Abstract-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-␤-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 ϩ/Ϫ 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. (Circ Res. 2007;100:914-922.) Key Words: autophagy Ⅲ AMP-activated protein kinase (AMPK) Ⅲ beclin 1 Ⅲ ischemia/reperfusion A utophagy is an intracellular bulk degradation process whereby cytosolic, long-lived proteins and organelles are degraded and recycled. 1 Autophagy occurs at basal levels but can be further induced by stresses, such as nutrient depletion. 2 Autolysosomal degradation of membrane lipids and proteins generates free fatty acids and amino acids, which can be reused to maintain mitochondrial ATP production and protein synthesis, and promote cell survival. Disruption of this pathway prevents cell survival in diverse organisms. 2 Interestingly, autophagy also promotes programmed cell death in some circumstances. 3,4 Thus, autophagy has a dual role in cell survival, although, in cardiac myocytes, it remains to be elucidated whether autophagy is required for survival, and is thereby salutary, or whether it mediates cell death, and is detrimental during pathologically relevant stresses, such as ischemia and reperfusion.The mTOR (mammalian target of rapamycin) pathway is a key regulator of cell growth and proliferation and integrates signals regarding nutrie...

Abstract-Angiotensin II (AII) is involved in the pathogenesis of both hypertension and insulin resistance, though few studies have examined the relationship between the two. We therefore investigated the effects of chronic AII infusion on blood pressure and insulin sensitivity in rats fed a normal (0.3% NaCl) or high-salt (8% NaCl) diet. AII infusion for 12 days significantly elevated blood pressure and significant insulin resistance, assessed by a hyperinsulinemic-euglycemic clamp study and glucose uptake into isolated muscle and adipocytes. High-salt loading exacerbated the effects of AII infusion significantly. Despite the insulin resistance, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates, activation of phosphatidylinositol (PI) 3-kinase, and phosphorylation of Akt were all enhanced by AII infusion. Subsequently, to investigate whether oxidative stress induced by AII contributes to insulin resistance, the membrane-permeable superoxide dismutase mimetic, tempol, was administered to AII-infused rats. Chronic AII infusion induced an accumulated plasma cholesterylester hydroperoxide levels, indicating the increased oxidative stress, whereas the treatment with tempol normalized plasma cholesterylester hydroperoxide levels in AII-infused rats. In addition, the treatment with tempol normalized insulin resistance in AII-infused rats, shown as a decreased glucose infusion rate in the hyperinsulinemic euglycemic clamp study and a decreased insulin-induced glucose uptake into isolated skeletal muscle, as well as enhanced insulin-induced PI 3-kinase activation to those in the control rats. These results strongly suggest that AII-induced insulin resistance cannot be attributed to impairment of early insulin-signaling steps and that increased oxidative stress, possibly through impaired insulin signaling located downstream from PI 3-kinase activation, is involved in AII-induced insulin resistance. Key Words: angiotensin II Ⅲ insulin resistance Ⅲ oxidative stress Ⅲ glucose clamp technique Ⅲ sodium Ⅲ kinase S everal lines of evidence point to an association between hypertension and insulin resistance, 1,2 eg, hypertensive individuals are more likely to become diabetic than normotensive ones. 3 It is therefore notable that angiotensin II (AII) is reportedly involved in the development of both hypertension and insulin resistance, 4 -7 and agents that inhibit the action of AII, ie, angiotensin-converting enzyme inhibitors and type 1 AII (AT1) receptor antagonists, not only reduce blood pressure but also restore insulin sensitivity. 8 -14 It has been suggested that crosstalk between AII-and insulinsignaling pathways underlies AII-induced insulin resistance. According to that model, AII induces tyrosine phosphorylation of insulin receptor substrate (IRS)-1 by Janus kinase 2 (JAK2) associated with the AT1 receptor, thereby attenuating insulin-induced activation of phosphatidylinositol (PI) 3-kinase associated with IRS-1, which in turn diminishes insulin sensitivity. 15,16 However, ...

Resistin is a hormone secreted by adipocytes that acts on skeletal muscle myocytes, hepatocytes, and adipocytes themselves, reducing their sensitivity to insulin. In the present study, we investigated how the expression of resistin is affected by glucose and by mediators known to affect insulin sensitivity, including insulin, dexamethasone, tumor necrosis factor-␣ (TNF-␣), epinephrine, and somatropin. We found that resistin expression in 3T3-L1 adipocytes was significantly upregulated by high glucose concentrations and was suppressed by insulin. Dexamethasone increased expression of both resistin mRNA and protein 2.5-to 3.5-fold in 3T3-L1 adipocytes and by ϳ70% in white adipose tissue from mice. In contrast, treatment with troglitazone, a thiazolidinedione antihyperglycemic agent, or TNF-␣ suppressed resistin expression by ϳ80%. Epinephrine and somatropin were both moderately inhibitory, reducing expression of both the transcript and the protein by 30 -50% in 3T3-L1 adipocytes. Taken together, these data make it clear that resistin expression is regulated by a variety of hormones and that cytokines are related to glucose metabolism. Furthermore, they suggest that these factors affect insulin sensitivity and fat tissue mass in part by altering the expression and eventual secretion of resistin from adipose cells.

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.

Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast twohybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APEinduced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.The serine/threonine protein kinase PKB 1 (also called Akt) is thought to be a key mediator of signal transduction. Upon growth factor stimulation, a family of lipid kinases known as class 1 phosphoinositide 3-kinases (PI 3-kinases) is recruited to the plasma membrane. PI 3-kinases phosphorylate phosphatidylinositol 4,5-bisphosphate at the D-3 position of the inositol ring, converting it to phosphatidylinositol 3,4,5-trisphosphate. Following the activation of PI 3-kinase, PKBs are recruited to the plasma membrane through direct contact of the pleckstrin homology (PH) domain with phosphatidylinositol 3,4,5-trisphosphate and are phosphorylated at Thr 308 by PDK1 and at Ser 473 by PDK2, a kinase of which the molecular structure has not yet been identified (1, 2). AGC kinases other than PKB are also known to be regulated by PI 3-kinase, and PKB acts downstream from PI 3-kinase to regulate numerous biological processes, such as proliferation, antiapoptosis, cell growth, and glucose metabolism (1, 2).PKB has a wide range of substrates, including GSK-3, FKHR (FoxO1), FKHR-L1 (FoxO3), AFX (FoxO4), and eNOS, all of which have the consensus motif RXRXX(S/T) (3, 4). Protein kinases do not generally form stable complexes with their substrates, although PKB has been shown to exist in a stable complex with several of its substrates including MDM2, p21 Cip1 /WAF1, an...

Glucocorticoids reportedly induce insulin resistance. In this study, we investigated the mechanism of glucocorticoid-induced insulin resistance using 3T3-L1 adipocytes in which treatment with dexamethasone has been shown to impair the insulin-induced increase in glucose uptake. In 3T3-L1 adipocytes treated with dexamethasone, the GLUT1 protein expression level was decreased by 30%, which possibly caused decreased basal glucose uptake. On the other hand, dexamethasone treatment did not alter the amount of GLUT4 protein in total cell lysates but decreased the insulin-stimulated GLUT4 translocation to the plasma membrane, which possibly caused decreased insulin-stimulated glucose uptake. Dexamethasone did not alter tyrosine phosphorylation of insulin receptors, and it significantly decreased protein expression and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Interestingly, however, protein expression and tyrosine phosphorylation of IRS-2 were increased. To investigate whether the reduced IRS-1 content is involved in insulin resistance, IRS-1 was overexpressed in dexamethasone-treated 3T3-L1 adipocytes using an adenovirus transfection system. Despite protein expression and phosphorylation levels of IRS-1 being normalized, insulin-induced 2-deoxy-D-[ 3 H]glucose uptake impaired by dexamethasone showed no significant improvement. Subsequently, we examined the effect of dexamethasone on the glucose uptake increase induced by overexpression of GLUT2-tagged p110␣, constitutively active Akt (myristoylated Akt), oxidative stress (30 mU glucose oxidase for 2 h), 2 mmol/l 5-aminoimidazole-4-carboxamide ribonucleoside for 30 min, and osmotic shock (600 mmol/l sorbitol for 30 min). Dexamethasone treatment clearly inhibited the increases in glucose uptake produced by these agents. Thus, in conclusion, the GLUT1 decrease may be involved in the dexamethasone-induced decrease in basal glucose transport activity, and the mechanism of dexamethasone-induced insulin resistance in glucose transport activity (rather than the inhibition of phosphatidylinositol 3-kinase activation resulting from a decreased IRS-1 content) is likely to underlie impaired glucose transporter regulation.

AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3␤ (GSK3␤) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3␤ within 15 min. Stimulation with AICAR or the GSK3␤ inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3␤ to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3␤(S9G) but not wild-type GSK3␤. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3␤ phosphorylation were very similar. Furthermore, AMPK-mediated GSK3␤ phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3␤ is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3␤ by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.

TNFalpha, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest.