Integrated quantum chemical and in vitro investigation of Capsanthin antioxidant activity: Mechanism (HAT), cultivar variability, enhanced bioavailability, and key gene expression in peppers.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Metabolic scaling, the inverse correlation of metabolic rates to body mass, has been appreciated for more than 80 years. Studies of metabolic scaling have largely been restricted to mathematical modeling of caloric intake and oxygen consumption, and mostly rely on computational modeling. The possibility that other metabolic processes scale with body size has not been comprehensively studied. To address this gap in knowledge, we employed a systems approach including transcriptomics, proteomics, and measurement of in vitro and in vivo metabolic fluxes. Gene expression in livers of five species spanning a 30,000-fold range in mass revealed differential expression according to body mass of genes related to cytosolic and mitochondrial metabolic processes, and to detoxication of oxidative damage. To determine whether flux through key metabolic pathways is ordered inversely to body size, we applied stable isotope tracer methodology to study multiple cellular compartments, tissues, and species. Comparing C57BL/6 J mice with Sprague-Dawley rats, we demonstrate that while ordering of metabolic fluxes is not observed in in vitro cell-autonomous settings, it is present in liver slices and in vivo. Together, these data reveal that metabolic scaling extends beyond oxygen consumption to other aspects of metabolism, and is regulated at the level of gene and protein expression, enzyme activity, and substrate supply. Editor's evaluation Key metabolic processes have been shown to scale inversely with the body mass of different animals. This study provides direct evidence for metabolic scaling of key metabolic fluxes in the livers of mice and rats, as well as species-specific differences in the transcription and expression of enzymes involved in energy metabolism that could contribute to metabolic scaling. The finding suggests that metabolic scaling likely reflects multiple levels of regulation and have broad implications for studying animal metabolism and physiology. https://doi.org/10.7554/eLife.78335.sa0 Decision letter Reviews on Sciety eLife's review process Introduction In 1932, Max Kleiber published a seminal study (Kleiber, 1932), integrating prior reports demonstrating a phenomenon that came to be termed 'Kleiber's law,' or the principle of metabolic scaling. Metabolic scaling refers to the phenomenon that the metabolic processes in many animals, if not all, scale inversely to three-quarters of their body mass (West et al., 1997). In simpler terms, there is a reduction in metabolic rate as body size increases. For example, an elephant is 25 million times larger than a fruit fly, yet its energy expenditure is only 20 thousand times higher; thus, from the fruit fly to elephant, the metabolic rate per gram of body weight scales down 1250 times. While there is experimental evidence for metabolic scaling from bacteria to large mammals, data have been generated almost exclusively from observations of caloric intake and oxygen consumption, with gene and protein expression, and substrate fluxes almost entirely unexplored. The concept of hierarchical regulation, whereby gene expression initiates the cascade that allows for the flux of metabolic pathways (Rossell et al., 2005; Suarez and Moyes, 2012), provides a systems framework to begin to understand scaling. Beginning at the transcriptional level, we studied liver gene expression across five species: mice (Mus musculus), rats (Rattus norvegicus), monkeys (Macaca mulatta), humans (Homo sapiens), and cattle (bos taurus), species with a 30,000-fold range of average body weight in adults (from 30 g in mice, to 900 kg in cattle). Numerous metabolic genes related to glycolysis, gluconeogenesis, fatty acid metabolism, oxygen consumption, electron transport, and redox function, and detoxification of oxidative damage, were expressed at levels inverse to body size. Further analysis of liver proteomics revealed that approximately half of the genes in the liver that were expressed inversely proportionally to body size at the transcriptional level, were also expressed at levels inversely proportional to body size at the level of protein expression. To determine if gene and protein expression would correlate with enzyme activity and metabolic flux, we performed a comprehensive assessment of liver metabolism in vivo and in vitro using modified Positional Isotopomer NMR Tracer Analysis (PINTA) (Perry et al., 2017b) and stable isotope-derived turnover (Perry et al., 2015) methods. Our analysis shows that rats exhibit lower metabolic rates when compared to mice in and ex vivo; however, no significant differences were observed when we isolated hepatocytes and cultured them in vitro under identical conditions. Taken together, this study demonstrates the variation of metabolic fluxes according to body size, extending prior studies of metabolic scaling, and provides unique insight into the regulation of metabolic flux across species. Results Genes within the liver that are expressed inversely proportional to body weight are predominantly metabolic genes We examined gene expression in livers from mice (Mus musculus), rats (Rattus norvegicus), monkeys (Macaca mulatta), humans (Homo sapiens), and cattle (Bos taurus). Using recent advances in high throughput mRNA sequencing and bioinformatics tools that allow for intra-species data preprocessing (Bray et al., 2016; Conesa et al., 2016; Ritchie et al., 2015), we searched for a set of genes in the liver, the metabolic hub of mammals, whose expression correlates inversely with body mass. After normalizing for differences in transcript length and abundance across species, we filtered out genes that followed the pattern of mouse >rat > monkey >human > cow. The genes that met these criteria were predominantly related to metabolic pathways, including pyruvate metabolism, amino acid metabolism, and glucose metabolism (Figure 1A). Genes from this list were further restricted to genes involved in amino acid, carbohydrate, energy, lipid, vitamin, and TCA cycle metabolism, and demonstrated a range of degrees of inverse correlation with body mass, with only TCA cycle genes clustering together (Figure 1B). Figure 1 Download asset Open asset Genes that follow the pattern of allometric scaling are most strongly related to metabolism. (A) KEGG Pathway enrichment of all genes that are expressed with an inverse correlation to body mass, and (B) clustering heatmap of scaled genes that belong to one of six Reactome metabolic superpathways. All samples were obtained from males. For clarity, the human gene (and style of writing human gene names) are shown. RAPGEF, rap guanine nucleotide exchange factor; ELOVL2, Elongation of Very Long Chain Fatty Acids-Like 2; MDH1, malate dehydrogenase 1; LIPE, hormone-sensitive lipase E; PANK1, pantothenate kinase 1; PGK1, phosphoglycerate kinase 1; SDC4, syndecan 4; ALDH7A1, aldehyde dehydrogenase 7 family member A1; GPX1, glutathione peroxidase 1; GLUL, glutamate-ammonia ligase; SORD, sorbitol dehydrogenase; TDO2, tryptophan 2,3-dioxygenase; DLST, dihydrolipoamide S-succinyltransferase; ACACA, acetyl-CoA carboxylase-alpha; ADIPOR1, adiponectin receptor-1; GPT, glutamic-pyruvate transaminase; HS3ST3B1, heparan sulfate-glucosamine 3-sulfotransferase 3B1; PSMD5, proteasome 26 S subunit, non-ATPase-5; COX8A, cytochrome c oxidase subunit 8 A; NDUFA9, NADH:ubiquinone oxidoreductase subunit A9. Genes encoding enzymes involved in hepatic metabolism are expressed inversely proportionally to body mass and involve metabolite detoxification, intertissue metabolism, substrate metabolism, electron transport, and NAD metabolism In order to further understand the functional aspects of the metabolic genes that are expressed inversely proportionally to body size, the gene list from Figure 1B was categorized into several functional categories, converging on optimizing energy provision, oxidative metabolism, and damage control from oxidative stress and ammonia (Figure 2). Furthermore, to understand whether or not certain genes that are expressed inversely proportionally to body size involved anabolic or catabolic processes, they were further classified by their properties to be energy suppliers or consumers. Eleven of sixteen critical metabolic enzymes that scaled required molecular oxygen, NAD+/NADH, or ATP/ADP for function, possibly indicating exquisite regulation of energy-consuming processes at the individual gene level. Genes involved in the detoxication of lipid peroxidation-derived aldehydes (ALDH7A1), hydrogen peroxide (GPX1), and ammonia (GLUL) suggest scaling of damage control mechanisms that are associated with increased oxidative metabolism across species (Figure 2A). The inverse correlation between body size and expression of genes that are associated with interorgan crosstalk is consistent with scaling in vivo which would not be expected in plated cells. For example, the differentially expressed genes include GPT1, which is involved in recycling skeletal muscle-derived alanine back to liver-derived glucose (Felig and Wahren, 1971; Petersen et al., 2019), and the adiponectin receptor (ADIPOR1), which binds an adipose tissue-derived hormone that regulates gluconeogenesis and fatty acid oxidation (Lin and Accili, 2011; Li et al., 2020 Figure 2B). Genes involved in fatty acid metabolism included the rate-limiting steps of the synthesis of CoA (PANK1), of de novo fatty acid synthesis (ACACA), and of fatty acid elongation (ELOVL2), in addition to the oxidation of diacylglycerols (LIPE) (Figure 2C). NAD and ATP-dependent genes involved in glycolysis (PDK1), fructose/glucose metabolism (SORD1), and DLST of the TCA cycle also correlated inversely with body size (Figure 2D–E). Differentially regulated genes also couple oxygen consumption to NAD provision (MDH1, TDO2), and are involved with the function of the electron transport chain (subunits of complex I, NDUFA9, and complex IV, COX8A, which catalyzes oxygen accepting the final electrons of the electron transport chain) (Figure 2F–G). Figure 2 with 1 supplement see all Download asset Open asset Metabolic genes that are expressed inversely proportionally to body size implicate key pathways in substrate and nucleotide supply, glucose and fatty acid flux, oxygen consumption, and detoxification pathways. mRNA expression of key regulatory genes related to metabolite detoxication (A), intertissue metabolism (B), fatty acid metabolism (C), glucose metabolism (D), tricarboxylic acid (TCA) cycle, NAD metabolism (F), and the electron transport chain (G) in mice, rats, monkeys, humans, and cattle. Bars denote expression levels by an organism, following the same order shown in the cartoon of organisms. Expression was normalized to counts per million and was then further normalized for sequencing depth and transcript length. All genes met an adjusted p-value threshold of 0.01 using a one-way ANOVA with the Bonferroni correction for multiple comparisons. All samples were obtained from males (n=2 replicates per species). ALDH7A1, aldehyde dehydrogenase 7 family member A1; GPX1, glutathione peroxidase 1; GLUL, glutamate-ammonia ligase; GPT, glutamic-pyruvate transaminase; ADIPOR1, adiponectin receptor-1; LIPE, hormone-sensitive lipase E; PANK1, pantothenate kinase 1; ACACA, acetyl-CoA carboxylase-alpha; ELOVL2, Elongation of Very Long Chain Fatty Acids-Like 2; SORD, sorbitol dehydrogenase; PGK1, phosphoglycerate kinase 1; DLST, dihydrolipoamide S-succinyltransferase; TDO2, tryptophan 2,3-dioxygenase; MDH1, malate dehydrogenase 1; NDUFA9, NADH:ubiquinone oxidoreductase subunit A9; COX8A, cytochrome c oxidase subunit 8 A. Figure 2—source data 1 Source data for Figure 2 and Figure 2—figure supplement 1. https://cdn.elifesciences.org/articles/78335/elife-78335-fig2-data1-v1.xlsx Download elife-78335-fig2-data1-v1.xlsx To examine the possibility that the inverse correlation between body mass and gene expression observed in the transcriptomics analysis could be a consequence of global alterations in mRNA (for example, as a consequence of alterations in RNA turnover rates), we performed targeted quantitative polymerase chain reaction (qPCR), measuring in liver tissue abundance of mRNA encoding several enzymes that were found to scale in the five-species transcriptomics analysis, relative to the common housekeeping gene β-actin (Actb). We found that all three enzymes (Glul, Lipe, and Dlst) scaled relative to Actb (Figure 2—figure supplement 1A–C), whereas structural genes (collagenase 3 [Mmp3] and Larp1) did not (Figure 2—figure supplement 1D–E), indicating that the differences in metabolic gene expression observed across species is likely not a result of global changes in RNA levels. In addition to transcriptomics, we assessed proteomics data to evaluate the protein levels corresponding to the genes that were found to be expressed inversely proportionally to body size at the level of mRNA expression. Our proteomics data were limited to mouse, rat, and human, as all the open-source proteomic databases that we identified lacked data from monkey or cow. An important limitation for finding such data is that even with careful post-processing, we cannot combine data from different studies, because differences in methods of tissue preparation may influence results. Therefore, we were limited to a single experiment that had generated proteomics data for mouse, rat, and human using the same experimental procedures. The dataset contained protein expression corresponding to eight of the twenty genes identified to scale in our transcriptomics data analysis. Of these, three (GLUL, GPX1, and MDH1) were found to follow a reverse correlation with body size (Figure 3A–C). Interestingly, one of these proteins (GLUL) was also found to be expressed inversely proportionally to body size in the left ventricle of the heart (Figure 3D). Additionally, we measured liver transaminase concentrations and observed that both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) exhibited lower concentrations in humans as compared to rats and rats as compared to mice (Figure 3E–F), consistent with scaling at the level of protein expression as well as mRNA expression. Finally, we utilized established enzymatic assays to measure the activity of peroxidase and pyruvate carboxylase in the livers of mice and rats. 30–40% lower activity of each enzyme per mg tissue was observed in rats as compared to mice (Figure 3—figure supplement 1A–B), suggesting scaling at the level of metabolic enzyme activity. Figure 3 with 1 supplement see all Download asset Open asset Proteomics reveals a negative correlation between body size and the expression of some liver proteins. Liver (A) glutamate-ammonia ligase (GLUL), (B) glutathione peroxidase 1 (GPX1), and (C) malate dehydrogenase 1 (MDH1) protein expression. (D) GLUL protein expression in the left ventricle of the heart. The proteomics analysis was performed on n=1 per species, so statistical comparisons were not possible. (E) Plasma alanine aminotransferase (ALT) and (F) aspartate aminotransferase (AST) concentrations (for both transaminases, n=5 per species). *p<0.05, ***p<0.001, ****p<0.0001. Figure 3—source data 1 Source data for Figure 3 and Figure 3—figure supplement 1. https://cdn.elifesciences.org/articles/78335/elife-78335-fig3-data1-v1.xlsx Download elife-78335-fig3-data1-v1.xlsx Metabolic rates of mouse vs. rat hepatocytes in vitro are not significantly different Considering prior data reporting higher oxygen consumption per unit body mass in smaller as compared to larger animals (Gilman et al., 2013; Brody, 1945; Urbina and Glover, 2013), we first asked whether these differences were cell-intrinsic, or whether in vivo or hepatocyte-extrinsic signals are required. We incubated plated hepatocytes in [3-13C] lactate and first validated that the data met the assumptions of PINTA, including reaching steady-state in [13C] lactate and glucose enrichment, and producing glucose at a linear rate throughout the 6 hr incubation (Figure 4—figure supplement 1A–C). Consistent with the possibility that hepatocyte-extrinsic signals are primarily responsible for metabolic scaling, when we used PINTA to assess cytosolic and mitochondrial fluxes, we observed no significant differences between species in any of the fluxes measured in plated hepatocytes: glucose production, VPC, VCS, the contribution of glucose or fatty acids to the tricarboxylic acid (TCA) cycle, or lipolysis (Figure 4A–H, Figure 4—figure supplement 1D–F). Similarly, a mitochondrial stress test in plated hepatocytes revealed no difference in any parameter: neither basal mitochondrial and non-mitochondrial respiration, ATP production, maximal (uncoupled) respiration, spare respiratory capacity, nor proton leak differed between plated hepatocytes from mice and rats (Figure 4I). Previous studies have demonstrated scaling in vitro in cell suspensions only when analyzed immediately after hepatocyte isolation (Porter and Brand, 1995), and have suggested that the phenomenon of scaling gradually disappears around 24 hr post removal (Brown et al., 2007), similar to the conditions in which we performed these studies. Most prior in vitro studies have also demonstrated an absence of scaling, in contrast to in vivo (Glazier, 2015), and we extend these results to gluconeogenic and lipolytic fluxes in hepatocytes, glucose production in liver slices, and multimodal flux analysis in vivo. Figure 4 with 1 supplement see all Download asset Open asset Metabolic fluxes are not different between mouse and rat hepatocytes in vitro. (A) Study design. This figure was made using Biorender.com. (B) Tracer labeling strategy. (C) Glucose production. (D) Gluconeogenesis from pyruvate (pyruvate carboxylase flux, VPC). (E) Citrate synthase flux (VCS), i.e., mitochondrial oxidation. (F) Pyruvate dehydrogenase flux (VPDH), i.e., the contribution of glucose via glycolysis to total mitochondrial oxidation. (G) Non-esterified fatty acid (NEFA) production. (H) The contribution of fatty acid oxidation to citrate synthase flux. (I) Oxygen consumption rate (OCR) during a mitochondrial stress test. In all panels, hepatocytes from wild-type males were studied, and groups were compared using the two-tailed unpaired Student's t-test. No significant differences were observed. In all panels, the mean ± SEM. of six biological replicates (averaged from three technical replicates per biological replicate) is shown. Figure 4—source data 1 Source data for Figure 4 and Figure 4—figure supplement 1. https://cdn.elifesciences.org/articles/78335/elife-78335-fig4-data1-v1.xlsx Download elife-78335-fig4-data1-v1.xlsx Glucose production per gram tissue is higher ex vivo in liver slices from mice than in rats Next, considering that hepatocytes comprise approximately 70–80% of liver mass and that their culture in vitro does not replicate in vivo conditions (Krebs, 1950), we asked whether glucose production would be different between mice and rats in slices of liver. Indeed, we found that liver glucose production per gram liver mass was threefold greater in mouse liver slices as compared to rats (Figure 5A–B), suggesting that hepatocyte-extrinsic signals (for example, from other liver cell types) are involved in liver metabolic scaling. Figure 5 Download asset Open asset Glucose production scales ex vivo in liver slices. (A) Study design. This figure was made using Biorender.com. (B) Glucose production. Groups were compared by the two-tailed unpaired Student's t-test. Liver slices from male, wild-type animals (n=4 mice and 2 rats, three technical replicates per biological replicate) were studied. Figure 5—source data 1 Source data for Figure 5. https://cdn.elifesciences.org/articles/78335/elife-78335-fig5-data1-v1.xlsx Download elife-78335-fig5-data1-v1.xlsx Metabolic rates in multiple tissue types are higher in vivo in mice relative to rats We utilized multimodal stable isotope metabolic flux analysis to compare rats and mice with respect to a panel of metabolic fluxes (Figure 6A). First, we validated tracer assumptions in vivo, including the metabolic and isotopic steady state in plasma and negligible liver glycogen concentrations, although in the recently hepatic was higher in mice than that in rats (Figure supplement Using PINTA, we found that both glucose production and gluconeogenesis from pyruvate per gram liver were more than higher in mice than rats (Figure although the contribution of pyruvate to gluconeogenesis did not between mice and rats (Figure supplement oxidation scaled threefold in mice as compared to rats studied under the same to in both glucose oxidation (pyruvate dehydrogenase flux, and fatty acid oxidation (Figure associated with an in the of pyruvate carboxylase to citrate synthase flux any difference in the of flux by glucose through (Figure supplement While we did not have the to measure liver fluxes in larger in the glucose production, VPC, and measured using PINTA were lower in humans than in rats et al., 2019), a liver size of g in differences in metabolic fluxes according to body size applied not only to liver metabolism also to adipose tissue fatty acid was higher in mice than in rats (Figure No differences were observed in any of the measured fluxes (Figure supplement Taken together, these data the of common in vitro methods as a of in vivo whereas in vivo mitochondrial oxidation cycle was threefold higher in mice than in rats, in vitro of oxygen consumption throughout a mitochondrial stress TCA cycle flux, and glucose production were not different between the species (Figure Figure 6 with 1 supplement see all Download asset Open asset Analysis of metabolic fluxes suggests in vivo metabolic scaling in mice vs. rats. (A) Study design. (B) glucose production. (C) Gluconeogenesis from pyruvate (D) VCS, i.e., mitochondrial oxidation. (E) i.e., the contribution of glucose via glycolysis to total mitochondrial oxidation. (F) (G) The contribution of fatty acid oxidation to citrate synthase flux. In all panels, groups were compared using the two-tailed unpaired Student's t-test. (n=4 mice and 6 were studied. Figure data 1 Source data for Figure 6 and Figure supplement 1. Download clustering species-specific on in vivo metabolic fluxes not in vitro fluxes clustering was applied to our in vitro flux data and no clustering between species (Figure the in vivo metabolic flux data to clustering of rats and mice (Figure a analysis of in vitro in vivo metabolic flux. Figure 7 Download asset Open asset of in vitro and in vivo results. (A) Study (B) heatmap demonstrating the absence of metabolic differences in vitro. (C) heatmap demonstrating metabolic differences between mice and rats in vivo. In (B) and (C), mouse and rat to the species to the on the of each pyruvate carboxylase flux, citrate synthase flux, pyruvate dehydrogenase flux, fatty acid fatty acid All data in Figures 3 and 5 were utilized in the clustering analysis and are included in this Discussion Oxygen consumption has been shown to scale inversely with body mass in species in mass across 20 of from to et al., et al., et al., et al., et al., et al., This phenomenon has been most studied in mammals, is also been shown to in and et al., et al., et al., et al., et al., et al., et al., et al., and et al., and et al., et al., and (Glazier, et al., 2013; et al., a limitation of prior studies in this has been that observations have been largely limited to oxygen consumption and caloric other metabolic processes unexplored. This study to address this by the of the inverse between body mass and metabolic using both experimental and databases that have not been employed in this is important to that the metabolic processes which we observed to be higher in mice as compared to rats did not to the metabolic scaling with metabolic rates proportional to three-quarters of body mass. This to the that the scaling is it is entirely that oxygen consumption could be proportional to three-quarters of body mass, while other metabolic processes may exhibit a different scaling Further studies across species beyond be required to address this The possibility that gene expression, as by mRNA may also scale with body mass has not been We observed that the expression of key genes in glycolysis, gluconeogenesis, fatty acid metabolism, NAD synthesis and transport, mitochondrial oxygen consumption, and from oxidative damage scale with body mass. however, is the that genes for which an inverse of expression with body mass is are not across the the of genes whose expression is inversely correlated with body mass is for genes related to metabolic processes, and whose corresponding enzymatic are by the of or The that body mass is a related to the level of expression of certain genes has not been as an of metabolic scaling. it be that metabolic scaling cannot be at the transcriptional level, because many rate-limiting enzymes in the metabolic processes measured in vivo did not scale at the transcriptional level, and only approximately half of genes that scaled at the level of mRNA scaled at the level of it is likely that both transcriptional and other mechanisms such as enzyme activity are responsible for in metabolic flux per unit mass, inversely proportionally to body size. Additionally, the data not allow to assess whether the expression of certain of key metabolic enzymes scales differentially across species. is also to contrast the of an inverse between body size and metabolic fluxes per tissue weight consumption, mitochondrial and glucose production in measured in the in vitro to our in vivo all fluxes in mice than in This the to tracer methods in vivo to a comprehensive of differences in metabolic fluxes between species. Our that measurement of oxygen consumption in vitro may to any influence of scaling processes present in vivo. Glucose production was threefold higher in mouse liver slices relative to rat liver slices, did not significantly between plated hepatocytes from mice and rats. studies using metabolic flux analysis may have the further to as to the and of metabolic scaling For example, our data not allow to whether differences in oxygen consumption metabolic as has been suggested in the of et al., or metabolic alterations changes in oxygen Additionally, there are to the that metabolic flux studies were performed only in the most related species included in the transcriptomics analysis monkey and human Our does not have the to flux analysis in larger or smaller species, we that our beyond the range in body size between mice and

Short-term, high-fat diet increases the expression of key intestinal genes involved in lipoprotein metabolism in healthy men

The role of miRNA and miRNA biogenesis genes in the adult brain is just beginning to be explored. In this study we have performed a comprehensive analysis of the expression, genetic regulation, and co-expression of major components of the miRNA biogenesis pathway using human and mouse data sets and resources available on the GeneNetwork web site (genenetwork.org). We found a wide range of variation in expression in both species for key components of the pathway—Drosha, Pasha, and Dicer. Across species, tissues, and expression platforms all three genes are generally well-correlated. No single genetic locus exerts a strong and consistent influence on the expression of these key genes across murine brain regions. However, in mouse striatum, many members of the miRNA pathway are correlated—including Dicer, Drosha, Pasha, Ars2 (Srrt), Eif2c1 (Ago1), Eif2c2 (Ago2), Zcchc11, and Snip1. The expression of these genes may be partly influenced by a locus on Chromosome 9 (105.67–106.32 Mb). We explored ~1500 brain phenotypes available for the C57BL/6J × DBA/2J (BXD) genetic mouse population in order to identify miRNA biogenesis genes correlated with traits related to addiction and psychiatric disorders. We found a significant association between expression of Dicer and Drosha in several brain regions and the response to many drugs of abuse, including ethanol, cocaine, and methamphetamine. Expression of Dicer, Drosha, and Pasha in most of the brain regions explored is strongly correlated with the expression of key members of the dopamine system. Drosha, Pasha, and Dicer expression is also correlated with the expression of behavioral traits measuring depression and sensorimotor gating, impulsivity, and anxiety, respectively. Our study provides a global survey of the expression and regulation of key miRNA biogenesis genes in brain and provides preliminary support for the involvement of these genes and their product miRNAs in addiction and psychiatric disease processes.

Dynamic and Temporal Transcriptomic Analysis Reveals Ferroptosis-Mediated Antileukemia Activity of S-Dimethylarsino-Glutathione: Insights into Novel Therapeutic Strategy

Dietary saturated fat (SFA) and trans fatty acids (TFA) have been linked to an increased risk of cardiovascular disease mainly by increasing plasma LDL-C levels. The modulation of cholesterol and fatty acids homeostasis by SFA and TFA is thought to be mediated by changes in expression of key intestinal genes involved in lipid and lipoprotein metabolism. However, the short-term impact of dietary fat intake on expression of these genes has not been fully investigated. To test whether short-term changes in SFA and TFA intake affects expression of key intestinal genes involved in lipid and lipoprotein metabolism, we conducted a randomized, double-blind, cross-over study using an intensive dietary modification in 12 nonobese healthy men with normal plasma lipid profile. Participants were subjected to 2 isocaloric 3-day diets: 1) high-fat diet (37% energy from fat, 15% from SFA, 3.5% from TFA and 50% energy from carbohydrate) and 2) low-fat diet (25% energy from fat, 6% from SFA, 0% from TFA and 62% energy from carbohydrate) in random order, each separated by a two-week washout period. Fasting plasma lipid levels were determined and expression of key genes involved in lipid and lipoprotein metabolism was compared by real-time PCR quantification in duodenal biopsy specimens obtained in the fasted state after 3 days of feeding on each diet. Following the 3-day high-fat diet, plasma-C (+7.4%, P=0.02), LDL-C (+16.9%, P=0.005) and HDL-C (+9.3%, P=0.002) levels were significantly increased as compared to low-fat diet. Plasma triglycerides (-31.7%, P=0.001) and apolipoprotein B-48 (-39.6%, P=0.003) levels were significantly decreased after the high-fat diet relative to the low-fat diet. The high-fat diet also resulted in significant increases in intestinal mRNA expression levels of SREBP-2, HNF-4α, PPAR-α, PPAR-γ, NPC1L1, ABCG8, FABP-2, ACAC-α, SCD-1, ELOVL5, DGAT-2, apolipoprotein B, MTTP, SAR1β and LDL receptor. These findings suggest that short-term exposure to a high-SFA and TFA diet upregulates the expression of key genes involved in lipid and lipoprotein metabolism at the enterocyte level.

Simple SummaryDetailed knowledge of the molecular mechanisms of immunoglobulin synthesis appears necessary for a better understanding of foal immunity maturity and its influencing factors. At the same time, it encourages studies regarding the influence of the signaling cascade’s proteins on the primary immunological response, which provides an opportunity to develop extremely precise methods of regulating acquired immunity. The results revealed that the expression of theTLR3 and TLR4 genes, as well as the levels of immunoglobulins and interleukins, can be modulated by stimulation with the pharmacological agent, and that the expression of the TLR3 and TLR4genes in peripheral blood cells is dependent on age.The aim of this study was to investigate the molecular mechanisms leading to the identification of pathogens by congenital immune receptors in foals up to 60 days of age. The study was conducted on 16 foal Polish Pony Horses (Polish Konik) divided into two study groups: control (n = 9) and experimental (n = 7). Foals from the experimental group received an intramuscular duplicate injection of 5 mL of Biotropina (Biowet) at 35 and 40 days of age. The RNA isolated from venous blood was used to evaluate the expression of theTLR3, TLR4, and TLR7 genes using RT-PCR. The results of the experiment demonstrated a statistically significant increase in the level of TLR3 gene expression and a decrease in the level ofTLR4 gene expression with foal aging. The level of TLR7 gene expression did not show age dependence. Immunostimulation with Biotropina had a significant impact on the level of the genes’ expression for Toll-like receptors. It increased the level of TLR4 expression and decreased TLR3 expression. Thus, it was concluded that the expression of theTLR3 and TLR4genes in peripheral blood cells is dependent on age. This experiment demonstrated a strong negative correlation between TLR3 and TLR4 gene expression.

PDF HTML阅读 XML下载 导出引用 引用提醒 盐胁迫对雨生红球藻虾青素累积、虾青素合成相关酶基因表达和抗氧化指标的影响 DOI: 作者: 作者单位: 1. 河南师范大学水产学院, 河南 新乡 453007;2. 宁德师范学院 生物系, 福建 宁德 352100;3. 河南师范大学 生命科学学院, 河南 新乡 453007 作者简介: 江红霞(1974-),女,副教授,研究方向为水生生物资源与环境.E-mail:jianghongxia2007@126.com 通讯作者: 中图分类号: S96 基金项目: 河南省重点科技攻关项目（102102210181）；河南省水产学重点学科基金项目（2012）. Effects of salt stress on astaxanthin accumulation, gene expression of astaxanthin synthesis-related enzymes, and antioxidant indices in Haematococcus pluvialis Author: Affiliation: 1. College of Fisheries, Henan Normal University, Xinxiang 453007, China;2. Department of Biology, Ningde Normal University, Ningde 352100, China;3. College of Life Sciences, Henan Normal University, Xinxiang 453007, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为探讨盐胁迫对雨生红球藻（）虾青素合成的影响与机理，以及雨生红球藻各抗氧化机制之间的关系，本研究采用生化和分子生物学方法研究了不同浓度（0.04 mol/L、0.08 mol/L、0.12 mol/L和0.16 mol/L）和不同时间（3 d、6 d和9 d）的盐（NaCl）胁迫对雨生红球藻生长、虾青素积累、番茄红素-胡萝卜素酮化酶（Bkt）基因表达、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性以及丙二醛（MDA）含量的影响。结果表明，各胁迫时间的雨生红球藻的密度均随着盐胁迫浓度的增加而不断下降，在盐胁迫的第9天，雨生红球藻的死亡率和孢子比例均随着盐胁迫浓度的增加而不断升高；雨生红球藻虾青素含量、基因表达量均随着盐胁迫浓度和时间的增加而不断提高；雨生红球藻SOD、CAT和GSH-Px活性以及MDA含量在不同浓度和不同时间的盐胁迫下与对照组（0.00 mol/L NaCl）相比均升高，且在不同时间的0.12 mol/L NaCl胁迫下与对照组相比均显著升高（基因表达量在盐胁迫的早期（第3天）和中期（第6天）阶段较低，在盐胁迫的后期（第9天）阶段较高，而SOD、CAT和GSH-Px活性以及MDA含量在盐胁迫的早期和中期阶段较高，在盐胁迫的后期阶段较低。实验结果说明了适当浓度和时间的盐胁迫能促进雨生红球藻累积虾青素，雨生红球藻在盐胁迫下主要是通过提高虾青素合成相关酶基因的转录水平来促进虾青素的合成，其虾青素和抗氧化酶的抗氧化活性可能互为补充，共同保护雨生红球藻免受盐胁迫的氧化损伤。 Abstract:(Chlorophyceae, Order Volvocales), a freshwater green microalgal species, has commercial value owing to its ability to accumulate high concentrations of astaxanthin (up to 5% of dry weight). Astaxanthin (3,3'-dihydroxy--carotene-4,4'-dione) is a red ketocarotenoid which has many important biological functions, including antioxidant activity, regulation of immune responses, and disease resistance, and has the potential for application in the aquacultural, nutraceutical, pharmaceutical, and cosmetic industries. has a distinctive lifecycle as it exhibits a green motile stage and a red non-motile resting stage called an aplanospore. In general, astaxanthin accumulation in is restricted to the aplanospore stage. Astaxanthin is accumulated in extra-plastidic lipid vesicles as a secondary carotenoid, and it is believed to be synthesized in response to oxidative stress in the red aplanospore stage under unfavorable environmental conditions such as high light, temperature, and salinity, or low nutrient availability. Several enzymes such as lycopene -carotene ketolase (Bkt) are involved in the astaxanthin bios-y-n-thesis pathway in -carotene from lycopene, and CrtR-B and Bkt cat-alyze further steps leading to astaxanthin synthesis. Changes in expression of the genes encoding these three enzymes can critically affect the biosynthesis and accumulation of astaxanthin in . In addition, various ant-i-oxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) also have important protective effects for combating oxidative stress under unfavourable environmental conditions. We aimed to explore the effect of salt stress on astaxanthin accumulation in , by examining the mechanism of astaxanthin synthesis and the relationship between the different antioxidant mechanisms in at high salinity. We examined its growth rate; astaxanthin content; gene expression levels; SOD, CAT, and GSH-Px activities; and malondialdehyde (MDA) content at four salinity levels (0.04 mol/L, 0.08 mol/L, 0.12 mol/L, and 0.16 mol/L) over three timescales (3 days, 6 days, and 9 days). Our results showed that the density of decreased under increasing salinity over different periods of time, while its mortality rate and aplanospore proportion increased with increasing salt stress concentration by the ninth day of stress. Asta-xanthin content, gene expressions increased over time with increasing salinity. SOD, CAT, and GSH-Px activities and MDA content also increased in comparison to grown at the control level of 0 mol/L NaCl, and significantly increased at the 0.12 mol/L NaCl level ( gene expressions were lower during the early and mid-stages of salt stress (e.g., on the third and sixth days of observation), and increased by the ninth day. Meanwhile, SOD, CAT, and GSH-Px activities and MDA content were higher during early and mid-stage stress, but were lower by the ninth day. These results suggest that salt stress can improve astaxanthin accumulation over time in at the appropriate level of salt stress, despite its negative effects on growth. Astaxanthin synthesis in is promoted mainly through an increase in the transcription level of astaxanthin synthesis-related enzyme genes under salt stress, and the antioxid-ant activities of astaxanthin and antioxidant enzymes complement one other to protect from oxidative damage under salt stress. This study provides a new insight into the astaxanthin synthesis and antioxidant mechanisms in . 参考文献 相似文献 引证文献

To assess blood leucocytes gene profiling during recovery phase of septic shock; to test the relation between encoding gene expression and protein level. Gene expression levels were studied at days 0, 1, 7 and 28 (D0, 1, 7 and 28) on a dedicated microarray of 340 genes involved in inflammatory processes. 16-bed intensive care unit, Lariboisière University hospital. Seventeen septic shock patients enrolled when at least one additional organ dysfunction occurred. Changes over time were compared with D0 via the ratio Dx/D0. The time-related gene expression study showed significant changes in ten genes. Among them, S100A8 and S100A12 had a reduced expression over time compared with D0, whereas CD74's expression increased. The microarray results were validated by RT-qPCR for four genes. The S100A8 plasma levels decrease along recovery in parallel with the gene expression decrease. The CD74 gene expression evolution significantly correlated with HLA-DR monocyte expression. These results are the first description of variations in expression of key inflammatory genes in the course of the septic shock recovery period.

Cancer is a deadly and fast-spreading disease that is a growing health problem worldwide due to a lack of comprehensive screening and appropriate medication. However, natural products derived from medicinal plants have gained attention as potential sources of bioactive compounds that selectively remove cancerous lesions and are nontoxic and safe. Pancreatic cancer (PC) is a major therapeutic challenge and is predicted to surpass breast cancer as the third leading cause of cancer death. This study investigated the cytotoxic effects of methanolic extracts from five Iranian medicinal plants, Cuscuta epithymum, Achillea millefolium, Salvia officinalis, Salvia hydrangea, and Teucrium polium, on pancreatic cancer cell lines (MIA PaCa-2 and PaTu8902). Additionally, we examined the changes in the expression of key genes following treatment with C. epithymum extract. The findings revealed that the plant extracts had a dose-dependent effect on the cell viability of the lines, with the C. epithymum extract exhibiting the greatest cytotoxic effect (IC50 values of 85.03 µg/mL for MIA PaCa-2 and 156.57 µg/mL for PaTu 8902). GC‒MS analysis revealed 25 bioactive compounds in C. epithymum, with quinic acid (14.13%), p-vinylphenol (13.22%), and valeraldehyde (11.21%) as the most abundant. The study also investigated the changes in the expression of the STAT4, PIK3CD, EMP1, and RAB11FIP3 genes in MIA PaCa-2 and PaTu 8902 pancreatic cancer cell lines after treatment with the extract from C. epithymum. A correlation was detected between the expression levels of PIK3CD, STAT4, EMP1, and RAB11FIP3 and various concentrations of C. epithymum extract. The results revealed that the extract increased the mRNA levels of STAT4, PIK3CD, and EMP1, whereas RAB11FIP3 was reduced in the treated cells. Accordingly, C. epithymum extract has strong cytotoxic effects on pancreatic cancer cells and influences the expression of key cancer-related genes, suggesting its potential as a therapeutic candidate for PC treatment. Further in vivo studies are recommended to explore its mechanisms of action and clinical applicability.

Klotho, which was originally identified as an antiaging gene, forms a complex with fibroblast growth factor 23 receptor in the kidney, with subsequent signaling that regulates mineral metabolism. Other biological activities of Klotho, including antiaging effects such as protection from various types of cellular stress, have been shown; however, the precise mechanism of these effects of Klotho gene in the healthy human kidney is not well understood. In this study, we examined the relationships of Klotho and antioxidative stress gene expression levels in zero-hour biopsy specimens from 44 donors in kidney transplantation and verified them in animal models whose Klotho gene expression levels were varied. The nitrotyrosine expression level in the kidney was evaluated in these animal models. Expression levels of Klotho gene were positively correlated with the p53 gene and antioxidant enzyme genes such as catalase, superoxide dismutase 1 (SOD1), SOD2, peroxiredoxin 3 (PRDX3), and glutathione peroxidase 1 (GPX1) but not clinical parameters such as age and renal function or pathological features such as glomerulosclerosis and interstitial fibrosis tubular atrophy. The expression levels of all genes were significantly higher in mice with Klotho overexpression than in wild-type mice, and those except for catalase, PRDX3, and GPX1 were significantly lower in Klotho-deficient mice than in wild-type littermate mice. Nitrotyrosine-positive bands of various sizes were observed in kidney from Klotho-deficient mice only. The preservation of Klotho gene expression might induce the antioxidative stress mechanism for homeostasis of healthy human kidney independently of its general condition, including age, renal function, and histological findings.

Accumulation of lipid in nonadipose tissues plays a critical role in the pathogenesis of a diverse array of chronic diseases, including diabetes and heart failure. Normal cellular homeostasis relies on a critical balance of fuel uptake and utilization that is controlled by elaborate transcriptional networks, which ensure that cellular energy needs are consistently met and toxic intermediates do not accumulate. This process is particularly important for the heart, which has a continuous high energy demand. The heart meets this demand by dynamically shifting its preference for glucose and fatty acids in various physiologic and pathologic circumstances. This change is driven by alterations in signaling networks and regulated expression of key metabolic genes. While the heart predominantly relies on fatty acids as a fuel source, under normal conditions the myocardium is not a site of significant lipid storage. Moreover, conditions that promote fatty acid excess, such as insulin resistance and diabetes, are associated with detrimental effects on cardiac function. Evidence has emerged that fatty acid overload may damage the myocardium through excessive fatty acid oxidation and accumulation of toxic lipid species within the myocardium. These toxic effects have been termed “lipotoxicity” (1). A number of investigators have implicated specific lipid species, such as long-chain saturated fatty acids (palmitate), in the pathogenesis of cardiomyocyte dysfunction (2, 3), while unsaturated fatty acids (oleate) are considered cardioprotective (4). The mechanism whereby some fatty acid species exert more toxic effects than others is poorly understood. In this issue of The Journal of Lipid Research, Lockridge et al. (5) report microarray data of the differential effects of several fatty acid species in adult cardiac myocytes. Previous investigations have primarily evaluated direct effects of individual fatty acid species in neonatal rat cardiac myocytes, where the expression of key metabolic regulators is still changing. Thus, the work by Lockridge et al. provides new insight into effects in a mature cardiac myocyte and does this in a systematic and unbiased manner. The most striking finding in this study was the dramatic effect of the long-chain saturated fatty acid palmitate on gene expression for enzymes involved in the endoplasmic reticulum (ER) stress response and apoptosis pathways as well as markers of DNA damage. These findings are consistent with other recent data in CHO cells, in which palmitate was found to be trafficked to the ER and to have dramatic effects on ER structure and integrity as well as to disrupt mitochondrial function (6). Furthermore, ER stress has been implicated in apoptosis of pancreatic b cells from type 2 diabetic models (7). Taken together, this recent evidence strongly implicates ER stress in the cellular dysfunction associated with palmitate exposure. Further studies specifically evaluating the ER stress response in cardiac myocytes will be important in understanding the mechanisms of cardiac dysfunction associated with lipid overload and in targeting potential therapies to alleviate cardiomyopathy associated with diabetes. Interestingly, Borradaile et al. (6) also found that induction of fatty acid oxidation minimized the effects on ER integrity upon exposure to palmitate. Lockridge et al. (5) demonstrated differential effects of oleate versus palmitate on gene expression for a number of enzymes that play a role in upregulating mitochondrial fatty acid oxidation pathways (e.g., Cpt1a, Fatp1, and Ucp3). While palmitate also upregulated many components of mitochondrial oxidative pathways, the more robust effect of oleate on some of these genes is intriguing. One of the genes the authors highlight that is specifically induced by oleate is Ucp3. They suggest that upregulation of Ucp3 may result in increased recycling of free CoA and thus promote the oxidation of oleate, whereas palmitate may be more likely to accumulate in the mitochondria, leading to cytotoxic effects. Further studies to evaluate this hypothesis will be important for understanding the fate of fatty acid species within the cell and potentially to assist in identifying targets for cellular rescues. One such study might investigate the potential for unsaturated fatty acids to serve as preferential ligands for the peroxisome proliferator-activated receptor family, which is known to modulate fatty acid uptake and oxidation.

Objective: To investigate the protective mechanism of ginsenoside Rb-1 on the brain in a rat model of Alzheimer's disease. Methods: Fifty-six male Sprague-Dawley rats were randomly divided into control group, model group, low-dose Rb-1 group (Rb-1: 25 mg•kg(-1)•d(-1)) and high-dose Rb-1 group (Rb-1:50 mg•kg(-1)•d(-1)). Morris water maze was designed to observe the changes of learning and memory ability in rats. Flow cytometry was used to detect the apoptosis of hippocampal neurons. Immunohistochemistry and Western blot were employed to detect the expression levels of apoptosis-related genes (p53, Bax, cytochrome C (Cyto C), Caspase-3 and caspase-9) and anti-oxidative stress-associated genes (nuclear Factor-E2-related factor 2 (Nrf2), kelch-like ECH-associated protein 1 (keap-1), heme oxygenase 1(HO-1) and NADPH quinone dehydrogenase 1 (NQO1)).The activities of catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were detected by relevant kits. ANOVA and Tukey-Kramer test were used for statistical analysis. Results: The learning and memory ability of rats in the model group was lower than that of the control group (P<0.01).The learning and memory ability of rats in the high-dose Rb-1 treatment group was significantly higher than that of the model group [(80±8) s vs (100±11) s, t=5.390, P<0.01]. The expression levels of apoptosis-related genes (p53, Bax, Cyto C, caspase-3 and caspase-9) in the model group were significantly higher than those in the control group (P<0.01), while the expression levels of these genes in low-dose and high-dose Rb-1 groups were significantly lower than those of the model group (P<0.01). The expression levels of Nrf2, HO-1 and NQO1 genes in the model group were significantly lower than those in the control group (P<0.05), while the expression of these genes in low-dose and high-dose Rb-1 groupswere significantly higher than those of the model group (P<0.01). The activities of CAT, GSH-Px and SOD in the model group were lower than those in the control group (P<0.01), however the activities of CAT, GSH-Px and SOD in low-dose and high-dose Rb-1 groups were higher than those of model group (P<0.05). Conclusions: Both low-dose and high-dose Rb-1 have protective effect on memory and cognitive function of Alzheimer's disease rats by reducing the damage and apoptosis of hippocampal neurons, down-regulating the expression levels of p53, Bax, Cyto C, caspase-3 and caspase-9, up-regulating the expression of Nrf2, HO-1 and NQO1 genes, and increasing the activities of CAT, GSH-Px and SOD. Moreover, the protective effect of Rb-1 on rat brain may be dose-dependent.

Association of PKLR gene copy number, expression levels and enzyme activity with 2,3,7,8-TCDD exposure in individuals exposed to Agent Orange/Dioxin in Vietnam

'Shannongsu' pear is a new high-quality cultivar. To ascertain the storage characteristics of 'Shannongsu' pears at low temperatures (0 ± 0.5 °C), the following parameters were determined: fruit firmness, ethylene, aromatic compounds, sugar content, acidity, ascorbic acid, and the expression levels of ethylene-related genes and texture-softening genes. The firmness of 'Shannongsu' pears changed less than that of the control, decreasing by only 18.8% after 170 days of storage. Low temperatures suppressed the expression of key genes associated with PbACS1a and PbACO1. Moreover, the expression of key genes related to fruit softening (PbPG1, PbXET, PbPME, and Pbα-L-Af) was suppressed during storage at low temperatures and remained at low levels. Therefore, the low levels of ethylene biosynthesis and the expression of key genes involved in fruit softening might play a major role in the excellent storage characteristics of the 'Shannongsu' cultivar. After 170 days of storage, 'Shannongsu' pears did not show significant changes in key quality dimensions such as firmness, sugar, acid, sugar-acid ratio, and ascorbic acid content. Therefore, low temperatures could help maintain the freshness, flavor, and nutritional quality of the 'Shannongsu' pear. Our findings reveal for the first time the low-temperature storage characteristics of 'Shannongsu' pears, providing a new scientific theoretical basis for pear production and marketing.

Today, consumers are very concerned regarding food quality, nutritional composition and positive health effects of consumed foods. In this context, the preference and consumption of organic products has been increasing worldwide. In the present work, sweet peppers in two maturation stages (i.e., green and red peppers) from organic and conventional production systems were evaluated in regards to phenolic composition and antioxidant activity. Nine phenolic compounds were identified and quantified by a high-performance liquid chromatography-diode-array detector (HPLC-DAD), namely resveratrol, meta-coumaric acid, ortho-coumaric acid, clorogenic acid, caffeic acid, myricetin, rutin, luteolin-7-O-glucoside and quercitin-3-O-rhamnoside. In contrast to the production system, the maturation stage showed a pronounced significant effect on the phenolic composition of the studied sweet peppers; in general, green peppers possessed higher contents than red ones. Meta-coumaric acid, ortho-coumaric acid and quercitin-3-O-rhamnoside were more abundant in green conventional peppers and chlorogenic acid, caffeic acid and rutin were found in higher levels in red organic peppers. Regarding the antioxidant activity, green conventional peppers showed the highest DPPH, ABTS•+ and total reducing capacities, while red conventional peppers had higher TEAC values. Finally, principal component analysis showed that the phenolic composition together with the antioxidant capacities could be used to differentiate the production system and the maturation stage of sweet peppers. This finding confirmed that both factors influenced the peppers’ phenolic composition and antioxidant capacity, allowing their possible use as maturation–production biomarkers.