Tomoichiro Asano

Distinct Roles of Autophagy in the Heart During Ischemia and Reperfusion: Roles of AMP-Activated Protein Kinase and Beclin 1 in Mediating Autophagy

Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-&bgr;-d-arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1+/− mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1–dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion.

Neuronal Pathway from the Liver Modulates Energy Expenditure and Systemic Insulin Sensitivity

Coordinated control of energy metabolism and glucose homeostasis requires communication between organs and tissues. We identified a neuronal pathway that participates in the cross talk between the liver and adipose tissue. By studying a mouse model, we showed that adenovirus-mediated expression of peroxisome proliferator–activated receptor (PPAR)–g2 in the liver induces acute hepatic steatosis while markedly decreasing peripheral adiposity. These changes were accompanied by increased energy expenditure and improved systemic insulin sensitivity. Hepatic vagotomy and selective afferent blockage of the hepatic vagus revealed that the effects on peripheral tissues involve the afferent vagal nerve. Furthermore, an antidiabetic thiazolidinedione, a PPARg agonist, enhanced this pathway. This neuronal pathway from the liver may function to protect against metabolic perturbation induced by excessive energy storage.

Adenosine Monophosphate-Activated Protein Kinase Suppresses Vascular Smooth Muscle Cell Proliferation Through the Inhibition of Cell Cycle Progression

Vascular smooth muscle cell (VSMC) proliferation is a critical event in the development and progression of vascular diseases, including atherosclerosis. We investigated whether the activation of adenosine monophosphate-activated protein kinase (AMPK) could suppress VSMC proliferation and inhibit cell cycle progression. Treatment of human aortic smooth muscle cells (HASMCs) or isolated rabbit aortas with the AMPK activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) induced phosphorylation of AMPK and acetyl Co-A carboxylase. AICAR significantly inhibited HASMC proliferation induced by both platelet-derived growth factor-BB (PDGF-BB) and fetal calf serum (FCS). Treatment with AICAR inhibited the phosphorylation of retinoblastoma gene product (Rb) induced by PDGF-BB or FCS, and increased the expression of cyclin-dependent kinase inhibitor p21CIP but not that of p27KIP. Pharmacological inhibition of AMPK or overexpression of dominant negative-AMPK inhibited both the suppressive effect of AICAR on cell proliferation and the phosphorylation of Rb, suggesting that the effect of AICAR is mediated through the activation of AMPK. Cell cycle analysis in HASMCs showed that AICAR significantly increased cell population in G0/G1-phase and reduced that in S- and G2/M-phase, suggesting AICAR induced cell cycle arrest. AICAR increased both p53 protein and Ser-15 phosphorylated p53 in HASMCs, which were blocked by inhibition of AMPK. In isolated rabbit aortas, AICAR also increased Ser-15 phosphorylation and protein expression of p53 and inhibited Rb phosphorylation induced by FCS. These data suggest for the first time that AMPK suppresses VSMC proliferation via cell cycle regulation by p53 upregulation. Therefore, AMPK activation in VSMCs may be a therapoietic target for the prevention of vascular diseases.

Regulation of Pancreatic β Cell Mass by Neuronal Signals from the Liver

Metabolic regulation in mammals requires communication between multiple organs and tissues. The rise in the incidence of obesity and associated metabolic disorders, including type 2 diabetes, has renewed interest in interorgan communication. We used mouse models to explore the mechanism whereby obesity enhances pancreatic β cell mass, pathophysiological compensation for insulin resistance. We found that hepatic activation of extracellular regulated kinase (ERK) signaling induced pancreatic β cell proliferation through a neuronal-mediated relay of metabolic signals. This metabolic relay from the liver to the pancreas is involved in obesity-induced islet expansion. In mouse models of insulin-deficient diabetes, liver-selective activation of ERK signaling increased β cell mass and normalized serum glucose levels. Thus, interorgan metabolic relay systems may serve as valuable targets in regenerative treatments for diabetes.

Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowmans capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.

Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats

Prolonged activation of p70 S6 kinase (S6K) by insulin and nutrients leads to inhibition of insulin signaling via negative feedback input to the signaling factor IRS-1. Systemic deletion of S6K protects against diet-induced obesity and enhances insulin sensitivity in mice. Herein, we present evidence suggesting that hypothalamic S6K activation is involved in the pathogenesis of diet-induced hepatic insulin resistance. Extending previous findings that insulin suppresses hepatic glucose production (HGP) partly via its effect in the hypothalamus, we report that this effect was blunted by short-term high-fat diet (HFD) feeding, with concomitant suppression of insulin signaling and activation of S6K in the mediobasal hypothalamus (MBH). Constitutive activation of S6K in the MBH mimicked the effect of the HFD in normal chow-fed animals, while suppression of S6K by overexpression of dominant-negative S6K or dominant-negative raptor in the MBH restored the ability of MBH insulin to suppress HGP after HFD feeding. These results suggest that activation of hypothalamic S6K contributes to hepatic insulin resistance in response to short-term nutrient excess.

Stat3-induced apoptosis requires a molecular switch in PI(3)K subunit composition.

Physiological apoptosis is induced by a switch from survival to death signalling. Dysregulation of this process is frequently associated with cancer. A powerful model for this apoptotic switch is mammary gland involution, during which redundant milk-producing epithelial cells undergo apoptosis. Signal transducer and activator of transcription 3 (Stat3) is an essential mediator of this switch but the mechanism has not yet been defined. Stat3-dependent cell death during involution can be blocked by activation of Akt/protein kinase B (PKB), a downstream effector of the phosphoinositide-3-OH kinase (PI(3)K) pathway. Here we show that expression of the PI(3)K regulatory subunits p55α and p50α is induced by Stat3 during involution. In the absence of Stat3 in vivo, upregulation of p55α and p50α is abrogated, levels of activated Akt are sustained and apoptosis is prevented. Chromatin immunoprecipitation assays show that Stat3 binds directly to the p55α and p50α promoters in vivo. Overexpression of either p55α or p50α reduces levels of activated Akt. We propose a novel mechanism in which Stat3 regulates apoptosis by inducing expression of distinct PI(3)K regulatory subunits to downregulate PI(3)K-Akt-mediated survival signalling.

Sirt3 protects in vitro–fertilized mouse preimplantation embryos against oxidative stress–induced p53-mediated developmental arrest

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3-/- embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

Blockade of the Nuclear Factor-κB Pathway in the Endothelium Prevents Insulin Resistance and Prolongs Life Spans

Background— Nuclear factor-&kgr;B (NF-&kgr;B) signaling plays critical roles in physiological and pathological processes such as responses to inflammation and oxidative stress. Methods and Results— To examine the role of endothelial NF-&kgr;B signaling in vivo, we generated transgenic mice expressing dominant-negative I&kgr;B under the Tie2 promoter/enhancer (E-DNI&kgr;B mice). These mice exhibited functional inhibition of NF-&kgr;B signaling specifically in endothelial cells. Although E-DNI&kgr;B mice displayed no overt phenotypic changes when young and lean, they were protected from the development of insulin resistance associated with obesity, whether diet- or genetics-induced. Obesity-induced macrophage infiltration into adipose tissue and plasma oxidative stress markers were decreased and blood flow and mitochondrial content in muscle and active-phase locomotor activity were increased in E-DNI&kgr;B mice. In addition to inhibition of obesity-related metabolic deteriorations, blockade of endothelial NF-&kgr;B signaling prevented age-related insulin resistance and vascular senescence and, notably, prolonged life span. These antiaging phenotypes were also associated with decreased oxidative stress markers, increased muscle blood flow, enhanced active-phase locomotor activity, and aortic upregulation of mitochondrial sirtuin-related proteins. Conclusions— The endothelium plays important roles in obesity- and age-related disorders through intracellular NF-&kgr;B signaling, thereby ultimately affecting life span. Endothelial NF-&kgr;B signaling is a potential target for treating the metabolic syndrome and for antiaging strategies.

Specific Roles of the p110α Isoform of Phosphatidylinsositol 3-Kinase in Hepatic Insulin Signaling and Metabolic Regulation

The class I(A) phosphatidylinsositol 3-kinases (PI3Ks) form a critical node in the insulin metabolic pathway; however, the precise roles of the different isoforms of this enzyme remain elusive. Using tissue-specific gene inactivation, we demonstrate that p110alpha catalytic subunit of PI3K is a key mediator of insulin metabolic actions in the liver. Thus, deletion of p110alpha in liver results in markedly blunted insulin signaling with decreased generation of PIP(3) and loss of insulin activation of Akt, defects that could not be rescued by overexpression of p110beta. As a result, mice with hepatic knockout of p110alpha display reduced insulin sensitivity, impaired glucose tolerance, and increased gluconeogenesis, hypolipidemia, and hyperleptinemia. The diabetic syndrome induced by loss of p110alpha in liver did not respond to metformin treatment. Together, these data indicate that the p110alpha isoform of PI3K plays a fundamental role in insulin signaling and control of hepatic glucose and lipid metabolism.