Accumulation Of Nonesterified Fatty Acids Research Articles

Combined Effect of Quercetin and Fish Oil on Oxidative Stress in the Liver of Mice Fed a Western-Style Diet.

To study the combined effect of the flavonoid quercetin and fish oil containing ω-3 fatty acids on preventing diet-induced metabolic syndrome, we fed mice with a control diet, a high-fat, high-sucrose, and high-cholesterol Western-style diet (Western diet), a Western diet supplemented with 0.05% quercetin, a Western diet containing 5% fish oil rich in docosahexaenoic acid (DHA) (DHA diet), or a DHA diet supplemented with 0.05% quercetin. After 18 weeks of feeding, fish oil potentiated the suppression of lipid peroxidation by quercetin in the liver but not in the epididymal adipose tissue. Fish oil but not quercetin suppressed the accumulation of non-esterified fatty acids and the expression of fatty acid synthase in the liver of Western-diet-fed mice. Thus, the combination of quercetin and DHA-rich fish oil may partly alleviate non-alcoholic fatty liver disease by reducing oxidative stress and suppressing fatty acid synthesis.

Puerarin ameliorated pressure overload-induced cardiac hypertrophy in ovariectomized rats through activation of the PPARα/PGC-1 pathway.

Estrogen deficiency induces cardiac dysfunction and increases the risk of cardiovascular disease in postmenopausal women and in those who underwent bilateral oophorectomy. Previous evidence suggests that puerarin, a phytoestrogen, exerts beneficial effects on cardiac function in patients with cardiac hypertrophy. In this study, we investigated whether puerarin couldprevent cardiac hypertrophy and remodeling in ovariectomized, aortic-banded rats. Female SD rats subjected to bilateral ovariectomy (OVX) plus abdominal aortic constriction (AAC). The rats were treated withpuerarin (50 mg·kg-1 ·d-1, ip) for 8 weeks. Then echocardiography was assessed, and the rats were sacrificed, their heart tissues were extracted and allocated for further experiments. We showed that puerarinadministration significantly attenuated cardiac hypertrophy and remodeling in AAC-treated OVX rats, which could be attributed to activation of PPARα/PPARγ coactivator-1 (PGC-1) pathway. Puerarinadministration significantly increased the expression of estrogen-related receptor α, nuclear respiratory factor 1, and mitochondrial transcription factor A in hearts.Moreover, puerarinadministrationregulated the expression of metabolic genes in AAC-treated OVX rats. Hypertrophic changes could be inducedin neonatal rat cardiomyocytes (NRCM) in vitro by treatment with angiotensin II (Ang II, 1 μM), which was attenuated by co-treatemnt withpuerarin (100 μM). We further showed that puerarin decreased Ang II-induced accumulation of non-esterified fatty acids (NEFAs) and deletion of ATP, attenuated the Ang II-induced dissipation of the mitochondrial membrane potential, and improved the mitochondrial dysfunction in NRCM. Furthermore, addition of PPARα antagonist GW6471 (10 μM) partially abolished the anti-hypertrophic effects and metabolic effects of puerarinin NRCM. In conclusion,puerarin prevents cardiac hypertrophy in AAC-treated OVX rats through activation of PPARα/PGC-1 pathway and regulation of energy metabolism remodeling. This may provide a new approach to prevent the development of heart failure in postmenopausal women.

PPARA/RXRA signalling regulates the fate of hepatic non-esterified fatty acids in a sheep model of maternal undernutrition

Maternal undernutrition during late gestation accelerates body fat mobilization to provide more energy for foetal growth and development, which unbalances metabolic homeostasis and results in serious lipid metabolism disorder. However, detailed regulatory mechanisms are poorly understood. Here, a sheep model was used to explore the regulatory role of PPARA/RXRA signalling in hepatic lipid metabolism in undernutrition based on RNA sequencing and cell experiments. KOG function classification showed that lipid transport and metabolism was markedly altered in an undernourished model. In detail, when compared with the controls, fatty acid transport and oxidation and triglyceride metabolism were up-regulated in an undernourished model, while fatty acid synthesis, steroid synthesis, and phospholipid metabolism were down-regulated. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis demonstrated that PPARA/RXRA signalling pathway was altered. Moreover, PPARA signalling associated genes were positively correlated with hepatic non-esterified fatty acid (NEFA) levels, while retinol metabolism associated genes were negatively correlated with blood beta-hydroxybutyric acid (BHBA) levels. Results of primary hepatocytes showed that NEFAs could activate PPARA signalling and facilitate fatty acid oxidation (FAO) and ketogenesis, while BHBA could inhibit RXRA signalling and repress FAO and ketogenesis. Excessively accumulated NEFAs in hepatocytes promoted triglyceride synthesis. Furthermore, activation of PPARA/RXRA signalling by WY14643 and 9-cis-retinoic acid could enhance FAO and ketogenesis and reduce NEFAs accumulation and esterification. Our findings elucidate the regulatory mechanisms of NEFAs and BHBA on lipid metabolism as well as the potential role of the PPARA/RXRA signalling pathway in hepatic lipid metabolism, which may contribute to exploring new strategies to maintain lipid metabolic homeostasis in human beings.

Impact of different ectopic fat depots on cardiovascular and metabolic diseases.

A growing body of evidence is pointing out the pathophysiological role of fat accumulation in different organs. Ectopic fat depots within heart, liver, skeletal muscle, kidney, and pancreas as well as around blood vessels might be more associated to cardiometabolic risk than classical variables, such as body mass index. Among different mechanisms, lipid metabolism appears to be particularly influenced by ectopic fat depots. Indeed, intracellular accumulation of nonesterified fatty acids, and triglycerides promotes endoplasmic reticulum stress, mitochondrial uncoupling, oxidative stress, and altered membrane composition/function, finally promoting inflammatory response and cell death. The dysfunctional adipose tissue was shown to induce both local and systemic effects, with relevant clinical consequences. Epicardial fat and myocardial steatosis have been associated with the development of atrial fibrillation and ventricular dysfunction. Similarly perivascular adipose tissue appears to trigger atherosclerosis and hypertension. Nonalcoholic fatty liver disease has been recognized both as the hepatic manifestation of metabolic syndrome and as a cardiovascular (CV) risk factor. Importantly, the renal sinus fat emerged as a potential player in kidney dysfunction. Finally, both skeletal muscle and pancreatic fat depots have been indicated as potential endocrine modulators of insulin resistance. Considering the global rise in the prevalence of obesity, the understanding of mechanisms underlying ectopic fat accumulation represents an urgent need, with potential clinical implications for CV risk stratification. Here, we attempt to update the current knowledge of the different ectopic fat depots, focusing on underlying mechanisms and potential clinical implications.

Function and Dysfunction of Adipose Tissue.

Adipose tissue is an endocrine organ which is responsible for postprandial uptake of glucose and fatty acids, consequently producing a broad range of adipokines controlling several physiological functions like appetite, insulin sensitivity and secretion, immunity, coagulation, and vascular tone, among others. Many aspects of adipose tissue pathophysiology in metabolic diseases have been described in the last years. Recent data suggest two main factors for adipose tissue dysfunction: accumulation of nonesterified fatty acids and their secondary products and hypoxia. Both of these factors are thought to be on the basis of low-grade inflammatory activation, further increasing metabolic dysregulation in adipose tissue. In turn, inflammation is involved in the inhibition of substrate uptake, alteration of the secretory profile, stimulation of angiogenesis, and recruitment of further inflammatory cells, which creates an inflammatory feedback in the tissue and is responsible for long-term establishment of insulin resistance.

Some pathophysiologic mechanisms of cardiovascular diseases in patients with chronic prostatitis

Aim. To study the relation of autonomic disorders severity, energy and lipid metabolism parameters, immune, hormonal disorders and structural and functional changes of the myocardium in patients with chronic prostatitis. Methods. The study included 75 patients with chronic prostatitis, who were divided into two groups: the first group consisted of 48 patients with the sympathetic autonomous nervous system hyper activation, the second group - of 27 patients with autonomic balance. Echocardiography, 24-hour ECG monitoring with heart rate variability analysis were performed in all patients. The overall serum level of non-esterified fatty acids and glycerol, red blood cells levels of macroergic substances, lipid and long-chain fatty acids fractions were measured. Hormonal profile included estimation of total testosterone level, sex hormone-binding globulin, calculated free testosterone index. Serum cytokines profile was also studied. Results. In patients with sympathetic activity and prostatitis, most of heart rate variability parameters were significantly lower compared to patients with autonomic balance and controls. Patients with left ventricle diastolic dysfunction had higher serum concentrations of pro-and anti-inflammatory cytokines. Dysautonomia with prolonged sympathetic autonomous nervous system hyper activation was frequently found in patient with chronic prostatitis (64% of cases), contributing to low fatty acids utilization in myocardium and reduced levels of glycerol (by 32.3%) and adenosine triphosphate (by 49.5%) in erythrocytes. These disorders were accompanied by left ventricular diastolic dysfunction in 19.2% of patients and by arrhythmias in over than a half of patients. Conclusion. Autonomic disorders (sympathetic autonomous nervous system hyper activation associated with decreased parasympathetic control of the cardiovascular system) are quite common in patients with chronic prostatitis, leading to fatty acids utilization impairment, resulting in blood accumulation of non-esterified fatty acids, low serum glycerol levels, adenine nucleotide imbalance) accompanied by low unbound testosterone level, serum cytokines profile imbalance, reduction of cardiac hemodynamic indicators and development of cardiac arrhythmias.

Insulin Resistance and Progression of Heart Failure in the Older Adult

Heart failure is a disease of advancing age. Despite the advent of effective therapies, the overall outcome among the elderly has not been altered significantly. Aging is also associated with whole body and myocardial insulin resistance. Insulin resistance is accompanied by increased sympathetic nervous system activation and increased nonesterified fatty acid (NEFA) uptake in excess of NEFA oxidation, leading to NEFA accumulation within cardiac myocytes and mitochondria. Myocardial mitochondria demonstrate increased reactive oxygen species and calcium sequestration and decreased protein expression leading to impaired energetic reserves. Although these alterations in myocardial mitochondrial function lead to an accelerated course of heart failure, these changes can be modified and protect against exacerbation of heart failure. In particular, glucagon-like peptide-1, a naturally occurring incretin, has been shown to improve left ventricular ejection fraction and functional status in patients. Further investigation must be pursued for this promising novel therapeutic target for heart failure.

The role of nonesterified fatty acids in pathogenesis of cardiovascular diseases

The paper reviews publications concerned the role of nonesterifi ed fatty acids (NEFA) in pathogenesis of cardiovascular diseases. NEFAs are four and more carbons chain length carbonic acids and they are presented in free form (nonesterifi ed) in human body. Plasma NEFAs are produced by the adipose tissue triglyceride lipolysis, another source are lipoproteins such as chylomicrons, very low density lipoproteins and intermediate density lipoproteins. Elevated NEFA concentrations in plasma are the risk factor of cardiovascular diseases and type 2 diabetes mellitus and the independent risk factor of hypertension and sudden death. NEFA plasma concentration is elevated in atherosclerosis, acute myocardial infarction, diabetes mellitus, obesity, hypertension, and often in metabolic syndrome. A probable cause of NEFAs accumulation in plasma may be overeating and low physical activity, which result in increase of adipose tissue mass, lipolysis intensifi cation and elevation of NEFAs concentration in plasma. The role of elevated plasma NEFA concentration in a number of conditions (abdominal obesity, atherogenic dyslipidemia, insulin resistance, type 2 diabetes mellitus, endothelial dysfunction, vascular infl ammation, atherosclerosis, hypertension, ischemic heart disease, rhythm disturbances, sudden death) and possible ways of their correction are discussed.

Regulation of the mitochondrial permeability transition in kidney proximal tubules and its alteration during hypoxia-reoxygenation.

Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg(2+). To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg(2+) greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg(2+). Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg(2+), or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

Antitumor Effects of JBD57 in Human Multiple Myeloma Cells in Vitro

JBD57 is a nucleoside/nucleotide analogue that in human cells causes depletion of mitochondrial DNA by disrupting oxidative phosphorylation pathways leading to toxic accumulation of nonesterified fatty acids, dicarboxylic acids and free radicals. Human 26S proteasome is also a target for JBD57. Here we evaluated JBD57 citotoxicity in several human tumor cell lines in vitro. Human MM cell line RPMI 8226/S (CCL-155), human T-cell lymphoblastic-like (Jurkat) and human T-cell leukemia (1301) were grown in RPMI 1640 medium; uterine sarcoma (MES-S (CRL-1976) cells were grown in McCoy medium; HUV-EC-C (CRL-1730) cells were grown in 199/EBSS medium. Media were supplemented with 10 % FBS. Cells were incubated at 37°C in a water-jacketed incubator with 5 % CO2. To evaluate JBD57 citotoxicity in RPMI 8226/S, MES-S, Jurkat, 1301 and HUV-EC-C cells, 104cells/well were grown in flat-bottomed 96-well tissue culture plates for 24, 48 and 72 hr; JBD57 was added to the media in several concentrations (0μM, 32.25μM, 62.5μM, 125μM, 250μM and 500μM). At the end of the experimental periods, cell viability was determined by the MTT method. JBD57 inhibited the growth of MM cell line RPMI 8226/S in a dose- and time-dependent manner. Cell viability decreased progressively with increasing concentrations of JBD57 as well as with increasing time periods. The IC50 (inhibitory concentration at 50%) was 125 μM at 72 hr. The viability of the MM cells after 72 hr incubation with JBD57 500μM was 33%, whereas 100% viability was observed when no drug was added. On the other hand, JBD57 did not affect cell viability of any of the other studied cell lines (uterine sarcoma, Jurkat, 1301 and HUVEC-C). JBD57 promotes a significant human MM cell death in a dose and time dependent manner but do not affect neither normal cell HUV-EC-C nor the tumoral cells MES-S, Jurkat and 1301, at least in the studied conditions. These results suggest that the potent antitumoral activity of JBD57 observed against MM cells could be potentially useful in the treatment of multiple myeloma.

Evidence for involvement of nonesterified fatty acid-induced protonophoric uncoupling during mitochondrial dysfunction caused by hypoxia and reoxygenation

Proximal tubules subjected to hypoxia in vitro under conditions relevant to ischaemia in vivo develop an energetic deficit that is not corrected even after full reoxygenation. We have provided evidence that accumulation of nonesterified fatty acids (NEFA) is the primary reason for this energetic deficit. In this study, we have further investigated the mechanism for the NEFA-induced energetic deficit. Mitochondrial membrane potential (Deltapsi) was measured in digitonin-permeabilized, freshly isolated proximal tubules by safranin O uptake. Addition of the potassium/proton exchanger nigericin enables the determination of the mitochondrial proton motive force (Deltap) and the proton gradient (DeltapH). ATP was measured luminometrically and NEFA colorimetrically. Tubule ATP content was depleted after hypoxia and recovered incompletely, even after full reoxygenation. Mitochondrial safranin O uptake was decreased in proximal tubules after hypoxia and reoxygenation (H/R). This decrease was attenuated by delipidated bovine serum albumin (dBSA) or citrate. Addition of nigericin increased safranin O uptake of mitochondria in normoxic proximal tubules, but not in proximal tubules after H/R. Addition of dBSA restored the effect of nigericin to increase mitochondrial safranin O uptake. Addition of the NEFA oleate had the same impact on mitochondrial safranin O uptake as subjecting proximal tubules to H/R. The mechanism of the NEFA-induced energetic deficit in freshly isolated rat proximal tubules induced by H/R is characterized by impaired ATP production after full reoxygenation, impaired recovery of Deltapsi and Deltap, abrogation of DeltapH and sensitivity to citrate, consistent with involvement of the tricarboxylate carrier. The data support the concept that protonophoric uncoupling by NEFA movement on anion carriers plays a critical role in proximal tubule mitochochondrial dysfunction after H/R.

Metabolomics as an Extension of Proteomic Analysis: Study of Acute Kidney Injury

Although proteomics studies the global expression of proteins, metabolomics characterizes and quantifies their end products: the metabolites, produced by an organism under a certain set of conditions. From this perspective it is apparent that proteomics and metabolomics are complementary and when joined allow a fuller appreciation of an organism's phenotype. Our studies using (1)H-nuclear magnetic resonance spectroscopic analysis showed the presence of glucose, amino acids, and trichloroacetic acid cycle metabolites in the urine after 48 hours of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also showed the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, despite increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of fibrates. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity. These studies serve to illustrate that metabolomic studies add insight into pathophysiology not provided by proteomic analysis alone.

Maleate nephrotoxicity: mechanisms of injury and correlates with ischemic/hypoxic tubular cell death

Maleate injection causes dose-dependent injury in proximal tubular cells. This study sought to better define underlying pathogenic mechanisms and to test whether maleate toxicity recapitulates critical components of the hypoxic/ischemic renal injury cascade. CD-1 mice were injected with maleate or used as a source for proximal tubule segments (PTS) for in vitro studies. Maleate induced dose-dependent PTS injury [lactate deydrogenase (LDH) release, ATP reductions, nonesterified fatty acid (NEFA) accumulation]. These changes were partially dependent on maleate metabolism (protection conferred by metabolic inhibitors: succinate, acetoacetate). Maleate toxicity reproduced critical characteristics of the hypoxia/ATP depletion-induced injury cascade: 1) glutathione (GSH) conferred protection, but due to its glycine, not cysteine (antioxidant), content; 2) ATP reductions reflected decreased production, not Na-K-ATPase-driven increased consumption; 3) cell death was completely blocked by extracellular acidosis (pH 6.6); 4) intracellular Ca(2+) chelation (BAPTA) mitigated cell death; 5) maleate and hypoxia each caused plasma membrane cholesterol shedding and in both instances, this was completely glycine suppressible; 6) maleate + hypoxia caused neither additive NEFA accumulation nor LDH release, implying shared pathogenic pathways; and 7) maleate, like ischemia, induced renal cortical cholesterol loading; increased HMG CoA reductase (HMGCR) activity (statin inhibitable), increased HMGCR mRNA levels, and increased RNA polymerase II recruitment to the HMGCR locus (chromatin immunoprecipitation, ChIP, assay) were involved. These results further define critical determinants of maleate nephrotoxicity and suggest that it can serve as a useful adjunct for studies of ischemia/ATP depletion-induced, proximal tubule-specific, cell death.

Metabolomic study of cisplatin-induced nephrotoxicity

We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARalpha ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARalpha ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARalpha ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity.

Accumulation of nonesterified fatty acids causes the sustained energetic deficit in kidney proximal tubules after hypoxia-reoxygenation

Kidney proximal tubules exhibit decreased ATP and reduced, but not absent, mitochondrial membrane potential (Deltapsi(m)) during reoxygenation after severe hypoxia. This energetic deficit, which plays a pivotal role in overall cellular recovery, cannot be explained by loss of mitochondrial membrane integrity, decreased electron transport, or compromised F1F0-ATPase and adenine nucleotide translocase activities. Addition of oleate to permeabilized tubules produced concentration-dependent decreases of Deltapsi(m) measured by safranin O uptake (threshold for oleate = 0.25 microM, 1.6 nmol/mg protein; maximal effect = 4 microM, 26 nmol/mg) that were reversed by delipidated BSA (dBSA). Cell nonesterified fatty acid (NEFA) levels increased from <1 to 17.4 nmol/mg protein during 60- min hypoxia and remained elevated at 7.6 nmol/mg after 60 min reoxygenation, at which time ATP had recovered to only 10% of control values. Safranin O uptake in reoxygenated tubules, which was decreased 85% after 60-min hypoxia, was normalized by dBSA, which improved ATP synthesis as well. dBSA also almost completely normalized Deltapsi(m) when the duration of hypoxia was increased to 120 min. In intact tubules, the protective substrate combination of alpha-ketoglutarate + malate (alpha-KG/MAL) increased ATP three- to fourfold, limited NEFA accumulation during hypoxia by 50%, and lowered NEFA during reoxygenation. Notably, dBSA also improved ATP recovery when added to intact tubules during reoxygenation and was additive to the effect of alpha-KG/MAL. We conclude that NEFA overload is the primary cause of energetic failure of reoxygenated proximal tubules and lowering NEFA substantially contributes to the benefit from supplementation with alpha-KG/MAL.

Fatty-acid-binding proteins do not protect against induced cytotoxicity in a kidney cell model

Intracellular accumulation of fatty acids (FAs) is a well-described consequence of renal ischaemia and may lead to lethal cell injury. Fatty-acid-binding proteins (FABPs) are small cytosolic proteins with high affinity for FAs. They may protect vital cellular functions by binding to and promoting the metabolism of FAs, thereby reducing their intracellular concentration. In this study we investigated the putative cytoprotective role of FABPs in a Madin–Darby canine kidney (MDCK) cell model for renal damage. We studied the effects of transfection with cDNA encoding heart FABP, adipocyte FABP or liver FABP on cytotoxicity induced by chemical anoxia or FAs. Transfection of MDCK type II cells with these cDNA types caused a 5–20-fold increase in FABP content, but did not change the rate or extent of palmitate uptake. After 1h of incubation with KCN, all cell types showed reduced viability and cellular ATP content and an intracellular accumulation of non-esterified FAs. High extracellular concentrations of oleate, but not palmitate, caused a markedly decreased cell viability and cellular ATP content. Oleate accumulated in non-esterified form in these cells. Simultaneous addition of glucose ameliorated the damaging effects of KCN or oleate, indicating that glycolytic ATP could substitute for uncoupled oxidative phosphorylation. No significant differences in the effects of chemical anoxia or oleate were observed between non-transfected, mock-transfected and FABP-cDNA-transfected cells. Non-esterified FA accumulation was not reduced in any of the FABP-cDNA-transfected cell lines. In conclusion, our data do not provide evidence for a cytoprotective role of FABP in this kidney cell model.

Fatty-acid-binding proteins do not protect against induced cytotoxicity in a kidney cell model.

Intracellular accumulation of fatty acids (FAs) is a well-described consequence of renal ischaemia and may lead to lethal cell injury. Fatty-acid-binding proteins (FABPs) are small cytosolic proteins with high affinity for FAs. They may protect vital cellular functions by binding to and promoting the metabolism of FAs, thereby reducing their intracellular concentration. In this study we investigated the putative cytoprotective role of FABPs in a Madin-Darby canine kidney (MDCK) cell model for renal damage. We studied the effects of transfection with cDNA encoding heart FABP, adipocyte FABP or liver FABP on cytotoxicity induced by chemical anoxia or FAs. Transfection of MDCK type II cells with these cDNA types caused a 5-20-fold increase in FABP content, but did not change the rate or extent of palmitate uptake. After 1 h of incubation with KCN, all cell types showed reduced viability and cellular ATP content and an intracellular accumulation of non-esterified FAs. High extracellular concentrations of oleate, but not palmitate, caused a markedly decreased cell viability and cellular ATP content. Oleate accumulated in non-esterified form in these cells. Simultaneous addition of glucose ameliorated the damaging effects of KCN or oleate, indicating that glycolytic ATP could substitute for uncoupled oxidative phosphorylation. No significant differences in the effects of chemical anoxia or oleate were observed between non-transfected, mock-transfected and FABP-cDNA-transfected cells. Non-esterified FA accumulation was not reduced in any of the FABP-cDNA-transfected cell lines. In conclusion, our data do not provide evidence for a cytoprotective role of FABP in this kidney cell model.

An alternative function for human uncoupling protein 3: protection of mitochondria against accumulation of nonesterified fatty acids inside the mitochondrial matrix.

The physiological function of the human uncoupling protein 3 UCP3, which was discovered in 1997, is unknown. Here we evaluate the available data on human UCP3 expression and show that UCP3 is up-regulated in situations where fatty acid delivery to the mitochondria exceeds oxidative capacity, whereas down-regulation of UCP3 is observed when oxidative capacity is enhanced. With a surplus of fatty acid delivery, accumulation of nonesterified fatty acids in the cytoplasm is likely to occur. Although the inner mitochondrial membrane provides a barrier for nonesterified fatty acids, neutral nonesterified fatty acids can partition into the phospholipid bilayer and flip-flop to the other side of the membrane, where they can be released into the mitochondrial matrix. Due to pH differences, these nonesterified fatty acids will be protonated. Because fatty acid anions can neither be metabolized inside the matrix or cross the inner mitochondrial membrane, accumulation of nonesterified fatty acids inside the matrix might occur. Therefore, we postulate that UCP3 is required for the outward translocation of fatty acids from the mitochondrial matrix. In this way, UCP3 is involved in the protection of mitochondria against accumulation of nonesterified fatty acids inside the mitochondrial matrix.

Phospholipase A2 is not responsible for lysophosphatidylcholine-induced damage in cardiomyocytes.

Lysophosphatidylcholine (LPC) is known to increase the intracellular concentration of Ca2+ ([Ca2+]i), leading to cell damage. In the present study we examined whether LPC activates phospholipase A2 (PLA2) and whether the activation of PLA2 is responsible for the LPC-induced cell damage in isolated rat cardiomyocytes. LPC (15 microM) produced an increase in [Ca2+]i, a change in cell shape from rod to round, and the release of creatine kinase (CK) accompanied by a significant elevation of the cellular level of nonesterified fatty acids (NEFA), especially arachidonic acid. Three PLA2 inhibitors, 7, 7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA), 3-(4-octadecylbenzoyl)acrylic acid (OBAA), and manoalide, attenuated the LPC-induced accumulation of unsaturated NEFA to a similar degree. Nevertheless, whereas both DEDA and OBAA attenuated the LPC-induced increase in [Ca2+]i, change in cell shape, and release of CK, manoalide attenuated none of them. In the Ca2+-free solution, LPC did not increase [Ca2+]i with significantly less accumulation of NEFA, but it changed the cell shape from rod to round and increased the release of CK. These results suggest that exogenous LPC increases the PLA2 activity, which, however, may not be responsible for the LPC-induced damage in cardiomyocytes.

Spontaneous transfer of monoacyl amphiphiles between lipid and protein surfaces

The kinetics of transfer of natural and fluorescent nonesterified fatty acids (NEFA) and lysolecithins (lysoPC) from phospholipid and protein surfaces were measured. The kinetics of transfer of 12-(1-pyrenyl)dodecanoic acid, from liquid crystalline and gel phase single unilamellar phospholipid vesicles, very low, low, and high density lipoproteins, human serum albumin, and rat liver fatty acid-binding protein, were first-order and characterized by similar rate constants. The halftimes (t1/2) of NEFA transfer from lipids and proteins were dependent on the acyl chain structure according to log t1/2 = -0.62n + 0.59m + 12.0, where n and m, respectively, are the numbers of carbon atoms and double bonds. The structure of the donor surface had a measurable but smaller effect on transfer rates. The kinetics of NEFA and lysoPC transfer are slow relative to the lipolytic processes that liberate them. Therefore, one would predict a transient accumulation of NEFA and lysoPC during lipolysis and an attendant modulation of many metabolic processes within living cells and within the plasma compartment of blood. These data will be useful in the refinement of current models of membrane and lipoprotein function and in the selection of fluorescent NEFA analogs for studying transport in living cells.