Subunit Of AMP-activated Protein Kinase Research Articles

Expression of messenger RNA encoding two cellular metabolic regulators, AMP-activated protein kinase (AMPK) and O-GlcNAc transferase (OGT), in channel catfish: Their tissue distribution and relationship with changes in food intake

AMP-activated protein kinase (AMPK) is considered as the master cellular metabolism regulator that activates various proteins, including O-GlcNAc transferase (OGT). Physiological roles of AMPK and OGT, including the relationship between their mRNA expression and food intake, are poorly understood in channel catfish. This study examined the tissue distribution of AMPK and OGT mRNA and changes in their expression in response to changes in food intake in channel catfish. Expression of all AMPK subunit and OGT mRNA was detectable in the whole brain, liver, heart, spleen, white muscle, and kidney of channel catfish. The OGT mRNA was highly localized in the brain compared to other tissues. 28-day fasting increased hepatic expression of AMPK α1, β1, and OGT mRNA while refeeding fish for 14 days after the 14-day fast decreased their expression to the level similar to that of fish that were fed daily. No changes were noted in the expression of muscle and brain AMPK mRNA or OGT mRNA by fasting and refeeding. Hepatic AMPK α1, α2 and β1 mRNA decreased in response to increased feeding frequency, whereas no changes in the expression of AMPK or OGT mRNA were noted in the brain or the muscle. Results of the current study indicated that the hepatic expression of AMPK and OGT mRNA appeared to be more sensitive to changes in food intake in channel catfish. However, further studies are needed to clearly demonstrate if food intake influences the expression of AMPK and OGT mRNA in various tissues, including the hypothalamus.

848 Genes implicated in lipid & metabolic regulation downregulated in CCCA

Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring hair loss in black women. It is characterized by fibrosis out of proportion to the clinical and histologic signs of inflammation, which may lead to progressive end stage fibrosis. Previous work by our group showed that affected CCCA scalp is characterized by an increased expression of profibrotic genes seen in other conditions of aberrant scarring, including hepatic fibrosis. While the cause of this fibrosis is unknown, downregulation of adenosine monophosphate activated protein kinase (AMPK) have been implicated as a primary mediator of hepatic fibrosis, as well as other conditions of scarring. Furthermore, disruption in fatty acid metabolism, including linoleic acid, has been implicated in excess fibrosis including enhanced proliferation of fibroblasts and collagen type I. We performed gene expression microarrays on paired affected and unaffected scalp samples in five patients with CCCA to understand the role of metabolic dysregulation on the pathogenesis of CCCA. Transcriptome-wide analysis of over 20,000 human genes was performed, analyzing all known exons and the 200 (0.9%) most downregulated genes based on fold change were identified. We revealed widespread alterations of genes regulating lipid metabolism (p=2.3 x 10-3) and fatty acid biosynthesis (p=5.3 x 10-6) genes. In particular, the mRNA transcript of protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2), which partially encodes for the a subunit of AMPK, was shown to be downregulated by 34% in our CCCA affected scalp compared to the unaffected scalp (p=6.4 x 10-3). Linoleic acid metabolism was also disproportionately dysregulated in our dataset (p=9.1 x 10-3). Our work signifies a potential role that metabolic dysregulation may play in the pathogenesis of CCCA.

Absence of the β1 subunit of AMP-activated protein kinase reduces myofibroblast infiltration of the kidneys in early diabetes.

Activation of the heterotrimeric energy-sensing kinase AMP-activated protein kinase (AMPK) has been reported to improve experimental diabetic kidney disease. We examined the effect of type 1 diabetes in wild-type (WT) mice and mice lacking the β1 subunit of AMPK (AMPK β1-/- mice), which have reduced AMPK activity in kidneys and other organs. Diabetes was induced using streptozotocin (STZ) and the animals followed up for 4weeks. Hyperglycaemia was more severe in diabetic AMPK β1-/- mice, despite the absence of any difference in serum levels of insulin, adiponectin and leptin. There was no change in AMPK activity in the kidneys of diabetic WT mice by AMPK activity assay, or phosphorylation of either the αT172 activation site on the α catalytic subunit of AMPK or the AMPK-specific phosphosite S79 on acetyl CoA carboxylase 1 (ACC1). Phosphorylation of the inhibitory αS485 site on the α subunit of AMPK was significantly increased in the WT diabetic mice compared to non-diabetic controls. Despite increased plasma glucose levels in the diabetic AMPK β1-/- mice, there were fewer myofibroblasts in the kidneys compared to diabetic WT mice, as evidenced by reduced α-smooth muscle actin (α-SMA) protein by Western blot, mRNA by qRT-PCR and fewer α-SMA-positive cells by immunohistochemical staining. Albuminuria was also reduced in the AMPK β1-/- mice. In contrast to previous studies, therefore, myofibroblasts were reduced in the kidneys of AMPK β1-/- diabetic mice compared to diabetic WT mice, despite increased circulating glucose, suggesting that AMPK can worsen renal fibrosis in type 1 diabetes.

The relationship between food exposure frequency and expression of AMP‐activated protein kinase (AMPK) mRNA in the brain, liver, and muscle of channel catfish

Obesity poses serious challenges to our healthcare system with serious consequences of associated diseases. Our laboratory has been examining how the expression of genes involved in food intake and tissue nutrient metabolism is associated with the development of obese‐like phenotypes in channel catfish. The objective of the study was to examine changes in expression of AMPK mRNA in response to changes in feeding frequency. Fish were fed every 12 (overfed), 24 (control), or 48 (underfed) hours (n= 4 tanks per treatment, 8 fish per tank) for 28 days. Brain, liver, and muscle tissue were collected at the end of the study. The expression of the AMPK mRNA was examined using quantitative RT‐PCR. The muscle expression of all AMPK subunit mRNA was unaffected by feeding frequency. In contrast, brain expression of alpha 1 and beta 1 subunit mRNA had a linear tendency for increase in response to increased feeding frequency. Hepatic expression of alpha 2 and beta 1 mRNA was lower in the overfeed group compared to those assigned as control or underfeed. Hepatic expression of alpha 1 mRNA decreased linearly in response to increased feeding frequency. Our results demonstrated that the hepatic and the brain expression of AMPK mRNA was influenced by increase in exposure to food in a transcript‐specific manner. Given that the AMPK serves as a regulator of tissue nutrient sensing and metabolism in mammals, it is possible that AMPK may share a similar function in the brain and liver but not in the muscle of channel catfish.Support or Funding InformationKansas IDeA Network of Biomedical Research Excellence Network (NIH/NIGMS grant number P20GM103418)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LCAL1 enhances lung cancer survival via inhibiting AMPK-related antitumor functions.

Lung cancer is one of the most common cancers and has been the most common cause of cancer deaths for several decades. Recently, lung cancer-associated lncRNA 1 (LCAL1) has been identified to be overexpressed in lung cancer tissues, while inhibiting LCAL1 expression has shown potential to inhibiting lung cancer growth. However, the molecular mechanism between LCAL1 and lung cancer cell survival remains poorly understood. In the present study, we provided the first evidence that LCAL1 may support lung cancer survival via inhibiting the activity of AMP-activated protein kinase (AMPK). According to our results, LCAL1 may physically interact with the catalytic subunit of tumor suppressor AMPK, prevent AMPK activation by upstream kinase (liver kinase B1), and thus inhibit the downstream AMPK signaling network. Our study revealed that overexpressed LCAL1 may induce aerobic glycolysis in lung cancer cells through AMPK/HIF1α axis, enhance protein synthesis through AMPK/mTOR/S6K axis, and suppress autophagic cell death through AMPK/ULK1 pathway. All these alterations supported rapid proliferation of lung cancer cells, while knockdown of LCAL1 expression demonstrated the potential of inhibiting lung cancer growth by reversing the tumorigenic phenotypes triggered by the loss of AMPK activity, and could become a promising therapeutic strategy for lung cancer treatment.

A Polygenic Risk Score of Lipolysis-Increasing Alleles Determines Visceral Fat Mass and Proinsulin Conversion.

Primary dysregulation of adipose tissue lipolysis caused by genetic variation and independent of insulin resistance could explain unhealthy body fat distribution and its metabolic consequences. To analyze common single nucleotide polymorphisms (SNPs) in 48 lipolysis-, but not insulin-signaling-related genes, to form polygenic risk scores of lipolysis-associated SNPs, and to investigate their effects on body fat distribution, glycemia, insulin sensitivity, insulin secretion, and proinsulin conversion. SNP array, anthropometric, and metabolic data were available from up to 2789 participants without diabetes of the Tübingen Family study of type 2 diabetes characterized by oral glucose tolerance tests. In a subgroup (n = 942), magnetic resonance measurements of body fat stores were available. We identified insulin-sensitivity-independent nominal associations (P < 0.05) of SNPs in 10 genes with plasma free fatty acids (FFAs), in 7 genes with plasma glycerol and in 6 genes with both, plasma FFAs and glycerol. A score formed of the latter SNPs (in ADCY4, CIDEA, GNAS, PDE8B, PRKAA1, PRKAG2) was associated with plasma FFA and glycerol measurements (1.4*10-9 ≤ P ≤ 1.2*10-5), visceral adipose tissue mass (P = 0.0326), and proinsulin conversion (P ≤ 0.0272). The more lipolysis-increasing alleles a subject had, the lower was the visceral fat mass and the lower the proinsulin conversion. We found evidence for a genetic basis of adipose tissue lipolysis resulting from common SNPs in CIDEA, AMP-activated protein kinase subunits, and cAMP signaling components. A genetic score of lipolysis-increasing alleles determined lower visceral fat mass and lower proinsulin conversion.

Cyclin-dependent kinase 1-mediated AMPK phosphorylation regulates chromosome alignment and mitotic progression.

AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase and cellular metabolic sensor, has been found to regulate cell cycle checkpoints in cancer cells in response to energetic stress, to harmonize proliferation with energy availability. Despite AMPK's emergent association with the cell cycle, it still has not been fully delineated how AMPK is regulated by upstream signaling pathways during mitosis. We report, for the first time, direct CDK1 phosphorylation of both the catalytic α1 and α2 subunits, as well as the β1 regulatory subunit, of AMPK in mitosis. We found that AMPK-knockout U2OS osteosarcoma cells have reduced mitotic indexes and that CDK1 phosphorylation-null AMPK is unable to rescue the phenotype, demonstrating a role for CDK1 regulation of mitotic entry through AMPK. Our results also denote a vital role for AMPK in promoting proper chromosomal alignment, as loss of AMPK activity leads to misaligned chromosomes and concomitant metaphase delay. Importantly, AMPK expression and activity was found to be critical for paclitaxel chemosensitivity in breast cancer cells and positively correlated with relapse-free survival in systemically treated breast cancer patients.

EBV-miR-BART1-5P activates AMPK/mTOR/HIF1 pathway via a PTEN independent manner to promote glycolysis and angiogenesis in nasopharyngeal carcinoma.

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. The occurrence of both events involves many key molecular changes including miRNA. However, EBV encoded miRNAs are rarely mentioned as capable of regulating tumor metabolism and tumor angiogenesis. Here, we reported that one of the key miRNAs encoded by EBV, EBV-miR-Bart1-5P, can significantly promote nasopharyngeal carcinoma (NPC) cell glycolysis and induces angiogenesis in vitro and in vivo. Mechanistically, EBV-miR-Bart1-5P directly targets the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1) and consequently regulates the AMPK/mTOR/HIF1 pathway which impelled NPC cell anomalous aerobic glycolysis and angiogenesis, ultimately leads to uncontrolled growth of NPC. Our findings provide new insights into metabolism and angiogenesis of NPC and new opportunities for the development of targeted NPC therapy in the future.

Postnatal induction of muscle fatty acid oxidation in mice differing in propensity to obesity: a role of pyruvate dehydrogenase.

Adaptation to the extrauterine environment depends on a switch from glycolysis to catabolism of fatty acids (FA) provided as milk lipids. We sought to learn whether the postnatal induction of muscle FA oxidation in mice could reflect propensity to obesity and to characterize the mechanisms controlling this induction. Experiments were conducted using obesity-resistant A/J and obesity-prone C57BL/6J (B6) mice maintained at 30 °C, from 5 to 28 days after birth. At day 10, both A/J and B6 mice with genetic ablation (KO) of α2 subunit of AMP-activated protein kinase (AMPK) were also used. In skeletal muscle, expression of selected genes was determined using quantitative real-time PCR, and AMPK subunits content was evaluated using Western blotting. Activities of both AMPK and pyruvate dehydrogenase (PDH), as well as acylcarnitine levels in the muscle were measured. Acylcarnitine levels and gene expression indicated transient increase in FA oxidation during the first 2 weeks after birth, with a stronger increase in A/J mice. These data correlated with (i) the surge in plasma leptin levels, which peaked at day 10 and was higher in A/J mice, and (ii) relatively low activity of PDH linked with up-regulation of PDH kinase 4 gene (Pdk4) expression in the 10-day-old A/J mice. In contrast with the Pdk4 expression, transient up-regulation of uncoupling protein 3 gene was observed in B6 but not A/J mice. AMPK activity changed during the development, without major differences between A/J and B6 mice. Expression of neither Pdk4 nor other muscle genes was affected by AMPK-KO. Our results indicate a relatively strong postnatal induction of FA oxidation in skeletal muscle of the obesity-resistant A/J mice. This induction is transient and probably results from suppression of PDH activity, linked with a postnatal surge in plasma leptin levels, independent of AMPK.

The Role of AMPK in Chemotherapy Response in AML

Introduction: Acute myeloid leukemia is an aggressive malignancy characterized by relapse and resistance to therapy. When treated with standard chemotherapy 60-80% of patients will achieve a remission however most will suffer a relapsed and once relapsed the disease is much more resistant to repeated treatment. Despite the central role of resistance little is known about the mechanism of chemotherapy responsiveness in AML. Our laboratory has previous shown a metabolic shift occurs in AML cells following chemotherapy exposure with increased mitochondrial oxygen consumption leading to resistance. Adenosine monophosphate activated protein kinase (AMPK) is a central metabolic regulator and leading candidate to coordinate this metabolic shift. However, the role of AMPK in chemotherapy response in AML is unknown.Methods: A Cas9 expressing AML model was generated and partially infected with a lentiviral vector coding an sgRNA targeting the alpha 1 subunit of AMPK, PRKAA1. This subunit was chosen as cell line and clinical expression datasets demonstrated that AML cells expressed little or none of the second alpha subunit PRKAA2. PRKAA1 deleted clones (AMPK KO) were isolated and loss of PRKAA1 expression and AMPK activity were confirmed. AMPK KO cells were characterized for responsiveness to chemotherapy, metabolic inhibitors and the DNA damage response.Results: Consistent with a role in response to chemotherapy AML cells treated with cytarabine demonstrated a significant increase in AMPK activation. When partially AMPK deleted populations were treated with cytarabine or doxorubicin a significant enrichment was seen in the deleted cells suggesting that loss of AMPK promotes therapy resistance. To confirm this finding isogeneic AML cell lines were treated with chemotherapy. AMPK KO cells were significantly more resistant to doxorubicin and cytarabine compared to isogeneic controls expressing an sgRNA targeting the ROSA26 (ROSA KO) safe harbor locus. When the mechanism of this resistance was explored AMPK KO cells were found to have diminished H2AX, p53 and p21 induction following exposure to doxorubicin culminating in a decrease in apoptosis. To assess if this resistant phenotype was present in vivo, AMPK KO or ROSA KO cells were tail vein injected into sub-lethally irradiated mice. Upon confirmation of engraftment mice were treated with a combination of cytarabine and doxorubicin. AMPK KO mice had a significantly decreased survival following treatment then treated ROSA controls. Additionally, AMPK KO and ROSA KO cells were treated with the glycolysis inhibitor 2-Deoxyglucose, the autophagy inhibitor chloroquine and the fatty acid oxidation inhibitor extomoxir. In all cases AMPK KO cells were significantly more resistant to these agents than the ROSA KO cells suggesting a proapoptotic role of AMPK following exposure to metabolic inhibitors. Finally, the clinical relevance of these findings was assessed by examining outcomes of AML patients treated with chemotherapy in relationship to their levels of PRKAA1 expression. Patients with the lowest expression of PRKAA1 had a significantly worse overall survival consistent with our findings.Conclusions: In AML cells activated AMPK provides pro-apoptotic signaling in response to DNA damaging agents allowing incorporation of metabolic status into cell fate decisions following treatment. Utilization of metabolic inhibitors that activate AMPK in combination with chemotherapy should be explored in the treatment of AML. DisclosuresPardee:Karyopharm: Research Funding; Novartis: Speakers Bureau; Rafael Pharmaceuticals: Employment; Celgene: Speakers Bureau; Amgen: Speakers Bureau.

Role of the α2 subunit of AMP-activated protein kinase and its nuclear localization in mitochondria and energy metabolism-related gene expressions in C2C12 cells

BackgroundAMP-activated protein kinase (AMPK), a heterotrimer with α1 or α2 catalytic subunits, acts as an energy sensor and regulates cellular homeostasis. Whereas AMPKα1 is necessary for myogenesis in skeletal muscle, the role of AMPKα2 in myogenic differentiation and energy metabolism-related gene expressions has remained unclear. We here examined the specific roles of AMPKα1 and AMPKα2 in the myogenic differentiation and mitochondria and energy metabolism-related gene expressions in C2C12 cells. Materials and MethodsStable C2C12 cell lines expressing a scramble short hairpin RNA (shRNA) or shRNAs specific for AMPKα1 (shAMPKα1), AMPKα2 (shAMPKα2), or both AMPKα1 and AMPKα2 (shPanAMPK) were generated by lentivirus infection. Lentiviruses encoding wild-type AMPKα2 (WT-AMPKα2) or AMPKα2 with a mutated nuclear localization signal (ΔNLS-AMPKα2) were also constructed for introduction into myoblasts. Myogenesis was induced by culture of C2C12 myoblasts for 6 days in differentiation medium. ResultsThe amount of AMPKα2 increased progressively, whereas that of AMPKα1 remained constant, during the differentiation of myoblasts into myotubes. Expression of shPanAMPK or shAMPKα1, but not that of shAMPKα2, attenuated the proliferation of myoblasts as well as the phosphorylation of both acetyl-CoA carboxylase and the autophagy-initiating kinase ULK1 in myotubes. Up-regulation of myogenin mRNA, a marker for the middle stage of myogenesis, was attenuated in differentiating myotubes expressing shPanAMPK or shAMPKα1. In contrast, up-regulation of gene expression for muscle creatine kinase (MCK), a late-stage differentiation marker, as well as for genes related to mitochondrial biogenesis including the transcriptional coactivator peroxisome proliferator–activated receptor–γ coactivator–1α1 and α4 (PGC-1α1 and PGC-1α4) and mitochondria-specific genes such as cytochrome c were attenuated in myotubes expressing shAMPKα2 or shPanAMPK. The diameter of myotubes expressing shPanAMPK or shAMPKα2 was reduced, whereas that of those expressing shAMPKα1 was increased, compared with myotubes expressing scramble shRNA. A portion of AMPKα2 became localized to the nucleus during myogenic differentiation. The AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) and 2-deoxyglucose (2DG) each induced the nuclear translocation of WT-AMPKα2, but not that of ΔNLS-AMPKα2. Finally, expression of WT-AMPKα2 increased the mRNA abundance of PGC-1α1 and MCK mRNAs as well as cell diameter and tended to increase that of PGC-1α4, whereas that of ΔNLS-AMPKα2 increased only the abundance of MCK mRNA, in myotubes depleted of endogenous AMPKα2. ConclusionTAMPKα1 and AMPKα2 have distinct roles in myogenic differentiation of C2C12 cells, with AMPKα1 contributing to the middle stage of myogenesis and AMPKα2 to the late stage. AMPKα2 regulates gene expressions including MCK, PGC-1α1 and PGC-1α4 and mitochondria-specific genes such as cytochrome c during the late stage of differentiation. Furthermore, the nuclear translocation of AMPKα2 is necessary for maintenance of PGC-1α1 mRNA during myogenesis.

Myricanol mitigates lipid accumulation in 3T3-L1 adipocytes and high fat diet-fed zebrafish via activating AMP-activated protein kinase

Myricanol is a diarylheptanoid isolated from Chinese bayberry. Through virtual docking strategy, myricanol was discovered as an AMP-activated protein kinase (AMPK) activator among a series of structural analogs, with high affinity for the γ subunit of AMPK. Myricanol was also evaluated for regulatory effects on lipid accumulation and insulin sensitivity in 3T3-L1 adipocytes and adiposity in high-fat diet-fed zebrafish. Myricanol suppressed lipid accumulation in 3T3-L1 cells in the initial stage (days 0–2) by suppressing adipogenesis and in the terminal stage (days 4–7) by inducing lipolysis and lipid combustion through activating AMPK. Moreover, myricanol enhanced insulin-stimulated glucose uptake by activating the insulin signaling pathway. In high-fat diet-fed zebrafish, myricanol inhibited lipid accumulation by suppressing adipogenic factors including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). In summary, the results indicate that myricanol could be a potential therapeutic agent against obesity by activating the AMPK signaling pathway.

Raspberry alleviates obesity-induced inflammation and insulin resistance in skeletal muscle through activation of AMP-activated protein kinase (AMPK) \u03b11

OBJECTIVEThrough dynamic means, etiological factors, including chronic inflammation and insulin resistance have the potential to perpetuate metabolic incidences such as type 2 diabetes and obesity. Abatement of such syndromes can be achieved by complex mechanisms initiated through bioactive compounds such as polyphenols derived from fruits. Using a whole-fruit approach, the effects of dietary red raspberry, which is rich in polyphenols, on inflammatory responses and insulin resistance in the skeletal muscles of Mus musculus were studied along with the potential role of AMP-activated protein kinase (AMPK) to act as a key mediator.SUBJECTSWild-type (WT) mice and mice deficient in the catalytic subunit (α1) of AMPK (AMPKα1−/−) were fed with a high-fat diet (HFD) or HFD supplemented with raspberry (5% dry weight) for 10 weeks. Factors involved in inflammatory responses, insulin signaling transduction, and mitochondrial biogenesis were evaluated.RESULTSDietary raspberry reduced ectopic lipid storage, alleviated inflammation responses, improved whole-body insulin sensitivity, and promoted mitochondrial biogenesis in the skeletal muscle of WT mice, but not AMPKα1−/− mice.CONCLUSIONSAMPKα1 is an important mediator for the beneficial effects of raspberry through alleviating inflammatory responses and sensitizing insulin signaling in skeletal muscle of HFD-fed mice.

AMPK-Mediated Regulation of Lipid Metabolism by Phosphorylation.

AMP-activated protein kinase (AMPK) is a metabolic sensor in mammals that is activated when ATP levels in the cell decrease. AMPK is a heterotrimeric protein that comprises 3 subunits, each of which has multiple phosphorylation sites that play critical roles in the regulation of either anabolism or catabolism by directly phosphorylating proteins or modulating gene transcription in multiple pathways, such as synthesis, oxidation and lipolysis of lipid. Research focused on the phosphorylation sites that are involved in lipid metabolism will lead to a better recognition of the role of AMPK in therapeutics for several common diseases. In this review, close attention is paid to the recent research on the structure, and multisite phosphorylation of AMPK subunits, as well as AMPK regulation of lipid metabolism via phosphorylation of related molecules.

The Effect of Resveratrol on Reducing Neointimal Growth after Femoral Artery Injury in AMPKα2 Knockout Mice Given High-Fat Diet

The rates of restenosis after percutaneous transluminal angioplasty are higher in diabetic than nondiabetic patients. Insulin is protective against restenosis, but these effects are diminished in the case of insulin resistance, which develops with high fat diet (HFD). Therefore, it makes sense to use an insulin-sensitizing compound which also has vasculoprotective effects such as resveratrol. Resveratrol is a phenolic compound found in plant tissues such as the skin of grapes, and has been hypothesized to account for the lower rates of cardiovascular disease among countries with high wine consumption. We have shown that resveratrol decreases neointimal formation in both mouse and rat models of arterial injury, and also that these effects are exerted through eNOS (endothelial nitric oxide synthase). We hypothesized that resveratrol acts though Sirt1, a NAD+-dependent deacetylase, to activate eNOS and decrease neointimal formation, but did not see any difference between neointimal growth of control and Sirt1-/- mice given HFD. AMPK (AMP-activated protein kinase) is an energy sensor which also protects against neointimal hyperplasia in the vasculature via directly activating eNOS and inhibiting the migration of vascular smooth muscle cells. Knocking out the α2 subunit of AMPK in particular has been shown to increase neointimal growth in mouse carotid artery in response to injury. We investigated the effect of resveratrol on neointimal growth after femoral arterial injury of wild type and AMPKα2 knock-out mice given HFD. Resveratrol was given at 0.0375% in the diet two days after wire injury. After 28 days, arteries were harvested and histomorphometric analysis was performed on the cross-sections. We found that neointimal growth was significantly higher in AMPKα2 knock-out mice given resveratrol compared to wild type mice given resveratrol (p&amp;lt;0.05). These results suggest that the vasculoprotective effects of resveratrol are mediated through AMPK. Disclosure L. Zhou: None. J. Guo: None. H. Zhang: None. S. Heximer: None. A. Giacca: None.

AMP‐Activated Protein Kinase (AMPK) Modulates Myoendothelial Junctions (MEJ) in Murine Microvessels

Myoendothelial junctions (MEJ) in microvessels are not only the site of direct cell communication via gap junctions but also represent a signaling microdomain critical for regulating vascular processes such as endothelium dependent hyperpolarization (EDH). The 5′‐adenosine monophosphate‐activated protein kinase (AMPK) has been shown to influence vascular tone. It has not yet been studied, however, whether AMPK could also affect MEJ. Thus, the aim of this project was to elucidate the impact of AMPK on MEJ dynamics and its potential mechanism of action.We studied isolated small mesenteric and skeletal muscle resistance arteries of mice using a pressure myograph system developed in our laboratory. Using confocal and two photon excitation fluorescence microscopy we identified the internal elastic lamina, which in these vessels exhibits small holes that allow direct contact between endothelial and smooth muscle cells. On average, we found that in about 35% of the holes, structures with a bright actin core as assessed by using F‐actin fluorescence in LifeAct mice or by staining with phalloidin which we could define as sites of MEJ. These structures showed expression of Cx37 and Cx43 suggesting that gap junctions (MEGJs) were also localized in these structures. Over observation times of up to 2 hours, we found that the number and localization of these MEJ varied, while they always positioned in the area of holes of IEL only. Thus, for the first time we could investigate MEJ dynamics. AMPK negatively regulates MEJ formation, since in AMPK α1, but not α2 knockout (KO) mice, the number of MEJ was significantly increased. In accordance, in wild type mice, incubation with the AMPK inhibitory compound C (CC) significantly augmented the number of MEJ. Furthermore, KO of AMPK α1, in intact mice as well as in cultured human and porcine smooth muscle cells, went along with an increase in PAI‐1 expression. Accordingly, incubation of arteries with exogenous PAI‐1 also increased the number of MEJ. The negative effect of AMPK on PAI‐1 could be explained by enhanced expression of the silencer of the PAI‐1 gene, small heterodimer partner (SHP). Since MEJ are also involved in EDH, we studied whether an enhanced formation of MEJ correlated with the magnitude of EDH induced vasodilation. Indeed, a higher amount of MEJs in α1 KO mice went along with a left shift of acetylcholine (ACh) ‐induced dilation dose effect curve.This work describes for the first time a role of the α1 subunit of AMPK as a potent modulator of MEJ dynamics which is probably mediated by controlling PAI‐1 expression. Our data also for the first time demonstrate a dynamic regulation of MEJ expression in intact blood vessels.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic studies of a patient harbouring a novel S487L mutation in the catalytic subunit of AMPK

AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.

Influence of Single-Nucleotide Polymorphisms in PPAR-δ, PPAR-γ, and PRKAA2 on the Changes in Anthropometric Indices and Blood Measurements through Exercise-Centered Lifestyle Intervention in Japanese Middle-Aged Men.

The purpose of the current study was to examine the influence of single-nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptor-δ (PPAR-δ), PPAR-γ, and α2 isoforms of the catalytic subunit of AMP-activated protein kinase (PRKAA2) on the extent of changes in anthropometric indices and blood measurements through exercise-centered lifestyle intervention in middle-aged men. A total of 109 Japanese middle-aged male subjects (47.0 ± 0.4 years) participated in the baseline health checkup, 6-month exercise-centered lifestyle intervention, and second checkup conducted several months after the subject completed the intervention. The body mass index (BMI), waist circumference, and clinical measurements, including hemoglobin Alc (HbA1c), triglyceride (TG), alanine aminotransferase (ALT), and γ-glutamyl-transpeptidase (γ-GTP), were measured at the baseline and second checkup. The three SNPs of PPAR-δ A/G (rs2267668), PPAR-γ C/G (rs1801282), and PRKAA2 A/G (rs1418442) were determined. Blunted responses in the reduction in the BMI and waist circumference were observed in A/A carriers of PPAR-δ SNP compared with G allele carriers (all p < 0.05). The A/A carriers also displayed less-marked improvements in HbA1c, TG, ALT, and γ-GTP (all p < 0.05). The current results suggest that A/A carriers of PPAR-δ SNP (rs2267668) may enjoy fewer beneficial effects of exercise-centered lifestyle intervention on anthropometric indices and blood measurements.

Uncarboxylated Osteocalcin Enhances Glucose Uptake Ex Vivo in Insulin-Stimulated Mouse Oxidative But Not Glycolytic Muscle.

Uncarboxylated osteocalcin (ucOC) stimulates muscle glucose uptake in mice EDL and soleus muscles. However, whether ucOC also exerts a similar effect in insulin-stimulated muscles in a muscle type-specific manner is currently unclear. We aimed to test the hypothesis that, with insulin stimulation, ucOC per se has a greater effect on oxidative muscle compared with glycolytic muscle, and to explore the underlying mechanisms. Mouse (C57BL6, male 9-12weeks) extensor digitorum longus (EDL) and soleus muscles were isolated and longitudinally split into halves. Muscle samples were treated with varying doses of recombinant ucOC (0, 0.3, 1, 3, 30ng/ml), followed by insulin addition. Muscle glucose uptake, protein phosphorylation and total expression of protein kinase B (Akt), Akt substrate of 160kDa (AS160), extracellular signal-regulated kinase isoform 2 (ERK2), and adenosine monophosphate-activated protein kinase subunit α (AMPKα) were assessed. ucOC treatment at 30ng/ml enhanced muscle glucose uptake in insulin-stimulated soleus, a mainly oxidative muscle (17.5%, p < 0.05), but not in EDL-a mostly glycolytic muscle. In insulin-stimulated soleus only, ucOC treatment (3 and 30ng/ml) increased phosphorylation of AS160 and ERK2, but not Akt or AMPKα. The ucOC-induced increase in ERK2 phosphorylation in soleus was not associated with the increase in glucose uptake or AS160 phosphorylation. ucOC enhances glucose uptake and AS160 phosphorylation in insulin-stimulated oxidative but not glycolytic muscle, via upstream mechanisms which appear to be independent of ERK or AMPK.

AMPK Protein Interaction Analyses by Yeast Two-Hybrid.

Mammalian AMP-activated protein kinase (AMPK) is a Ser/Thr protein kinase that acts as a crucial energy sensor in the cell. Since AMPK plays a key role in a multitude of different pathways in the cell, major efforts have been concentrated to elucidate its signaling network, mainly by the identification of AMPK downstream targets. In this chapter we describe a yeast two-hybrid method for the direct evaluation of the interaction between an AMPK subunit and putative substrates.