Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae.

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

Dosage-dependent effects of Akt1/protein kinase Balpha (PKBalpha) and Akt3/PKBgamma on thymus, skin, and cardiovascular and nervous system development in mice.

Akt/protein kinase B (PKB) plays a critical role in the regulation of metabolism, transcription, cell migration, cell cycle progression, and cell survival. The existence of viable knockout mice for each of the three isoforms suggests functional redundancy. We generated mice with combined mutant alleles of Akt1 and Akt3 to study their effects on mouse development. Here we show that Akt1-/- Akt3+/- mice display multiple defects in the thymus, heart, and skin and die within several days after birth, while Akt1+/- Akt3-/- mice survive normally. Double knockout (Akt1-/-) Akt3-/-) causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems. Increased apoptosis was found in the developing brain of double mutant embryos. These data indicate that the Akt1 gene is more essential than Akt3 for embryonic development and survival but that both are required for embryo development. Our results indicate isoform-specific and dosage-dependent effects of Akt on animal survival and development.

Urocortins improve dystrophic skeletal muscle structure and function through both PKA- and Epac-dependent pathways.

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Ly49E-dependent inhibition of natural killer cells by urokinase plasminogen activator.

The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.

NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.

Evidence that extrapancreatic GLUT2-dependent glucose sensors control glucagon secretion.

GLUT2-/- mice reexpressing GLUT1 or GLUT2 in their beta-cells (RIPGLUT1 x GLUT2-/- or RIPGLUT2 x GLUT2-/- mice) have nearly normal glucose-stimulated insulin secretion but show high glucagonemia in the fed state. Because this suggested impaired control of glucagon secretion, we set out to directly evaluate the control of glucagonemia by variations in blood glucose concentrations. Using fasted RIPGLUT1 x GLUT2-/- mice, we showed that glucagonemia was no longer increased by hypoglycemic (2.5 mmol/l glucose) clamps or suppressed by hyperglycemic (10 and 20 mmol/l glucose) clamps. However, an increase in plasma glucagon levels was detected when glycemia was decreased to < or =1 mmol/l, indicating preserved glucagon secretory ability, but of reduced sensitivity to glucopenia. To evaluate whether the high-fed glucagonemia could be due to an abnormally increased tone of the autonomic nervous system, fed mutant mice were injected with the ganglionic blockers hexamethonium and chlorisondamine. Both drugs lead to a rapid return of glucagonemia to the levels found in control fed mice. We conclude that 1) in the absence of GLUT2, there is an impaired control of glucagon secretion by low or high glucose; 2) this impaired glucagon secretory activity cannot be due to absence of GLUT2 from alpha-cells because these cells do not normally express this transporter; 3) this dysregulation may be due to inactivation of GLUT2-dependent glucose sensors located outside the endocrine pancreas and controlling glucagon secretion; and 4) because fed hyperglucagonemia is rapidly reversed by ganglionic blockers, this suggests that in the absence of GLUT2, there is an increased activity of the autonomic nervous system stimulating glucagon secretion during the fed state.

Macrophage migration inhibitory factor deficiency leads to age-dependent impairment of glucose homeostasis in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic beta-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIF-/-) mice. Compared with wild-type (WT) mice, MIF-/- mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIF-/- mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIF-/- mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIF-/- mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIF-/- mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIF-/- mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIF-/- mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein¿Raf1 and the Raichu-KRas probe, we identified for the first time in vivo¿active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14¿MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27Kip1

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection.

α-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr(317)ProThr(319)ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.

A-kinase anchoring proteins: Molecular regulators of the cardiac stress response

In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

D-3 2009 Dependent Adult Abuse Report, July - December 2009.

D-3 Dependent Adult Abuse Report