AMPKα2 Deletion Research Articles

Metabolic reprogramming and AMPK activation: Key players in the therapeutic effects of Cooling Blood and Detoxicating Formular on psoriasis

Ethnopharmacological relevanceCooling Blood and Detoxicating Formular (CBDF) based on the theory of cooling blood and dosing detoxification, is a useful traditional Chinese medicine (TCM) medication for psoriasis with blood-heat syndrome. Aim of the studyInvestigate the active constituents and mechanisms of the CBDF for the treatment of psoriasis. Materials and methodsUPLC-Q-Orbitrap-HRMS technique was used to analyse the ingredients of CBDF absorbed into plasma and skin tissue. The therapeutic efficacy of CBDF was evaluated in treating an imiquimod (IMQ)-induced mouse model was assessed. Transcriptome analysis and gene enrichment analysis were used to explore the changes in gene expression and pathways following treatment with the CBDF. Validation was performed using western blotting, quantitative RT-PCR, flow cytometry, gene knockout and molecular docking in vitro and in vivo. Results26 compounds were identified in the plasma of IMQ-induced psoriasis-like mouse with CBDF treatment, and higher levels of cimifugin in the lesion. CBDF improved the pathological changes of psoriasis, with inhibition of TNF-α, IL-23, and IL-17A and upregulation of IL-10. Gene enrichment analysis showed that the therapeutic effect of CBDF was related to AMPK pathway. In psoriasis lesions, the AMPK and fatty acid oxidation were suppressed, and glycolysis was enhanced. The Prkaa2, encoding AMPKα2 was down-regulated in psoriasis patients. CBDF inhibited glycolysis while stimulating fatty acid oxidation by the activating AMPK, thereby exerting an inhibitory effect on inflammation. CBDF inhibited MHCII, CD80, and CD86 on dendritic cells of skin drainage lymph node. In vitro, CBDF inhibited bone marrow-derived DCs secrete IL-23, TNF-α, and lactate, while enhanced fatty acid oxidation and AMPK activity. However, the therapeutic effect was weakened in AMPKα2 deletion. Additionally, psoriasis lesions and dendritic cells activation were significantly aggravated after AMPKα2 knockout. The key ingredients of the CBDF, cimifugin, rutin, astilbin, quercetin, and prim-O-glucosylcimifugin, all exhibit a notable affinity towards AMPKα2 binding. ConclusionsCBDF ameliorates psoriasis symptoms and inhibit dendritic cells maturation by regulating metabolic reprogramming in an AMPK-dependent mechanism.

AMPK deficiency in smooth muscles causes persistent pulmonary hypertension of the new-born and premature death

AMPK has been reported to facilitate hypoxic pulmonary vasoconstriction but, paradoxically, its deficiency precipitates pulmonary hypertension. Here we show that AMPK-α1/α2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the new-born. Accordingly, dual AMPK-α1/α2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularisation and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no oedema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes were markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation was also evident. Importantly, there was no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction was attenuated by AMPK-α1 or AMPK-α2 deletion without triggering pulmonary hypertension.

Retraction of: AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells.

HomeCirculation ResearchVol. 129, No. 6Retraction of: AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells Free AccessRetractionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessRetractionPDF/EPUBRetraction of: AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells Originally published2 Sep 2021https://doi.org/10.1161/RES.0000000000000502Circulation Research. 2021;129:e120This article is retracted byAMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle CellsThe following Circulation Research article is being retracted:Song P, Wang S, He C, Wang S, Liang B, Viollet B, Zou MH. AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells. Circulation Research. 2011;109:1230−1239. DOI: http://dx.doi.org/10.1161/CIRCRESAHA.111.250423After a confidential investigation conducted in accordance with the University’s policy and federal regulations, the University of Oklahoma Health Sciences Center determined that the following figures were reused by the first author:Duplication of the β-actin blot in Figures 1D, 2A, and 3ADuplication of β-actin blot in online Figure IV with β-actin blot in Figure 2DDuplication of β-actin blot in Figure 6E and online Figure IIBThe first author claims to have the repeated experiments and data and he stands by his results and conclusion; however, the editors are retracting this article based on the findings of the university investigation and in agreement with the University of Oklahoma Health Sciences Center and the authors. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesAMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle CellsPing Song, et al. Circulation Research. 2011;109:1230-1239 September 3, 2021Vol 129, Issue 6 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000502PMID: 34473534 Originally publishedSeptember 2, 2021 PDF download Advertisement

Expression of Concern for: AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells

Expression of Concern for: AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells

P6276Impact of the cardiac specific deletion of AMPKalpha2 on the contractile and metabolic phenotype of the heart in male and female mice

Abstract Introduction Mitochondrial dysfunction plays a major role in the Heart Failure (HF) pathophysiology.The AMP activated protein kinase (AMPK) is activated by a high AMP-ADP/ATP ratio and regulates a number of metabolic pathways. Many studies have highlighted a protective role of AMPK in HF, but its relevance to cardiac tissue, its metabolic part and its sex specificity are not well established. Purpose Then, the aim of this study is to determine the role of AMPK in the healthy and failing heart in male and female mice. Methods We developed and validated a mouse strain with an adult-inducible cardiac-specific deletion of AMPKα2, the major cardiac isoform, using the Cre-Lox system (40mg/kg tamoxifen injection on two consecutive days at adult age). At four months after the deletion, cardiac contractility, morphology and metabolism were studied in control and KO mice from both sexes. Results We observed only in male KO mice a decrease of left ventricular ejection fraction (−10%), an increase of the total fibrosis (+64%) and defects in mitochondrial structures. Male KO mice also showed a reduced (−28%) mitochondrial respiration via complex I associated with a different cardiolipin species distribution. Conclusion Our results reveal in adult healthy hearts, a sex-specificity in the effects of AMPKα2 deletion, leading to impaired contractile function related to metabolic and non-metabolic alterations only in male mice.

Myeloid-Specific Deletion of the AMPKα2 Subunit Alters Monocyte Protein Expression and Atherogenesis.

The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.

Metabolic and non-metabolic effects of cardiac-specific and inducible deletion of the AMPKalpha2 in female and male mice

Mitochondrial dysfunction plays a major role in the Heart Failure (HF) pathophysiology. The AMP activated protein kinase (AMPK) is activated by a high AMP-ADP/ATP ratio and regulates a number of metabolic pathways. Many studies have highlighted a protective role of AMPK in HF, but its relevance to cardiac tissue, its metabolic part and its sex-specificity are not well established. The aim of this study is to determine the role of AMPK in the healthy and failing heart in male and female mice. We developed and validated a mouse strain with an adult-inducible cardiac-specific deletion of AMPKα2, the major cardiac isoform, using the Cre-Lox system (40 mg/kg tamoxifen injection on two consecutive days at adult age). At four months after the deletion, cardiac contractility, morphology and metabolism were studied in control and KO mice from both sexes. We observed only in male KO mice a decrease of left ventricular ejection fraction (− 10%), an increase of the total fibrosis (+ 64%) and defects in mitochondrial structures. Male KO mice also showed a reduced (− 28%) mitochondrial respiration via complex I associated with a different cardiolipin species distribution. Our results reveal in adult healthy hearts, a sex-specificity in the effects of AMPKα2 deletion, leading to impaired contractile function related to metabolic and non-metabolic alterations only in male mice.

The LKB1-AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria.

Hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical "oxygen-sensing" K+ channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia.

AMPK-α1 or AMPK-α2 Deletion in Smooth Muscles Does Not Affect the Hypoxic Ventilatory Response or Systemic Arterial Blood Pressure Regulation During Hypoxia.

The hypoxic ventilatory response (HVR) is markedly attenuated by AMPK-α1 deletion conditional on the expression of Cre-recombinase in tyrosine hydroxylase (TH) expressing cells, precipitating marked increases in apnea frequency and duration. It was concluded that ventilatory dysfunction caused by AMPK deficiency was driven by neurogenic mechanisms. However, TH is transiently expressed in other cell types during development, and it is evident that central respiratory depression can also be triggered by myogenic mechanisms that impact blood supply to the brain. We therefore assessed the effect on the HVR and systemic arterial blood pressure of AMPK deletion in vascular smooth muscles. There was no difference in minute ventilation during normoxia. However, increases in minute ventilation during severe hypoxia (8% O2) were, if affected at all, augmented by AMPK-α1 and AMPK-α2 deletion in smooth muscles; despite the fact that hypoxia (8% O2) evoked falls in arterial SpO2 comparable with controls. Surprisingly, these mice exhibited no difference in systolic, diastolic or mean arterial blood pressure during normoxia or hypoxia. We conclude that neither AMPK-α1 nor AMPK-α2 are required in smooth muscle for the regulation of systemic arterial blood pressure during hypoxia, and that AMPK-α1 deficiency does not impact the HVR by myogenic mechanisms.

Chronic Exercise Training Improved Aortic Endothelial and Mitochondrial Function via an AMPKα2-Dependent Manner.

Chronic exercise training is known to protect the vasculature; however, the underlying mechanisms remain obscure. The present study hypothesized that exercise may improve aortic endothelial and mitochondrial function through an adenosine monophosphate-activated protein kinase α2 (AMPKα2)-dependent manner. Ten-week-old AMPKα2 knockout (AMPKα2−/−) mice and age-matched wild-type (WT) mice were subjected to daily treadmill running for 6 weeks, and the thoracic aorta from these mice were used for further examination. Our results showed that exercise significantly promoted vasodilatation and increased expression and phosphorylation of endothelial nitric oxide synthase (eNOS), concomitant with increased AMPKα2 expression in WT mice. These effects were not observed in AMPKα2−/− mice. Furthermore, exercise training increased thoracic aortic mitochondrial content as indicated by increased Complex I and mitochondrial DNA (mtDNA) in WT mice but not in AMPKα2−/− mice. This may be caused by decreased mitochondrial autophagy since the expression of BH3 domain-containing BCL2 family members BNIP3-like (BNIP3L) and LC3B were decreased in WT mice with exercise. And these changes were absent with AMPKα2 deletion in mice. Importantly, exercise increased the expression of manganous superoxide dismutase (MnSOD) and catalase, suggesting that mitochondrial antioxidative capacity was increased. Notably, the improved antioxidative capacity was lost in AMPKα2−/− mice with exercise. In conclusion, this study illustrated that AMPKα2 plays a critical role in exercise-related vascular protection via increasing endothelial and mitochondrial function in the artery.

AMP-Activated Protein Kinase Alpha 2 Deletion Induces VSMC Phenotypic Switching and Reduces Features of Atherosclerotic Plaque Stability.

AMP-activated protein kinase (AMPK) has been reported to play a protective role in atherosclerosis. However, whether AMPKα2 controls atherosclerotic plaque stability remains unknown. The aim of this study was to evaluate the impact of AMPKα2 deletion on atherosclerotic plaque stability in advanced atherosclerosis at the brachiocephalic arteries and to elucidate the underlying mechanisms. Features of atherosclerotic plaque stability and the markers for contractile or synthetic vascular smooth muscle cell (VSMC) phenotypes were monitored in the brachiocephalic arteries from Apoe(-/-)AMPKα2(-/-) mice or VSMC-specific AMPKα2(-/-) mice in an Apoe(-/-) background (Apoe(-/-)AMPKα2(sm-/-)) fed Western diet for 10 weeks. We identified that Apoe(-/-)AMPKα2(-/-) mice and Apoe(-/-)AMPKα2(sm-/-) mice exhibited similar unstable plaque features, aggravated VSMC phenotypic switching, and significant upregulation of Kruppel-like factor 4 (KLF4) in the plaques located in the brachiocephalic arteries compared with those found in Apoe(-/-) and Apoe(-/-)AMPKα2(sm+/+) control mice. Pravastatin, an AMPK activator, suppressed VSMC phenotypic switching and alleviated features of atherosclerotic plaque instability in Apoe(-/-)AMPKα2(sm+/+) mice, but not in Apoe(-/-)AMPKα2(sm-/-) mice. VSMC isolated from AMPKα2(-/-) mice displayed a significant reduction of contractile proteins(smooth muscle actin-α, calponin, and SM-MHC [smooth muscle-mysion heavy chain]) in parallel with increased detection of synthetic proteins (vimentin and osteopontin) and KLF4, as observed in vivo. KLF4-specific siRNA abolished AMPKα2 deletion-induced VSMC phenotypic switching. Furthermore, pharmacological or genetic inhibition of nuclear factor-κB significantly decreased KLF4 upregulation in VSMC from AMPKα2(-/-) mice. Finally, we found that AMPKα2 deletion markedly promoted the binding of nuclear factor-κBp65 to KLF4 promoter. This study demonstrated that AMPKα2 deletion induces VSMC phenotypic switching and promotes features of atherosclerotic plaque instability in nuclear factor-κB-KLF4-dependent manner.

AMP-activated Protein Kinase Deficiency Blocks the Hypoxic Ventilatory Response and Thus Precipitates Hypoventilation and Apnea.

Modulation of breathing by hypoxia accommodates variations in oxygen demand and supply during, for example, sleep and ascent to altitude, but the precise molecular mechanisms of this phenomenon remain controversial. Among the genes influenced by natural selection in high-altitude populations is one for the adenosine monophosphate-activated protein kinase (AMPK) α1-catalytic subunit, which governs cell-autonomous adaptations during metabolic stress. We investigated whether AMPK-α1 and/or AMPK-α2 are required for the hypoxic ventilatory response and the mechanism of ventilatory dysfunctions arising from AMPK deficiency. We used plethysmography, electrophysiology, functional magnetic resonance imaging, and immediate early gene (c-fos) expression to assess the hypoxic ventilatory response of mice with conditional deletion of the AMPK-α1 and/or AMPK-α2 genes in catecholaminergic cells, which compose the hypoxia-responsive respiratory network from carotid body to brainstem. AMPK-α1 and AMPK-α2 deletion virtually abolished the hypoxic ventilatory response, and ventilatory depression during hypoxia was exacerbated under anesthesia. Rather than hyperventilating, mice lacking AMPK-α1 and AMPK-α2 exhibited hypoventilation and apnea during hypoxia, with the primary precipitant being loss of AMPK-α1 expression. However, the carotid bodies of AMPK-knockout mice remained exquisitely sensitive to hypoxia, contrary to the view that the hypoxic ventilatory response is determined solely by increased carotid body afferent input to the brainstem. Regardless, functional magnetic resonance imaging and c-fos expression revealed reduced activation by hypoxia of well-defined dorsal and ventral brainstem nuclei. AMPK is required to coordinate the activation by hypoxia of brainstem respiratory networks, and deficiencies in AMPK expression precipitate hypoventilation and apnea, even when carotid body afferent input is normal.

Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts

Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, delays aging process. However, the molecular mechanisms by which AMPKα isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to the accelerated cell senescence by inducing p16INK4A (p16) expression thereby arresting cell cycle. The markers of cellular senescence, cell cycle proteins, and reactive oxygen species (ROS) were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1−/−, AMPKα2−/−) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPKα2, the minor isoform of AMPKα, but not AMPKα1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-β-galactosidase (SA-β-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1γ). It was shown here that AMPKα2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPKα2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein 1 (HBP1) partially blocked the cellular senescence of AMPKα2-deleted MEFs via the reduction of p16. Finally, dermal cells senescence, including fibroblasts senescence evidenced by the staining of p16, HBP1, and Ki-67, in the skin of aged AMPKα2−/− mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPKα2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence.

AMPKα1 Deficiency in Fibroblasts Promotes Angiogenesis in Athymic Nude Mice

Angiogenesis is required and essential for tumor development. Increasing evidence suggests that adenosine monophosphate‐activated protein kinase (AMPK), an energy sensor and modulator of redox system, is associated with cancer development. However, the effect of AMPK on tumor development is controversial. Mouse embryo fibroblasts (MEF) were isolated from AMPKα1 knock out (AMPKα1‐KO), AMPKα2‐KO, and wild type (WT) C57BL/6J mouse embryos, and immortalized by using standard 3T3 protocol to investigate the mechanisms of fibroblast‐mediated angiogenesis. Xenograft model data revealed that knock out of AMPKα1, but not AMPKα2, stimulated the cellular proliferation of immortalized MEFs, angiogenesis, and tumor development in athymic nude mice in vivo. Consistent with this finding, the migration of human microvascular endothelial cells (hmvEC) was enhanced by co‐culturing with AMPKα1‐KO MEFs when compared to either WT or AMPKα2‐KO MEFs. Moreover, the network formation of hmvEC was stimulated by conditioned medium from AMPKα1‐KO MEFs, but not from WT or AMPKα2‐KO MEFs. Mechanistically, we found that the protein level of noncanonical nuclear factor kappa B2 (NF‐ĸB2)/p52 was elevated in AMPKα1‐KO MEFs and was responsible for induction of erythropoietin (Epo) expression. Finally, the most conclusive evidence for AMPKα1‐dependent inhibition of EC migration and consequent angiogenesis as well as tumor progression was that the xenograft growth of the inoculated AMPKα1‐KO MEFs was ameliorated by treatment with Epo neutralization antibody. This compelling evidence indicates that AMPKα1, rather than AMPKα2 deletion in fibroblasts, instigates EC migration, angiogenesis, and the resultant tumor development.

Abstract 9547: AMPKa2 Deletion Promotes Vascular Smooth Muscle Cell Migration via Skp2 Upregulation and E-cadherin Downregulation

The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the progression of several vasculopathologies. Adenosine monophosphate-activated protein kinase (AMPK) has been shown to play a pivotal role in cellular proliferation and migration. However, the exact role of AMPK in VSMCs migration and its underlying molecular mechanisms remain elusive. We cultured mouse VSMCs isolated from wild-type (WT) C57BL/6J mice and either AMPKα2 or AMPKα1 homozygous-deficient (AMPKα2 -/- , AMPKα1 -/- ) mice to investigate the molecular mechanisms of VSMCs migration. Deletion of AMPKα2, but not AMPKα1, led to increased phosphorylation of IκB kinase α (IκKα) and its downstream target nuclear factor κB2 (NFκB2)/p100 at serine 866/870. This resulted in pp100-S866/870 association with E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP), which contributed to NFκB2/p52 induction from the proteolytic processing of the p100 precursor. Interestingly, acetylation of histone H3 at lysine 56 (AcH3-K56) resulted in association with histone deacetylase 3 (HDAC3) which was significantly enhanced in AMPKα2 -/- VSMCs compared with either WT or AMPKα1 -/- VSMCs. Moreover, the augmented association of p52/AcH3-K56 with the promoter of ubiquitin E3 ligase S-phase kinase-associated protein 2 (Skp2) was shown in AMPKa2 -/- VSMCs by ChIP assay. Furthermore, AMPKα2 deletion caused E-cadherin downregulation, which is mediated by Skp2 and the ubiquitin-proteasome system. Skp2 siRNA abolished the increased migration of AMPKα2 -/- VSMCs via E-cadherin upregulation. Finally, neointima formation after ligation of carotid artery was increased in AMPKα2 -/- , but not AMPKα1 -/- , mice and was associated with increased AcH3-K56 as well as reduction of both HDAC3 and E-cadherin. This compelling evidence indicates that deletion of AMPKα2, rather than AMPKα1, mediates VSMCs migration and the resultant neointimal formation after vascular injury. These events appear to occur via the NFκB2/Skp2/E-cadherin pathway.

Adenosine Monophosphate–Activated Protein Kinase-α2 Deficiency Promotes Vascular Smooth Muscle Cell Migration via S-Phase Kinase–Associated Protein 2 Upregulation and E-Cadherin Downregulation

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the progression of several vasculopathologies. Adenosine monophosphate-activated protein kinase (AMPK) has been shown to play a pivotal role in cellular proliferation and migration. However, the roles of AMPK in VSMC migration and its underlying molecular mechanisms remain elusive. VSMC migration and the neointima formation were studied in cultured mouse VSMCs or in carotid artery ligation of wild-type C57BL/6J mice, AMPKα2, AMPKα1 homozygous-deficient (AMPKα2(-/-), AMPKα1(-/-)) mice. Deletion of AMPKα2, but not AMPKα1, led to increased phosphorylation of both IкB kinase α and its downstream target nuclear factor кB2/p100 at serine 866/870. Consequently, phosphor-p100 at S866/870 bound with E3 ubiquitin ligase β-transducin repeat-containing protein resulting in the proteolytic processing of the p100 precursor and nuclear factor кB2/p52 induction. Interestingly, acetylation of histone H3 at lysine 56 mediated by histone deacetylase-3 reduction was enhanced significantly in AMPKα2(-/-) VSMCs compared with wild-type or AMPKα1(-/-) VSMCs. Moreover, the augmented association of p52/acetylation of histone H3 at lysine 56 with the promoter of ubiquitin E3 ligase, S-phase kinase-associated protein 2, was shown in AMPKα2(-/-) VSMCs by chromatin immunoprecipitation assay. Furthermore, AMPKα2 deletion caused S-phase kinase-associated protein 2-mediated E-cadherin downregulation. S-Phase kinase-associated protein 2 siRNA abolished the increased migration of AMPKα2(-/-) VSMCs via E-cadherin upregulation. Finally, neointima formation after ligation of carotid artery was increased in AMPKα2(-/-), but not AMPKα1(-/-), mice. We conclude that deletion of AMPKα2 causes aberrant VSMC migration with accelerated neointima formation in vivo.

AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells

Adenosine monophosphate-activated protein kinase (AMPK), a metabolic and redox sensor, is reported to suppress cell proliferation of nonmalignant and tumor cells. Whether AMPKα alters vascular neointima formation induced by vascular injury is unknown. The aim of this study was to determine the roles of AMPKα in the development of vascular neointima hyperplasia and to elucidate the underlying mechanisms. Vascular smooth muscle cell (VSMC) proliferation and neointimal hyperplasia were evaluated in cultured VSMCs and wire-injured mouse carotid arteries from wild-type (WT, C57BL/6J), AMPKα2(-/-), and AMPKα1(-/-) mice. Mouse VSMCs derived from aortas of AMPKα2(-/-) mice exhibited increased proliferation compared with either WT or AMPKα1(-/-) VSMCs. Further, deletion of AMPKα2 but not AMPKα1 reduced the level of p27(Kip1), a cyclin-dependent kinase inhibitor, and increased the level of S-phase kinase-associated protein 2 (Skp2), a known E3 ubiquitin ligase for p27(Kip1), through activation of p52 nuclear factor kappa B (NF-κB)-2. Moreover, either pharmacological (ie, through compound C) or genetical (ie, through AMPKα2-specific siRNA) inhibition of AMPK decreased p27(Kip1) levels but increased the abundance of Skp2 in human VSMCs. Furthermore, gene silencing of Skp2 reversed the levels of p27(Kip1) and VSMCs proliferation. Finally, neointima formation after mechanical arterial injury was increased in AMPKα2(-/-) but not AMPKα1(-/-) mice. These findings indicate that deletion of AMPKα2 through p52-Skp2-mediated ubiquitination and degradation of p27(Kip1) accentuates neointimal hyperplasia in response to wire injury.

AMPKα2 deletion leads to marked alterations in SIRT1 activity, CPT1 expression and basal FA muscle metabolism in high‐fat‐fed mice

Because AMPKα2 is a critical signal in the regulation of muscle metabolism, we sought to determine if muscle AMPKα2 deletion would exacerbate the loss of insulin sensitivity, induced by high fat diet (HFD), as it pertains to the regulation of fatty acid (FA) metabolism. Male C57BL/6 mice (8–12 wks), wild‐type (WT) or dominant negative (DN) AMPKα2 in heart and skeletal muscle, were fed control diet (CD; 12%fat) or HFD (60%fat). After 6 wks, hindlimbs of CD (n=10) and HFD (n=10) mice were perfused (550μM palmitate, 6mM glucose, [1‐14C]palmitate) ±450μU insulin (IS). CD (n=8) and HFD (n=8) muscles were used for basal protein expression. In CD, AMPKα2 deletion did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 81% and α2 deletion prevented the IS increase in FAU and decrease in FAO. HFD abolished the IS increase in FAU and decrease in FAO in WT. In HFD, α2 deletion increased basal FAU by 139% (P<0.05) but did not affect IS FAU nor FAO (P>0.05). IS SIRT1 activity was higher in HFD‐DN than HFD‐WT (P<0.05). Neither DN nor HFD affected PGC1α content. HFD increased (P<0.05) PTP1b, SIRT1, and CD36 content and α2 deletion prevented the HFD‐induced increases in PTP1b and SIRT1. In HFD, α2 deletion decreased (P<0.05) CPT1 content. These data show that AMPKα2 may be more critical for the regulation of FAU and FAO under basal than IS conditions and that it affects CPT1, PTP1b, and SIRT1 content and IS SIRT1 activity with HFD.

AMPKα2 Deletion Causes Aberrant Expression and Activation of NAD(P)H Oxidase and Consequent Endothelial Dysfunction In Vivo

AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established. The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress, and endothelial function. The markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91(phox), p47(phox), p67(phox), NOX1 to -4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IkappaBalpha degradation, and nuclear translocation of nuclear factor (NF)-kappaB (p50 and p65) were examined in cultured human umbilical vein endothelial cells and mouse aortas isolated from AMPKalpha2 deficient mice. Compared to the wild type, acetylcholine-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKalpha2(-/-) mice. Further, pretreatment of aorta with either superoxide dismutase (SOD) or tempol or apocynin significantly improved acetylcholine-induced endothelium-dependent relaxation in AMPKalpha2(-/-) mice. Analysis of aortic endothelial cells from AMPKalpha2(-/-) mice and human umbilical vein endothelial cells expressing dominant negative AMPK or AMPKalpha2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91(phox), p47(phox), p67(phox), NOX1 and -4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IkappaBalpha degradation, and nuclear translocation of NF-kappaB (p50 and p65), whereas AMPK activation by AICAR or overexpression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKalpha2 in low-density lipoprotein receptor knockout (LDLr(-/-)) strain markedly increased 26S proteasome activity, IkappaB degradation, NF-kappaB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, and endothelial dysfunction, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor. We conclude that AMPKalpha2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way, AMPK maintains the nonatherogenic and noninflammatory phenotype of endothelial cells.

Role of the α2-isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia

AMP-activated protein kinase (AMPK) is a major sensor and regulator of the energetic state of the cell. Little is known about the specific role of AMPKalpha(2), the major AMPK isoform in the heart, in response to global ischemia. We used AMPKalpha(2)-knockout (AMPKalpha(2)(-/-)) mice to evaluate the consequences of AMPKalpha(2) deletion during normoxia and ischemia, with glucose as the sole substrate. Hemodynamic measurements from echocardiography of hearts from AMPKalpha(2)(-/-) mice during normoxia showed no significant modification compared with wild-type animals. In contrast, the response of hearts from AMPKalpha(2)(-/-) mice to no-flow ischemia was characterized by a more rapid onset of ischemia-induced contracture. This ischemic contracture was associated with a decrease in ATP content, lactate production, glycogen content, and AMPKbeta(2) content. Hearts from AMPKalpha(2)(-/-) mice were also characterized by a decreased phosphorylation state of acetyl-CoA carboxylase during normoxia and ischemia. Despite an apparent worse metabolic adaptation during ischemia, the absence of AMPKalpha(2) does not exacerbate impairment of the recovery of postischemic contractile function. In conclusion, AMPKalpha(2) is required for the metabolic response of the heart to no-flow ischemia. The remaining AMPKalpha(1) cannot compensate for the absence of AMPKalpha(2).