Altered Enzyme Expression Research Articles

Reorganization of 3D genome architecture provides insights into pathogenesis of early fatty liver disease in laying hens

BackgroundFatty liver disease causes huge economic losses in the poultry industry due to its high occurrence and lethality rate. Three-dimensional (3D) chromatin architecture takes part in disease processing by regulating transcriptional reprogramming. The study is carried out to investigate the alterations of hepatic 3D genome and H3K27ac profiling in early fatty liver (FLS) and reveal their effect on hepatic transcriptional reprogramming in laying hens.ResultsResults show that FLS model is constructed with obvious phenotypes including hepatic visible lipid deposition as well as higher total triglyceride and cholesterol in serum. A/B compartment switching, topologically associating domain (TAD) and chromatin loop changes are identified by high-throughput/resolution chromosome conformation capture (HiC) technology. Targeted genes of these alternations in hepatic 3D genome organization significantly enrich pathways related to lipid metabolism and hepatic damage. H3K27ac differential peaks and differential expression genes (DEGs) identified through RNA-seq analysis are also enriched in these pathways. Notably, certain DEGs are found to correspond with changes in 3D chromatin structure and H3K27ac binding in their promoters. DNA motif analysis reveals that candidate transcription factors are implicated in regulating transcriptional reprogramming. Furthermore, disturbed folate metabolism is observed, as evidenced by lower folate levels and altered enzyme expression.ConclusionOur findings establish a link between transcriptional reprogramming changes and 3D chromatin structure variations during early FLS formation, which provides candidate transcription factors and folate as targets for FLS prevention or treatment.

Intravenous MSC-Treatment Improves Impaired Brain Functions in the R6/2 Mouse Model of Huntington's Disease via Recovered Hepatic Pathological Changes.

Huntington's disease (HD), a congenital neurodegenerative disorder, extends its pathological damages beyond the nervous system. The systematic manifestation of HD has been extensively described in numerous studies, including dysfunction in peripheral organs and peripheral inflammation. Gut dysbiosis and the gut-liver-brain axis have garnered greater emphasis in neurodegenerative research, and increased plasma levels of pro-inflammatory cytokines have been identified in HD patients and various in vivo models, correlating with disease progression. In the present study, we investigated hepatic pathological markers in the liver of R6/2 mice which convey exon 1 of the human mutant huntingtin gene. Furthermore, we evaluated the impact of intravenously administered Mesenchymal Stromal Cells (MSCs) on the liver enzymes, changes in hepatic inflammatory markers, as well as brain pathology and behavioral deficits in R6/2 mice. Our results revealed altered enzyme expression and increased levels of inflammatory mediators in the liver of R6/2 mice, which were significantly attenuated in the MSC-treated R6/2 mice. Remarkably, neuronal pathology and altered motor activities in the MSC-treated R6/2 mice were significantly ameliorated, despite the absence of MSCs in the postmortem brain. Our data highlight the importance of hepatic pathological changes in HD, providing a potential therapeutic approach. Moreover, the data open new perspectives for the search in blood biomarkers correlating with liver pathology in HD.

Effects of chronic dichlorodiphenyldichloroethylene exposure on testosterone secretion and steroidogenic pathway in the male gonad.

Endocrine disrupting chemicals are present in the environment and/or in consumer products. These agents have the capacity to mimic and/or antagonize endogenous hormones and thus perturb the endocrine axis. The male reproductive tract expresses steroid hormone (androgen and estrogen) receptors at high levels and is a major target for endocrine disrupting chemicals. In this study, Long-Evans male rats were exposed to dichlorodiphenyldichloroethylene, a metabolite of dichlorodiphenyltrichloroethane and a chemical present in the environment, in drinking water at 0.1 and 10μg/L for 4 weeks. At the end of exposure, we measured steroid hormone secretion and analyzed steroidogenic proteins, including 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, steroidogenic acute regulatory protein, aromatase, and the LH receptor. We also analyzed Leydig cell apoptosis (poly-(ADP-ribose) polymerase) and caspase-3 in the testes. Testicular testosterone (T) and 17β-estradiol (E2) were both affected by exposure to dichlorodiphenyldichloroethylene by displaying altered steroidogenic enzyme expression. Dichlorodiphenyldichloroethylene exposure also increased the expression of enzymes mediating the pathway for programmed cell death, including caspase 3, pro-caspase 3, PARP, and cleaved PARP. Altogether, the present results demonstrate that dichlorodiphenyldichloroethylene directly and/or indirectly can target specific proteins involved in steroid hormone production in the male gonad and suggest that exposure to environmentally relevant dichlorodiphenyldichloroethylene levels has implications for male reproductive development and function.

No Back-up Plan: Loss of Isozyme Diversity as a Promising Therapeutic Strategy for Cancer

Metabolic rewiring in cancer cells supports many aspects of tumor growth. Understanding the mechanisms that result in metabolic rewiring, such as altered enzyme expression, is key to identifying therapeutic vulnerabilities that selectively target cancer cells. In this issue of Cancer Research, Marczyk and colleagues analyze matched tumor-normal enzyme expression across 14 different cancer types and report that cancer cells exhibit a general loss of isozyme diversity (LID) relative to corresponding normal tissue. The authors hypothesized that the presence of a cancer dominant isozyme may reduce metabolic plasticity and uniquely sensitize cancer cells to isozyme-specific inhibitors. Several LID targets were identified, including acetyl-CoA carboxylase 1 (ACC1), which the authors validated using a clinically available inhibitor of ACC1/2. This study is the first to systematically evaluate isozymes affected by LID, which represents a promising strategy to target the unique metabolic demands of cancer. See related article by Marczyk et al., p. 1698.

Glucocorticoid Production in Lymphoid Organs: Acute Effects of Lipopolysaccharide in Neonatal and Adult Mice.

Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.

Quantitative proteomic analysis reveals altered enzyme expression profile in Zea mays roots during the early stages of colonization by Herbaspirillum seropedicae.

The use of plant growth-promoting bacteria as agricultural inoculants of plants should be encouraged because of their prominent role in biological nitrogen fixation, the increase of nutrient uptake by roots, abiotic stress mitigation, and disease control. The complex mechanisms underlying the association between plant and beneficial bacteria have been increasingly studied, and proteomic tools can expand our perception regarding the fundamental molecular processes modulated by the interaction. In this study, we investigated the changes in protein expression in maize roots in response to treatment with the endophytic diazotrophic Herbaspirillum seropedicae and the activities of enzymes related to nitrogen metabolism. To identify maize proteins whose expression levels were altered in the presence of bacteria, a label-free quantitative proteomic approach was employed. Using this approach, we identified 123 differentially expressed proteins, of which 34 were upregulated enzymes, in maize roots cultivated with H. seropedicae. The maize root colonization of H. seropedicae modulated the differential expression of enzymes involved in the stress response, such as peroxidases, phenylalanine ammonia-lyase, and glutathione transferase. The differential protein profile obtained in the inoculated roots reflects the effect of colonization on plant growth and development compared with control plants.

Dysregulated metabolism of polyunsaturated fatty acids in eosinophilic allergic diseases

Polyunsaturated fatty acids (PUFAs), represented by the omega-6 fatty acid arachidonic acid (AA) and omega-3 fatty acid docosahexaenoic acid (DHA), are essential components of the human body. PUFAs are converted enzymatically into bioactive lipid mediators, including AA-derived cysteinyl leukotrienes (cys-LTs) and lipoxins and DHA-derived protectins, which orchestrate a wide range of immunological responses. For instance, eosinophils possess the biosynthetic capacity of various lipid mediators through multiple enzymes, including 5-lipoxygenase and 15-lipoxygenase, and play central roles in the regulation of allergic diseases. Dysregulated metabolism of PUFAs is reported, especially in severe asthma, aspirin-exacerbated respiratory disease, and eosinophilic chronic rhinosinusitis (ECRS), which is characterized by the overproduction of cys-LTs and impaired synthesis of pro-resolving mediators. Recently, by performing a multi-omics analysis (lipidomics, proteomics, and transcriptomics), we demonstrated the metabolic derangement of eosinophils in inflamed tissues of patients with ECRS. This abnormality occurred subsequent to altered enzyme expression of gamma-glutamyl transferase-5. In this review, we summarize the previous findings of dysregulated PUFA metabolism in allergic diseases, and discuss future prospective therapeutic strategies for correcting this imbalance.

Saturated and monounsaturated fatty acids in membranes are determined by the gene expression of their metabolizing enzymes SCD1 and ELOVL6 regulated by the intake of dietary fat

PurposeWe investigated the effect of dietary fats on the incorporation of saturated (SAFAs) and monounsaturated dietary fatty acids (MUFAs) into plasma phospholipids and the regulation of the expression of lipid-metabolizing enzymes in the liver.MethodsMice were fed different diets containing commonly used dietary fats/oils (coconut fat, margarine, fish oil, sunflower oil, or olive oil) for 4 weeks (n = 6 per diet group). In a second experiment, mice (n = 6 per group) were treated for 7 days with synthetic ligands to activate specific nuclear hormone receptors (NHRs) and the hepatic gene expression of CYP26A1 was investigated. Hepatic gene expression of stearoyl-coenzyme A desaturase 1 (SCD1), elongase 6 (ELOVL6), and CYP26A1 was examined using quantitative real-time PCR (QRT-PCR). Fatty acid composition in mouse plasma phospholipids was analyzed by gas chromatography (GC).ResultsWe found significantly reduced hepatic gene expression of SCD1 and ELOVL6 after the fish oil diet compared with the other diets. This resulted in reduced enzyme-specific fatty acid ratios, e.g., 18:1n9/18:0 for SCD1 and 18:0/16:0 and 18:1n7/16:1n7 for ELOVL6 in plasma phospholipids. Furthermore, CYP26A1 a retinoic acid receptor-specific target was revealed as a new player mediating the suppressive effect of fish oil-supplemented diet on SCD1 and ELOVL6 hepatic gene expression.ConclusionPlasma levels of MUFAs and SAFAs strongly reflect an altered hepatic fatty acid-metabolizing enzyme expression after supplementation with different dietary fats/oils.

Role of Metabolic Reprogramming in Epithelial⁻Mesenchymal Transition (EMT).

Activation of epithelial–mesenchymal transition (EMT) is thought to be an essential step for cancer metastasis. Tumor cells undergo EMT in response to a diverse range of extra- and intracellular stimulants. Recently, it was reported that metabolic shifts control EMT progression and induce tumor aggressiveness. In this review, we summarize the involvement of altered glucose, lipid, and amino acid metabolic enzyme expression and the underlying molecular mechanisms in EMT induction in tumor cells. Moreover, we propose that metabolic regulation through gene-specific or pharmacological inhibition may suppress EMT and this treatment strategy may be applied to prevent tumor progression and improve anti-tumor therapeutic efficacy. This review presents evidence for the importance of metabolic changes in tumor progression and emphasizes the need for further studies to better understand tumor metabolism.

F199. O-GLCNAC DYSREGULATION IN SCHIZOPHRENIA CORTEX

BackgroundO-linked β-linked N-acetylglucosamine (O-GlcNAc) is a post-translational glycosylation modification with ubiquitous functions in cell biology. The attachment of GlcNAc to serine or threonine (S/T) residues transiently adorns thousands of nuclear, cytosolic, and mitochondrial proteins and modulates protein function and localization via dynamic cooperation with kinase and phosphatase enzymes. O-GlcNAc transferase (OGT) exists in complex with S/T phosphatase subunits PP1β and PP1γ and, along with the activity of O-GlcNAcase (OGA), facilitates rapid cycling between O-GlcNAcylation and phosphorylation states to serve as an “on/off switch” for substrate activation. Altered levels of OGT, OGA, and/or O-GlcNAc have been shown to influence many pathways pertinent to schizophrenia (SZ) pathophysiology. Notably, elevated O-GlcNAc and enhanced O-GlcNAcylation mediate glucose tolerance and insulin resistance which can lead to diabetes, an illness often found comorbid with SZ. Elevated O-GlcNAcylation can also produce mitochondrial abnormalities consistent with those identified in SZ. In an exploratory study of glycosylation enzyme transcript expression, our lab found OGT mRNA levels 253% higher in SZ than non-psychiatrically ill comparison (COMP) subjects (p < 0.0001). Based on this evidence, we hypothesized that OGT protein levels or the ratio of OGT:OGA enzymes are elevated in SZ brain.MethodsExpression of OGT and OGA were measured using western blots of superior temporal gyrus (STG; Brodmann Area 22) homogenates from sex- and age-matched pairs of SZ and COMP subjects (N = 17). Standard immunoblotting methods and commercially available antibodies were used to detect the targets of interest and protein levels were normalized to intralane valosin containing protein (VCP) expression; VCP expression has previously been found to be unchanged in SZ STG.ResultsIn the current study, we found OGA protein levels reduced 18% in SZ (p < 0.01) and SZ subjects demonstrate a trend toward increased ratios of OGT:OGA (p = 0.05). OGT was not different between groups (p = 0.77).DiscussionOur current results partially support our original hypothesis that an altered ratio of OGT:OGA may contribute to abnormalities of O-GlcNAcylation and consequent cellular metabolic abnormalities in SZ. A trend toward increased OGT:OGA along with decreased expression of OGA would produce the same functional outcome as the originally predicted OGT increase: upregulation of protein O-GlcNAcylation. Given that the mRNA study used samples of dorsolateral prefrontal cortex (DLPFC) while our protein-level measures were from STG, it is not inconceivable that potential O-GlcNAc dysregulation could arise from upregulated OGT in one brain region, but downregulated OGA in another. To elaborate on these findings, we will investigate OGT and OGA expression in the DLPFC and will assess total O-GlcNAcylation in both brain regions to determine functional consequences of altered enzyme expression.

Susceptibility to Temephos and Spinosad in Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

We examined the susceptibility to temephos and spinosad (Natular EC) of eight Aedes aegypti (L.) populations from Puerto Rico, following WHO method (WHO 2005). Enzyme activity was measured for alpha- and beta-esterases, multiple function oxidases, glutathione-s-transferases, and insensitive acetylcholinesterase and was tested for correlation with the susceptibility level. The results showed that larval populations from Puerto Rico obtained during 2014 were found to be susceptible to both larvicides, with low (resistance factor) RRLC50 values (<5 fold) and altered and incipiently altered enzyme expression for all populations, except the insensitive acetylcholinesterase enzyme, where only the population of Ponce showed overexpression (53.3%) above the threshold established with the New Orleans susceptible strain. We recommend the use of both larvicides for mosquito control in the study area and encourage further susceptibility monitoring.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism.

BackgroundEicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment.MethodsSimultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification.ResultsThe limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases.ConclusionsQuantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Enzyme-responsive nanomaterials for controlled drug delivery.

Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials used for controlled drug release have achieved significant development and have been studied as an important class of drug delivery strategies in nanomedicine. In this review, we describe enzymes such as proteases, phospholipases and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area.

Selective and Cytokine-Dependent Regulation of Hepatic Transporters and Bile Acid Homeostasis during Infectious Colitis in Mice

Various disease models have been shown to alter hepatic drug-metabolizing enzyme (DME) and transporter expression and to induce cholestasis through altered enzyme and transporter expression. Previously, we detailed the regulation of hepatic DMEs during infectious colitis caused by Citrobacter rodentium infection. We hypothesized that this infection would also modulate hepatic drug transporter expression and key genes of bile acid (BA) synthesis and transport. Mice lacking Toll-like receptor 4 (TLR4), interleukin-6 (IL-6), or interferon-gamma (IFNγ) and appropriate wild-type animals were orally infected with C. rodentium and sacrificed 7 days later. In two wild-type strains, drug transporter mRNA expression was significantly decreased by infection for Slc22a4, Slco1a1, Slco1a4, Slco2b1, and Abcc6, whereas the downregulation of Abcc2, Abcc3, and Abcc4 were strain-dependent. In contrast, mRNA expressions of Slco3a1 and Abcb1b were increased in a strain-dependent manner. Expression of Abcb11, Slc10a1, the two major hepatic BA transporters, and Cyp7a1, the rate-limiting enzyme of BA synthesis, was also significantly decreased in infected animals. None of the above effects were caused by bacterial lipopolysaccharide, since they still occurred in the absence of functional TLR4. The downregulation of Slc22a4 and Cyp7a1 was absent in IFNγ-null mice, and the downregulation of Slco1a1 was abrogated in IL-6-null mice, indicating in vivo roles for these cytokines in transporter regulation. These data indicate that C. rodentium infection modulates hepatic drug processing through alteration of transporter expression as well as DMEs. Furthermore, this infection downregulates important genes of BA synthesis and transport and may increase the risk for cholestasis.

Altered growth and enzyme expression profile of ZnO nanoparticles exposed non-target environmentally beneficial bacteria

The extensive production and usage of nanoparticles with ultimate disposal in the environment leads to unintentional exposure of non-target environmentally beneficial bacteria thereby posing a serious threat to the native soil inhabitants. Soil microflora is an important link in the biogeochemical cycling of nutrients, affecting ecosystem functioning and productivity. This study evaluates the effect of one of the widely used nanoparticles, zinc oxide on two predominant soil bacteria, Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa with respect to their biocatalytic activities. Growth profiles of these bacteria in the presence of zinc oxide nanoparticles (ZnONPs) at a concentration of 20 ppm exhibited a prolonged lag phase in B. subtilis, whereas no significant effect was observed in the case of P. aeruginosa even at 200 ppm. Interestingly, the enzymatic profile of both the organisms was affected at non-lethal ZnONPs concentrations. The most pronounced effect was on the enzymes associated with amylolytic activity, denitrification and urea degradation wherein total inhibition of activity was noted in B. subtilis. The enzyme activities were lowered in the case of P. aeruginosa. The results presented here reiterate a critical need for exposure assessment and risk characterization of nanomaterial disposal on soil microflora while formalizing waste management strategies.

DIFFERING PATTERNS OF PECTIN DISASSEMBLY AND PERICARP SOFTENING IN RIPENING AND CHILL-INJURED TOMATO FRUIT WITH DOWNREGULATED POLYGALACTURONASE ACTIVITY

ABSTRACT Pectin disassembly was examined in transgenic tomato fruit with reduced polygalacturonase (PG). Mature-green tomato fruit were stored at 5 and 12C for 21 days and subsequently transferred to 18C for an additional 5 days. Pericarp softened during storage at 12C and the softening rate increased after fruit were transferred to 18C. Firmness remained unchanged during storage at 5C but decreased after transfer of chilled fruit to 18C. Water-soluble pectins increased five- and ninefold during ripening of PG-antisense and wild-type fruit, respectively, while trans-1,2-cyclohexanediaminetetraacetic acid-soluble pectins increased only slightly. Solubilization of pectins was very restricted in fruit stored at 5C and upon subsequent transfer to 18C. Pectins exhibited a PG-mediated reduction in molecular mass during ripening at 12C, but no depolymerization was observed in chill-injured fruit. Chill-injured tomato fruit softened even though pectin solubilization and molecular mass distribution remained unchanged, suggesting that the softening mechanism differs in ripening and chill-injured tomato fruit. PRACTICAL APPLICATIONS Chilling injury poses serious problems in the supply chain of sensitive commodities. Abnormal textural changes are one of the chilling-injury symptoms described in sensitive fruit, including tomato, but the mechanism for the accelerated softening rate and development of mealiness is not yet fully understood. Although ripening-related softening and pectin metabolism have been extensively characterized, the pectic metabolism in chill-injured fruit remains elusive. Transgenic plants with altered enzyme expression are excellent tools to test hypothesis regarding the physiological role of the enzymes. Because polygalacturonase (PG) expression is inhibited during low-temperature storage, it is possible that, at least in part, the differences in textural changes between chill-injured and normally ripening fruit are related to this enzyme. The data presented here indicate that PG plays an even smaller role in the softening of chilled fruit than in that of ripening fruit. Tomato can provide a model for the understanding of the relationships between cell wall metabolism and texture that applies to other chilling-sensitive fruit.

Association of Hypoxia with Impaired Remodeling Mechanisms in the Abdominal Aortic Aneurysm (AAA) Wall

AAA rupture occurs when wall stresses exceed the wall strength. We tested two hypotheses: 1) the AAA wall near thick intraluminal thrombus (ILT) exhibits impaired remodeling compared to wall near thin ILT and 2) macrophages (mΦ) cultured under hypoxic conditions exhibit altered enzyme expression compared to mΦ in normoxic conditions. For 1, pairs of AAA wall, selected based on disparate thicknesses of near ILT, were snap frozen. QPCR was used to compare the gene expression. Total wall thickness was measured on H&amp;E sections. For 2, peripheral mononuclear cells were isolated from human buffy coats and cultured under hypoxic (2% O2) or normoxic (20% O2) conditions in serum‐free medium. Media levels of MMP‐9 were determined using ELISA. We found a down‐regulation of MMP‐2, MMP‐9, uPA, tPA, CD68, and collagen IV, and an up‐regulation of HO‐1, ORP‐150, ICAM‐1, IL‐12, IFN‐γ, and iNOS in AAA wall near thick ILT compared to AAA wall near thin ILT. AAA wall thickness was positively correlated (ρ=0.476) to ILT thickness. MΦ cultured in hypoxic conditions showed a marked decrease in secreted MMP‐9 activity and protein levels relative to mΦ cultured in normoxic conditions on collagen I or fibrin. The results support hypothesis 1, suggesting that compared to AAA wall near thin ILT, the AAA wall adjacent to a thick ILT has a more activated immune component and impaired remodeling mechanisms. Our results also suggest that the hypoxic environment of the AAA wall may lead to an alteration in protein expression by mΦ. This work was supported by NIH R01‐HL79313.

Phosphoenolpyruvate Carboxykinase Overexpression Selectively Attenuates Insulin Signaling and Hepatic Insulin Sensitivity in Transgenic Mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.

Potato hexokinase 2 complements transgenic Arabidopsis plants deficient in hexokinase 1 but does not play a key role in tuber carbohydrate metabolism.

Potato plants (Solanum tuberosum L. cv. Désirée) transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 2 exhibited altered enzyme activities and expression of hexokinase 2 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed an 11-fold variation in leaf (from 48% of the wild-type activity in antisense transformants to 446% activity in sense transformants) and an 8-fold variation in developing tubers (from 35% of the wild-type activity in antisense transformants to 212% activity in sense transformants). Despite the wide range of hexokinase activities, no substantial change was found in the fresh weight yield, starch, sugar and metabolite levels of transgenic tubers. However, both potato hexokinases 1 and 2 were able to complement the hyposensitivity of antisense hexokinase 1 Arabidopsis transgenic plants to glucose. In an in vitro bioassay of seed germination in a medium with high glucose levels, double transformants showed the same sensitivity to glucose as that of the wild-type ecotype, displaying a stunted phenotype in hypocotyls, cotyledons and roots.

Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants but not to significant changes in tuber carbohydrate metabolism.

Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants). Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers. However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period. Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content. These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.