Chronic Akt Activation Research Articles

High USP6NL Levels in Breast Cancer Sustain Chronic AKT Phosphorylation and GLUT1 Stability Fueling Aerobic Glycolysis.

USP6NL, also named RN-tre, is a GTPase-activating protein involved in control of endocytosis and signal transduction. Here we report that USP6NL is overexpressed in breast cancer, mainly of the basal-like/integrative cluster 10 subtype. Increased USP6NL levels were accompanied by gene amplification and were associated with worse prognosis in the METABRIC dataset, retaining prognostic value in multivariable analysis. High levels of USP6NL in breast cancer cells delayed endocytosis and degradation of the EGFR, causing chronic AKT (protein kinase B) activation. In turn, AKT stabilized the glucose transporter GLUT1 at the plasma membrane, increasing aerobic glycolysis. In agreement, elevated USP6NL sensitized breast cancer cells to glucose deprivation, indicating that their glycolytic capacity relies on this protein. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing cell proliferation exclusively in breast cancer cells that harbored increased levels of USP6NL. Overall, these findings argue that USP6NL overexpression generates a metabolic rewiring that is essential to foster the glycolytic demand of breast cancer cells and promote their proliferation.Significance: USP6NL overexpression leads to glycolysis addiction of breast cancer cells and presents a point of metabolic vulnerability for therapeutic targeting in a subset of aggressive basal-like breast tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3432/F1.large.jpg Cancer Res; 78(13); 3432-44. ©2018 AACR.

Abstract IA08: Identifying and overcoming metabolic checkpoints to antitumor immunity

Abstract It has now become clear that the immune system is not oblivious to cancer initiation and progression, but rather is rendered dysfunctional through a wide array of tolerogenic mechanisms. One of these is the generation of a potently immunosuppressive microenvironment, characterized by altered populations of suppressive cells, secretion of suppressive cytokines, and upregulation of inhibitory ligands. The tumor microenvironment also possesses, due to the deregulated energetics of cancer cells, an altered metabolic profile: hypoxia, acidosis, and a dearth of glucose and other carbon sources. T cell activation and effector function are metabolically demanding, requiring elevated glucose processing and mitochondrial activity to keep up with the bioenergetic demands of proliferation, cytokine secretion, and cytolytic capacity. Thus, we sought to determine whether the metabolism of the tumor microenvironment acts as a barrier to antitumor immunity and resistance mechanism to immunotherapy. We have demonstrated that T cells infiltrating murine and human tumors exhibit a phenotype of metabolic insufficiency, characterized by decreased glucose uptake and a crippling loss of mitochondrial function. We show that this loss of oxidative function is specific to the chronic activation observed in the tumor microenvironment, and occurs rapidly upon entry into the tumor. This loss of mitochondrial activity and mass is due, in part, to the repression of PGC1α, a transcriptional co-activator that programs mitochondrial biogenesis. Further, we have identified chronic Akt activation and consequent Foxo inactivation as a major driver of PGC1α repression. Importantly, metabolic reprogramming of tumor-specific T cells to enforce mitochondrial biogenesis results in dramatically increased antitumor immunity, characterized by robust proliferation, cytokine production, and tumor regression and clearance. Our data support a model in which mitochondrial insufficiency represents a primordial checkpoint inhibiting antitumor immunity in addition to immunologic suppression. Further supporting this notion are additional studies from our laboratory suggesting that selective boosting of T cell mitochondrial activity through mitigation of tumor hypoxia dramatically enhances immunotherapies like checkpoint blockade and adoptive cell therapy. Our data suggest not only that the metabolic nature of the tumor microenvironment may be predictive of a patient's response to various immunotherapies, but that metabolic reprogramming of T cells or the microenvironment itself may be an important means to improve the immunotherapeutic treatment of cancer. Citation Format: Greg M. Delgoffe. Identifying and overcoming metabolic checkpoints to antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA08.

Partial Loss of the Glutamate Transporter GLT-1 Alters Brain Akt and Insulin Signaling in a Mouse Model of Alzheimer's Disease.

The glutamate transporter GLT-1 (also called EAAT2 in humans) plays a critical role in regulating extracellular glutamate levels in the central nervous system (CNS). In Alzheimer's disease (AD), EAAT2 loss is associated with neuropathology and cognitive impairment. In keeping with this, we have reported that partial GLT-1 loss (GLT-1+/-) causes early-occurring cognitive deficits in mice harboring familial AD AβPPswe/PS1ΔE9 mutations. GLT-1 plays important roles in several molecular pathways that regulate brain metabolism, including Akt and insulin signaling in astrocytes. Significantly, AD pathogenesis also involves chronic Akt activation and reduced insulin signaling in the CNS. In this report we tested the hypothesis that GLT-1 heterozygosity (which reduces GLT-1 to levels that are comparable to losses in AD patients) in AβPPswe/PS1ΔE9 mice would induce sustained activation of Akt and disturb components of the CNS insulin signaling cascade. We found that partial GLT-1 loss chronically increased Akt activation (reflected by increased phosphorylation at serine 473), impaired insulin signaling (reflected by decreased IRβ phosphorylation of tyrosines 1150/1151 and increased IRS-1 phosphorylation at serines 632/635 - denoted as 636/639 in humans), and reduced insulin degrading enzyme (IDE) activity in brains of mice expressing familial AβPPswe/PS1ΔE9 AD mutations. GLT-1 loss also caused an apparent compensatory increase in IDE activity in the liver, an organ that has been shown to regulate peripheral amyloid-β levels and expresses GLT-1. Taken together, these findings demonstrate that partial GLT-1 loss can cause insulin/Akt signaling abnormalities that are in keeping with those observed in AD.

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca2+ properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca2+ release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca2+ properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca2+ anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.

Biphasic Action of Aldosterone on Akt Signaling in Cardiomyocytes

Both aldosterone and Akt signaling play pivotal roles in the pathogenesis of heart failure. However, little is known about the correlation between them. We herein investigated whether aldosterone interacts with Akt signaling in a coordinated manner in cardiomyocytes. Neonatal rat cardiomyocytes were stimulated with aldosterone for either a short (10-min) or long (24-h) time. The phosphorylation of Akt and its downstream effector, GSK3β, were transiently increased after short-term stimulation, which was blocked by either PI3K or Na(+)/H(+) exchanger inhibitors, but not by the mineralocorticoid receptor antagonist, eplerenone. Long-term stimulation also significantly increased Akt-GSK3β phosphorylation and this effect was reduced by eplerenone. Thus, these results suggest that aldosterone activates Akt signaling via a biphasic reaction that occurs through different cascades. To understand the significance of the rapid action of aldosterone, cardiomyocytes were exposed to hydrogen peroxide for from 10 to 60 min. A short-term aldosterone stimulation (for up to 30 min) significantly protected cardiomyocytes from oxidative stress-induced cellular damage. Eplerenone did not abrogate this beneficial effect, while a PI3K inhibitor did. Therefore, during the early phase, aldosterone has favorable effects on cardiomyocytes, partly by acute activation of a mineralocorticoid receptor-independent cascade through the Na(+)/H(+) exchanger, PI3K, and Akt. In contrast, its persistent activity produces pathological effects partly by chronic Akt activation in a mineralocorticoid receptor-dependent manner.

Abstract P305: Detrimental Effects of Chronic Activation of AKT in Hearts from Ozone-Exposed Healthy Adult Rats

Death of cardiomyocytes results in decreased cardiac function, which correlates with overall morbidity and mortality in many different clinical settings. For these reasons, understanding the intracellular signaling pathways that control cardiomyocyte survival has significant clinical implications. Each year in the US 60,000 cardiovascular deaths have been linked to environmental pollutants such as ozone (O 3 ). Earlier studies from our laboratory have shown that myocardial dysfunction, subsequent to chronic O 3 exposure, in normal adult rats. This attenuation of cardiac function was associated with increased myocardial TNF-alpha (TNF-α) levels. Progressive increases in the expression of myocardial TNF-α in 4 and 8 week O 3 exposed animals were followed by decreases in cardiac caveolin-1 levels. Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). Akt is activated in samples from patients with chronic heart failure, however, less is known about the chronic effects of Akt activation in the hearts from ozone exposed patients. Although caveolin-1 has been shown to negatively regulate AKT expression; a cell survival pathway, regulatory role of AKT in hearts from ozone exposed subjects has never been studied. Sprague Dawley rats were exposed 8 hr/day for 28 and 56 days to filtered air or 0.8 ppm O 3 . In order to assess the chronic effects to O 3 , expressions of AKT and p-AKT were determined by wetern blotting in hearts extracted from rats, 24 hr after termination of air or O 3 exposure. Compared to rats exposed to filtered air, total AKT levels were significantly increased in all O 3 exposed animals. Similar results were seen with pAKT levels. Since earlier studies have determined detrimental effects of chronic activation of AKT in ischemia reperfusion injury, our novel findings suggest the interesting possibility of a mechanism by which chronic Akt activation can become maladaptive after exposure to chronic levels of ozone. It is possible that PI3K-dependent but Akt-independent effectors are required for full cardioprotection. Studies are currently in progress to prove this hypothesis and complete study will be presented at the meeting in New Orleans in 2011.

RETRACTED: Thapsigargin triggers cardiac contractile dysfunction via NADPH oxidase-mediated mitochondrial dysfunction: Role of Akt dephosphorylation

ER stress triggers myocardial contractile dysfunction although the underlying mechanism is still elusive. Given that NADPH oxidase was recently implicated in ER stress-induced tissue injury, this study was designed to examine the role of NADPH oxidase in ER stress-induced cardiac mechanical defects and the impact of Akt activation on ER stress-induced cardiac anomalies. Wild-type and transgenic mice with cardiac-specific overexpression of an active mutant of Akt (MyAkt) were subjected to the ER stress inducer thapsigargin (1 and 3mg/kg, ip, for 48h). Thapsigargin compromised echocardiographic parameters, including elevating LVESD and reducing fractional shortening; suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, and cell survival; and enhanced carbonyl formation, apoptosis, superoxide production, NADPH oxidase expression, and mitochondrial damage. Interestingly, these anomalies were attenuated or mitigated by chronic Akt activation. Treatment with thapsigargin also dephosphorylated Akt and its downstream signal GSK3β (leading to activation of GSK3β), the effect of which was abrogated in MyAkt hearts. Knockdown of the cytosolic subunit of NADPH oxidase, p47(phox), using siRNA abrogated thapsigargin-induced apoptosis and cell death in H9C2 myoblasts. In vitro exposure to thapsigargin induced murine cardiomyocyte dysfunction reminiscent of the in vivo setting, the effects of which were ablated by the NADPH oxidase inhibitor apocynin and the mitochondrial Ca(2+) uptake inhibitor Ru360. In addition, apocynin abrogated thapsigargin-induced loss of mitochondrial membrane potential and permeability transition pore opening, similar to chronic Akt activation. In summary, these data suggest that ER stress interrupts cardiac contractile and intracellular Ca(2+) homeostasis, cell survival, and mitochondrial integrity through an Akt dephosphorylation- and NADPH oxidase-dependent mechanism.

Chronic akt activation accentuates aging-induced cardiac hypertrophy and myocardial contractile dysfunction: role of autophagy

Aging is often accompanied with geometric and functional changes in the heart, although the underlying mechanisms remain unclear. Recent evidence has described a potential role of Akt and autophagy in aging-associated organ deterioration. This study was to examine the impact of cardiac-specific Akt activation on aging-induced cardiac geometric and functional changes and underlying mechanisms involved. Cardiac geometry, contractile and intracellular Ca(2+) properties were evaluated using echocardiography, edge-detection and fura-2 techniques. Level of insulin signaling and autophagy was evaluated by western blot. Our results revealed cardiac hypertrophy (enlarged chamber size, wall thickness, myocyte cross-sectional area), fibrosis, decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca(2+) release and clearance in aged (24-26month-old) mice compared with young (3-4month-old) mice, the effects of which were accentuated by chronic Akt activation. Aging enhanced Akt and mTOR phosphorylation while reducing that of PTEN, AMPK and ACC with a more pronounced response in Akt transgenic mice. GSK3β phosphorylation and eNOS levels were unaffected by aging or Akt overexpression. Levels of beclin-1, Atg5 and LC3-II-to-LC3-I ratio were decreased in aged hearts, the effect of which with the exception of Atg 5 was exacerbated by Akt overactivation. Levels of p62 were significantly enhanced in aged mice with a more pronounced increase in Akt mice. Neither aging nor Akt altered β-glucuronidase activity and cathepsin B although aging reduced LAMP1 level. In addition, rapamycin reduced aging-induced cardiomyocyte contractile and intracellular Ca(2+) dysfunction while Akt activation suppressed autophagy in young but not aged cardiomyocytes. In conclusion, our data suggest that Akt may accentuate aging-induced cardiac geometric and contractile defects through a loss of autophagic regulation.

Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3β-mediated suppression of mitochondrial permeation pore opening.

The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved. Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA. This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time. Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

Characterization of AKT independent effects of the synthetic AKT inhibitors SH-5 and SH-6 using an integrated approach combining transcriptomic profiling and signaling pathway perturbations

BackgroundSignal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, analogs of phosphatidyl inositol phosphates (PIAs) were designed as new small drugs to block AKT activity for cancer treatment. Here we characterize the biological effects of the PIAs SH-5 and SH-6 in colorectal cancer cell lines.MethodsSerum-starved or serum-supplemented human colorectal cancer cell lines SW480, HT29 and HCT116 were exposed to SH-5 and SH-6. AKT activation was determined by western blotting. Cell viability was assessed using a colorimetric XTT-based assay, apoptosis and cell cycle changes were monitored by FACS analysis. The dynamics of cell morphology alterations was evaluated by confocal and time-lapse microscopy. Transcriptional changes due to inhibitor treatment were analyzed using Affymetrix HG-U133A microarrays and RT-PCR.ResultsWhile the PIAs clearly reduce AKT phosphorylation in serum starved cells, we did not observe a significant reduction under serum supplemented conditions, giving us the opportunity to analyze AKT independent effects of these compounds. Both inhibitors induce broadly the same morphological alterations, in particular changes in cell shape and formation of intracellular vesicles. Moreover, we observed the induction of binucleated cells specifically in the SW480 cell line. Gene expression analysis revealed transcriptional alterations, which are mostly cell line specific. In accordance to the phenotype we found a gene group associated with mitosis and spindle organization down regulated in SW480 cells, but not in the other cell lines. A bioinformatics analysis using the Connectivity Map linked the gene expression pattern of the inhibitor treated SW480 cells to PKC signaling. Using confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation.ConclusionsThe PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment.

Protein Tyrosine Nitration and Glutathionylation Impairs Pro‐Survival Mechanisms of Akt in CryABmut Cardiomyopathy in Mice

BackgroundSerine‐threonine Akt affords powerful cellular protection but the role of this pro‐survival pathway in hR120GCryAB‐TG cardiomyopathy remains poorly defined. We hypothesized that chronic oxidative modifications and sequestration of Akt could impair the pro‐survival mechanisms in the hR120GCryAB‐TG leading to heart failure.MethodsTo gain further insights, we have determined biochemical markers of oxidative modifications.ResultsWestern blot and immunohistochemical analyses indicated increased protein kinase‐B (PKB/Akt) is sequestered into the protein aggregates containing hR120GCryAB in myopathic hearts compared with the non‐transgenic (NTG) controls. Increased S‐glutathionylation and tyrosine nitrosylation were present in the 3 and 6 months old TG mice, suggesting potential role in inactivation of pro‐survival mechanisms of Akt. Surprisingly, we found increased Akt kinase (substrate: GSK3‐α/β) activity in vitro in the hR120GCryAB‐TG hearts compared with the NTG, suggesting chronic Akt activation is coupled with cardiac hypertrophy and progression of cardiaomyopathy.ConclusionsTaken together, these findings implicate chronic activation and sequestration of Akt in the pathogenesis of hR120GCryAB‐induced cardiotoxicity.

Potential Redox‐Sensitive Akt Activation by Dopamine Activates Bad and Promotes Cell Death in Melanocytes

Dopamine (DA) is a well known oxidative neurotoxin. In addition, Akt has been reported to deliver a survival signal that inhibits apoptosis. However, it has also been reported that chronic Akt activation leads to apoptosis in response to oxidative stress. The objective of the present study was to investigate the possible role of the Akt pathway in vitiligo and its possible relationship with DA‐induced cell death using Mel‐Ab cells. Cultured Mel‐Ab cells were treated with DA with and without N‐Acetyl‐L‐cysteine (NAC), which is known to have antioxidative properties. Cell viability was then assessed by a crystal violet assay and Annexin staining was performed. The changes in the expression of Akt were analyzed by western blot analysis. The cell viability was reduced by approximately 60% in response to treatment with 500 µM DA, and NAC effectively prevented this cytotoxic effect. Likewise, treatment with DA produced numerous Annexin positive cells, while treatment with NAC prevented this apoptotic cell death. Akt was slowly phosphorylated after treatment with DA, while NAC clearly inhibited the DA‐induced Akt activation. Western blot analysis also showed that treatment with DA induced the activation of Bad. Finally, LY294002 exerted a protective effect against DA‐induced apoptotic cell death. DA may induce redox‐sensitive Akt activation and increase the level of Bad, which can promote cell death by heterodimerization with survival proteins. Moreover, NAC effectively protects against DA‐induced melanocyte death via inhibition of DA‐induced Akt activation.

Important role for AMPKαl in limiting skeletal muscle cell hypertrophy

Activation of AMP-activated protein kinase (AMPK) inhibits protein synthesis through the suppression of the mammalian target of rapamycin complex 1 (mTORC1), a critical regulator of muscle growth. The purpose of this investigation was to determine the role of the AMPKalpha1 catalytic subunit on muscle cell size control and adaptation to muscle hypertrophy. We found that AMPKalpha1(-/-) primary cultured myotubes and myofibers exhibit larger cell size compared with control cells in response to chronic Akt activation. We next subjected the plantaris muscle of AMPKalpha1(-/-) and control mice to mechanical overloading to induce muscle hypertrophy. We observed significant elevations of AMPKalpha1 activity in the control muscle at days 7 and 21 after the overload. Overloading-induced muscle hypertrophy was significantly accelerated in AMPKalpha1(-/-) mice than in control mice [+32 vs. +53% at day 7 and +57 vs. +76% at day 21 in control vs. AMPKalpha1(-/-) mice, respectively]. This enhanced growth of AMPKalpha1-deficient muscle was accompanied by increased phosphorylation of mTOR signaling downstream targets and decreased phosphorylation of eukaryotic elongation factor 2. These results demonstrate that AMPKalpha1 plays an important role in limiting skeletal muscle overgrowth during hypertrophy through inhibition of the mTOR-signaling pathway.

Atorvastatin impairs the myocardial angiogenic response to chronic ischemia in normocholesterolemic swine

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, used routinely in patients with coronary disease, can improve endothelial function but can have biphasic and dose-dependent effects on angiogenesis. In vitro evidence suggests that the proangiogenic effects of statins are linked to activation of Akt, a mediator of endothelial cell survival and an activator of endothelial nitric oxide synthase. We investigated the functional and molecular effects of atorvastatin supplementation on microvascular function and the endogenous angiogenic response to chronic myocardial ischemia in normocholesterolemic swine. Yucatan miniswine were fed a normal diet with (ATOR, n = 7) or without (control, n = 8) atorvastatin (1.5 mg/kg/d) for 20 weeks. Chronic ischemia was induced by ameroid constrictor placement around the circumflex artery. Myocardial perfusion was assessed at 3 and 7 weeks using isotope-labeled microspheres. In vitro microvessel relaxation responses and myocardial protein expression were evaluated. Endothelium-dependent relaxation to adenosine diphosphate and endothelium-independent relaxation to sodium nitroprusside were intact in both groups. The ATOR group demonstrated impaired microvessel relaxation to vascular endothelial growth factor (53% +/- 3% vs 70% +/- 7%, ATOR vs NORM at 10(-10) mol/L, P = .05) and fibroblast growth factor-2 (35% +/- 3% vs 57% +/- 5%, ATOR vs NORM at 10(-10) mol/L, P = .04). Baseline-adjusted myocardial perfusion in the ischemic circumflex territory was significantly reduced in the ATOR group (-0.29 +/- 0.10 mL/min/g vs NORM, P = .009). Phosphorylation of Akt was significantly increased in the ATOR group (+235% +/- 72%, P = .009 vs NORM), as was the myocardial expression of endostatin, an antiangiogenic protein (+51% +/- 9%, P < .001 vs NORM). Expression of vascular endothelial growth factor, Tie-2, fibroblast growth factor receptor-1, and endothelial nitric oxide synthase was similar in both groups. Atorvastatin supplementation is associated with impaired growth factor-mediated microvessel relaxation and a significant reduction in collateral-dependent perfusion. Chronic Akt activation, increased myocardial expression of endostatin, and impaired growth factor signaling may account for the diminished endogenous angiogenic response observed with atorvastatin treatment.

High-dose atorvastatin is associated with impaired myocardial angiogenesis in response to vascular endothelial growth factor in hypercholesterolemic swine

The disappointing results of myocardial angiogenic therapy have been attributed, in part, to endothelial dysfunction present in patients with coronary disease. Statins have established proendothelial properties but seem to have dose-dependent effects on angiogenesis. We investigated the functional and molecular effects of high-dose atorvastatin on vascular endothelial growth factor-induced myocardial angiogenesis in hypercholesterolemic swine. Yucatan miniswine (20-30 kg) were fed either a normal (ND group, n = 8) or high-cholesterol diet, with (HC-ATOR group, n = 8) or without (HC group, n = 8) atorvastatin (3 mg x kg(-1) x d(-1)), for 13 weeks. Chronic ischemia was induced by ameroid constrictor placement around the circumflex artery, followed 3 weeks later by perivascular vascular endothelial growth factor administration (2 microg over 4 weeks) with a sustained release osmotic pump. Microvessel relaxation responses, myocardial perfusion, and myocardial expression of angiogenic mediators were assessed 4 weeks later. Hypercholesterolemic swine demonstrated impaired microvessel relaxation to vascular endothelial growth factor (P < .01 vs ND group) and adenosine diphosphate (P < .001 vs ND group), which was normalized in the HC-ATOR group. After perivascular vascular endothelial growth factor administration, collateral-dependent myocardial perfusion was significantly increased in the ND group but decreased in both the HC and HC-ATOR groups (both P < .01 vs the ND group). The animals in the HC-ATOR group demonstrated increased myocardial expression of the antiangiogenic protein endostatin and increased Akt phosphorylation without significant changes in Akt and endothelial nitric oxide synthase expression. Atorvastatin treatment reverses hypercholesterolemia-induced endothelial dysfunction without appreciable improvements in collateral-dependent myocardial perfusion in response to vascular endothelial growth factor treatment. Increased myocardial endostatin expression and chronic Akt activation, associated with atorvastatin therapy, might account for the lack of improvement in the angiogenic response to vascular endothelial growth factor therapy.

High-Dose Atorvastatin Improves Hypercholesterolemic Coronary Endothelial Dysfunction Without Improving the Angiogenic Response

Although 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) can restore endothelial function in coronary disease, in vitro and murine studies have shown their effects on myocardial angiogenesis to be biphasic and dose dependent. We investigated the functional and molecular effects of high-dose atorvastatin on the endogenous angiogenic response to chronic myocardial ischemia in hypercholesterolemic swine. Yucatan pigs were fed either a normal (NORM group; n=7) or high-cholesterol diet, with (CHOL-ATR group; n=7) or without (CHOL group; n=6) atorvastatin (3 mg/kg per day) for 13 weeks. Chronic ischemia was induced by ameroid constrictor placement around the circumflex artery. Seven weeks later, microvessel relaxation responses, myocardial perfusion, and myocardial protein expression were assessed. The CHOL group demonstrated impaired microvessel relaxation to adenosine diphosphate (29+/-3% versus 61+/-6%, CHOL versus NORM; P<0.05), which was normalized in the CHOL-ATR group (67+/-2%; P=NS versus NORM). Collateral-dependent myocardial perfusion, adjusted for baseline, was significantly reduced in the CHOL group (-0.27+/-0.07 mL/min per gram versus NORM; P<0.001) as well as the CHOL-ATR group (-0.35+/-0.07 mL/min per gram versus NORM; P<0.001). Atorvastatin treatment was associated with increased phosphorylation of Akt (5.7-fold increase versus NORM; P=0.001), decreased vascular endothelial growth factor expression (-68+/-8%; P<0.001 versus NORM), and increased expression of the antiangiogenic protein endostatin (210+/-48%; P=0.004 versus NORM). Atorvastatin improves hypercholesterolemia-induced endothelial dysfunction without appreciable changes in collateral-dependent perfusion. Increased myocardial expression of endostatin, decreased expression of vascular endothelial growth factor, and chronic Akt activation associated with atorvastatin treatment may account for the diminished angiogenic response.

PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury

PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury

Effects of chronic Akt activation on glucose uptake in the heart

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.

Highlights from the Literature

Highlights from the Literature

PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury

Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). However, less is known about the chronic effects of Akt activation in the heart, and, paradoxically, Akt is activated in samples from patients with chronic heart failure. We generated Tg mice with cardiac-specific expression of either activated (myristoylated [myr]) or dominant-negative (dn) Akt and assessed their response to IRI in an ex vivo model. While dn-Akt hearts demonstrated a moderate reduction in functional recovery after IRI, no function was restored in any of the myr-Akt-Tg hearts. Moreover, infarcts were dramatically larger in myr-Akt-Tg hearts. Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of PI3K activity through both proteasome-dependent degradation of insulin receptor substrate-1 (IRS-1) and inhibition of transcription of IRS-1 as well as that of IRS-2. To test the functional significance of these signaling changes, we performed in vivo cardiac gene transfer with constitutively active PI3K in myr-Akt-Tg mice. Restoration of PI3K rescued function and reduced injury after IRI. These data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection and suggest a mechanism by which chronic Akt activation can become maladaptive.