Resistance to <Emphasis Type="Italic">Soil-borne cereal mosaic virus</Emphasis> in durum wheat is controlled by a major QTL on chromosome arm 2BS and minor loci

Soil-borne cereal mosaic (SBCM) is a viral disease, which seriously affects hexaploid as well as tetraploid wheat crops in Europe. In durum wheat (Triticum durum Desf.), the elite germplasm is characterized by a wide range of responses to SBCMV, from susceptibility to almost complete resistance. In this study, the genetic analysis of SBCMV resistance was carried out using a population of 181 durum wheat recombinant inbred lines (RILs) obtained from Meridiano (resistant) × Claudio (moderately susceptible), which were profiled with SSR and DArT markers. The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection during 2007 and 2008. A wide range of disease reactions (as estimated by symptom severity and DAS-ELISA) was observed. A large portion of the variability for SBCMV response was explained by a major QTL (QSbm.ubo-2BS) located in the distal telomeric region of chromosome 2BS near the marker triplet Xbarc35–Xwmc661–Xgwm210, with R ² values ranging from 51.6 to 91.6%. The favorable allele was contributed by Meridiano. Several QTLs with minor effects on SBCMV response were also detected. Consistently with the observed transgressive segregation, the resistance alleles at minor QTLs were contributed by both parents. The presence and effects of QSbm.ubo-2BS were validated through association mapping in a panel of 111 elite durum wheat accessions.     

Immunopurification and characterization of rat adipocyte caveolae suggest their dissociation from insulin signaling

Adipocytes play an important role in the insulin-dependent regulation of organismal fuel metabolism and express caveolae at levels as high or higher than any other cell type. Recently, a link between insulin signaling and caveolae has been suggested; nevertheless, adipocyte caveolae have been the subject of relatively few studies, and their contents have been minimally characterized. With the aid of a new monoclonal antibody, we developed a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein, flotillin, and insulin receptors. Immunogold labeling and electron microscopy of the adipocyte plasma membrane confirmed the lack of insulin receptors in caveolae. In addition to caveolins, the structural components of caveolae, their major protein constituents, are the semicarbazide-sensitive amine oxidase and the scavenger lipoprotein receptor CD36. The results are consistent with a role for caveolae in lipid flux in and of adipocytes.     

Dynamics of protein-tyrosine phosphatases in rat adipocytes

Protein-tyrosine phosphatases (PTPases) play a key role in maintaining the steady-state tyrosine phosphorylation of the insulin receptor (IR) and its substrate proteins such as insulin receptor substrate 1 (IRS-1). However, the PTPase(s) that inactivate IR and IRS-1 under physiological conditions remain unidentified. Here, we analyze the subcellular distribution in rat adipocytes of several PTPases thought to be involved in the counterregulation of insulin signaling. We found that the transmembrane enzymes, protein-tyrosine phosphatase (PTP)-alpha and leukocyte common antigen-related (LAR), were detected predominantly in the plasma membrane and to a lesser extent in the heavy microsomes, a distribution similar to that of insulin receptor. PTP-1B and IRS-1 were present in light microsomes and cytosol, whereas SHPTP2/Syp was exclusively cytosolic. Insulin induced a redistribution of PTP-alpha from the plasma membrane to the heavy microsomes in a parallel fashion with the receptor. The distribution of PTP-1B in the light microsomes from resting adipocytes was similar to that of IRS-1 as determined by sucrose velocity gradient fractionation. Analysis of the catalytic activity of partially purified rat adipocyte PTP-alpha and LAR and recombinant PTP-1B showed that all three PTPases dephosphorylate IR. When a mix of IR/IRS-1 was used as a substrate, PTP-1B was particularly effective in dephosphorylating IRS-1. Considering that IR and IRS-1 can be dephosphorylated in internal membrane compartments from rat adipocytes (Kublaoui, B., Lee, J., and Pilch, P.F. (1995) J. Biol. Chem. 270, 59-65) and that PTP-alpha and PTP-1B are the respective PTPases in these fractions, we conclude that these PTPases are responsible for the counterregulation of insulin signaling there, whereas both LAR and PTP-alpha may act upon cell surface insulin receptors.     

Dissociation of insulin receptor expression and signaling from caveolin-1 expression

The presence of cell surface caveolin/caveolae has been postulated to influence the localization, expression levels, and kinase activity of numerous receptors, including the insulin receptor. However, there are conflicting data concerning the effects of caveolin on insulin receptor expression and function. To help clarify this issue, we created a gain of function situation by expressing caveolin-1 at various levels in HEK-293 cells where the endogenous level of caveolin-1 is very low. We generated four permanent lines of this cell expressing amounts of caveolin-1 ranging from 10 to 40 times that of parental cells. The amount of caveolin-1 in the human embryonic kidney cells expressing the highest caveolin levels is comparable with that of adipocytes, cells that naturally express one of the highest levels of caveolin-1. We measured insulin receptor amount and insulin-dependent receptor autophosphorylation as well as insulin receptor substrate 1 (IRS1) tyrosine phosphorylation as an index of insulin signaling. We found that the insulin receptor level was essentially the same in the parental and all four derived cell lines. Likewise, we determined that insulin-dependent insulin receptor and IRS1 tyrosine phosphorylation was not significantly different in the four cell lines representing parental, low, medium, and high levels of caveolin-1 expression. We conclude that insulin receptor expression and ligand-dependent signaling is independent of caveolin-1 expression.     

Acyl Coenzyme A Synthetase Regulation: Putative Role in Long‐Chain Acyl Coenzyme A Partitioning

Objective: Long‐chain acyl coenzyme A synthetase (ACSL) converts free fatty acids (FFAs) into their metabolizable long‐chain acyl coenzyme A (LC‐CoA) derivatives that are essential for FFA conversion to CO₂, triglycerides, or complex lipids. ACSL‐1 is highly expressed in adipose tissue with broad substrate specificity. We tested the hypothesis that ACSL localization, and resulting local generation of LC‐CoA, regulates FFA partitioning. Research Methods and Procedures: These studies used cell fractionation of rat adipocytes to measure ACSL activity and mass and compared cells from young, mature, fed, fasted, and diabetic rats. Functional studies included measurement of FFA oxidation, complex lipid synthesis, and LC‐CoA levels. Results: High ACSL specific activity was expressed in the mitochondria/nuclei (M/N), high‐density microsomes (HDM), low‐density microsomes (LDM), and plasma membrane (PM) fractions. We show here that, during fasting, total FFA oxidation increased, and, although total ACSL activity decreased, a greater percentage of activity (43 ± 1.5%) was associated with the M/N fraction than in the fed state (23 ± 0.3%). In the fed state, more ACSL activity (34 ± 0.5%) was associated with the HDM than in the fasted state (25 ± 0.9%), concurrent with increased triglyceride formation from FFA. Insulin increased LC‐CoA and ACSL activity associated with the PM. The changes in ACSL activity in response to insulin were associated with only minor changes in mass as determined by Western blotting. Discussion: It is hypothesized that ACSL plays an important role in targeting FFA to specific metabolic pathways or acylation sites in the cell, thus acting as an important control mechanism in fuel partitioning. Localization of ACSL at the PM may serve to decrease FFA efflux and trap FFA within the cell as LC‐CoA.     

Insulin-dependent phosphorylation of a 70-kDa protein in light microsomes from rat adipocytes

In order to discover possibly novel insulin receptor substrates and/or downstream targets in the insulin signaling pathway, we established a cell-free system for this purpose using purified insulin receptor and subcellular fractions from rat adipocytes as a sourse of cellular substrates. Under these conditions, we have found a 70-kDa protein (pp70) in fat cells that is tyrosine-phosphorylated by the activated insulin receptor. Using sucrose velocity gradient sedimentation we also show that pp70 cofractionate a particulate fraction containing IRS-1 but not with GLUT-4 vesicle-enriched fractions. Our results suggest that pp70 may be an endogenous substrate for the insulin receptor tyrosine kinase.     

Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles

Skeletal muscle denervation decreases insulin-sensitive glucose uptake into this tissue as a result of marked GLUT-4 protein downregulation ( approximately 20% of controls). The process of insulin-stimulated glucose transport in muscle requires the movement or translocation of intracellular GLUT-4-rich vesicles to the cell surface, and it is accompanied by the translocation of several additional vesicular cargo proteins. Thus examining GLUT-4 translocation in muscles from denervated animals allows us to determine whether the loss of a major cargo protein, GLUT-4, affects the insulin-dependent behavior of the remaining cargo proteins. We find no difference, control vs. denervated, in the insulin-dependent translocation of the insulin-responsive aminopeptidase (IRAP) and the receptors for transferrin and insulin-like growth factor II/mannose 6-phosphate, proteins that completely (IRAP) or partially co-localize with GLUT-4. We conclude that 1) denervation of skeletal muscle does not block the specific branch of insulin signaling pathway that connects receptor proximal events to intracellular GLUT-4-vesicles, and 2) normal levels of GLUT-4 protein are not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment. Muscle denervation also causes a twofold increase in GLUT-1. In normal muscle, all GLUT-1 is present at the cell surface, but in denervated muscle a significant fraction (25.1 +/- 6.1%) of this transporter is found in intracellular vesicles that have the same sedimentation coefficient as GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption. These GLUT-1-containing vesicles respond to insulin and translocate to the cell surface. Thus the formation of insulin-sensitive GLUT-1-containing vesicles in denervated muscle may be a compensatory mechanism for the decreased level of GLUT-4.     

High molecular weight glutenin subunit variation in Triticum turgidum var. dicoccum

Summary Variation in high molecular weight (HMW) glutenin subunit composition among 167 accessions of dicoccum wheat (Triticum turgidum L. var. dicoccum Schrank) of diverse origins was investigated using one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). A total of 20 alleles were identified, and 9 of them were found to be different from those previously detected by Payne and Lawrence (1983 b) in hexaploid wheat (Triticum aestivum L.). The newly discovered alleles enhance the genetic variability available to improve the industrial quality of wheats and some of them may facilitate basic research on the relationship of industrial quality with HMW glutenin subunit number. The novel variants include a GLU-A1 encoded subunit which has higher molecular mass than any other so far described in tetraploid and hexaploid wheats, and a null GLU-B1 allele. Dicoccums containing neither GLU-A1- nor GLU-B1-encoded subunits were also identified. A comparison of the mean number of HMW glutenin subunits contained in various primitive and modern domesticated wheats of different ploidy levels and the identification of wheats containing no HMW glutenin subunits suggest that the occurrence of null GLU-1 alleles in these species depends on chance rather on an inherent tendency on the part of modern polyploid wheats to suppress the activity of redundant GLU-1 genes.     

UCP-3 expression in skeletal muscle: effects of exercise, hypoxia, and AMP-activated protein kinase

Uncoupling protein 3 (UCP-3), a member of the mitochondrial transporter superfamily, is expressed primarily in skeletal muscle where it may play a role in altering metabolic function under conditions of fuel depletion caused, for example, by fasting and exercise. Here, we show that treadmill running by rats rapidly (30 min) induces skeletal muscle UCP-3 mRNA expression (sevenfold after 200 min), as do hypoxia and swimming in a comparably rapid and substantial fashion. The expression of the mitochondrial transporters, carnitine palmitoyltransferase 1 and the tricarboxylate carrier, is unaffected under these conditions. Hypoxia and exercise-mediated induction of UCP-3 mRNA result in a corresponding four- to sixfold increase in rat UCP-3 protein. We treated extensor digitorum longus (EDL) muscle with 5'-amino-4-imidazolecarboxamide ribonucleoside (AICAR), a compound that activates AMP-activated protein kinase (AMPK), an enzyme known to be stimulated during exercise and hypoxia. Incubation of rat EDL muscle in vitro for 30 min with 2 mM AICAR causes a threefold increase in UCP-3 mRNA and a 1.5-fold increase of UCP-3 protein compared with untreated muscle. These data are consistent with the notion that activation of AMPK, presumably as a result of fuel depletion, rapidly regulates UCP-3 gene expression.