Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8+ T cells.

CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.

Improved developmental competence in embryos treated with lycopene during in vitro culture system.

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Selenium significantly inhibits adipocyte hypertrophy and abdominal fat accumulation in OLETF rats via induction of fatty acid ß-oxidation.

A combination of selenium (Se) with other trace element is associated with partially modulate fatty acid distribution as well as reduction of the body weight and feed efficiency. To investigate whether or not Se treatment has an impact on lipid metabolism, we examined the levels of lipid metabolism-related factors, including abdominal fat, adiponectin, cholesterol, very long chain dehydrogenase (VLCAD), and medium chain acyl-CoA dehydrogenase (MCAD) in 20-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats following sodium selenite treatment for 2 weeks. Herein, we observed that (a) Se treatment induced insulin-like effects by lowering the serum glucose level in rats; (b) Se-treated rats showed significance values decreases in abdominal fat mass, adipocyte size, and adiponectin, which are associated with lipid metabolism; (c) Se treatment led to reduced levels of cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; (d) fat tissue in Se-treated rats displayed significantly lower expression of adipocyte marker genes along with increased expression of VLCAD and MCAD; and (e) fatty liver formation and ß-oxidation gene expression were both significantly reduced in liver tissue of Se-treated rats. Therefore, our results suggest that Se may induce inhibition of adipocyte hypertrophy and abdominal fat accumulation along with suppression of fatty liver formation by the differential regulation of the gene expression for fatty acid ß-oxidation in the OLETF model.

Long-term follow-up of bezafibrate treatment in patients with the myopathic form of carnitine palmitoyltransferase 2 deficiency.

Carnitine palmitoyltransferase 2 (CPT2) deficiency is a rare mitochondrial fatty acid oxidation (FAO) disorder characterized by myalgia, exercise intolerance, and rhabdomyolysis. We evaluate the efficacy of bezafibrate (BZ), a hypolipidemic drug, as a treatment for this form of CPT2 deficiency. A pilot trial was conducted with BZ in six patients for 6 months. There was a follow-up period of 3 years. The oxidation rates of the long-chain fatty acid derivative palmitoyl-CoA, measured in the mitochondria of the patients' muscles, were markedly lower than normal before treatment and increased significantly (+39 to +206%; P = 0.028) in all patients after BZ treatment. The evaluation of the therapeutic effects by the patients themselves (using the Short Form Health Survey (SF-36)), as well as by the physicians, indicated an improvement in the condition of the patients; there was an increase in physical activity and a decline in muscular pain. The results suggest that BZ has a therapeutic effect in the muscular form of CPT2 deficiency.

Role of hormone-sensitive lipase in beta-adrenergic remodeling of white adipose tissue.

Free fatty acids (FFA) are important extracellular and intracellular signaling molecules and are thought to be involved in beta-adrenergic-induced remodeling of adipose tissue, which involves a transient inflammatory response followed by mitochondrial biogenesis and increased oxidative capacity. This work examined the role of hormone-sensitive lipase (HSL), a key enzyme of acylglycerol metabolism, in white adipose tissue (WAT) remodeling using genetic inactivation or pharmacological inhibition. Acute treatment with the beta(3)-adrenergic agonist CL-316,243 (CL) induced expression of inflammatory markers and caused extravasation of myeloid cells in WAT of wild-type (WT) mice. HSL-knockout (KO) mice had elevated inflammatory gene expression in the absence of stimulation, and acute injection of CL did not further recruit myeloid cells, nor did it further elevate inflammatory gene expression. Acute pharmacological inhibition of HSL with BAY 59-9435 (BAY) had no effect on inflammatory gene expression in WAT or in cultured 3T3-L1 adipocytes. However, BAY prevented induction of inflammatory cytokines by beta-adrenergic stimulation in WAT in vivo and in cultured 3T3-L1 adipocytes. Chronic CL treatment stimulated mitochondrial biogenesis, expanded oxidative capacity, and increased lipid droplet fragmentation in WT mice, and these effects were significantly impaired in HSL-KO mice. In contrast to HSL-KO mice, mice with defective signaling of Toll-like receptor 4, a putative FFA receptor, showed normal beta-adrenergic-induced remodeling of adipose tissue. Overall, results reveal the importance of HSL activity in WAT metabolic plasticity and inflammation.

Reduced capacity for fatty acid oxidation in rats with inherited susceptibility to diet-induced obesity.

High-fat, energy-dense diets promote weight gain and obesity in humans and other animals, but the mechanisms underlying such diet-induced obesity remain elusive. To determine whether a reduced capacity to oxidize fat is involved in the etiology of diet-induced obesity, we examined different measures of fatty acid oxidation in rats selectively bred for susceptibility (DIO) or resistance (DR) to dietary obesity before and after they were fed a high-fat diet and became obese. DIO rats eating a low-fat diet oxidized less dietary fatty acid in vivo and had lower levels of plasma ketone bodies during fasting compared with DR rats. Lean DIO rats fed a low-fat diet showed reduced liver messenger RNA expression of CD36, which transports fatty acids across cell membranes, and long-chain acyl-coenzyme A dehydrogenase (ACADL), which catalyzes the first step in the mitochondrial beta-oxidation of fatty acids. The deficit in CD36 and ACADL messenger RNA expression was also seen in obese DIO rats that had been eating a high-fat diet and, in addition, was accompanied by reduced expression of liver carnitine palmitoyl transferase I, the enzyme that mediates transport of long-chain fatty acids into mitochondria. No differences were found in the expression of liver enzymes involved in fat synthesis; however, in muscle, DIO rats fed the low-fat, but not high-fat, diet showed greater expression of diacylglycerol O-acyltransferase 1 and lipoprotein lipase than did DR rats. Expression of muscle enzymes involved in fatty acid oxidation was similar in the 2 groups. These findings provide a metabolic mechanism for the development of diet-induced obesity and thus suggest potential targets for intervention strategies to treat or prevent it.

Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system.

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first enzymatic step in the mitochondrial beta-oxidation of fatty acids 14-20 carbons in length. More than 100 cases of VLCAD deficiency have been reported with the disease varying from a severe, often fatal neonatal form to a mild adult-onset form. VLCAD is distinguished from matrix-soluble acyl-CoA dehydrogenases by its unique C-terminal domain, homodimeric structure, and localization to the inner mitochondrial membrane. We have for the first time expressed and purified VLCAD using a bacterial system. Recombinant VLCAD had similar biochemical properties to those reported for native VLCAD and the bacterial system was used to study six previously described disease-causing missense mutations including the two most common mild mutations (T220M, V243A), a mutation leading to the severe disease phenotype (R429W), and three mutations in the C-terminal domain (A450P, L462P, and R573W). Of particular interest was the finding that the A450P and L462P bacterial extracts had normal or increased amounts of VLCAD antigen and activity. In the pure form L462P had roughly 30% of wild-type activity while A450P was normal. Using computer modeling both mutations were mapped to a predicted charged surface of VLCAD that we postulate interacts with the mitochondrial membrane. In a membrane pull down assay both mutants showed greatly reduced mitochondrial membrane association, suggesting a mechanism for the disease in these patients. In summary, the bacterial expression system developed here will significantly advance our understanding of both the clinical aspects of VLCAD deficiency and the basic biochemistry of the enzyme.

Potential of fibrates in the treatment of fatty acid oxidation disorders: revival of classical drugs?

Exposure to fibrates leads to normalization of fatty acid oxidation (FAO) in fibroblasts from patients with myopathic forms of CPT2 deficiency or VLCAD deficiency. Correction of FAO is related to a drug-induced increase of residual enzyme activity, and this could provide a new treatment strategy for these disorders.

In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of fatty acid beta-oxidation that can present at any age with cardiomyopathy, rhabdomyolysis, hepatic dysfunction, and/or nonketotic hypoglycemia. Through the expansion of newborn screening programs an increasing number of individuals with VLCAD deficiency are being identified prior to the onset of symptoms allowing early initiation of therapy. The development of a safe, durable, and effective VLCAD gene delivery system for use at the time of diagnosis could result in a significant improvement in the quality and duration of life for patients with VLCAD deficiency. To this end, we developed a construct containing the human VLCAD cDNA under the control of the strong CMV promoter (pCMV-hVLCAD). A novel rabbit polyclonal anti-VLCAD antibody was prepared using a 24 amino-acid peptide unique to the human VLCAD protein to study human VLCAD expression in immune competent mice. Antibody specificity was demonstrated in Western blots of human VLCAD deficient fibroblasts and in pCMV-hVLCAD transiently transfected VLCAD deficient fibroblasts. Transfected fibroblasts showed correction of the metabolic block as demonstrated by normalization of C14- and C16-acylcarnitine species in cell culture media and restoration of VLCAD activity in cells. Following tail vein injection of pCMV-hVLCAD into mice, we demonstrated expression of hVLCAD in liver. Altogether, these steps are important in the development of a durable gene therapy for VLCAD deficiency.

Leptin and the control of respiratory gene expression in muscle.

Leptin plays a central role in the regulation of fatty acid homeostasis, promoting lipid storage in adipose tissue and fatty acid oxidation in peripheral tissues. Loss of leptin signaling leads to accumulation of lipids in muscle and loss of insulin sensitivity secondary to obesity. In this study, we examined the direct and indirect effects of leptin signaling on mitochondrial enzymes including those essential for peripheral fatty acid oxidation. We assessed the impact of leptin using the JCR:LA-cp rat, which lacks functional leptin receptors. The activities of marker mitochondrial enzymes citrate synthase (CS) and cytochrome oxidase (COX) were similar between wild-type (+/?) and corpulent (cp/cp) rats. In contrast, several tissues showed variations in the fatty acid oxidizing enzymes carnitine palmitoyltransferase II (CPT II), long-chain acyl-CoA dehydrogenase (LCAD) and 3-hydroxyacyl-CoA dehydrogenase (HOAD). It was not clear if these changes were due to loss of leptin signaling or to insulin insensitivity. Consequently, we examined the effects of leptin on cultured C(2)C(12) and Sol8 cells. Leptin (3 days at 0, 0.2, or 2.0 nM) had no direct effect on the activities of CS, COX, or fatty acid oxidizing enzymes. Leptin treatment did not affect luciferase-based reporter genes under the control of transcription factors involved in mitochondrial biogenesis (nuclear respiratory factor-1 (NRF-1), nuclear respiratory factor-2 (NRF-2)) or fatty acid enzyme expression (peroxisome proliferator-activated receptors (PPARs)). These studies suggest that leptin exerts only indirect effects on mitochondrial gene expression in muscle, possibly arising from insulin resistance.

Cloning and expression of an acyl-CoA dehydrogenase from Mycobacterium tuberculosis.

A gene from Mycobacterium tuberculosis coding for acyl-CoA dehydrogenase was cloned, overexpressed and characterized on the basis of enzyme activity with various chain length substrates. The results show that the protein is a medium chain acyl-CoA dehydrogenase (MCADH). The mycobacterium protein expressed appears to be unique, since by comparison, the active site glutamic acid of the protein does not lie in the same position as other well characterized MCADH, but in a position present in long chain and isovaleryl acyl-CoA dehydrogenases (LCADH and IVDH).

Regulation of the human long chain acyl-CoA dehydrogenase gene by nuclear hormone receptor transcription factors.

Mitochondrial fatty acid oxidation provides most of the energy required for myocardial function after birth. Long chain acyl-CoA dehydrogenase (LCAD) catalyzes the first step in the beta-oxidation spiral. Our objective was to define regulatory elements of the human LCAD gene required for high levels of expression in mature heart and to locate elements suppressing gene expression in the fetus. We characterized the human LCAD gene structure and used in vitro transfection into cardiomyocytes and hepatoma cells of LCAD genomic fragments fused to a reporter gene to examine the effects of putative regulatory elements on transcription. Binding of transcription factors to nuclear hormone receptor consensus DNA binding domains was studied by gel shift experiments. The 200 bp of the human LCAD gene immediately upstream of the transcription initiation site are sufficient to act as a minimal promoter for the gene and provide some tissue-specific positive regulatory elements. The region from -1800 bp to -250 bp contains elements which markedly suppress transcription, including nuclear hormone receptor response elements. The dominant interaction is with the repressor factor, chicken ovalbumin upstream promoter transcription factor. We conclude that the developmental and tissue-specific regulation of the human LCAD gene is mediated, in part, by these nuclear hormone receptor transcription factors.

Effects of short-chain acyl-CoA dehydrogenase deficiency on development expression of metabolic enzyme genes in the mouse.

Patients with an acyl-CoA dehydrogenase deficiency share the disease features of hypoglycemia, hyperammonemia, tissue fatty change, hypoketonemia, carnitine deficiency, and organic acidemia due to apparent disruption of normal fatty acid, glucose, and urea metabolism. Most of the acute clinical episodes occur in young children. These episodes are precipitated by fasting and are often fatal, with the in vivo mechanisms essentially unknown. Since the genes of the rate controlling enzymes of these pathways are tissue and developmentally regulated at the transcriptional level, we measured, throughout neonatal development, the steady-state mRNA levels of long-chain, medium-chain, and short-chain (SCAD) acyl-CoA dehydrogenases, pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), carbamyl phosphate synthetase I (CPS), ornithine transcarbamylase (OTC), and argininosuccinate synthetase (AS) in fed or fasted SCAD-deficient BALB/ByJ mice compared to BALB/cBy controls. Overall, our results showed no major effects on expression of acyl-CoA dehydrogenases due to SCAD deficiency, regardless of age or fasting. In SCAD-deficient mice we found depressed mRNA expression and enzyme activity for the urea cycle enzymes CPS and AS at 6 days of age, and found no apparent effects on expression of gluconeogenic enzymes PC or PEPCK. There was a period of overall lower gene expression for most genes at 6 and 15 days, which appears to be in parallel with the developmental period when children with these diseases are most severely affected.

RNA expression and chromosomal location of the mouse long-chain acyl-CoA dehydrogenase gene.

The cDNA for mouse long-chain acyl-CoA dehydrogenase (Acadl, gene symbol; LCAD, enzyme) was cloned and characterized. The cDNA was obtained by library screening and reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence showed a high degree of homology to both the rat and the human LCAD sequence. Northern analysis of multiple tissues using the mouse Acadl cDNA as a probe showed two bands in all tissues examined. We found a total of three distinct mRNAs for Acadl. These three mRNAs were encoded by a single gene that we mapped to mouse chromosome 1. The three transcripts differed in the 3' untranslated region due to use of alternative polyadenylation sites. Quantitative evaluation of a multitissue Northern blot showed a varied ratio of the larger transcript as compared with the smaller transcripts.

Biosynthesis of four rat liver mitochondrial acyl-CoA dehydrogenases: in vitro synthesis, import into mitochondria, and processing of their precursors in a cell-free system and in cultured cells.

The synthesis, translocation, processing, and assembly of rat liver short chain acyl-CoA, medium chain acyl-CoA, long chain acyl-CoA, and isovaleryl-CoA dehydrogenases were studied. These four acyl-CoA dehydrogenases are homotetrameric flavoproteins which are located in the mitochondrial matrix. They were synthesized in a cell-free rabbit reticulocyte lysate system, programmed by rat liver polysomal RNA, as precursor polypeptides which are 2-4 kDa larger than their corresponding mature subunits (Mr 41,000-45,000). When the radiolabeled precursors were incubated with intact rat liver mitochondria, they appeared to bind tightly to the mitochondrial outer membrane. At this stage they were completely susceptible to the action of exogenous trypsin. The precursors bound to mitochondria at 0 degrees C were translocated into the mitochondria and processed when the temperature was raised to 30 degrees C. No reaction occurred when the temperature was kept at 0 degrees C, however, suggesting that the binding of the precursors is temperature independent while the subsequent steps of the pathway are energy dependent. Indeed, the translocation reaction was inhibited by compounds such as dinitrophenol and rhodamine 6G which inhibit mitochondrial energy metabolism. The newly imported (mature) enzymes were inaccessible to the proteolytic action of added trypsin. The processing of the precursors to mature subunits was proteolytically carried out in the mitochondrial matrix, and the processed mature subunits mostly assembled to their respective tetrameric forms. Newly synthesized larger precursors of each of the four acyl-CoA dehydrogenases were recovered from intact, cultured Buffalo rat liver cells in the presence of dinitrophenol. When dinitrophenol was removed in a pulse-chase protocol, the accumulated precursors were rapidly (t1/2 3-5 min) converted to their corresponding mature subunits. On the other hand, when the chase was performed in the presence of the inhibitor, the labeled precursors disappeared with t1/2 of greater than 4 h for long chain acyl-CoA dehydrogenase and 1-2 h for the other three enzyme precursors.