Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.

Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain

A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3′-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5′-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 μM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.

Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent γ-glutamyl carboxylation during protein processing and block the secretion of under-γ-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-γ-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FIIRHH and FIIHRR, FIIRRH and FIIHHR, FIIRHR and FIIHRH) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-γ-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FIIHrhH and FIIHhrH, FIIRrhR, and FIIRhrR) was used to determine that the first of the two kringles plays a more important role in the recognition process.

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.

Effects of Short- and Long-Term Elevated CO2 on the Expression of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes and Carbohydrate Accumulation in Leaves of Arabidopsis thaliana (L.) Heynh.1

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 μL L−1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35–40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

Developmentally related responses of maize catalase genes to salicylic acid.

The response of the maize catalase genes (Cat1, Cat2, and Cat3) to salicylic acid (SA) was examined at two distinct developmental stages: embryogenesis and germination. A unique, germination-related differential response of each maize catalase gene to various doses of SA was observed. During late embryogenesis, total catalase activity in scutella increased dramatically with 1 mM SA treatment. The accumulation of Cat2 transcript and CAT-2 isozyme protein provided the major contribution to the observed increase in total catalase activity. This increase was paralleled by the enhanced growth of germinated embryos at that stage. In a CAT-2 null mutant line, a full compensation of total catalase activity by the CAT-1 isozyme was observed in the presence of SA. This suggests that catalase is important for maintenance of normal cellular processes under stress conditions. SA at 1 mM, which enhances growth of precociously germinated embryos, appeared to inhibit seed germination at 1 day after inhibition. Furthermore, Cat2 transcript accumulation was inhibited at this stage. SA is probably not a direct signal for the induction of the catalase genes. Other signals, possibly germination-related regulator(s), might be responsible for the induction of the catalase genes. The effect of SA on the activity of purified catalase protein was also examined.

Regulation of Oleoresinosis in Grand Fir (Abies grandis)1 : Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.

Differential Expression of a Novel Gene in Response to Coronatine, Methyl Jasmonate, and Wounding in the Coi1 Mutant of Arabidopsis1

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Drosophila host defense: Differential induction of antimicrobial peptide genes after infection by various classes of microorganisms

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.

Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes

Insulin and guanosine-5′-O-(3-thiotriphosphate) (GTPγS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (∼30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPγS response was significantly attenuated (∼85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPγS-stimulated GLUT4 translocation by ∼40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPγS-stimulated GLUT4 translocation but inhibited the insulin response by ∼40%. GTPγS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (∼50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPγS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.