Reduced Citrate Synthase Activity Research Articles

Altered mitochondrial function in fibroblast cell lines derived from disease carriers of spinal muscular atrophy

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive childhood-onset neuromuscular disease with a carrier frequency of ~1:50. Mitochondrial abnormalities are widespread in patients with SMA. Disease carriers for SMA (i.e., the parents of patients with SMA) are viewed as asymptomatic for SMA disease. As far as we are aware, mitochondria have not been previously examined in SMA carriers, yet as they are maternally inherited, mitochondrial function in SMA carriers has putative implications for disease pathogenesis.MethodsFibroblast cell lines derived from SMA carriers and controls were obtained from two different sources and cultured under standard conditions. The mitochondrial membrane potential, reactive oxygen species (ROS) production, citrate synthase activity, and bioenergetic analysis were examined as measures of mitochondrial function. The mitochondrial genome was also sequenced in a subset of the fibroblast cell lines to identify any mitochondrial DNA variants.ResultsHere, we show a depolarized mitochondrial membrane potential, increased levels of reactive oxygen species, and reduced citrate synthase activity in SMA carriers compared with controls. A likely pathogenic variant in the MT-CO3 gene (which encodes subunit III of cytochrome c oxidase) was also identified in a paternal carrier.ConclusionsThis study was conducted as a preliminary investigation of mitochondrial function in SMA carriers. Our findings suggest that disease carriers of SMA show differences in mitochondrial function, indicative of a subclinical mitochondrial phenotype. Further investigation in a larger sample set is warranted.

Multiple-stressor effects of ocean acidification, warming and predation risk cues on the early ontogeny of a rocky-shore keystone gastropod

To understand how climate change stressors might affect marine organisms and support adequate projections it is important to know how multiple stressors may be modulated by the presence of other species. We evaluated the direct effects of ocean warming (OW) and ocean acidification (OA) together with non-consumptive effects (NCEs) of the predatory crab Acanthocyclus hassleri on early ontogeny fitness-related traits of the commercially important rocky-shore keystone gastropod Concholepas concholepas. We measured the response of nine traits to these stressors at either the organismal level (survival, growth, feeding rates, tenacity, metabolic rate, calcification rate) or sub-organismal level (nutritional status, ATP-supplying capacity, stress condition). C. concholepas survival was not affected by any of the stressors. Feeding rates were not affected by OW or OA; however, they were reduced in the presence of crab NCEs compared with control conditions. Horizontal tenacity was affected by the OA × NCEs interaction; in the presence of NCEs, OA reduced tenacity. The routine metabolic rate, measured by oxygen consumption, increased significantly with OW. Nutritional status assessment determined that carbohydrate content was not affected by any of the stressors. However, protein content was affected by the OA × NCEs interaction; in the absence of NCEs, OA reduced protein levels. ATP-supplying capacity, measured by citrate synthase (CS) activity, and cellular stress condition (HSP70 expression) were reduced by OA, with reduction in CS activity found particularly at the high temperature. Our results indicate C. concholepas traits are affected by OA and OW and the effects are modulated by predator risk (NCEs). We conclude that some C. concholepas traits are resilient to climate stressors (survival, growth, horizontal tenacity and nutritional status) but others are affected by OW (metabolic rate), OA (ATP-supplying capacity, stress condition), and NCEs (feeding rate). The results suggest that these negative effects can adversely affect the associated community.

Isometric training improves fatigue resistance in dystrophin-deficient muscle

Eccentric contractions, in which the muscle is stretched during contraction, cause substantially greater damage than isometric (ISO) contractions, in which the length of the muscle does not change during contraction. Here, we tested the hypothesis that ISO training improves fatigue resistance in skeletal muscle from dystrophin-deficient mdx52 mice (15-22 wk old). ISO training (100 Hz stimulation frequency, 0.25-s contractions every 0.5 s, 6 sets of 60 contractions) was performed on the left plantar flexor muscles in vivo with supramaximal electrical stimulation every other day for 4 wk. Compared with the normal control muscle, resistance to fatigue was reduced in the nontrained muscle from mdx52 mice, which was accompanied by a reduction in citrate synthase activity and the LC3BII/I ratio and an increase in the phosphorylation levels of Akt Ser473 and the expression levels of p62. ISO training restored these alterations and markedly increased in vivo fatigue resistance and PGC-1α expression in mdx52 muscles. Moreover, an increased number of Evans Blue dye-positive fibers was significantly reduced by ISO training in mdx52 muscles. In contrast, ISO training did not restore a reduction in the amount of SH3 and cysteine-rich domain 3 in mdx muscles. Thus, our data suggest that mitochondrial function is impaired in dystrophin-deficient muscles, which is likely to be induced by the defective autophagy due to persistent activation of Akt. ISO training inhibits the aberrant activation of Akt presumably by up-regulating the PGC-1α expression, which results in improved mitochondrial function and thus fatigue resistance in dystrophin-deficient muscles.

Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM. We performed a comprehensive multiomics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts). Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites (ATP, ADP, and phosphocreatine) and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance. Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.

Mitochondrial dysfunction in skeletal muscle contributes to the development of acute insulin resistance in mice.

BackgroundAlthough mounting evidence indicates that insulin resistance (IR) co‐occurs with mitochondrial dysfunction in skeletal muscle, there is no clear causal link between mitochondrial dysfunction and IR pathogenesis. In this study, the exact role of mitochondria in IR development was determined.MethodsSix‐week‐old C57BL/6 mice were fed a high‐fat diet for 2 weeks to induce acute IR or for 24 weeks to induce chronic IR (n = 8 per group). To characterize mitochondrial function, we measured citrate synthase activity, ATP content, mitochondrial DNA (mtDNA) content, and oxygen consumption rate in gastrocnemius and liver tissues. We intraperitoneally administered mitochondrial division inhibitor 1 (mdivi‐1) to mice with acute IR and measured mitochondrial adaptive responses such as mitophagy, mitochondrial unfolded protein response (UPRmt), and oxidative stress (n = 6 per group).ResultsAcute IR occurred coincidently with impaired mitochondrial function, including reduced citrate synthase activity (−37.8%, P < 0.01), ATP production (−88.0%, P < 0.01), mtDNA (−53.1%, P < 0.01), and mitochondrial respiration (−52.2% for maximal respiration, P < 0.05) in skeletal muscle but not in liver. Administration of mdivi‐1 attenuated IR development by increasing mitochondrial function (+58.5% for mtDNA content, P < 0.01; 4.06 ± 0.69 to 5.84 ± 0.95 pmol/min/mg for citrate synthase activity, P < 0.05; 13.06 ± 0.70 to 34.87 ± 0.70 pmol/min/g for maximal respiration, P < 0.001). Western blot analysis showed acute IR resulted in increased autophagy (mitophagy) and UPRmt induction in muscle tissue. This adaptive response was inhibited by mdivi‐1, which reduced the mitochondrial oxidative stress of skeletal muscle during acute IR.ConclusionsAcute IR induced mitochondrial oxidative stress that impaired mitochondrial function in skeletal muscle. Improving mitochondrial function has important potential for treating acute IR.

Metabolic Reprogramming of the Retinal Pigment Epithelium Drives TGFβ2‐Induced Epithelial‐Mesenchymal Transition

Transforming growth factor-beta 2 (TGFβ2) is a key orchestrator of retinal wound healing through induction of epithelial-mesenchymal-transition (EMT) in retinal pigment epithelial cells (RPE). Here we describe a previously unrecognized function of TGFβ2 in modulating mitochondrial morphology and metabolic function in human RPE cells. Treating ARPE-19 (human RPE cell line) with TGFβ2 (10 ng/ml) induced defects in mitochondrial network integrity with increased sphericity and fragmentation. Correspondingly, TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial oxidative phosphorylation (OXPHOS) levels consistent with reduced expression of NDUFB5, a key gene of Complex I of the electron transport chain. The reduced mitochondrial respiration was associated with a compensatory increase in gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA) and glycolytic reserve. TGFβ2 induced a severe suppression of PGC-1α gene expression and treatment with the selective small molecule activator of PGC-1α, ZLN005, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a profound metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. The PGC-1α promoter, ZLN005, effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment for subretinal fibrosis.

Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells.

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

Bisphenols alter thermal responses and performance in zebrafish (Danio rerio).

Plastic pollutants are novel environmental stressors that are now persistent components of natural ecosystems. Endocrine disrupting chemicals such as bisphenols that leach out of plastics can modify physiological responses of animals by interfering with hormone signalling. Here, we tested whether three commonly produced bisphenols, bisphenol A (BPA), bisphenol F (BPF) and bisphenol S (BPS), impair thermal acclimation of swimming performance and metabolic enzyme [citrate synthase (CS) and lactate dehydrogenase (LDH)] activities in adult zebrafish (Danio rerio). We found that exposure to 30-μgl-1 BPF and BPS, but not BPA, reduced swimming performance, and no interactions between bisphenol exposure and acclimation (3weeks to 18°C and 28°C) or acute test (18°C and 28°C) temperatures were found. BPA interacted with acclimation and acute test temperatures to determine CS activity, an indicator of mitochondrial density and aerobic metabolic capacity. BPS reduced CS activity and an interaction (at a one-tailed significance) between acclimation temperature and BPF exposure determined CS activity. LDH activity reflects anaerobic ATP production capacity, and BPA and BPF altered the effects of thermal acclimation and acute test temperatures on LDH activity. Our data show that all bisphenols we tested at ecologically relevant concentrations can disrupt the thermal responses of fish. BPS and BPF are used as environmentally safer alternatives to BPA, but our data show that these bisphenols are also of concern, particularly in thermally variable environments.

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.

A single muscle contusion promotes an immediate alteration in mitochondrial bioenergetics response in skeletal muscle fibres with different metabolism

Skeletal muscle is the most abundant tissue in the human body and mechanical injuries are common; these are frequently of mechanical origins, such as contusion. However, the immediate mitochondrial response to injury and energetic substrate utilisation is still unclear. We evaluated the acute response in mitochondrial function after a single muscle contusion, either in fast twitch fibres (glycolytic metabolism), fast and slow twitch (oxidative and glycolytic metabolism), or slow twitch fibres (oxidative metabolism). Rats were assigned to two groups: control and Lesion (muscle contusion). We collected the gastrocnemius and soleus muscles. The fibres were analysed for mitochondrial respiration, lactate dehydrogenase (LDH), citrate synthase (CS) activity, Ca2+ uptake, and H2O2 production. We found that muscle injury was able to increase ATP synthesis-dependent and OXPHOS oxygen flux in the oxidative fibres when stimulated by complex I + II substrates. On the other hand, the muscle injury increased hydrogen peroxide (H2O2) production when compared to control fibres, and reduced citrate synthase activity; however, it did not change Ca2+ uptake. Surprisingly, injury in mixed fibres increased the OXPHOS and ATP synthesis oxygen consumption, and H2O2 production, but it reduced Ca2+ uptake. The injury in glycolytic fibres did not affect oxygen flux coupled to ATP synthesis, citrate synthase, and lactate dehydrogenase activity, but did reduce Ca2+ uptake. Finally, we demonstrated distinct mitochondrial responses between the different muscle fibres, indicating that the mitochondrial dynamics is related to flexibilities in metabolism, and that reactive oxygen species directly affect physiology and normal function.

Metabolomics reveals tepotinib-related mitochondrial dysfunction in MET-activating mutations-driven models.

Genetic aberrations in the hepatocyte growth factor receptor tyrosine kinase MET induce oncogenic addiction in various types of human cancers, advocating MET as a viable anticancer target. Here, we report that MET signaling plays an important role in conferring a unique metabolic phenotype to cellular models expressing MET-activating mutated variants that are either sensitive or resistant toward MET small molecule inhibitors. MET phosphorylation downregulated by the specific MET inhibitor tepotinib resulted in markedly decreased viability and increased apoptosis in tepotinib-sensitive cells. Moreover, prior to the induction of MET inhibition-dependent cell death, tepotinib also led to an altered metabolic signature, characterized by a prominent reduction of metabolite ions related to amino sugar metabolism, gluconeogenesis, glycine and serine metabolism, and of numerous TCA cycle-related metabolites such as succinate, malate, and citrate. Functionally, a decrease in oxygen consumption rate, a reduced citrate synthase activity, a drop in membrane potential, and an associated misbalanced mitochondrial function were observed exclusively in MET inhibitor-sensitive cells. These data imply that interference with metabolic state can be considered an early indicator of efficient MET inhibition and particular changes reported here could be explored in the future as markers of efficacy of anti-MET therapies.

Metabolic regulation by the PGC-1α and PGC-1β coactivators in larval zebrafish (Danio rerio)

The peroxisome proliferator activated receptor γ coactivator-1 (PGC-1) family is composed of three coactivators whose role in regulating mammalian bioenergetics regulation is clear, but is much less certain in other vertebrates. Current evidence suggests that in fish, PGC-1α and PGC-1β may exhibit much less redundancy in the control of fatty acid oxidation and mitochondrial biogenesis compared to mammals. To assess these roles directly, we knocked down PGC-1α and PGC-1β expression with morpholinos in zebrafish embryos, and we investigated the resulting molecular and physiological phenotypes. First, we found no effects of either morpholinos on larval hatching, heart rates and oxygen consumption over the first few days of development. Next, at 3 days post fertilization (dpf), we confirmed by real time PCR a specific knock down of both coactivators, that resulted in a significant reduction in the transcript levels of citrate synthase (CS), 3-hydroxyacyl-CoA dehydrogenase (HOAD), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in both morphant groups. However, there was no effect on transcription factors' gene expression except for a marked reduction in estrogen related receptor α (ERRα) transcripts in PGC-1α morphants. Finally, we assessed whole embryonic enzyme activity for CS, cytochrome oxidase (COX), HOAD and carnitine palmitoyltransferase I (CPT-1) at 4 dpf. The only significant effect of the knockdown was a reduced CS activity in PGC-1α morphants and a counterintuitive increase of cytochrome oxidase activity in PGC-1β morphants. Overall, our results indicate that in larval zebrafish, PGC-1α and PGC-1β both play a role in regulating expression of important mitochondrial genes potentially through ERRα.

Low Citrate Synthase Activity Is Associated with Glucose Intolerance and Lipotoxicity.

Citrate synthase (CS) is a key mitochondrial enzyme. The aim of this study was to test the hypothesis that low CS activity impairs the metabolic health of mice fed a high fat diet (HFD) and promotes palmitate-induced lipotoxicity in muscle cells. C57BL/6J (B6) mice and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A), a strain which carries the A/J allele of CS on the B6 strain background, were fed HFD (45% kcal from fat) for 12 weeks. C2C12 mouse muscle cells were used to investigate effects of CS knockdown on cell viability and signalling after incubation in 0.8 mM palmitate. CS activity, but not that of β-hydroxyacyl-coenzyme-A dehydrogenase was lower in the gastrocnemius muscle and heart of B6.A mice compared to B6 mice (P < 0.001). During HFD feeding, glucose tolerance of mice decreased progressively and to a greater extent in B6.A females compared to B6 females, with males showing a similar trend. Body weight and fat gain did not differ between B6.A and B6 mice. After an 18 h incubation in 0.8 mM palmitate C2C12 muscle cells with ∼50% shRNA mediated reduction in CS activity showed lower (P < 0.001) viability and increased (P < 0.001) levels of cleaved caspase-3 compared to the scramble shRNA treated C2C12 cells. A/J strain variant of CS is associated with low enzyme activity and impaired metabolic health. This could be due to impaired lipid metabolism in muscle cells.

Hepatic Mitochondrial Defects in a Nonalcoholic Fatty Liver Disease Mouse Model Are Associated with Increased Degradation of Oxidative Phosphorylation Subunits

Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic mitochondrial dysfunction characterized by reduced ATP synthesis. We applied the 2H2O-metabolic labeling approach to test the hypothesis that the reduced stability of oxidative phosphorylation proteins contributes to mitochondrial dysfunction in a diet-induced mouse model of NAFLD. A high fat diet containing cholesterol (a so-called Western diet (WD)) led to hepatic oxidative stress, steatosis, inflammation and mild fibrosis, all markers of NAFLD, in low density cholesterol (LDL) receptor deficient (LDLR-/-) mice. In addition, compared with controls (LDLR-/- mice on normal diet), livers from NAFLD mice had reduced citrate synthase activity and ATP content, suggesting mitochondrial impairment. Proteome dynamics study revealed that mitochondrial defects are associated with reduced average half-lives of mitochondrial proteins in NAFLD mice (5.41 ± 0.46 versus 5.15 ± 0.49 day, p < 0.05). In particular, the WD reduced stability of oxidative phosphorylation subunits, including cytochrome b-c1 complex subunit 1 (5.9 ± 0.1 versus 3.4 ± 0.8 day), ATP synthase subunit α (6.3 ± 0.4 versus 5.5 ± 0.4 day) and ATP synthase F(0) complex subunit B1 of complex V (8.5 ± 0.6 versus 6.5 ± 0.2 day) (p < 0.05). These changes were associated with impaired complex III and F0F1-ATP synthase activities. Markers of mitophagy were increased, but proteasomal degradation activity were reduced in NAFLD mice liver, suggesting that ATP deficiency because of reduced stability of oxidative phosphorylation complex subunits contributed to inhibition of ubiquitin-proteasome and activation of mitophagy. In conclusion, the 2H2O-metabolic labeling approach shows that increased degradation of hepatic oxidative phosphorylation subunits contributed to mitochondrial impairment in NAFLD mice.

224 Burn Injury Induced Functional and Morphological Alterations in Skeletal Mitochondria in a Metabolically Characterized Animal Model

Altered mitochondrial biology and function is considered as an underlying mechanism of burn injury induced hypermetabolism and insulin resistance. Our previous work has characterized a burn mouse model with insulin resistance by in vivo clamp method accompanied with muscle insulin signal proteins in skeletal muscle. The present study aimed at exploring the morphology and functional changes of muscle mitochondrial in this characterized burn animal model. C57BL/6 mice (10–12 weeks) received 30% total body surface area (TBSA) full thickness burn injury or sham burn injury. Gastrocnemius and quadriceps muscles were collected on days 1, 3, and 7 after burn injury. Mitochondrial function was assessed by citrate synthase activity, mitochondrial membrane potential and NAD+/NADH ratio. Gene expression related to mitochondrial biogenesis, dynamics and mitochondrial DNA contents were quantified with real time quantitative PCR. The morphological changes were assessed by a quantitative 2-dimensional transmission electron microscopy (TEM). Our studies demonstrated that the major changes occurred on day 3. Mitochondrial dysfunction was revealed with significantly reduced citrate synthase activity (fig A, B, C), mitochondrial membrane potential and NAD+/NADH ratio. This was accompanied with reduced mitochondrial DNA contents and mRNA expression of cytochrome C (CytC), mitochondrial transcription factor A (TFAM), nuclear respiratory factor 1 (NRF1) and fission protein 1(Fis1) (fig D, E). TEM demonstrated that mitochondria became longer and branched with disrupted cristae as compared to sham burn animals (fig F, G). There was a time dependent reduction to number of cristae time-dependently (fig H). Since CytC is the major component of electron transport chain, NRF1 regulates transcription rregulating cellular growth and nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication, the latter is also regulated by TFAM and Fis1 is one of the mitochondrial fission factors. We conclude that the above molecular and morphological changes mitochondria are part of the mechanisms of metabolic aberrations caused by burn injury.

RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain

BackgroundThe construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host.ResultsTo demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously. Four-different single genes (the pyc, gltA, idsA, and glgC genes) repressions were successfully performed using the CRISPRi vectors, resulting significant mRNA reductions of the targets compared to a control. Subsequently, the phenotypes for the target gene-repressed strains were analyzed, showing the expected cell growth behaviors with different carbon sources. In addition, double gene repression (the idsA and glgC genes in a different order) by the CRISPRi resulted in an independent gene repression to each target gene simultaneously. To demonstrate an industrial application of the CRISPRi, citrate synthase (CS)-targeting DM1919 (l-lysine producer) strains with a sgRNA-gltA-r showed reduced CS activity, resulting in the improvement of l-lysine yield by 1.39-fold than the parental DM1919 (a lysine producer).ConclusionsSingle or double gene repression were successfully performed using the CRISPRi vectors and sequence specific sgRNAs. The CRISPRi can be applied for multiplex metabolic engineering to enhanced lysine production and it will promote the further rapid development of microbial cell factories of C. glutamicum.

H55N polymorphism is associated with low citrate synthase activity which regulates lipid metabolism in mouse muscle cells.

The H55N polymorphism in the Cs gene of A/J mice has been linked to low activity of the enzyme in skeletal muscles. The aim of the study was to test this hypothesis and examine effects of low citrate synthase (CS) activity on palmitate metabolism in muscle cells. Results of the study showed that carriers of the wild type (WT) Cs (C57BL/6J and Balb/cByJ mouse strains) had higher CS activity (p < 0.01) than carriers of the A/J variant (B6.A-(rs3676616-D10Utsw1)/KjnB6 and A/J mouse strains) in the heart, liver and gastrocnemius muscle. Furthermore, the recombinant CS protein of WT showed higher CS activity than the A/J variant. In C2C12 muscle cells the shRNA mediated 47% knockdown of CS activity reduced the rate of fatty acid oxidation compared to the control cells. In summary, our results are consistent with the hypothesis that H55N substitution causes a reduction in CS activity. Furthermore, low CS activity interferes with metabolic flexibility of muscle cells.

METABOLISM ANALYSIS IN MICE WITH REDUCED CITRATE SYNTHASE ACTIVITY

Background. Citrate synthase (CS) plays an important role in the regulation of carbohydrate oxidation. Variation in citrate synthase activity has an influence on metabolic changes. We tested the hypothesis that reduced mitochondrial CS activity could affect energy expenditure (EE) and respiratory quotient (RQ) in mouse model with an emphasizing on gender differences between tested strains. Methods. 16-week of age wild-type C57Bl/6J (B6) mouse strain, B6.A-(rs3676616-D10Utsw1)/Kjn (B6.A) and C57BL/6J-Chr 10A/J/NaJ (B6.A10) strains with reduced CS activity were studied in physiocage by the “Panlab” metabolism analysing equipment. The following parameters were calculated: EE (ml/min/kg^0.75), RQ, physical activity and rearing. Results. In female mice EE values were lower in B6.A10 strain compared to wild-type B6 strain. RQ values were similar in all tested mouse strains. In B6 mice EE was higher in females compared to males. Rearing was elevated in females of B6 mice compared to males. Conclusions. EE was lower in B6.A10 compared to B6 mice. Gender differences were noticed only in B6 mice: EE and rearing were significantly higher in female compared to male mice. Current study did not reveal any other association between reduced CS activity and EE or RQ variation in male and female mice.

Ovariectomized Highly Fit Rats Are Protected against Diet-Induced Insulin Resistance.

In the absence of exercise training, rats selectively bred for high intrinsic aerobic capacity (high-capacity running (HCR)) are protected against ovariectomy (OVX)-induced insulin resistance (IR) and obesity compared with those bred for low intrinsic aerobic capacity (low-capacity running (LCR)). This study determined whether OVX HCR rats remain protected with exposure to high-fat diet (HFD) compared with OVX LCR rats. Female HCR and LCR rats (n = 36; age, 27-33 wk) underwent OVX and were randomized to a standard chow diet (NC, 5% kcal fat) or HFD (45% kcal fat) ad libitum for 11 wk. Total energy expenditure, resting energy expenditure, spontaneous physical activity (SPA), and glucose tolerance were assessed midway, whereas fasting circulating metabolic markers, body composition, adipose tissue distribution, and skeletal muscle adenosine monophosphate-activated protein kinase (AMPK), and mitochondrial markers were assessed at sacrifice. Both HCR and LCR rats experienced HFD-induced increases in total and visceral adiposity after OVX. Despite similar gains in adiposity, HCR rats were protected from HFD-induced IR and reduced total energy expenditure observed in LCR rats (P < 0.05). This metabolic protection was likely attributed to a compensatory increase in SPA and associated preservation of skeletal muscle AMPK activity in HCR; however, HFD significantly reduced SPA and AMPK activity in LCR (P < 0.05). In both lines, HFD reduced citrate synthase activity, gene expression of markers of mitochondrial biogenesis (tFAM, NRF1, and PGC-1α), and protein levels of mitochondrial oxidative phosphorylation complexes I, II, IV, and V in skeletal muscle (all P < 0.05). After OVX, HCR and LCR rats differentially respond to HFD such that HCR increase while LCR decrease SPA. This "physical activity compensation" likely confers protection from HFD-induced IR and reduced energy expenditure in HCR rats.

Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice.

We have investigated the effects of in utero exposure to environmentally persistent free radicals (EPFRs) on growth, metabolism, energy utilization, and skeletal muscle mitochondria in a mouse model of diet-induced obesity. Pregnant mice were treated with laboratory-generated, combustion-derived particular matter (MCP230). The adult offspring were placed on a high-fat diet for 12 wk, after which we observed a 9.8% increase in their body weight. The increase in body size observed in the MCP230-exposed mice was not associated with increases in food intake but was associated with a reduction in physical activity and lower energy expenditure. The reduced energy expenditure in mice indirectly exposed to MCP230 was associated with reductions in skeletal muscle mitochondrial DNA copy number, lower mRNA levels of electron transport genes, and reduced citrate synthase activity. Upregulation of key genes involved in ameliorating oxidative stress was also observed in the muscle of MCP230-exposed mice. These findings suggest that gestational exposure to MCP230 leads to a reduction in energy expenditure at least in part through alterations to mitochondrial metabolism in the skeletal muscle.