Kinase AMP-activated Catalytic Subunit Alpha Research Articles

AMPK/MTOR/TP53 Signaling Pathway Regulation by Calcitonin Gene-Related Peptide Reduces Oxygen-Induced Lung Damage in Neonatal Rats through Autophagy Promotion.

Our previous studies indicated that calcitonin gene-related peptide (CGRP) alleviates hyperoxia-induced lung injury and suggested the possible involvement of autophagy in this process. Herein, we aimed to further explore the potential involvement of tumor protein p53 (TP53) and autophagy in the mode of action of CGRP against hyperoxia-induced lung injury in vitro and in vivo. The study conducted tests on type II alveolar epithelial cells (AECII) and rats that were subjected to hyperoxia treatment or combined treatment of hyperoxia with CGRP, CGRP inhibitor, rapamycin (an autophagy agonist), 3-methyladenine (3-MA, an autophagy inhibitor), TP53 silencing/inhibitor (pifithrin-α), or expression vector/activator (PRIMA-1 (2,2-bis(hydroxymethyl)-3-quinuclidinone)) and their corresponding controls. We found that oxidative stress, apoptosis, and autophagy were all increased by hyperoxia treatment in vitro. However, treating AECII cells with CGRP reversed hyperoxia-induced oxidative stress and apoptosis but further promoted autophagy. In addition, the combined treatment with rapamycin or TP53 silencing with CGRP promoted the effect of CGRP, while contrary results were obtained with combined therapy with 3-MA or TP53 overexpression. In vivo, the number of hyperoxia-induced autophagosomes was promoted in the lung tissue of neonatal rats. Furthermore, hyperoxia increased the expression levels of AMP-activated protein kinase (AMPK) alpha 1 (also known as protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1)) but inhibited TP53 and mechanistic target of rapamycin (MTOR); these expression trends were regulated by CGRP treatment. In conclusion, we showed that CGRP can attenuate hyperoxia-induced lung injury in neonatal rats by enhancing autophagy and regulating the TP53/AMPK/MTOR crosstalk axis.

Effect of different levels of Lactobacillus added to diets on fat deposition and meat quality of Sunit lambs

In this study, we examined the impact of adding Lactobacillus to the diet on fat distribution and meat quality of Sunit lambs. For 90 days, twenty-four lambs (19.31 ± 0.47 kg) were fed diets that contained 0 (NP), 6 (P1), 12 (P2), or 24 (P3) g of Lactobacillus casei/d. The results suggested that dietary supplementation with Lactobacillus decreased serum triglyceride in Sunit lambs (P < 0.001). The loin muscle area displayed notable increases in the P1 group (P < 0.05). Meanwhile, tail and visceral fat deposition of lambs were reduced when Lactobacillus was added to the diet (P < 0.05). Compared with the NP group, the values of shear force and cooking loss of in the P1 group exhibited a significant reduction, and intramuscular fat content increased significantly (P < 0.05). Additionally, the P1 group showed an increase in polyunsaturated fatty acids and a decrease in saturated fatty acids in the longissimus thoracis and biceps femoris muscles (P < 0.05). The P1 group showed downregulation of protein kinase AMP-activated catalytic subunit alpha 2 (AMPKα2) and carnitine palmitoyltransferase 1B expression in the longissimus thoracis muscle (P < 0.05). However, there was an upregulation of acetyl-CoA carboxylase (ACC), sterol regulatory element binding transcription factor 1 (SREBF1), and fatty acid synthase (FASN) expression (P < 0.05). In conclusion, feeding Sunit lambs 6 g/d of Lactobacillus as a dietary supplement may be a valuable way to improve fat distribution and meat quality. The AMPK/ACC and AMPK/SREBF1/FASN signaling pathways may be involved in this outcome.

ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA.

Tuberculosis has the highest mortality rate worldwide for a chronic infectious disease caused by a single pathogen. RNA-binding proteins (RBPs) are involved in autophagy - a key defense mechanism against Mycobacterium tuberculosis (M. tuberculosis) infection - by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during M. tuberculosis infection remain relatively unexplored. Zinc finger NFX1-type containing 1 (ZNFX1), a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in M. tuberculosis-infected macrophages. Here, we aimed to explore the immunoregulatory functions of ZNFX1 during M. tuberculosis infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased M. tuberculosis burden, progressive lung tissue injury, and chronic inflammation in M. tuberculosis-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing protein kinase AMP-activated catalytic subunit alpha 2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to M. tuberculosis growth suppression. These findings reveal a function of ZNFX1 in establishing anti-M. tuberculosis immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster antituberculosis immunotherapy.

Molecular mechanisms of AMPK/YAP/NLRP3 signaling pathway affecting the occurrence and development of ankylosing spondylitis

BackgroundInvestigate the AMPK (protein kinase AMP-activated catalytic subunit alpha 1)/YAP (Yes1 associated transcriptional regulator)/NLRP3 (NLR family pyrin domain containing 3) signaling pathway's role in ankylosing spondylitis (AS) development using public database analysis, in vitro and in vivo experiments.MethodsRetrieve AS dataset, analyze differential gene expression in R, conduct functional enrichment analysis, collect 30 AS patient and 30 normal control samples, and construct a mouse model. ELISA, IP, and knockdown experiments were performed to detect expression changes.ResultsNLRP3 was identified as a significant AS-related gene. Caspase-1, IL-1β, IL-17A, IL-18, IL-23, YAP, and NLRP3 were upregulated in AS patients. Overexpressing AMPK inhibited YAP's blockade on NLRP3 ubiquitination, reducing ossification in fibroblasts. Inhibiting AMPK exacerbated AS symptoms in AS mice.ConclusionAMPK may suppress YAP expression, leading to NLRP3 inflammasome inhibition and AS alleviation.

Gene expression of protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1), solute carrier family 2 member 1 (SLC2A1) and mechanistic target of rapamycin (MTOR) in metformin-treated type 2 diabetes patients with COVID-19: impact on inflammation markers.

The COVID-19 pandemic has resulted in a global health crisis that has severely impacted patients with type 2 diabetes (T2D). T2D patients have a higher risk of experiencing severe COVID-19 symptoms, hospitalization, and mortality compared to patients without diabetes. The dysregulated immune response in T2D patients can exacerbate the severity of COVID-19 symptoms. Insulin therapy, a common treatment for T2D patients, has been linked to increased mortality in COVID-19 patients with T2D. However, metformin, an anti-diabetic medication, has been shown to have anti-inflammatory properties that may mitigate the cytokine storm observed in severe COVID-19 cases. In this study, we investigated how the PRKAA1, SLC2A1, and MTOR genes contribute to inflammation markers in COVID-19 patients with T2D, who were receiving either insulin or metformin therapy. Our findings revealed that metformin treatment was associated with reduced expression of genes involved in Th1/Th17 cell differentiation. These results suggest that metformin could be a potential treatment option for T2D patients with COVID-19 due to its anti-inflammatory properties, which may improve patient outcomes.

Astragalus polysaccharide inhibits the development of urothelial carcinoma by activating AMPK signaling to induce BENC1-xCT complex formation.

In recent years, the incidence of urothelial carcinoma (UC) has been high in men. The aim of this study was to investigate whether astragalus polysaccharide (APS) could inhibit the development of UC and the specific molecular mechanism. Our data showed that APS inhibited the proliferation of UC cells in a dose-dependent manner, and APS reduced the migratory capacity of RT4 and T24 cells. Further studies revealed that the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed APS-induced cell death, intracellular Fe2+ and malondialdehyde (MDA) accumulation, and lipid peroxidation product deposition. The Western blot and immunofluorescence results showed that APS significantly inhibited the expression of glutathione peroxidase 4 (GPX4) but did not alter the protein level of solute carrier family 7 member 11 (xCT, SLC7A11). Further analysis revealed that APS reduced the activity of xCT in RT4 and T24 cells. Moreover, APS significantly increased the phosphorylation levels of protein kinase AMP-activated catalytic subunit alpha 1 (AMPK) and BECN1 in RT4 and T24 cells, which induced the formation of the BECN1-xCT complex. However, when AMPK was silenced in RT4 and T24 cells, APS-induced ferroptosis was reversed to some extent, indicating that APS-mediated ferroptosis involves AMPK signaling. Moreover, APS has been shown to inhibit tumor growth in nude mice in vivo. In summary, our study demonstrated for the first time that APS could promote the formation of the BECN1-xCT complex in UC cells by activating AMPK/BECN1 signaling, which inhibited the activity of xCT to reduce GPX4 expression, thereby inducing ferroptosis and ultimately inhibiting UC progression.

Anti-ferroptotic PRKAA2 serves as a potential diagnostic and prognostic marker for hepatoblastoma.

The incidence rate of hepatoblastoma (HB), which is the most prevalent malignant tumour among children, rises each year. According to recent studies, a number of neoplastic disorders and ferroptosis are intimately connected. This study aims to identify key ferroptosis-related genes in HB and explore new directions for the diagnosis and treatment of HB. Differentially expressed ferroptosis-related genes were identified using the Gene Expression Omnibus datasets. The functional annotation of candidate genes was evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Machine learning and receiver operating characteristic (ROC) curves revealed protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2), tribbles homolog 2 (TRIB2), and liver-type glutaminase (GLS2) as potential diagnostic genes of HB. By using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry, relative expression of PRKAA2 was examined. The effect of PRKAA2 on proliferation, apoptosis, and ferroptosis of HB cells was verified in vitro and in vivo. Fisher's exact test was used to evaluate the clinical significance of PRKAA2 in HB. The prognostic indicators had a substantial correlation with PRKAA2 expression, which rose dramatically in HB tissues. PRKAA2 promotes proliferation and inhibits ferroptosis in HB cells. PRKAA2 plays a role in ferroptosis by regulating hypoxia-inducible factor 1α (HIF-1α) and transferrin receptor 1 (TFR1). PRKAA2 functions as a tumor-promoting factor in HB by promoting cell proliferation and prohibiting ferroptosis. Ferroptosis-related genes PRKAA2 is a potential diagnostic and prognostic marker for HB as well as a novel therapeutic target in the future.

Apolipoprotein H: a novel regulator of fat accumulation in duck myoblasts

Apolipoprotein H (APOH) primarily engages in fat metabolism and inflammatory disease response. This study aimed to investigate the effects of APOH on fat synthesis in duck myoblasts (CS2s) by APOH overexpression and knockdown. CS2s overexpressing APOH showed enhanced triglyceride (TG) and cholesterol (CHOL) contents and elevated the mRNA and protein expression of AKT serine/threonine kinase 1 (AKT1), ELOVL fatty acid elongase 6 (ELOVL6), and acetyl-CoA carboxylase 1 (ACC1) while reducing the expression of protein kinase AMP-activated catalytic subunit alpha 1 (AMPK), peroxisome proliferator activated receptor gamma (PPARG), acyl-CoA synthetase long chain family member 1 (ACSL1), and lipoprotein lipase (LPL). The results showed that knockdown of APOH in CS2s reduced the content of TG and CHOL, reduced the expression of ACC1, ELOVL6, and AKT1, and increased the gene and protein expression of PPARG, LPL, ACSL1, and AMPK. Our results showed that APOH affected lipid deposition in myoblasts by inhibiting fatty acid beta-oxidation and promoting fatty acid biosynthesis by regulating the expression of the AKT/AMPK pathway. This study provides the necessary basic information for the role of APOH in fat accumulation in duck myoblasts for the first time and enables researchers to study the genes related to fat deposition in meat ducks in a new direction.

MCAD activation by empagliflozin promotes fatty acid oxidation and reduces lipid deposition in NASH.

Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the significant enzymes involved in the β-oxidation of mitochondrial fatty acids. MCAD deficiency affects the β-oxidation of fatty acid and leads to lipid deposition in multiple organs, but little is known about its importance in nonalcoholic steatohepatitis (NASH). Empagliflozin is revealed to effectively improve NASH by increasing research, whereas the specific mechanism still has to be explored. Human liver tissues of patients with or without NASH were obtained for proteomic analysis to screen proteins of interest. db/db mice were given empagliflozin by gavage for 8 weeks. The expression of MCAD and signaling molecules involved in hepatic lipid metabolism was evaluated in human liver, mice and HL7702 cells. We found that the MCAD levels in the liver were significantly reduced in NASH patients compared to patients without NASH. Protein-protein interaction network analysis showed that MCAD was highly correlated with forkhead box A2 (FOXA2) and protein kinase AMP-activated catalytic subunit alpha (PRKAA). AMPK/FOXA2/MCAD signaling pathway was detected to be inhibited in the liver of NASH patients. Decreased expression of MCAD was also observed in the livers of db/db mice and hepatocyte treated with palmitic acid and glucose. Of note, empagliflozin could upregulate MCAD expression by activating AMPK/FOXA2 signaling pathway, reduce lipid deposition and improve NASH in vivo and in vitro. This research demonstrated that MCAD is a key player of hepatic lipid deposition and its targeting partially corrects NASH. MCAD thus may be a potential therapeutic target for the treatment of NASH.

A significant quantitative trait locus on chromosome Z and its impact on egg production traits in seven maternal lines of meat-type chicken

BackgroundEgg production is economically important in the meat-type chicken industry. To better understand the molecular genetic mechanism of egg production in meat-type chicken, genetic parameter estimation, genome-wide association analyses combined with meta-analyses, Bayesian analyses, and selective sweep analyses were performed to screen single nucleotide polymorphisms (SNPs) and other genetic loci that were significantly associated with egg number traits in 11,279 chickens from seven material lines.ResultsYellow-feathered meat-type chickens laid 115 eggs at 43 weeks of age and white-feathered chickens laid 143 eggs at 60 weeks of age, with heritability ranging from 0.034–0.258. Based on meta-analyses and selective sweep analyses, one region (10.81–13.05 Mb) on chromosome Z was associated with egg number in all lines. Further analyses using the W2 line was also associated with the same region, and 29 SNPs were identified that significantly affected estimation of breeding value of egg numbers. The 29 SNPs were identified as having a significant effect on the egg number EBV in 3194 birds in line W2. There are 36 genes in the region, with glial cell derived neurotrophic factor, DAB adaptor protein 2, protein kinase AMP-activated catalytic subunit alpha 1, NAD kinase 2, mitochondrial, WD repeat domain 70, leukemia inhibitory factor receptor alpha, complement C6, and complement C7 identified as being potentially affecting to egg number. In addition, three SNPs (rs318154184, rs13769886, and rs313325646) associated with egg number were located on or near the prolactin receptor gene.ConclusionOur study used genomic information from different chicken lines and populations to identify a genomic region (spanning 2.24 Mb) associated with egg number. Nine genes and 29 SNPs were identified as the most likely candidate genes and variations for egg production. These results contribute to the identification of candidate genes and variants for egg traits in poultry.

557-P: Effect of Monocarbonyl Curcumin Analogues C66 and B2BrBC on Pancreatic Expression of Genes Related to Insulin Signaling Pathway and Oxidative Stress in Streptozotocin-Induced Diabetes

Curcumin has well documented beneficial effects against diabetes but its poor bioavailability limits its use. Here, we investigated the effects of two monocarbonyl curcumin analogues - (2E,6E) -2,6-bis[ (2-trifluoromethyl) benzylidene]cyclohexanone (C66) and (2E,6E) -2,6-bis (2-bromobenzylidene) cyclohexanone) (B2BrBC) - on the expression of genes involved in insulin signaling and oxidative stress in pancreas samples from streptozotocin (STZ) -induced diabetic rats. Animals were divided into two experiments: (Experiment 1) STZ injection (60 mg/kg) followed by 30 days treatment with C66 or B2BrBC (both 50 µmol/kg, daily) or (Experiment 2) single-dose treated with C66 or B2BrBC (125 µmol/kg) , 6 hours before STZ injection, and sacrifice after 72 hours. Quantitative RT-PCR analysis was used to assess the expression of: insulin (Ins1) , insulin receptor substrate 2 (Irs2) , Slc2a2 solute carrier family 2 member 2 (Slc2a2) (s. Glut2) , mitogen-activated protein kinase 14 (Mapk14) , protein kinase AMP-activated catalytic subunit alpha 1 (Prkaa1) , peroxisome proliferator-activator receptor gamma (Pparg) , BCL2 apoptosis regulator (Bcl2) , BCL2-associated X apoptosis regulator (Bax) , and NF-κB inhibitor alpha (Nfkbia) . Results demonstrated that when C66 and B2BrBC were administered at lower, chronic doses after STZ application (Experiment 1) , they both reversed the effect of STZ on Irs2 mRNA levels in the pancreas, with the effect of C66 being significantly higher. At higher, acute doses (Experiment 2) , C66 ameliorated Ins1 mRNA levels after STZ treatment. Also, although not statistically significant, at higher doses, both C66 and B2BrBC showed a trend to normalize the suppressing effect of STZ on Glut2 mRNA levels. Overall, our data demonstrate that these two monocarbonyl curcumin analogues have direct effects on the expression of insulin signaling pathway-related genes in the pancreas. Disclosure N. Hadzi-Petrushev: None. M. Mladenov: None. J.B. Bogdanov: None. L. Poretsky: None. D. Avtanski: None. Funding Gerald J. and Dorothy R. Friedman New York Foundation for Medical Research

Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells

ABSTRACT The maintenance of a strong IL21 production in memory CD4 T cells, especially in HIV-1-specific cells, represents a major correlate of natural immune protection against the virus. However, the molecular mechanisms underlying IL21 production during HIV-1 infection, which is only elevated among the naturally protected elite controllers (EC), are still unknown. We recently found out that lipophagy is a critical immune mediator that control an antiviral metabolic state following CD8A T cell receptor engagement, playing an important role in the natural control of HIV-1 infection. This led us to investigate whether the beneficial role of a strong macroautophagy/autophagy, could also be used to ensure effective IL21 production as well. Herein, we confirm that after both polyclonal and HIV-1-specific activation, memory CD4 T cells (Mem) from EC display enhanced activity of the autophagy-mediated proteolysis compared to ART. Our results indicate that the enhanced autophagy activity in EC was controlled by the energy-sensing PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1). We further confirmed the critical role of the autophagy-mediated proteolysis in the strong IL21 production in EC by using BECN1 gene silencing as well as protease, PRKAA1, and lysosomal inhibitors. Finally, we established that high autophagy-mediated proteolysis in EC fuels their cellular rates of mitochondrial respiration due to glutaminolysis. Our data confirm the critical role of autophagy in dictating the metabolic input, which is required not only to ensure protective cytotoxic CD8A T cell responses, but also to provide strong IL21 production among antiviral CD4 T cells. Abbreviations: AKG: alpha-ketoglutarate; ART: patients under antiretroviral therapy; ATG7: autophagy related 7; BaF: bafilomycin A1; BECN1: beclin 1; Chloro.: chloroquine; EC: elite controllers; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FOXO3: forkhead box O3; GLS: glutaminase; GLUD1: glutamate dehydrogenase 1; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MTOR: mechanistic target of rapamycin kinase; PBMC: peripheral blood mononuclear cells; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; SQSTM1: sequestosome 1; TCA: tricarboxylic acid cycle; ULK1: unc-51 like autophagy activating kinase

High Expression of Citron Kinase Contributes to the Development of Esophageal Squamous Cell Carcinoma.

ObjectiveThis study aimed to investigate the role and potential regulatory mechanism of citron kinase (CIT) in esophageal squamous cell carcinoma (ESCC).MethodsCitron kinase (CIT) expression in ESCC tissues was analyzed based on the microarray dataset GSE20347, and CIT expression in ESCC cell lines was analyzed. Eca-109 cells were lentivirally transfected with shRNA-CIT (LV-shCIT) to knock down CIT, followed by investigation of cell proliferation and apoptosis. Nude mouse xenograft experiments were performed to evaluate the tumorigenicity of CIT-knockdown Eca-109 cells. Microarray analysis of Eca-109 cells transfected with LV-shCIT or LV-shNC and subsequent Ingenuity Pathway Analysis (IPA) were performed to identify CIT-related differentially expressed genes (DEGs) and signaling pathways. Furthermore, the expression of key DEGs was validated using the clinical samples of ESCC.ResultsCitron kinase (CIT) was highly expressed in ESCC tissues and cell lines. Knockdown of CIT suppressed Eca-109 cell proliferation and promoted apoptosis in vitro. Moreover, CIT knockdown significantly reduced tumorigenicity of Eca-109 cells in vivo. Microarray and IPA analysis showed that signaling by the Rho family GTPases pathway was significantly activated, and CIT intrinsically interacted with the protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1), sequestosome 1 (SQSTM1), and interleukin 6 (IL6). Notably, the expression levels of PRKAA1 and SQSTM1 were upregulated in ESCC tissues, while the IL6 expression was downregulated.ConclusionOur findings confirm that CIT functions as an oncogene in ESCC. CIT may contribute to ESCC development by upregulating PRKAA1 and SQSTM1 as well as downregulating IL6. Citron kinase may serve as a promising therapeutic target for ESCC.

Manassantin B attenuates obesity by inhibiting adipogenesis and lipogenesis in an AMPK dependent manner.

Saururus chinensis (Schinensis) has been used as an herb to treat edema, jaundice, and gonorrhea. Manassantin B (MNSB), a dineolignan isolated from Schinensis, was identified as a potent adipogenesis/lipogenesis inhibitor (IC50 =9.3nM). To explore the underlying mechanism, both adipogenesis and lipogenesis were measured in differentiated 3T3-L1 preadipocytes, murine primary preadipocytes and adipose tissue explants upon MNSB treatment. Key regulators of adipogenesis/lipogenesis were downregulated by MNSB treatment, mainly resulting from increased phosphorylation of AMPK which was identified as a vital regulator of adipogenesis and lipogenesis. Moreover, MNSB did not increase AMPK phosphorylation in 3T3-L1 cells transfected with Prkaa1 (encoding protein kinase AMP-activated catalytic subunit alpha 1) siRNA or adipose tissue explants isolated from adipose-specific Prkaa1-disrupted mice (Prkaa1Δad ). In diet-induced obese C57BL/6N mice, MNSB displayed preventive and therapeutic effects on obesity accompanied by decreased adipocyte size. MNSB was also found to increase AMPK phosphorylation both in subcutaneous white adipose tissue and brown adipose tissue in vivo. These findings suggest that MNSB can be a new therapeutic agent for the prevention and treatment of obesity and other related metabolic disorders.

Upregulation of signal transducer and activator of transcription 4 promotes osteoblast activity by activating AMP-activated protein kinase based on cationic liposome transfection

Activation of Protein Kinase AMP-Activated Catalytic Subunit Alpha (AMPKα) is an important regulatory pathway for osteogenic differentiation. STAT4 acts as a transcriptional activity factor to regulate the transcription of many genes and is potentially a regulatory factor for AMPKα transcription activity. To confirm the regulatory effect of STAT4 on AMPKα and the effect of STAT4 on osteogenic differentiation, the promoter sequence of AMPKα was analyzed via bioinformatics, the STAT4 overexpression vector was constructed and transfected into human osteoblast-like cells MG-63 by cationic liposome, fluorescence quantitative PCR (RT-qPCR) and western blotting technologies were used to detect the effect of STAT4 on the expression of AMPKα. MTT and ALP activity assays were also used to verify the effect of STAT4 on the proliferation and maturation of osteoblasts by regulating AMPKα expression. Our results showed that STAT4 was a co-transcriptional regulator of AMPKα1 and AMPKα2, which combined the enrichment region of CpG on the promoter sequence of AMPKα1/2. Overexpression of STAT4 significantly increased the expression of AMPKα1 and AMPKα2, which promoted the proliferation and maturation of osteoblasts. We concluded that STAT4 was a transcriptional activator of AMPKα and promoting STAT4 expression enhances the proliferation and differentiation activity of AMPKα in osteoblasts.

Induction of autophagy and suppression of type I IFN secretion by CSFV

ABSTRACT Macroautophagy/autophagy plays an essential role in cellular responses to pathogens. However, the precise mechanisms and signaling pathways that modulate cellular autophagy in classical swine fever virus (CSFV)-infected host cells have not been confirmed. In this study, we showed that CSFV infection inhibits the phosphorylation of MTOR (mechanistic target of rapamycin kinase), subsequently leading to autophagy initiation. We also show that MAPK/ERK (mitogen-activated protein kinase) signaling is involved in CSFV-induced autophagy. The CSFV-induced inhibition of AKT/PKB (AKT serine/threonine kinase)-MTOR was observed to be partially responsible for the MTOR inactivation and subsequent autophagy initiation. Moreover, the CAMKK2/CaMKKβ (calcium/calmodulin dependent protein kinase kinase 2)-PRKAA/AMPK (protein kinase AMP-activated catalytic subunit alpha) axis was found to be involved in CSFV-induced autophagy. Meanwhile, CSFV non-structural protein NS5A induced autophagy via the CAMKK2-PRKAA-MTOR signaling pathway but not the AKT-MTOR or MAPK1/ERK2-MAPK3/ERK1-MTOR pathway. Although the AKT-MTOR pathway also plays an important role in the induction of autophagy by CSFV. We also found the interaction between HSP90AB1/HSPCB and NS5A by tandem affinity purification/liquid chromatography-mass spectrometry (LC-MS) and immunoprecipitation. Furthermore, the CSFV-induced [Ca2+]cyto increase potently induced autophagy through CAMKK2 and PRKAA. Moreover, we isolated and identified the BECN1/Beclin 1 protein complexes by tandem affinity purification/LC-MS and immunoprecipitation, the interaction between BECN1 and MAVS was confirmed by immunoprecipitation, laser scanning confocal microscope technology, and GST affinity-isolation experiments. Furthermore, CSFV-mediated autophagy suppressing type I IFN production is related to the interaction between MAVS and BECN1. Finally, the modulation of autophagy induction pathways by different autophagy regulatory factors significantly affected the replication of CSFV. Abbreviations: AKT: AKT serine/threonine kinase; AMPK: Adenosine monophosphate-activated protein kinase; CAMKK2: Calcium/calmodulin dependent protein kinase kinase 2; CSFV: Classical swine fever virus; HRP: Horseradish peroxidase; HSP90AB1: Heat shock protein 90 alpha family class B member 1; IFN: Interferon; ISGs: IFN-stimulated genes; LC-MS: Liquid chromatography-mass spectrometry; MAP1LC3/LC3: Microtubule associated protein 1 light chain 3; MAPK: Mitogen-activated protein kinase; MAVS: Mitochondrial antiviral signaling protein; MOI: Multiplicity of infection; MTOR: Mechanistic target of rapamycin kinase; PBS: Phosphate-buffered saline; PRKAA: Protein kinase AMP-activated catalytic subunit alpha; shRNA: short hairpin RNA.

M1-like macrophage-derived exosomes suppress angiogenesis and exacerbate cardiac dysfunction in a myocardial infarction microenvironment.

The roles and the underlying mechanisms of M1-type macrophages in angiogenesis and postmyocardial infarction (MI) cardiac repair have remained unclear. In this study, we investigated the role of M1-like macrophage-derived exosomes in a MI microenvironment. We found that the proinflammatory M1-like-type macrophages released an extensive array of proinflammatory exosomes (M1-Exos) after MI. M1-Exos exerted an anti-angiogenic effect and accelerated MI injury. They also exhibited highly expressed proinflammatory miRNAs, such as miR-155. miR-155 was transferred to endothelial cells (ECs), leading to the inhibition of angiogenesis and cardiac dysfunction by downregulating its novel target genes, including Rac family small GTPase 1 (RAC1), p21 (RAC1)-activated kinase 2 (PAK2), Sirtuin 1 (Sirt1), and protein kinase AMP-activated catalytic subunit alpha 2 (AMPKα2). M1-Exos depressed Sirt1/AMPKα2-endothelial nitric oxide synthase and RAC1-PAK2 signaling pathways by simultaneously targeting the five molecule nodes (genes), reduced the angiogenic ability of ECs, aggravated myocardial injury, and restrained cardiac healing. The elucidation of this mechanism provides novel insights into the functional significance of M1 macrophages and their derived exosomes on angiogenesis and cardiac repair. This mechanism can be used as a novel potential therapeutic approach for the prevention and treatment of MI.

Dehydroepiandrosterone protects against hepatic glycolipid metabolic disorder and insulin resistance induced by high fat via activation of AMPK-PGC-1α-NRF-1 and IRS1-AKT-GLUT2 signaling pathways.

Mitochondrial dysfunction, oxidative stress, or fatty liver are the key pathophysiological features for insulin resistance and obesity. Dehydroepiandrosterone (DHEA) can ameliorate obesity and insulin resistance; however, the mechanisms of these actions are poorly understood. The present study aimed to investigate the effect and possible mechanism of DHEA against glycolipid metabolic disorder and insulin resistance. Rats fed a high-fat diet (HFD) and palmitic acid (PA)-induced BRL-3A cells were employed to analyze the effect of DHEA on factors related to metabolic disorder and insulin resistance in vivo and in vitro. DHEA prevented lipid metabolism disorders by enhancing phospho (p)-protein kinase AMP-activated catalytic subunit alpha (AMPKα) (Thr172) protein level and its downstream lipid metabolism-related factors in liver of rats fed an HFD or in PA-induced BRL-3A cells. Meanwhile, DHEA ameliorated mitochondrial dysfunction through activation of the AMPK-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-nuclear respiratory factor-1 (NRF-1) pathway, which represented as the enhancing of the mtDNA copy number, ATP level, and membrane potential, and decreasing of reactive oxygen species production. Moreover, DHEA alleviated insulin resistance via increasing the phosphorylated insulin receptor substrate 1 (p-IRS1) (Tyr612) level and decreasing that of p-IRS1 (Ser307) level in liver of rats fed an HFD or in PA-induced BRL-3A cells, which subsequently enhanced p-protein kinase B (AKT) (Ser473) and membrane glucose transporter type 2 (GLUT2) expression levels. The protective effect of DHEA on high-fat-induced hepatic glycolipid metabolic disorder and insulin resistance are achieved through activation of the AMPK-PGC-1α-NRF-1 and IRS1-AKT-GLUT2 signaling pathways. The results provide compelling evidence for the mechanism by which DHEA prevents glycolipid metabolic disorder, and suggest its potential applications for controlling diabetes and obesity in animals and humans.

PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1)

PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1)

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.