Altered Expression Of Enzymes Research Articles

Increased Expression of Mevalonate Pathway-Related Enzymes in Angiotensin II-Induced Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is characterized by permanent luminal expansion and a high mortality rate due to aortic rupture. Despite the identification of abnormalities in the mevalonate pathway (MVA) in many diseases, including cardiovascular diseases, the potential impact of this pathway on AAA remains unclear. This study aims to investigate whether the expression of the MVA-related enzyme is altered during the progression of angiotensin II (Ang II) -induced AAA.Ang II 28D and Ang II 5D groups were continuously perfused with Ang II for 28 days and 5 days, respectively, and the Sham group was perfused with saline. The general and remodeling characteristics of AAA were determined by biochemical and histological analysis. Alteration of MVA-related enzyme expressions was revealed by western blot and single-cell RNA sequencing (scRNA-seq).The continuous Ang II infusion for 28 days showed significant aorta expansion and arterial remodeling. Although the arterial diameter slightly increased, the aneurysm formation was not found in Ang II induction for 5 days. MVA-related enzyme expression and activation of small GTP-binding proteins were significantly increased after Ang II-induced. As verified by scRNA-seq, the key enzyme gene expression was also higher in Ang II 28D. Similarly, it was detected that the expression levels of the above enzymes and the activity of small G proteins were elevated in the early stage of AAA as induced by Ang II infusion for 5 days.Continuous Ang II infusion-induced abdominal aortic expansion and arterial remodeling were accompanied by altered expression of key enzymes in the MVA.

SGLT2 inhibition leads to a restoration of hepatic and circulating metabolites involved in the folate cycle and pyrimidine biosynthesis.

Inhibition of sodium-glucose cotransporter 2 (SGLT2) by empagliflozin (EMPA) and other "flozins" can improve glycemic control under conditions of diabetes and kidney disease. Though they act on the kidney, they also offer cardiovascular and liver protection. Previously, we found that EMPA decreased circulating triglycerides and hepatic lipid and cholesterol esters in male TallyHo mice fed a high-milk-fat diet (HMFD). The goal of this study was to determine whether the liver protection is associated with a change in metabolic function by characterizing the hepatic and circulating metabolic and lipidomic profiles using targeted LC-MS. In both male and female mice, HMFD feeding significantly altered the circulating and hepatic metabolome compared with low-fat diet (LFD). Addition of EMPA resulted in the restoration of circulating orotate (intermediate in pyrimidine biosynthesis) and hepatic dihydrofolate (intermediate in the folate and methionine cycles) levels in males and acylcarnitines in females. These changes were partially explained by altered expression of rate-limiting enzymes in these pathways. This metabolic signature was not detected when EMPA was incorporated into an LFD, suggesting that the restoration requires the metabolic shift that accompanies the HMFD. Notably, the HMFD increased expression of 18 of 20 circulating amino acids in males and 11 of 20 in females, and this pattern was reversed by EMPA. Finally, we confirmed that SGLT2 inhibition upregulates ketone bodies including β-hydroxybutyrate. Collectively, this study highlights the metabolic changes that occur with EMPA treatment, and sheds light on the possible mechanisms by which this drug offers liver and systemic protection.NEW & NOTEWORTHY Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, have emerged as a new treatment option for individuals with type 2 diabetes that have positive impacts on kidney and cardiovascular disease. However, less is known about their impact on other tissues, including the liver. Here, we report that empagliflozin reduces hepatic steatosis that is associated with restoring metabolic intermediates in the folate and pyrimidine biosynthesis pathways. These changes may lead to new approaches to treat nonalcoholic fatty liver disease.

CAMP in budding yeast: Also a messenger for sucrose metabolism?

S. cerevisiae (or budding yeast) is an important micro-organism for sucrose-based fermentation in biotechnology. Yet, it is largely unknown how budding yeast adapts to sucrose transitions. Sucrose can only be metabolized when the invertase or the maltose machinery are expressed and we propose that the Gpr1p receptor signals extracellular sucrose availability via the cAMP peak to adapt cells accordingly. A transition to sucrose or glucose gave a transient cAMP peak which was maximally induced for sucrose. When transitioned to sucrose, cAMP signalling mutants showed an impaired cAMP peak together with a lower growth rate, a longer lag phase and a higher final OD600 compared to a glucose transition. These effects were not caused by altered activity or expression of enzymes involved in sucrose metabolism and imply a more general metabolic adaptation defect. Basal cAMP levels were comparable among the mutant strains, suggesting that the transient cAMP peak is required to adapt cells correctly to sucrose. We propose that the short-term dynamics of the cAMP signalling cascade detects long-term extracellular sucrose availability and speculate that its function is to maintain a fermentative phenotype at continuously low glucose and fructose concentrations.

Phosphate Limitation Responses in Marine Green Algae Are Linked to Reprogramming of the tRNA Epitranscriptome and Codon Usage Bias.

Marine algae are central to global carbon fixation, and their productivity is dictated largely by resource availability. Reduced nutrient availability is predicted for vast oceanic regions as an outcome of climate change; however, there is much to learn regarding response mechanisms of the tiny picoplankton that thrive in these environments, especially eukaryotic phytoplankton. Here, we investigate responses of the picoeukaryote Micromonas commoda, a green alga found throughout subtropical and tropical oceans. Under shifting phosphate availability scenarios, transcriptomic analyses revealed altered expression of transfer RNA modification enzymes and biased codon usage of transcripts more abundant during phosphate-limiting versus phosphate-replete conditions, consistent with the role of transfer RNA modifications in regulating codon recognition. To associate the observed shift in the expression of the transfer RNA modification enzyme complement with the transfer RNAs encoded by M. commoda, we also determined the transfer RNA repertoire of this alga revealing potential targets of the modification enzymes. Codon usage bias was particularly pronounced in transcripts encoding proteins with direct roles in managing phosphate limitation and photosystem-associated proteins that have ill-characterized putative functions in "light stress." The observed codon usage bias corresponds to a proposed stress response mechanism in which the interplay between stress-induced changes in transfer RNA modifications and skewed codon usage in certain essential response genes drives preferential translation of the encoded proteins. Collectively, we expose a potential underlying mechanism for achieving growth under enhanced nutrient limitation that extends beyond the catalog of up- or downregulated protein-encoding genes to the cell biological controls that underpin acclimation to changing environmental conditions.

The role of GCNT1 mediated O-glycosylation in aggressive prostate cancer

Prostate cancer is the most common cancer in men and a major cause of cancer related deaths worldwide. Nearly all affected men develop resistance to current therapies and there is an urgent need to develop new treatments for advanced disease. Aberrant glycosylation is a common feature of cancer cells implicated in all of the hallmarks of cancer. A major driver of aberrant glycosylation in cancer is the altered expression of glycosylation enzymes. Here, we show that GCNT1, an enzyme that plays an essential role in the formation of core 2 branched O-glycans and is crucial to the final definition of O-glycan structure, is upregulated in aggressive prostate cancer. Using in vitro and in vivo models, we show GCNT1 promotes the growth of prostate tumours and can modify the glycome of prostate cancer cells, including upregulation of core 2 O-glycans and modifying the O-glycosylation of secreted glycoproteins. Furthermore, using RNA sequencing, we find upregulation of GCNT1 in prostate cancer cells can alter oncogenic gene expression pathways important in tumour growth and metastasis. Our study highlights the important role of aberrant O-glycosylation in prostate cancer progression and provides novel insights regarding the mechanisms involved.

Maternal diabetes increases FOXO1 activation during embryonic cardiac development

Maternal diabetes is known to affect heart development, inducing the programming of cardiac alterations in the offspring's adult life. Previous studies in the heart of adult offspring have shown increased activation of FOXO1 (a transcription factor involved in a wide variety of cellular functions such as apoptosis, cellular proliferation, reactive oxygen species detoxification, and antioxidant and pro-inflammatory processes) and of target genes related to inflammatory and fibrotic processes. In this work, we aimed to evaluate the effects of maternal diabetes on FOXO1 activation as well as on the expression of target genes relevant to the formation of the cardiovascular system during organogenesis (day 12 of gestation). The embryonic heart from diabetic rats showed increased active FOXO1 levels, reduced protein levels of mTOR (a nutrient sensor regulating cell growth, proliferation and metabolism) and reduced mTORC2-SGK1 pathway, which phosphorylates FOXO1. These alterations were related to increases in the levels of 4-hydroxynonenal (an oxidative stress marker) and increased mRNA levels of inducible nitric oxide synthase, angiopoietin-2 and matrix metalloproteinase-2 (MMP2) (all FOXO1 target genes relevant for cardiac development). Results also showed increased extracellular and intracellular immunolocalization of MMP2 in the myocardium and its projection into the lumen of the cavity (trabeculations) together with decreased immunostaining of connexin 43, a protein relevant for cardiac function that is target of MMP2. In conclusion, increases in active FOXO1 induced by maternal diabetes initiate early during embryonic heart development and are related to increases in markers of oxidative stress and of proinflammatory cardiac development, as well to an altered expression of proteolytic enzymes that regulate connexin 43. These alterations may lead to an altered programming of cardiovascular development in the embryonic heart of diabetic rats.

High doses of rosuvastatin induce impaired branched-chain amino acid catabolism and lead to insulin resistance.

What is the central question of this study? Is there a risk of developing diabetes associated with statin treatment? What is the underlying mechanism of the increased incidence rate of new-onset diabetes in patients treated with rosuvastatin? What is the main finding and its importance? Rosuvastatin therapy reduced intraperitoneal glucose tolerance and changed the catabolism of branched-chain amino acid (BCAAs) in white adipose tissue and skeletal muscle. Protein phosphatase 2Cm knockdown completely abolished the effects of insulin and rosuvastatin on glucose absorption. This study provides mechanistic support for recent clinical data on rosuvastatin-related new-onset diabetes and underscores the logic for intervening in BCAA catabolism to prevent the harmful effects of rosuvastatin. Accumulating evidence indicates that patients treated with rosuvastatin have an increased risk of developing new-onset diabetes. However, the underlying mechanism remains unclear. In this study, we administered rosuvastatin (10mg/kg body weight) to male C57BL/6J mice for 12weeks and found that oral rosuvastatin dramatically reduced intraperitoneal glucose tolerance. Rosuvastatin-treated mice showed considerably higher serum levels of branched-chain amino acids (BCAAs) than control mice. They also showed dramatically altered expression of BCAA catabolism-related enzymes in white adipose tissue and skeletal muscle, including downregulated mRNA expression of BCAT2 and protein phosphatase 2Cm (PP2Cm) and upregulated mRNA expression of branched-chain ketoacid dehydrogenase kinase (BCKDK). The levels of BCKD in the skeletal muscle were reduced in rosuvastatin-treated mice, which was associated with lower PP2Cm protein levels and increased BCKDK levels. We also investigated the effects of rosuvastatin and insulin administration on glucose metabolism and BCAA catabolism in C2C12 myoblasts. We observed that incubation with insulin enhanced glucose uptake and facilitated BCAA catabolism in C2C12 cells, which was accompanied by elevated Akt and glycogen synthase kinase 3 β (GSK3β) phosphorylation. These effects of insulin were prevented by co-incubation of the cells with 25μM rosuvastatin. Moreover, the effects of insulin and rosuvastatin administration on glucose uptake and Akt and GSK3β signaling in C2C12 cells were abolished when PP2Cm was knocked down. Although the relevance of these data, obtained with high doses of rosuvastatin in mice, to therapeutic doses in humans remains to be elucidated, this study highlights a potential mechanism for the diabetogenic effects of rosuvastatin, and suggests that BCAA catabolism could be a pharmacological target for preventing the adverse effects of rosuvastatin.

Estrogens in Adipose Tissue Physiology and Obesity-Related Dysfunction.

Menopause-related decline in estrogen levels is accompanied by a change in adipose tissue distribution from a gynoid to an android and an increased prevalence of obesity in women. These unfavorable phenomena can be partially restored by hormone replacement therapy, suggesting a significant role for estrogen in the regulation of adipocytes' function. Indeed, preclinical studies proved the involvement of these hormones in adipose tissue development, metabolism, and inflammatory activity. However, the relationship between estrogen and obesity is bidirectional. On the one hand-their deficiency leads to excessive fat accumulation and impairs adipocyte function, on the other-adipose tissue of obese individuals is characterized by altered expression of estrogen receptors and key enzymes involved in their synthesis. This narrative review aims to summarize the role of estrogen in adipose tissue development, physiology, and in obesity-related dysfunction. Firstly, the estrogen classification, synthesis, and modes of action are presented. Next, their role in regulating adipogenesis and adipose tissue activity in health and the course of obesity is described. Finally, the potential therapeutic applications of estrogen and its derivates in obesity treatment are discussed.

Loss of autism-candidate CHD8 perturbs neural crest development and intestinal homeostatic balance.

Individuals with mutations in CHD8 present with gastrointestinal complaints, yet the underlying mechanisms are understudied. Here, using a stable constitutive chd8 mutant zebrafish model, we found that the loss of chd8 leads to a reduced number of vagal neural crest cells (NCCs), enteric neural and glial progenitors, emigrating from the neural tube, and that their early migration capability was altered. At later stages, although the intestinal colonization by NCCs was complete, we found the decreased numbers of both serotonin-producing enterochromaffin cells and NCC-derived serotonergic neurons, suggesting an intestinal hyposerotonemia in the absence of chd8 Furthermore, transcriptomic analyses revealed an altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. The tissue examination of chd8 mutants revealed a thinner intestinal epithelium accompanied by an accumulation of neutrophils and the decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole intestines showed a global disruption of the immune balance with a perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, NCC development, intestinal homeostasis, and autism-associated gastrointestinal complaints.

Role of a Disease-associated ST3Gal-4 in Non-small Cell Lung Cancer.

Sialylation promotes tumorigenesis by affecting various cancer-related events, including apoptosis inhibition, cell growth, invasion, migration, metastasis, chemo-resistance, and immunomodulation in favor of tumor progression. An altered expression of sialyltransferase enzymes is responsible for synthesizing various tumor-associated sialylated structures. In the present study, our findings have revealed a significant up-regulation of ST3Gal-4 transcript in the two major subtypes of NSCLC cell lines [squamous cell carcinoma cell line (NCI-H520) and adenocarcinoma cell line (A549)]. Thus, the role of the ST3Gal-4 gene was assessed on cancer-associated signal transduction pathways in these cells in view of proliferation, invasion, and migration. ST3Gal-4 was silenced by transfection of both the cell lines with esi-ST3Gal-4-RNA, which RT-PCR and western immunoblotting confirmed. Silencing of ST3Gal-4 resulted in a decreased expression of MAL-I interacting membrane-HSP60, identified earlier as an α2,3-sialylated glycoprotein, thus pointing towards the possible role of ST3Gal-4 in its sialylation. The proliferation, invasion, and migration of both types of NSCLC cells were reduced significantly in the ST3Gal-4 silenced cells. Our findings were substantiated by the down-regulation of β-catenin and E-cadherin, a reduced expression of activated AKT1, ERK1/2, and NF-ƙB in these cells. We propose that ST3Gal-4 may be the disease-associated sialyltransferase involved in α2,3 sialylation of the membrane proteins, including HSP60 of the NSCLC cells. This may lead to the conformational alteration of these proteins, required for the activation of E-cadherin/β-catenin, AKT, and ERK/NF-ƙB mediated signal transduction pathways in these cells, resulting in their proliferation, invasion, and migration.

Dual HDAC/BRD4 Inhibitors Relieves Neuropathic Pain by Attenuating Inflammatory Response in Microglia After Spared Nerve Injury

Despite the effort on developing new treatments, therapy for neuropathic pain is still a clinical challenge and combination therapy regimes of two or more drugs are often needed to improve efficacy. Accumulating evidence shows an altered expression and activity of histone acetylation enzymes in chronic pain conditions and restoration of these aberrant epigenetic modifications promotes pain-relieving activity. Recent studies showed a synergistic activity in neuropathic pain models by combination of histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) inhibitors. On these premises, the present study investigated the pharmacological profile of new dual HDAC/BRD4 inhibitors, named SUM52 and SUM35, in the spared nerve injury (SNI) model in mice as innovative strategy to simultaneously inhibit HDACs and BETs. Intranasal administration of SUM52 and SUM35 attenuated thermal and mechanical hypersensitivity in the absence of locomotor side effects. Both dual inhibitors showed a preferential interaction with BRD4-BD2 domain, and SUM52 resulted the most active compound. SUM52 reduced microglia-mediated spinal neuroinflammation in spinal cord sections of SNI mice as showed by reduction of IBA1 immunostaining, inducible nitric oxide synthase (iNOS) expression, p65 nuclear factor-κB (NF-κB) and p38 MAPK over-phosphorylation. A robust decrease of the spinal proinflammatory cytokines content (IL-6, IL-1ß) was also observed after SUM52 treatment. Present results, showing the pain-relieving activity of HDAC/BRD4 dual inhibitors, indicate that the simultaneous modulation of BET and HDAC activity by a single molecule acting as multi-target agent might represent a promise for neuropathic pain relief.

Loss of Pkm2 Alters Myocardial Metabolism and Increases Oxidative Stress After Infarction

IntroductionPyruvate kinase (PKM1) directs pyruvate to the Krebs cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia‐mediated switch to the alternatively spliced isoform PKM2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that upregulation of PKM2 can alter metabolic pathways by promoting glycolysis, and that after injury, this can preserve ATP production, protecting the heart from the stresses of hypoxia and injury.MethodsGlobal Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA‐seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semi‐quantitative real‐time PCR was used to confirm changes in selected genes of interest.ResultsLoss of Pkm2 moderately altered gene expression at baseline (q<0.05, FDR<0.05). Notably, the mitochondrial gene COX3 was downregulated in Pkm2 KO hearts. 68 genes were differentially expressed in Pkm2 KO hearts after MI, not observed in control MI hearts. MI of Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, mitochondrial uncoupling, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. Semi‐quantitative PCR of selected genes involved in glucose metabolism confirmed RNA‐seq results.ConclusionsRNA‐seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Pkm2 KO hearts also showed increased abundance of pro‐apoptotic markers which may be a result of increased oxidative stress.

Gene expression levels of DNA methyltransferase enzymes in Shank3-deficient mouse model of autism during early development.

Objectives. The balance between DNA methylation and demethylation is crucial for the brain development. Therefore, alterations in the expression of enzymes controlling DNA methylation patterns may contribute to the etiology of neurodevelopmental disorders, including autism. SH3 and multiple ankyrin repeat domains 3 (Shank3)-deficient mice are commonly used as a well-characterized transgenic model to investigate the molecular mechanisms of autistic symptoms. DNA methyltransferases (DNMTs), which modulate several cellular processes in neurodevelopment, are implicated in the pathophysiology of autism. In this study, we aimed to describe the gene expression changes of major Dnmts in the brain of Shank3-deficient mice during early development. Methods and Results. The Dnmts gene expression was analyzed by qPCR in 5-day-old homo-zygous Shank3-deficient mice. We found significantly lower Dnmt1 and Dnmt3b gene expression levels in the frontal cortex. However, no such changes were observed in the hippocampus. However, significant increase was observed in the expression of Dnmt3a and Dnmt3b genes in the hypothalamus of Shank3-deficient mice. Conclusions. The present data indicate that abnormalities in the Shank3 gene are accompanied by an altered expression of DNA methylation enzymes in the early brain development stages, therefore, specific epigenetic control mechanisms in autism-relevant models should be more extensively investigated.

Abstract P443: Loss Of Pkm2 Alters Myocardial Metabolism And Increases Oxidative Stress After Infarction

Introduction: Pyruvate kinase (Pkm1) directs pyruvate to the TCA cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia-mediated switch to the alternatively spliced isoform Pkm2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that the upregulation of Pkm2 alters metabolic pathways after injury to promote glycolysis and preserve ATP production in hypoxia, which protects the heart from the stresses of hypoxia and injury. Methods: Global Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA-seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semiquantitative real-time PCR (qPCR) was used to confirm changes in selected genes of interest. Results: Loss of Pkm2 did not alter gene expression substantially at baseline. 68 genes were differentially expressed in Pkm2 KO hearts after MI (q<0.05, FDR<0.05) not observed in control MI hearts. MI in Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. qPCR of selected genes involved in glucose metabolism confirmed RNA-seq results. Conclusions: RNA-seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Conversely, Pkm2 KO hearts showed increased abundance of pro-apoptotic markers which may be a result of increased oxidative stress.

Influence of Selected Carbon Nanostructures on the CYP2C9 Enzyme of the P450 Cytochrome.

Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.

Impact of obese levels on the hepatic expression of nuclear receptors and drug-metabolizing enzymes in adult and offspring mice

The prevalence of obesity-associated conditions raises new challenges in clinical medication. Although altered expression of drug-metabolizing enzymes (DMEs) has been shown in obesity, the impacts of obese levels (overweight, obesity, and severe obesity) on the expression of DMEs have not been elucidated. Especially, limited information is available on whether parental obese levels affect ontogenic expression of DMEs in children. Here, a high-fat diet (HFD) and three feeding durations were used to mimic different obese levels in C57BL/6 mice. The hepatic expression of five nuclear receptors (NRs) and nine DMEs was examined. In general, a trend of induced expression of NRs and DMEs (except for Cyp2c29 and 3a11) was observed in HFD groups compared to low-fat diet (LFD) groups. Differential effects of HFD on the hepatic expression of DMEs were found in adult mice at different obese levels. Family-based dietary style of an HFD altered the ontogenic expression of DMEs in the offspring older than 15 days. Furthermore, obese levels of parental mice affected the hepatic expression of DMEs in offspring. Overall, the results indicate that obese levels affected expression of the DMEs in adult individuals and that of their children. Drug dosage might need to be optimized based on the obese levels.

Abstract 197: Metabolic Syndrome Severely Alters Major Metabolic Pathways in the Pig Myocardium

Introduction: Obesity and metabolic syndrome (MetS) are rapidly expanding worldwide. One of the most concerning implications of this trend is the association between MetS and the increased risk of cardiovascular disease (CVD) . There is a little data on cross-talk between metabolomics alterations and myocardial functionality. Hypothesis: Because of the pleiotropic nature of CVD and of MetS, it is unlikely that single metabolite or metabolic pathway underlies the development of CVD in MetS. Understanding the global metabolic state of the MS myocardium using high throughput metabolomics and transcriptomics may provide novel insights. Methods: Yorkshire swine received obesogenic diet (MetS). Control (LD) animals received regular chow. Tissue samples from identical myocardial left ventricular territories are analyzed by: targeted LC/MS-MS for LV polar metabolites, MetaboAnalyst 4.0; RNA-seq. Results: MetS vs LD demonstrates: elevated blood fasting glucose, triglycerides, LDL, cholesterol, blood pressure. LC-MS-MS shows diet impact on 287 metabolites availability in MetS together with altered gene expression of traditional enzymes that are involved in C - glycolysis, fatty acids metabolism, ketone body formation, transport, and utilization. Conclusions: Transcriptionally controlled fuel gauge in the MetS heart enzymatically turns down glucose and fatty acids oxidation and activates nutritional ketolysis. This study represents a significant step towards characterizing the metabolic state of the heart in MetS and provides potential avenues for therapeutic advance.

Altered Antioxidant and Inflammatory Signaling by Fibroblasts in the Hypertensive Heart

In response to hypertensive stimuli, like angiotensin II (AngII), cardiac fibroblasts (CFs) transform into an activated phenotype, which triggers increased collagen deposition and pro‐inflammatory signals. Transforming growth factor beta 1 (TGF‐β1) and reactive oxygen species are important modulators of AngII‐induced cardiac fibrosis and CF activation. The extent to which the AngII‐induced changes in CF phenotype persist in the absence of ongoing stimulation is not known. The objective of the present study was to determine the persistent impact of chronic AngII infusion on CF phenotype. Specifically, we performed an in vitro characterization of CFs isolated from rats chronically treated with AngII (in vivo) or saline (vehicle) and investigated acute (in vitro) responses to AngII thereafter. Adult male SD rats were administered AngII (200 ng/kg/min) or saline for 4 weeks. In half of the rats in each group, we measured mean arterial pressure (MAP), weighed the left ventricle (LV), and fixed a section of LV for assessment of collagen deposition (Sirius Red). In remaining rats, CFs were isolated and passaged to P1. CFs were treated in vitro with AngII (1uM) or vehicle for 72 hr. TGF‐β1 and monocyte chemoattractant protein 1 (MCP‐1) were assessed in culture media. Expression of pro‐/anti‐oxidant proteins NADPH oxidase 2 (Nox2), catalase and superoxide dismutase 1 (SOD1) were determined by western blot. Ang II increased MAP (43%, p<0.05), LV/BW (30%, p<0.05), and LV collagen deposition. TGF‐β1 release was not different from CFs isolated from saline vs. AngII infused rats. However, in vitro incubation with AngII significantly increased TGF‐β1 secretion in all CFs. CFs isolated from AngII‐infused rats secreted elevated levels of MCP‐1 (p<0.05), when compared to control CFs. MCP‐1 levels were not further modified by acute AngII treatment. Basal expression of Nox2, SOD1, and catalase was similar between CFs isolated from control vs. AngII‐infused rats. Acute AngII tended to increase Nox2 expression in CFs isolated from vehicle and AngII‐treated rats, whereas AngII only increased SOD1 expression (46%, p<0.05) in CFs from AngII‐infused rats. In vitro stimulation with AngII tended to increase catalase in CFs isolated from control rats, but reduced (20%, p<0.05) expression in CFs isolated from AngII infused rats. Taken together, these data demonstrate that CFs isolated from an actively remodeling myocardium continue to secrete elevated levels of MCP‐1, but not TGF‐β1, in vitro. Moreover, CFs from AngII‐infused rats displayed altered expression of antioxidant enzymes following AngII in vitro, which may play a role in worsened inflammatory signaling and resultant pro‐fibrotic activity. Future studies will further elucidate the fibrotic and inflammatory characteristics of CFs from the remodeling myocardium and determine the extent to which this persistent phenotype alters the future responsiveness to a pathogenic stimulus.Support or Funding InformationSpringboard Grant, UA College of Medicine – PhoenixThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MMP-9-1562 C/T single nucleotide polymorphism associates with increased MMP-9 level and activity during papillary thyroid carcinoma progression

Papillary thyroid carcinoma (PTC), a common form of thyroid malignancy, displays significant variations in clinical features and outcome. The malignant transformation of the thyroid is driven by altered expression of many matrix-modulating enzymes, including matrix metalloproteinase-9 (MMP-9). A single nucleotide polymorphism in its promotor (-1562 C/T) is suspected to cause overexpression of MMP-9, which in turn contributes to development of a tumour unfavourable phenotype. The aim of this study was to investigate the impact of MMP-9 promotor genotype on MMP-9 expression in PTC samples, and to assess its value as a possible risk factor for developing PTC or its aggressive phenotype. A total of 105 PTC patients and 43 healthy controls were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In order to estimate MMP-9 expression, PTC tissue sections were stained immunohistochemically. Statistical analysis showed that PTC cases and controls did not differ significantly in genotype frequencies (OR= 2.27, CI= 0.854-6.022). In PTC samples, the presence of the T allele was accompanied by elevated MMP-9 expression (p= 0.047) as well as a higher risk of developing extrathyroid extensions (p= 0.037) and high TNM stages (p= 0.009). Moreover, we observed overexpression of MMP-9 in cases presenting with extrathyroid invasion (p= 0.001), lymph node metastasis (p= 0.028), large tumour size (p= 0.031) and advanced stage (p= 0.005) compared to indolent tumours, along with enhanced enzymatic activity demonstrated by in situ zymography. Data suggests that MMP-9 (-1562 C/T) does not facilitate predisposition for PTC but affects the disease course by modulating MMP-9 expression. Genotyping MMP-9 provides important information which may prove beneficial in risk stratification of PTC patients.

67P - Association between altered expression of key enzymes involved in kynurenine pathway with clinical outcome in patients with different grades of astrocytoma

67P - Association between altered expression of key enzymes involved in kynurenine pathway with clinical outcome in patients with different grades of astrocytoma