Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

Structural insights into the substrate recognition of serine palmitoyltransferase from Sphingobacterium multivorum.

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis, which catalyzes the pyridoxal-5'-phosphate-dependent decarboxylative condensation reaction of l-serine (l-Ser) and palmitoyl-CoA (PalCoA) to form 3-ketodihydrosphingosine called long chain base (LCB). SPT is also able to metabolize l-alanine (l-Ala) and glycine (Gly), albeit with much lower efficiency. Human SPT is a membrane-bound large protein complex containing SPTLC1/SPTLC2 heterodimer as the core subunits, and it is known that mutations of the SPTLC1/SPTLC2 genes increase the formation of deoxy-type of LCBs derived from l-Ala and Gly to cause some neurodegenerative diseases. In order to study the substrate recognition of SPT, we examined the reactivity of Sphingobacterium multivorum SPT on various amino acids in the presence of PalCoA. The S. multivorum SPT could convert not only l-Ala and Gly but also l-homoserine, in addition to l-Ser, into the corresponding LCBs. Furthermore, we obtained high-quality crystals of the ligand-free form and the binary complexes with a series of amino acids, including a nonproductive amino acid, l-threonine, and determined the structures at 1.40 to 1.55 Å resolutions. The S. multivorum SPT accommodated various amino acid substrates through subtle rearrangements of the active-site amino acid residues and water molecules. It was also suggested that non-active-site residues mutated in the human SPT genes might indirectly influence the substrate specificity by affecting the hydrogen-bonding networks involving the bound substrate, water molecules, and amino acid residues in the active site of this enzyme. Collectively, our results highlight SPT structural features affecting substrate specificity for this stage of sphingolipid biosynthesis.

Mechanisms and inhibition of Porcupine-mediated Wnt acylation.

Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1-3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5-7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Diabetic mitochondria are resistant to palmitoyl CoA inhibition of respiration, which is detrimental during ischemia.

The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP-stimulated state 3 respiration and increased the apparent Km for ADP twofold. The half maximal inhibitory concentration (IC50 ) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the Km for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose-dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31 P-magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.

Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT.

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

Insulin rapidly increases skeletal muscle mitochondrial ADP sensitivity in the absence of a high lipid environment.

Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60 min post I.P.) increased submaximal (100-1000 µM ADP) mitochondrial respiration ∼2-fold without altering maximal (>1000 µM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration ∼50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.

Palmitoyl-CoA effect on cytochrome c release, a key process of apoptosis, from liver mitochondria of rat with sucrose diet-induced obesity.

Cytochrome c (cyt-c) release from the mitochondria to the cytosol is a key process in the initiation of hepatocyte apoptosis involved in the progression of non-alcoholic fatty liver disease (NAFLD) to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatocyte apoptosis may be related to lipotoxicity due to the accumulation of palmitic acid and palmitoyl-CoA (Pal-CoA). Therefore, the aim of this study is to examine whether Pal-CoA induces cyt-c release from liver mitochondria of sucrose-fed rat (SF). Pal-CoA-induced cyt-c release was sensitive to cyclosporine A indicating the involvement of the mitochondrial membrane permeability transition (mMPT). In addition, cyt-c release from SF mitochondria remains significantly lower than C mitochondria despite the increased rate of H2O2 generation in SF mitochondria. The decreased cyt-c release from SF may be also related to the increased proportion of the palmitic acid-enriched cardiolipin, due to the high availibilty of palmitic acid in SF liver. The enrichment of cardiolipin molecular species with palmitic acid makes cardiolipin more resistant to peroxidation, a mechanism involved in the dissociation of cyt-c from mitochondrial inner membrane. These results suggest that Pal-CoA may participate in the progression of NAFLD to more severe disease through mechanisms involving cyt-c release and mMPT, a key process of apoptosis.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)*.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

Stearoyl-CoA Desaturase-2 in Murine Development, Metabolism, and Disease.

Stearoyl-CoA Desaturase-2 (SCD2) is a member of the Stearoyl-CoA Desaturase (SCD) family of enzymes that catalyze the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis. The MUFAs palmitoleoyl-CoA (16:1n7) and oleoyl-CoA (18:1n9) are the major products of SCD2. Palmitoleoyl-CoA and oleoyl-CoA have various roles, from being a source of energy to signaling molecules. Under normal feeding conditions, SCD2 is ubiquitously expressed and is the predominant SCD isoform in the brain. However, obesogenic diets highly induce SCD2 in adipose tissue, lung, and kidney. Here we provide a comprehensive review of SCD2 in mouse development, metabolism, and various diseases, such as obesity, chronic kidney disease, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. In addition, we show that bone mineral density is decreased in SCD2KO mice under high-fat feeding conditions and that SCD2 is not required for preadipocyte differentiation or the expression of PPARγ in vivo despite being required in vitro.

Long-chain fatty acyl-CoA esters regulate metabolism via allosteric control of AMPK ß1 isoforms.

Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for ß-oxidation2-6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) ß1-containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the ß-subunit. ß1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK ß1-selective activators and promote LCFA oxidation.

PvrA is a novel regulator that contributes to Pseudomonas aeruginosa pathogenesis by controlling bacterial utilization of long chain fatty acids.

During infection of a host, Pseudomonas aeruginosa orchestrates global gene expression to adapt to the host environment and counter the immune attacks. P. aeruginosa harbours hundreds of regulatory genes that play essential roles in controlling gene expression. However, their contributions to the bacterial pathogenesis remain largely unknown. In this study, we analysed the transcriptomic profile of P. aeruginosa cells isolated from lungs of infected mice and examined the roles of upregulated regulatory genes in bacterial virulence. Mutation of a novel regulatory gene pvrA (PA2957) attenuated the bacterial virulence in an acute pneumonia model. Chromatin immunoprecipitation (ChIP)-Seq and genetic analyses revealed that PvrA directly regulates genes involved in phosphatidylcholine utilization and fatty acid catabolism. Mutation of the pvrA resulted in defective bacterial growth when phosphatidylcholine or palmitic acid was used as the sole carbon source. We further demonstrated that palmitoyl coenzyme A is a ligand for the PvrA, enhancing the binding affinity of PvrA to its target promoters. An arginine residue at position 136 was found to be essential for PvrA to bind palmitoyl coenzyme A. Overall, our results revealed a novel regulatory pathway that controls genes involved in phosphatidylcholine and fatty acid utilization and contributes to the bacterial virulence.

Hedgehog Acyltransferase Promotes Uptake of Palmitoyl-CoA across the Endoplasmic Reticulum Membrane.

Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. Palmitoyl-coenzyme A (CoA), the palmitate donor, is produced in the cytosol and is not permeable across membrane bilayers. It is not known how palmitoyl-CoA crosses the ER membrane to access the active site of Hhat. Here, we use fluorescent and radiolabeled palmitoyl-CoA probes to demonstrate that Hhat promotes the uptake of palmitoyl-CoA across the ER membrane in microsomes and semi-intact cells. Reconstitution of purified Hhat into liposomes provided further evidence that palmitoyl-CoA uptake activity is an intrinsic property of Hhat. Palmitoyl-CoA uptake was regulated by and could be uncoupled from Hhat enzymatic activity, implying that Hhat serves a dual function as a palmitoyl acyltransferase and a conduit to supply palmitoyl-CoA to the luminal side of the ER.

A mitochondria-targeted fatty acid analogue influences hepatic glucose metabolism and reduces the plasma insulin/glucose ratio in male Wistar rats.

A fatty acid analogue, 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), was previously shown to have hypolipidemic effects in rats by targeting mitochondrial activity predominantly in liver. This study aimed to determine if 1-triple TTA could influence carbohydrate metabolism. Male Wistar rats were treated for three weeks with oral supplementation of 100 mg/kg body weight 1-triple TTA. Blood glucose and insulin levels, and liver carbohydrate metabolism gene expression and enzyme activities were determined. In addition, human myotubes and Huh7 liver cells were treated with 1-triple TTA, and glucose and fatty acid oxidation were determined. The level of plasma insulin was significantly reduced in 1-triple TTA-treated rats, resulting in a 32% reduction in the insulin/glucose ratio. The hepatic glucose and glycogen levels were lowered by 22% and 49%, respectively, compared to control. This was accompanied by lower hepatic gene expression of phosphenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, and Hnf4A, a regulator of gluconeogenesis. Gene expression of pyruvate kinase, catalysing the final step of glycolysis, was also reduced by 1-triple TTA. In addition, pyruvate dehydrogenase activity was reduced, accompanied by 10-15-fold increased gene expression of its regulator pyruvate dehydrogenase kinase 4 compared to control, suggesting reduced entry of pyruvate into the TCA cycle. Indeed, the NADPH-generating enzyme malic enzyme 1 (ME1) catalysing production of pyruvate from malate, was increased 13-fold at the gene expression level. Despite the decreased glycogen level, genes involved in glycogen synthesis were not affected in livers of 1-triple TTA treated rats. In contrast, the pentose phosphate pathway seemed to be increased as the hepatic gene expression of glucose-6-phosphate dehydrogenase (G6PD) was higher in 1-triple TTA treated rats compared to controls. In human Huh7 liver cells, but not in myotubes, 1-triple-TTA reduced glucose oxidation and induced fatty acid oxidation, in line with previous observations of increased hepatic mitochondrial palmitoyl-CoA oxidation in rats. Importantly, this work recognizes the liver as an important organ in glucose homeostasis. The mitochondrially targeted fatty acid analogue 1-triple TTA seemed to lower hepatic glucose and glycogen levels by inhibition of gluconeogenesis. This was also linked to a reduction in glucose oxidation accompanied by reduced PHD activity and stimulation of ME1 and G6PD, favouring a shift from glucose- to fatty acid oxidation. The reduced plasma insulin/glucose ratio indicate that 1-triple TTA may improve glucose tolerance in rats.

The effect of a physiological increase in temperature on mitochondrial fatty acid oxidation in rat myofibers.

We investigated the effect of temperature increase on mitochondrial fatty acid (FA) and carbohydrate oxidation in the slow-oxidative skeletal muscles (soleus) of rats. We measured mitochondrial respiration at 35°C and 40°C with the physiological substrates pyruvate + 4 mM malate (Pyr) and palmitoyl-CoA (PCoA) + 0.5 mM malate + 2 mM carnitine in permeabilized myofibers under nonphosphorylating (V˙0) or phosphorylating (V˙max) conditions. Mitochondrial efficiency was calculated by the respiratory control ratio (RCR = V˙max/V˙0). We used guanosine triphosphate (GTP), an inhibitor of uncoupling protein (UCP), to study the mechanisms responsible for alterations of mitochondrial efficiency. We measured hydrogen peroxide (H2O2) production under nonphosphorylating and phosphorylating conditions at both temperatures and substrates. We studied citrate synthase (CS) and 3-hydroxyl acyl coenzyme A dehydrogenase (3-HAD) activities at both temperatures. Elevating the temperature from 35°C to 40°C increased PCoA-V˙0 and decreased PCoA-RCR, corresponding to the uncoupling of oxidative phosphorylation (OXPHOS). GTP blocked the heat-induced increase of PCoA-V˙0. Rising temperature moved toward a Pyr-V˙0 increase, without significance. Heat did not alter H2O2 production, resulting from either PCoA or Pyr oxidation. Heat induced an increase in 3-HAD but not in CS activities. In conclusion, heat induced OXPHOS uncoupling for PCoA oxidation, which was at least partially mediated by UCP and independent of oxidative stress. The classically described heat-induced glucose shift may actually be mostly due to a less efficient FA oxidation. These findings raise questions concerning the consequences of heat-induced alterations in mitochondrial efficiency of FA metabolism on thermoregulation.NEW & NOTEWORTHY Ex vivo exposure of skeletal myofibers to heat uncouples substrate oxidation from ADP phosphorylation, decreasing the efficiency of mitochondria to produce ATP. This heat effect alters fatty acids (FAs) more than carbohydrate oxidation. Alteration of FA oxidation involves uncoupling proteins without inducing oxidative stress. This alteration in lipid metabolism may underlie the preferential use of carbohydrates in the heat and could decrease aerobic endurance.

In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities.

Hedgehog and Wnt proteins are modified by covalent attachment of the fatty acids palmitate and palmitoleate, respectively. These lipid modifications are essential for Hedgehog and Wnt protein signaling activities and are catalyzed by related, but distinct fatty acyltransferases: Hedgehog acyltransferase (Hedgehog) and Porcupine (Wnt). In this chapter, we provide detailed methods to directly monitor Hedgehog and Wnt protein fatty acylation in vitro. Palmitoylation of Sonic hedgehog (Shh), a representative Hedgehog family member, is assayed using purified Hedgehog acyltransferase (Hhat) or Hhat-enriched membranes, a recombinant 19 kDa Shh protein or C-terminally biotinylated Shh 10-mer peptide, and 125I-iodopalmitoyl CoA as the donor fatty acyl CoA substrate. The radiolabeled reaction products are quantified by SDS-PAGE and phosphorimaging or by γ-counting. To assay Wnt acylation, the reaction consists of a biotinylated, double disulfide-bonded Wnt peptide containing the sequence surrounding the Wnt3a acylation site, [125I] iodo-cis-9-pentadecenoyl CoA, and Porcupine-enriched membranes. Radiolabeled, biotinylated Wnt3a peptide is captured on streptavidin coated beads and the reaction product is quantified by γ-counting.

Mitochondrial-derived reactive oxygen species influence ADP sensitivity, but not CPT-I substrate sensitivity.

The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H2O2 emission rates, as exercise decreased the suppression of H2O2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

Reduced mitochondrial reactive oxygen species production in peripheral nerves of mice fed a ketogenic diet.

NEW FINDINGS: What is the central question of this study? Do peripheral sensory neurons metabolize fat-based fuel sources, and does a ketogenic diet modify these processes? What is the main finding and its importance We show that peripheral axons from mice fed a ketogenic diet respond to fat-based fuel sources with reduced respiration and H2 O2 emission compared with mice fed a control diet. These results add to our understanding of the responses of sensory neurons to neuropathy associated with poor diet, obesity and metabolic syndrome. These findings should be incorporated into current ideas of axonal protection and might identify how dietary interventions may change mitochondrial function in settings of sensory dysfunction. ABSTRACT: Metabolic syndrome and obesity are increasing epidemics that significantly impact the peripheral nervous system and lead to negative changes in sensation and peripheral nerve function. Research to understand the consequences of diet, obesity and fuel usage in sensory neurons has commonly focused on glucose metabolism. Here, we tested whether mouse sensory neurons and nerves have the capacity to metabolize fat-based fuels (palmitoyl-CoA) and whether these effects are altered by feeding of a ketogenic (90% kcal fat) diet compared with a control diet (14% kcal fat). Male C57Bl/6 mice were placed on the diets for 10 weeks, and after the mice were killed, the dorsal root ganglion (DRG) and sciatic nerve (SN) were placed in an Oroboros oxygraph-2K to examine diet-induced alterations in metabolism (respiration) of palmitoyl-CoA and H2 O2 emission (fluorescence). In addition, RNAseq was performed on the DRG of mice fed a control or a ketogenic diet for 12 weeks, and genes associated with mitochondrial respiratory function were analysed. Our results suggest that the sciatic nerves from mice fed a ketogenic diet display reduced O2 respiration and H2 O2 emission when metabolizing palmitoyl-CoA compared with mice fed a control diet. Assessments of changes in mRNA gene expression reveal alterations in genes encoding the NADH dehydrogenase complex and complex IV, which could alter production of reactive oxygen species. These new findings highlight the ability of sensory neurons and axons to oxidize fat-based fuel sources and show that these mechanisms are adaptable to dietary changes.

Palmitate induces neuroinflammation, ER stress, and Pomc mRNA expression in hypothalamic mHypoA-POMC/GFP neurons through novel mechanisms that are prevented by oleate.

Dietary fats can modulate brain function. How free fatty acids (FFAs) alter hypothalamic pro-opiomelanocortin (POMC) neurons remain undefined. The saturated FFA, palmitate, increased neuroinflammatory and ER stress markers, as well as Pomc mRNA levels, but did not affect insulin signaling, in mHypoA-POMC/GFP-2 neurons. This effect was mediated through the MAP kinases JNK and ERK. Further, the increase in Pomc was dependent on palmitoyl-coA synthesis, but not de novo ceramide synthesis, as inhibition of SPT enhanced palmitate-induced Pomc expression, while methylpalmitate had no effect. While palmitate concomitantly induces neuroinflammation and ER stress, these effects were independent of changes in Pomc expression. Palmitate thus has direct acute effects on Pomc, which appears to be important for negative feedback, but not directly related to neuroinflammation. The monounsaturated FFA oleate completely blocked the palmitate-mediated increase in neuroinflammation, ER stress, and Pomc mRNAs. This study provides insight into the complex central metabolic regulation by FFAs.

Mitochondrial respiration and ROS emission during ß-oxidation in the heart: An experimental-computational study.

Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze ß-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO2) and hydrogen peroxide (H2O2) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H2O2 emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of ß-oxidation of long-chain fatty acids and main energy-redox processes is able to simulate the relationship between VO2 and H2O2 emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H2O2 emission as a function of VO2. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply.