF-κB and CPT1. CONCLUSIONS: Ellagic acid derived from nuts and fruits such as raspberries and pomegranates may provide a useful dietary supplement to decrease the characteristic changes in metabolism and in cardiac and hepatic structure and function induced by a high-carbohydrate, high-fat diet by suppressing oxidative stress and inflammation.

RETRACTED: Reversion of hepatic steatosis by exercise training in obese mice: the role of sterol regulatory element-binding protein-1c.

AIM: The dysregulation of regulatory element-binding protein-1c (SREBP-1c) is associated with hepatic steatosis. However, effects of exercise on SREBP-1c protein level in liver have not been investigated. Thus, in this study we investigated if reversion of the hepatic steatosis-induced by exercise training is related with levels of SREBP-1c. MAIN METHODS: Mice were divided into two groups: control lean mice (CT), fed on standard rodent chow, and obese mice (HF), fed on a high-fat diet for 2months. After this period obese mice were divided in two groups: obese mice and obese mice submitted to exercise (HF+EXE). The HF+EXE group performed a running program of 50min per day, 5days per week, for 8weeks. Forty-eight hours after the last exercise session, biochemical, immunoblotting, histology and immunohistochemistry analyses were performed. KEY FINDINGS: Livers of HF mice showed increased SREBP-1c, FAS (Fatty Acid Synthase), SCD1 (Stearoyl-CoA Desaturase1) and CPT1 (Carnitine Palmitoyl Transferase1) protein levels (3.4, 5.0, 2.6 and 2.9 times, respectively), though ACC (Acetyl-CoA Carboxilase) phosphorylation dropped 4.2 times. In livers of HF+EXE, levels of SREBP-1c, FAS, SCDI and CPTI decreased 2.1, 1.9, 1.8, and 2.7 times, respectively), while ACC phosphorylation increased 3.0 times. Lower SREBP-1c protein levels after exercise were confirmed also by immunohistochemistry. Total liver lipids content was higher in HF (2.2 times) when compared to CT, and exercise training reduced it significantly (1.7 times). SIGNIFICANCE: Our study allows concluding that the reduction in SREBP-1c protein levels is associated with steatosis reversion induced by exercise training.

Alterations of fatty acid ß-oxidation capability in the liver of ketotic cows.

Dairy cows are highly susceptible to ketosis after parturition. In the present study, we evaluated the expression of fatty acid ß-oxidation-related enzymes in the liver of ketotic (n=6) and nonketotic (n=6) cows. Serum levels of nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), and glucose were determined by using standard biochemical techniques. The mRNA abundance and protein content of acyl-CoA synthetase long-chain (ACSL), carnitine palmitoyltransferase I (CPT I), carnitine palmitoyltransferase II (CPT II), acyl-CoA dehydrogenase long chain (ACADL), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), and acetyl-CoA carboxylase (ACC) were evaluated by real-time PCR and ELISA. We found that serum glucose levels were lower in ketotic cows than in nonketotic cows, but serum BHBA and NEFA concentrations were higher. Messenger RNA and protein levels of ACSL were significantly higher in livers of ketotic cows than those in nonketotic cows. In contrast, mRNA levels of CPT I and mRNA and protein levels of CPT II, ACADL, HMGCS, and ACC were decreased in the liver of ketotic cows. Serum NEFA concentration positively correlated with ACSL protein levels and negatively correlated with protein levels of CPT II, HMGCS, ACADL, and ACC. In addition, serum BHBA concentration negatively correlated with protein levels of CPT II, HMGCS, and ACADL. Overall, fatty acid ß-oxidation capability was altered in the liver of ketotic compared with nonketotic cows. Furthermore, high serum NEFA and BHBA concentrations play key roles in affecting pathways of fatty acid metabolism in the liver.

Hepatitis C virus protein and iron overload induce hepatic steatosis through the unfolded protein response in mice.

BACKGROUND/AIM: Hepatic iron overload and steatosis play critical roles in the progression of hepatitis C virus (HCV)-associated chronic liver disease. However, how these two pathophysiological features affect each other remains unknown. The aim of this study was to investigate how hepatic iron overload contributes to the development of hepatic steatosis in the presence of HCV proteins. METHODS: Male C57BL/6 transgenic mice expressing the HCV polyprotein and nontransgenic littermates were fed an excess-iron diet or a control diet. Mice in each group were assessed for the molecules responsible for fat accumulation in the liver. RESULTS: Hepatic iron levels were positively correlated with triglyceride concentrations in the liver for all mice. As compared with the livers of nontransgenic mice fed the control diet, the livers of transgenic mice fed the excess-iron diet showed a lower expression of carnitine palmitoyl transferase I, a higher expression of sterol-regulatory element-binding protein 1 and fatty acid synthetase and an activated unfolded protein response indicated by a higher expression of unspliced and spliced X-box DNA-binding protein 1 (XBP-1), phosphorylated eukaryotic initiation factor-2alpha (p-eIF2alpha), CCAAT/enhancer-binding protein homology protein (CHOP) and abundant autophagosomes concomitant with increased production of reactive oxygen species. Six-month treatment with the anti-oxidant N-acetyl cysteine dramatically reduced hepatic steatosis in transgenic mice fed the excess-iron diet through decreased expression of unspliced and spliced XBP-1, p-eIF2alpha, and CHOP. CONCLUSIONS: The iron-induced unfolded protein response appears to be one of the mechanisms responsible for fat accumulation in the liver in transgenic mice expressing the HCV polyprotein.

Post-translational modifications of mitochondrial outer membrane proteins.

In recent years, a wide variety of proteomic approaches using gel electrophoresis and mass spectrometry has been developed to detect post-translational modifications. Mitochondria are often a focus of these studies due to their important role in cellular function. Many of their crucial transport and oxidative-phosphorylation functions are performed by proteins residing in the inner and outer membranes of the mitochondria. Although proteomic technologies have greatly enhanced our understanding of regulation in cellular processes, analysis of membrane proteins has lagged behind that of soluble proteins. Herein, we present techniques to facilitate the detection of post-translational modifications of mitochondrial membrane proteins including the isolation of resident membranes as well as electrophoretic and immunological-based methods for identification of post-translational modifications.

Increased carnitine palmitoyl transferase 1 expression and decreased sterol regulatory element-binding protein 1c expression are associated with reduced intramuscular triglyceride accumulation after insulin therapy in high-fat-diet and streptozotocin-induced diabetic rats.

We previously reported that early insulin treatment reduced intramuscular triglyceride content in type 2 diabetes mellitus Sprague-Dawley rats; the underlying mechanisms are, however, not completely understood. Here we investigated the regulation of insulin on molecular expressions involved in lipid metabolism pathways in skeletal muscle of high-fat-diet and streptozotocin-induced diabetic Sprague-Dawley rats. Neutral protamine Hagedorn insulin and gliclazide were initiated at the third day after streptozotocin injection and lasted for 3 weeks. Compared with normal rats, untreated diabetic rats had a 30% and 61% increase in lipoprotein lipase protein expression and activity, which were decreased by insulin and gliclazide (P < .05). Fatty acid translocase protein was down-regulated by 45% in untreated diabetic rats, which was up-regulated by 31% and 26% with insulin and gliclazide, respectively (P < .05). Insulin failed to affect fatty acid transport protein 1 and fatty acid binding protein expressions. Carnitine palmitoyl transferase 1 had a 47% decrease in untreated diabetic rats, which was normalized by insulin (P < .05). Moreover, compared with normal rats, untreated diabetic rats had higher expressions of sterol regulatory element-binding protein 1c, tumor necrosis factor alpha, and Tyr(705) phosphorylation of signal transducer and activator of transcription 3 levels, which all were down-regulated after insulin treatment. These results suggested that early insulin reduced intramuscular triglyceride levels in diabetic rats potentially through amelioration of lipid dysfunction and inhibition of lipid synthesis.

Vitamin A deficiency modifies lipid metabolism in rat liver.

Liver fatty acid metabolism of male rats fed on a vitamin A-deficient diet for 3 months from 21 d of age was evaluated. Vitamin A restriction produced subclinical plasma and negligible liver retinol concentrations, compared with the control group receiving the same diet with 4000 IU vitamin A (8 mg retinol as retinyl palmitate)/kg diet. Vitamin A deficiency induced a hypolipidaemic effect by decreasing serum triacylglycerol, cholesterol and HDL-cholesterol levels. The decrease of liver total phospholipid was associated with low phosphatidylcholine synthesis observed by lower [14C]choline incorporation into phosphatidylcholine, compared with control. Also, liver fatty acid synthesis decreased, as was indicated by activity and mRNA expression of acetyl-CoA carboxylase (ACC), and incorporation of [14C]acetate into saponified lipids. A decrease of the PPARalpha mRNA expression was observed. Liver mitochondria of vitamin A-deficient rats showed a lower total phospholipid concentration coinciding with a decrease of the cardiolipin proportion, without changes in the other phospholipid fractions determined. The mitochondria fatty acid oxidation increased by 30 % of the control value and it was attributed to a high activity and mRNA expression of carnitine palmitoyltransferase-I (CPT-I). An increase in serum beta-hydroxybutyrate levels was observed in vitamin A-deficient rats. Vitamin A deficiency alters the mitochondria lipid composition and also enhances fatty acid oxidation by modifying the production of malonyl-CoA, the endogenous inhibitor of CPT-I, due to decreased activity of liver ACC. The incorporation of vitamin A into the diet of vitamin A-deficient rats reverted all the changes observed.

An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry.

Carnitine palmitoyl transferase I (CPTI), which converts acyl-CoA and carnitine into acyl-carnitine and free CoASH, is the rate limiting enzyme of hepatic mitochondrial beta-oxidation. CPTI-deficiency is a severe disorder characterized by Reye-like attacks with hypoketotic hypoglycemia, hepatomegaly, elevated liver enzymes and hyperammonemia. We developed a simple tandem-MS-based assay to measure CPTI activity in human fibroblasts. Surprisingly, a large part of the palmitoyl-carnitine formed in our assay by CPTI was degraded into C14- to C2-acyl-carnitines. Degradation of the product of CPTI leads to under estimation of the CPTI activity. When we used potassium cyanide to inhibit enzymes downstream of CPTI and thereby degradation of the product, we measured four times more CPTI activity than the previous methods. This inhibition is essential for correct calculation of CPTI activity. In fibroblasts of CPTI-deficient patients, CPTI activity was not detectable and this assay can be used for the diagnosis of CPTI-deficiency.