Inhibition Of Β-oxidation Research Articles

PTEN Down-Regulation Promotes β-Oxidation to Fuel Hypertrophic Liver Growth After Hepatectomy in Mice.

PTEN down-regulation after hepatectomy promotes the burning of TRAS-derived lipids to fuel hypertrophic liver regeneration. Therefore, the anabolic function of PTEN deficiency in resting liver is transformed into catabolic activities upon tissue loss. These findings portray PTEN as a node coordinating liver growth with its energy demands and emphasize the need of lipids for regeneration. (Hepatology 2017;66:908-921).

A Computational Model of Hepatic Energy Metabolism: Understanding Zonated Damage and Steatosis in NAFLD.

In non-alcoholic fatty liver disease (NAFLD), lipid build-up and the resulting damage is known to occur more severely in pericentral cells. Due to the complexity of studying individual regions of the sinusoid, the causes of this zone specificity and its implications on treatment are largely ignored. In this study, a computational model of liver glucose and lipid metabolism is presented which treats the sinusoid as the repeating unit of the liver rather than the single hepatocyte. This allows for inclusion of zonated enzyme expression by splitting the sinusoid into periportal to pericentral compartments. By simulating insulin resistance (IR) and high intake diets leading to the development of steatosis in the model, we identify key differences between periportal and pericentral cells accounting for higher susceptibility to pericentral steatosis. Secondly, variation between individuals is seen in both susceptibility to steatosis and in its development across the sinusoid. Around 25% of obese individuals do not show excess liver fat, whilst 16% of lean individuals develop NAFLD. Furthermore, whilst pericentral cells tend to show higher lipid levels, variation is seen in the predominant location of steatosis from pericentral to pan-sinusoidal or azonal. Sensitivity analysis was used to identify the processes which have the largest effect on both total hepatic triglyceride levels and on the sinusoidal location of steatosis. As is seen in vivo, steatosis occurs when simulating IR in the model, predominantly due to increased uptake, along with an increase in de novo lipogenesis. Additionally, concentrations of glucose intermediates including glycerol-3-phosphate increased when simulating IR due to inhibited glycogen synthesis. Several differences between zones contributed to a higher susceptibility to steatosis in pericentral cells in the model simulations. Firstly, the periportal zonation of both glycogen synthase and the oxidative phosphorylation enzymes meant that the build-up of glucose intermediates was less severe in the periportal hepatocyte compartments. Secondly, the periportal zonation of the enzymes mediating β-oxidation and oxidative phosphorylation resulted in excess fats being metabolised more rapidly in the periportal hepatocyte compartments. Finally, the pericentral expression of de novo lipogenesis contributed to pericentral steatosis when additionally simulating the increase in sterol-regulatory element binding protein 1c (SREBP-1c) seen in NAFLD patients in vivo. The hepatic triglyceride concentration was predicted to be most sensitive to inter-individual variation in the activity of enzymes which, either directly or indirectly, determine the rate of free fatty acid (FFA) oxidation. The concentration was most strongly dependent on the rate constants for β-oxidation and oxidative phosphorylation. It also showed moderate sensitivity to the rate constants for processes which alter the allosteric inhibition of β-oxidation by acetyl-CoA. The predominant sinusoidal location of steatosis meanwhile was most sensitive variations in the zonation of proteins mediating FFA uptake or triglyceride release as very low density lipoproteins (VLDL). Neither the total hepatic concentration nor the location of steatosis showed strong sensitivity to variations in the lipogenic rate constants.

Inhibition of β-oxidation is not a valid therapeutic tool for reducing oxidative stress in conditions of neurodegeneration

According to recent reports, systemic treatment of rats with methylpalmoxirate (carnitine palmitoyltransferase-1 inhibitor) decreased peroxidation of polyunsaturated fatty acids in brain tissue. This was taken as evidence of mitochondrial β-oxidation in brain, thereby contradicting long-standing paradigms of cerebral metabolism, which claim that β-oxidation of activated fatty acids has minor importance for brain energy homeostasis. We addressed this controversy. Our experiments are the first direct experimental analysis of this question. We fueled isolated brain mitochondria or rat brain astrocytes with octanoic acid, but octanoic acid does not enhance formation of reactive oxygen species, neither in isolated brain mitochondria nor in astrocytes, even at limited hydrogen delivery to mitochondria. Thus, octanoic acid or l-octanoylcarnitine does not stimulate H2O2 release from brain mitochondria fueled with malate, in contrast to liver mitochondria (2.25-fold rise). This does obviously not support the possible occurrence of β-oxidation of the fatty acid octanoate in the brain. We conclude that a proposed inhibition of β-oxidation does not seem to be a helpful strategy for therapies aiming at lowering oxidative stress in cerebral tissue. This question is important, since oxidative stress is the cause of neurodegeneration in numerous neurodegenerative or inflammatory disease situations.

Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction.

BackgroundImpaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.Methods and ResultsWe investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.ConclusionPLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers.

Lipids and oocyte developmental competence: the role of fatty acids and β-oxidation.

Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with quality of oocyte and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. The triglyceride and fatty acid composition of ovarian follicular fluid has been characterised for many species and is influenced by nutritional status (i.e. dietary fat, fasting, obesity and season) as well as lactation in cows. Lipid in oocytes is a primarily triglyceride of specific fatty acids which differ by species, stored in distinct droplet organelles that re-localise during oocyte maturation. The presence of lipids, particularly saturated vs unsaturated fatty acids, in in vitro maturation systems affects oocyte lipid content as well as developmental competence. Triglycerides are metabolised by lipases that have been localised to cumulus cells as well as oocytes. Fatty acids generated by lipolysis are further metabolised by β-oxidation in mitochondria for the production of ATP. β-oxidation is induced in cumulus-oocyte complexes (COCs) by the LH surge, and pharmacological inhibition of β-oxidation impairs oocyte maturation and embryo development. Promoting β-oxidation with l-carnitine improves embryo development in many species. Thus, fatty acid metabolism in the mammalian COC is regulated by maternal physiological and in vitro environmental conditions; and is important for oocyte developmental competence.

Propionyl-L-Carnitine is Efficacious in Ulcerative Colitis Through its Action on the Immune Function and Microvasculature

Objectives:Microvascular endothelial dysfunction characterizes ulcerative colitis (UC), the most widespread form of inflammatory bowel disease. Intestinal mucosal microvessels in UC display aberrant expression of cell adhesion molecules (CAMs) and increased inflammatory cell recruitment. Propionyl-L-carnitine (PLC), an ester of L-carnitine required for the mitochondrial transport of fatty acids, ameliorates propionyl-CoA bioavailability and reduces oxidative stress in ischemic tissues. The present study aimed to document the efficacy of anti-oxidative stress properties of PLC in counteracting intestinal microvascular endothelial dysfunction and inflammation.Methods:To evaluate the efficacy in vivo, we analyzed the effects in intestinal biopsies of patients with mild-to-moderate UC receiving oral PLC co-treatment and in rat TNBS-induced colitis; in addition, we investigated antioxidant PLC action in TNF-α-stimulated human intestinal microvascular endothelial cells (HIMECs) in vitro.Results:Four-week PLC co-treatment reduced intestinal mucosal polymorph infiltration and CD4+ lymphocytes, ICAM-1+ and iNOS+ microvessels compared with placebo-treated patients with UC. Oral and intrarectal administration of PLC but not L-carnitine or propionate reduced intestinal damage and microvascular dysfunction in rat TNBS-induced acute and reactivated colitis. In cultured TNF-α-stimulated HIMECs, PLC restored β-oxidation and counteracted NADPH oxidase 4-generated oxidative stress-induced CAM expression and leukocyte adhesion. Inhibition of β-oxidation by L-aminocarnitine increased reactive oxygen species production and PLC beneficial effects on endothelial dysfunction and leukocyte adhesion. Finally, PLC reduced iNOS activity and nitric oxide accumulation in rat TNBS-induced colitis and in HIMEC cultures.Conclusions:Our results show that the beneficial antioxidant effect of PLC targeting intestinal microvasculature restores endothelial β-oxidation and function, and reduces mucosal inflammation in UC patients.

A Stereospecific Pathway Diverts β-Oxidation Intermediates to the Biosynthesis of Rhamnolipid Biosurfactants

Rhamnolipids are multipurpose surface-active molecules produced by the bacterium Pseudomonas aeruginosa from L-rhamnose and R-3-hydroxyalkanoate (C₁₀±₂) precursors. R-3-hydroxyalkanoate precursor is believed to be synthesized de novo. We demonstrate, however, that β-oxidation is the predominant source of this precursor. Inhibition of β-oxidation sharply decreases rhamnolipids production, even when using a nonfatty acid carbon source (glycerol). Isotope tracing shows that β-oxidation intermediates are direct precursors of rhamnolipids. A mutant-based survey revealed an operon coding for enoyl-CoA hydratases/isomerases (ECH/I), named RhlYZ, implicated in rhamnolipids production via an axial role in 3-hydroxyalkanoate synthesis. In vitro, RhlZ is an R-ECH/I transforming 2-decenoyl-CoA, a β-oxidation intermediate, into R-3-hydroxydecanoyl-CoA, the potential rhamnolipids precursor. Interestingly, polyhydroxyalkanoates share with rhamnolipids the RhlYZ-generated R-3-hydroxyalkanoates pool, as demonstrated by the decrease of polyhydroxyalkanoates upon mutation of rhlYZ and the increase of rhamnolipids in a polyhydroxyalkanoates-defective mutant.

Elaidate, an 18‐Carbon trans‐Monoenoic Fatty Acid, Inhibits β‐Oxidation in Human Peripheral Blood Macrophages

Consumption of trans-unsaturated fatty acids promotes atherosclerosis, but whether degradation of fats in macrophages is altered by trans-unsaturated fatty acids is unknown. We compared the metabolism of oleate (C18:1Δ9-10 cis; (Z)-octadec-9-enoate), elaidate (C18:Δ9-10 trans; (E)-octadec-9-enoate), and stearate (C18:0, octadecanoate) in adherent peripheral human macrophages. Metabolism was followed by measurement of acylcarnitines in cell supernatants by MS/MS, determination of cellular fatty acid content by GC/MS, and assessment of β-oxidation rates using radiolabeled fatty acids. Cells incubated for 44 h in 100 µM elaidate accumulated more unsaturated fatty acids, including both longer- and shorter-chain, and had reduced C18:0 relative to those incubated with oleate or stearate. Both C12:1 and C18:1 acylcarnitines accumulated in supernatants of macrophages exposed to trans fats. These results suggested β-oxidation inhibition one reaction proximal to the trans bond. Comparison of [1-(14)C]oleate to [1-(14)C]elaidate catabolism showed that elaidate completed the first round of fatty acid β-oxidation at rates comparable to oleate. Yet, in competitive β-oxidation assays with [9,10-(3)H]oleate, tritium release rate decreased when unlabeled oleate was replaced by the same quantity of elaidate. These data show specific inhibition of monoenoic fat catabolism by elaidate that is not shared by other atherogenic fats.

Fed-batch production of MCL-PHA with elevated 3-hydroxynonanoate content

With no inhibition of β-oxidation, Pseudomonas putida KT2440 produces medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) with approximately 65 mol% 3-hydroxynonanoate (HN) from nonanoic acid. Production of PHA with higher HN content and an adjustable monomeric composition was obtained using acrylic acid, a fatty acid β-oxidation inhibitor, together with nonanoic acid and glucose as co-substrates in fed-batch fermentations. Different monomeric compositions were obtained by varying the feeding conditions to impose different specific growth rates and inhibitor feed concentrations. At a nonanoic acid: glucose: acrylic acid feed mass ratio of 1.25: 1: 0.05 and a specific growth rate of 0.15 h-1, 71.4 g L-1 biomass was produced containing 75.5% PHA with 89 mol% HN at a cumulative PHA productivity of 1.8 g L-1 h-1.

Metabolism of Xenobiotic Carboxylic Acids: Focus on Coenzyme A Conjugation, Reactivity, and Interference with Lipid Metabolism

While xenobiotic carboxylic acids (XCAs) have been studied extensively with respect to their enzymatic conversion to potentially reactive acyl glucuronides with implications to drug induced hepatotoxicity, the formation of xenobiotic-S-acyl-CoA thioesters (xenobiotic-CoAs) have been much less studied in spite of data indicating that such conjugates may be equally or more reactive than the corresponding acyl glucuronides. This review addresses enzymes and cell organelles involved in the formation of xenobiotic-CoAs, the reactivity of such conjugates toward biological macromolecules, and in vitro and in vivo methodology to assess consequences of such reactivity. Further, the propensity of xenobiotic-CoAs to interfere with endogenous lipid metabolism, e.g., inhibition of β-oxidation or depletion of the CoA or carnitine pools, adds to the complexity of the potential contribution of XCAs to hepatotoxicity by a number of mechanisms in addition to those in common with the corresponding acyl glucuronides. On the basis of our review of the literature on xenobiotic-CoA conjugates, there appear to be a number of gaps in our understanding of the bioactivation of XCA both with respect to the mechanisms involved and the experimental approaches to distinguish between the role of acyl glucuronides and xenobiotic-CoA conjugates. These aspects are focused upon and described in detail in this review.

Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization

Cell polarization requires increased cellular energy and metabolic output, but how these energetic demands are met by polarizing cells is unclear. To address these issues, we investigated the roles of mitochondrial bioenergetics and autophagy during cell polarization of hepatocytes cultured in a collagen sandwich system. We found that as the hepatocytes begin to polarize, they use oxidative phosphorylation to raise their ATP levels, and this energy production is required for polarization. After the cells are polarized, the hepatocytes shift to become more dependent on glycolysis to produce ATP. Along with this central reliance on oxidative phosphorylation as the main source of ATP production in polarizing cultures, several other metabolic processes are reprogrammed during the time course of polarization. As the cells polarize, mitochondria elongate and mitochondrial membrane potential increases. In addition, lipid droplet abundance decreases over time. These findings suggest that polarizing cells are reliant on fatty acid oxidation, which is supported by pharmacologic inhibition of β-oxidation by etomoxir. Finally, autophagy is up-regulated during cell polarization, with inhibition of autophagy retarding cell polarization. Taken together, our results describe a metabolic shift involving a number of coordinated metabolic pathways that ultimately serve to increase energy production during cell polarization.

Regulation of poly-(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis by the AtoSCDAEB regulon in phaCAB + Escherichia coli

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB (+) Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB (+) E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27g/l with a 3HV fraction of 25.5% wt. and biopolymer content of 75% w/w in a time-dependent process. The atoSC locus deletion in the ∆atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4% wt. The ∆atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ∆atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8g/l with 21% 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ∆atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB (+) E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.

Electrical Plasticity and Cardioprotection in Myocardial Ischemia—Role of Selective Sodium Channel Blockers

The concept of electrical protection of the ischemic myocardium is in constant evolution and has recently been supported by experimental and clinical studies. Historically, antiplatelet agents, angiotensin-converting enzyme inhibitors, β-blockers, and statins have been all proposed as drugs conferring anti-ischemic cardioprotection. This was supported by the evidence consistently indicating that all these drugs were capable of reducing mortality and the risk of repeat myocardial infarction. The electrical plasticity paradigm is, however, a novel concept that depicts the benefits of improved sodium channel blockade with drugs such as ranolazine and cariporide. Although it has been hypothesized that the protective role of ranolazine depends on decreased fatty acid β-oxidation affecting preconditioning, we speculate against such a hypothesis, because inhibition of β-oxidation requires higher concentrations of the drug, above the therapeutic range. Rather, we discuss the key role of calcium overload reduction through inhibition of the late sodium current (I(Na)). Mechanisms driving cardioprotection involve the block of a cascade of complex ionic exchanges that can result in intracellular acidosis, excess cytosolic calcium, myocardial cellular dysfunction, and eventually cell injury and death. In this review we discuss the studies that demonstrate how electrical plasticity through sodium channel blockers can promote cardioprotection against ischemia in coronary heart disease.

138 FATTY ACID β-OXIDATION REGULATES BOVINE EMBRYO OXYGEN METABOLISM

Mammalian oocytes are rich in endogenous lipid, which provides a potential source of metabolic energy during oocyte maturation and early embryo development after fertilisation. Despite this, studies on early embryo metabolism have focussed on consumption of substrates from the culture medium, with comparatively little consideration for endogenous stores. Additionally, the interaction between fatty acid β-oxidation and oxygen consumption during pre-implantation development has not been investigated. This study has investigated the relationship between embryo fatty acid metabolism and oxygen consumption rate (OCR), a marker of overall oxidative metabolic activity. The amount of oxygen consumption by bovine blastocysts coupled to ATP synthesis was assessed by measuring embryo OCR following acute treatment with oligomycin, which inhibits ATP synthase. Bovine embryos were then produced in vitro and cultured from zygote to blastocyst stage (8 days) in the presence of β-mercaptoacetate (BMA), a competitive inhibitor of fatty acid β-oxidation, or L-carnitine, a cofactor that promotes β-oxidation. OCR of individual embryos was measured using a noninvasive, highly sensitive oxygen probe (Unisense), whereas OCR of embryos in groups was determined using the Oxygen Biosensor (OBS) fluorimetric assay (BD Biosciences, Erembodegem, Belgium). Mean OCR measured by each technique correlated well. Data are presented as means ± SEM. The effect of acute inhibition were compared by a paired t-test, with pre-treated embryos acting as their own control, whereas the effect of L-carnitine and BMA on OCR was compared across treatment groups using ANOVA with Dunn’s test for unequal groups post hoc, using Sigmaplot (Systat Software Inc., San Jose, CA, USA). Treatment with oligomycin caused blastocyst OCR to fall from 23.5 ± 3.3 to 7.9 ± 2.5 pmol/embryo/h (n = 3; P = 0.03). Inhibition of β-oxidation by BMA led to a modest, but consistent increased OCR in cleavage stage embryos: 4-cell: 14.8 ± 7.2 (n = 12) v. 8.9 ± 1.1 pmol/embryo/h (n = 3) control; 8-cell: 15.8 ± 2.2 (n = 6) v. 11.9 ± 2.3 pmol/embryo/h (n = 3) control. At the morula stage, BMA led to a significant rise in OCR (26.8 ± 3.6 (n = 7) v. 7.8 ± 1.7 pmol/embryo/h (n = 3) control; P = 0.003) and at the blastocyst stage, OCR was significantly elevated in the presence of BMA; 41.3 ± 2.6 (n = 19) v. 16.9 ± 1.3 pmol/embryo/h (n = 14) control (P = 0.01). By contrast, when groups of embryos were cultured in the presence of L-carnitine, blastocyst OCR was reduced by 45% from 24.7 ± 5.1 (group control) to 15.9 ± 3.7 pmol/embryo/h (n = 30; P < 0.06). We report that 66.5% of oxygen consumption by bovine blastocysts is oligomycin sensitive, meaning that 33.5% of oxygen consumption is used for processes other than ATP synthesis. Surprisingly, inhibition of fatty acid β-oxidation caused total bovine embryo OCR to rise, although the reasons for this are unclear. It is possible that inhibiting β-oxidation leads to an accumulation of free fatty acids in the mitochondrion, which can disrupt the inner membrane and lead decoupling of ATP synthesis from oxygen consumption. Thus, manipulating fatty acid oxidation in the early embryo dysregulates oxidative metabolism; the implications of this are under further investigation.

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.

Distinct functions of JNK and c‐Jun in oxidant‐induced hepatocyte death

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death.

Involvement of the AtoSCDAEB regulon in the high molecular weight poly-(R)-3-hydroxybutyrate biosynthesis in phaCAB+Escherichia coli

AtoSC two-component system plays a pivotal role in many regulatory indispensable Escherichia coli processes. AtoSCDAEB regulon, comprising the AtoSC system and the atoDAEB operon, regulates the short-chain fatty acids catabolism. We report here, that AtoSC up-regulates the high-molecular weight PHB biosynthesis, in recombinant phaCAB+E. coli, with the Cupriavidus necator phaCAB operon. PHB accumulation was maximized upon the acetoacetate-mediated induction of AtoSC, under glucose 1% w/v, resulting in a yield of 1.73g/l with a biopolymer content of 64.5% w/w. The deletion of the atoSC locus, in the ΔatoSC strains, resulted in a 5 fold reduction of PHB accumulation, which was restored by the extrachromosomal introduction of the AtoSC system. The deletion of the atoDAEB operon triggered a significant decrease in PHB synthesis in ΔatoDAEB strains. However, the acetoacetate-induced AtoSC system in those strains increased PHB to 1.55g/l, while AtoC expression increased PHB to 1.4g/l upon acetoacetate. The complementation of the ΔatoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. The individual inhibition of β-oxidation and mainly fatty-acid biosynthesis pathways by acrylic acid or cerulenin respectively, reduced PHB biosynthesis. Under those conditions the introduction of the atoSC locus or the atoSCDAEB regulon was capable to up-regulate the biopolymer accumulation. The concurrent inhibition of both the fatty acids metabolic pathways eliminated PHB production. PHB up-regulation in phaCAB+E. coli, by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay, provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards the biotechnologically improved polyhydroxyalkanoates biosynthesis.

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds

SUMMARYLipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using 3H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [3H]arachidonic acid. In addition, pharmacological inhibition of β-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

Effect of inulin and oligofructose enrichment of the diet on rats suffering thiamine deficiency

Thiamine deficiency resulted in inhibition of two main pathways supplying energy to the tissues: glycolysis and β-oxidation. Glycolysis was found to be inhibited to 40% of initial value calculated on the basis of RBC trans-membrane transport of glucose. Prolongation of experiment cause lowering of uptake of this sugar. In rats, energy production from fatty acids (FA) seems to be less sensitive to thiamine deficiency than glycolysis. After 30 days of feeding, utilization of FA in rats was depressed to the 61% of initial value. Thiamine deficiency suppressed insulin secretion, and the changes were statistically significant. Feeding of rats with thiamine restricted diet for 1 month caused the reduction of serum insulin by 14%. In the same animals, trans-membrane glucose transport was reduced over two-times, what might suggest a decreased efficiency of insulin action in such conditions. The kind and concentration of non-digestible fructo-oligosaccharides (FOS) did not affect significantly serum insulin concentration in animals fed thiamine restricted diet. Substitution of a part of wheat starch with FOS has only insignificant compensatory effect on the uptake of glucose. A partial amelioration of the β-oxidation inhibition caused by feeding rats with thiamine deficient diet was observed in animals supplemented with FOS. However, this effect was statistically significant only in rats receiving diet containing 10% of inulin. The effect of supplemented FOS and their concentration on trans-membrane glucose transport in RBC was statistically significant, when pulled supplementation groups were used for statistical evaluation.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with ω-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid β-oxidation inhibition

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) ω-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL ω-aromatic fatty acid in the presence of salicylic acid that is known as a β-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to ∼10mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL–PHA with improved physicochemical properties in P. fluorescens BM07.