AMPKα2 Knockout Research Articles

Ampk regulates IgD expression but not energy stress with B cell activation

Ampk is an energy gatekeeper that responds to decreases in ATP by inhibiting energy-consuming anabolic processes and promoting energy-generating catabolic processes. Recently, we showed that Lkb1, an understudied kinase in B lymphocytes and a major upstream kinase for Ampk, had critical and unexpected roles in activating naïve B cells and in germinal center formation. Therefore, we examined whether Lkb1 activities during B cell activation depend on Ampk and report surprising Ampk activation with in vitro B cell stimulation in the absence of energy stress, coupled to rapid biomass accumulation. Despite Ampk activation and a controlling role for Lkb1 in B cell activation, Ampk knockout did not significantly affect B cell activation, differentiation, nutrient dynamics, gene expression, or humoral immune responses. Instead, Ampk loss specifically repressed the transcriptional expression of IgD and its regulator, Zfp318. Results also reveal that early activation of Ampk by phenformin treatment impairs germinal center formation but does not significantly alter antibody responses. Combined, the data show an unexpectedly specific role for Ampk in the regulation of IgD expression during B cell activation.

613-P: Preventive Effect of Sulforaphane on Type 2 Diabetes-Induced Diabetic Cardiomyopathy via AMPK-Mediated Activation of Glucose/Lipid Metabolism and Nrf2 Function

Dyslipidemia has been shown to play a crucial role in the development of diabetic cardiomyopathy (DCM) in type 2 diabetes (T2D). AMP-activated protein kinase (AMPK), particularly α2 isoform can improve energy metabolism in cardiomyopathy. Sulforaphane (SFN), a natural isothiocyanate in vegetables, is important in cardiac protection from diabetes. We investigated whether SFN can protect heart from T2D damage, which is mediated by AMPKα2 via improving energy metabolism and activating Nrf2-mediated anti-oxidative pathways. AMPKα2 knockout (AMPKα2-KO) mice and their wild type (WT, C57BL/6J) mice were fed high-fat diet (HFD) for 3 months to induce insulin resistance, and then intraperitoneal injection of streptozotocin to induce hyperglycemia as T2D model. Age-matched mice were fed with normal diet (ND) without streptozotocin as control. T2D and control mice were treated with or without SFN for 3 months (3m) and then stop SFN treatment, remained feeding either ND or HFD for additional three months (6m). Echocardiography were performed before sacrifice both in 3m and 6m group. SFN treatment improved cardiac remodeling and dysfunction induced by T2D both in 3m and 6m group. The cardiac pathogenic changes induced by T2D, including fibrosis, lipotoxicity, inflammation, oxidative stress/damage along with suppression of AMPK signaling pathways was prevented by SFN in WT diabetic mice, as evidenced by down-regulating FN, TGF-β PAI-1, TNF-α, SCD-1and up-regulating CPT1-B, PPARα, Nrf2. The cardiac protection by SFN for 3 months from T2D could last till 6 months. AMPKα2-KO mice are also susceptible to T2D-induced cardiomyopathy; however, the protective effect of SFN on the heart from diabetes was lost in diabetic AMPKα2-KO mice. These results suggest that SFN treatment can attenuate cardiac damage induced by T2D both in the 3m and 6m group, and AMPKα2 plays the pivotal role in the SNF-mediated cardiac protection. Disclosure Y. Sun: None. L. Cai: None. Z. Li: None. Funding American Diabetes Association (1-18-IBS-082 to L.C.)

516-P: Preventive Effect of Sulforaphane on Type 2 Diabetes-Induced Diabetic Nephropathy via AMPK-Mediated Activation of Glucose/Lipid Metabolism and Nrf2 Antioxidative Function

AMP-activated protein kinase (AMPK), particularly AMPK-α2 isoform, can attenuate the nephropathic pathogenesis by improving lipid metabolism. The objective of the present study was to define if sulforaphane (SFN) can protect type 2 diabetes (T2D)-induced nephropathy (DN) by activating AMPKα2 and Nrf2-mediated anti-oxidative pathways. AMPKα2 knockout (AMPKα2-KO) mice and their wild type (WT, C57BL/6J) mice were fed high-fat diet (HFD) for 3 months to induce insulin resistance, followed by an i.p. injection of streptozotocin (STZ) to induce hyperglycemia, i.e.:T2D. Control mice were fed normal diet without STZ. T2D and control mice were treated with or without SFN for 3 months and then stop SNF treatment, remained feeding either ND or HFD. These mice were collected 24-hour urine and then sacrificed at 3 and 6 months after diabetes onset to collect kidney’s tissue for pathological examination and molecular assays. Proteinuria was significantly increased in T2D mice at 3 and 6 months, but not in SFN-treated T2D mice. The destruction of kidney structure induced by T2D, including renal fibrosis, lipotoxicity, inflammation, oxidative stress/damage along with significant suppression of AMPK signaling pathways was prevented by SFN in WT diabetic mice. Specifically, T2D-induced, time-dependent from 3 to 6 months, increased accumulation of FN and COL-1, increased expression of TGF-β PAI-1, TNF-α and decreased phosphorylation of AMPKα and its downstream markers (ACC, CPT1-A, Sirt-1, PGC1α, PPARα, and Nrf2). All these pathogenic changes were prevented by SFN in WT diabetic mice, which was more at 6 months compared to 3 months. However, in AMPKα2-KO mice, suggesting that all these are AMPK-dependent. Therefore, the present study suggests that SFN protects against T2D-induced DN via AMPKα2-mediated improvement of lipid metabolisms and up-regulation of Nrf2-mediated anti-oxidative defense system. Disclosure Z. Li: None. L. Cai: None. Y. Sun: None.

GSK621 attenuates oxygen glucose deprivation/re-oxygenation-induced myocardial cell injury via AMPK-dependent signaling

Recent studies have implied that activation of AMP-dependent protein kinase (AMPK) could protect myocardial cells from oxygen glucose deprivation-re-oxygenation (OGD/R). The aim of the present study is to test whether GSK621, a novel and direct AMPK activator, could exert myocardial cell protection against OGD/R. We show that in AC16 human myocardial cells and primary murine myocardiocytes GSK621 dose-dependently activated AMPK signaling. GSK621 pretreatment potently inhibited OGD/R-induced viability reduction, cell death and apoptosis in AC16 cells and primary myocardiocytes. Furthermore, GSK621 attenuated OGD/R-induced reactive oxygen species production and oxidative injury in the myocardial cells. AMPKα1 knockdown (via targeted shRNA), knockout (via a CRISPR/Cas9 construct) or dominant negative mutation (T172A) not only blocked GSK621-induced AMPK activation, but also nullified GSK621-mediated myocardial cell protection against OGD/R. Further studies demonstrated that GSK621 activated AMPK downstream Nrf2 signaling. Contrarily, Nrf2 silencing by targeted shRNAs almost abolished GSK621-induced anti-OGD/R myocardial cell protection. We conclude that GSK621 protects myocardial cells from OGD/R through activation of AMPK-dependent signaling.

Metformin promotes autophagy in ischemia/reperfusion myocardium via cytoplasmic AMPKα1 and nuclear AMPKα2 pathways

AimsIn myocardial ischemia-reperfusion (MI/R) injury, impaired autophagy function worsens cardiomyocyte death. AMP-activated protein kinase (AMPK) is a heterotrimeric protein that plays an important role in cardioprotection and myocardial autophagic function. AMPKα1 and α2 are localized primarily in the cytoplasm and nucleus, respectively, in cardiomyocytes, but the isoform-specific autophagy regulation of AMPK during MI/R remains unclear. Materials and methodsAn MI/R model was built, and the protein expression of AMPKα1/α2, p-AMPK, mTOR, p-mTOR, TFEB, p-FoxO3a, SKP2, CARM1, TBP, Atg5, LAMP2, LC3B, and p62 during ischemia and reperfusion was determined by western blotting. Recombinant adeno-associated virus (serotype 9) vectors carrying tandem fluorescent-tagged LC3 or mRFP-GFP-LC3/GFP-LC3 were used to evaluate the autophagy status. AMPKα2 knockout mice were used for in vivo studies. Key findingsBoth cytoplasmic AMPKα1 and nuclear α2 subunit expression decreased during the reperfusion period, which led to AMPKα1-mTOR-TFEB and AMPKα2-Skp2-CARM1-TFEB signaling inhibition, respectively. The decreased TFEB level during reperfusion suppressed autophagy. Metformin could activate both the AMPKα1- and α2- mediated pathways, thus restoring autophagy flux during reperfusion. Nevertheless, in AMPKα2 knockout mice, nuclear α2-regulated Skp2-CARM1-TFEB signaling was inhibited, while α1-related signaling was comparatively unaffected, which partially impaired metformin-enhanced autophagy. SignificanceOur study suggests that metformin had the dual effects of promoting both cytoplasmic AMPKα1- and nuclear AMPKα2-related signaling to improve autophagic flux and restore cardiac function during MI/R.

Post-translational regulation of lipogenesis via AMPK-dependent phosphorylation of insulin-induced gene

Insulin-induced gene (Insig) negatively regulates SREBP-mediated de novo fatty acid synthesis in the liver. However, the upstream regulation of Insig is incompletely understood. Here we report that AMPK interacts with and mediates phosphorylation of Insig. Thr222 phosphorylation following AMPK activation is required for protein stabilization of Insig-1, inhibition of cleavage and processing of SREBP-1, and lipogenic gene expression in response to metformin or A769662. AMPK-dependent phosphorylation ablates Insig’s interaction with E3 ubiquitin ligase gp78 and represses its ubiquitination and degradation, whereas AMPK deficiency shows opposite effects. Interestingly, activation of AMPK by metformin causes an augmentation of Insig stability and reduction of lipogenic gene expression, and leads to the attenuation of hepatic steatosis in HFHS diet-fed mice. Moreover, hepatic overexpression of Insig-1 rescues hepatic steatosis in liver-specific AMPKα2 knockout mice fed with HFHS diet. These findings uncover a novel effector of AMPK. Targeting Insig may have the therapeutic potential for treating fatty liver disease and related disorders.

AMPKα2 knockout enhances tumour inflammation through exacerbated liver injury and energy deprivation-associated AMPKα1 activation.

Tissue damage and its associated‐inflammation act as tumour initiators or propagators. AMP‐activated protein kinase (AMPK) is activated by environmental or nutritional stress factors, such as hypoxia, glucose deprivation, and other cell injury factors, to regulate cell energy balance and differentiation. We previously have reported that AMPKα2 deficiency resulted in the energy deprivation in tumour‐bearing liver and the enhanced‐hepatocyte death. In this study, AMPKα2 knockout mice and the liver metastasis model of colon cancer cells were used to address the role of AMPKα isoforms in tumour inflammation. First, we found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages. Meanwhile, although compensatory expression of AMPKα1 was not significant after AMPKα2 knockout, AMPKα1 phosphorylation was elevated in remnant liver in AMPKα2 knockout mice, which was positively associated with the enhanced energy deprivation in the AMPKα2 deficient mice. Furthermore, the activated AMPKα1 in macrophage contributed to its polarizing to tumour‐associated phenotype. Thus, the enhanced tumour‐associated inflammation and activation of AMPKα1 in the AMPKα2 deficient mice may exacerbate the tumour development by affecting the tumour inflammatory microenvironment. Our study suggests that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development.

AMP-activated protein kinase regulates the expression of human telomerase reverse transcriptase.

The expression of hTERT in tumor cells contributes to oncogenic transformation by promoting immortalization. For this reason, hTERT is one of the major targets for cancer therapy, and an efficient method to downregulate hTERT expression is required for treatment of hTERT-positive cancer. In this report, we demonstrated that inhibition of AMP-activated protein kinase (AMPK) downregulates the expression of hTERT. We screened cell signaling pathways in AMPK α1 knockout cells and found that AMPKα1 is required for activity of the hTERT promoter. AMPKα1 knockout cells showed decreased expression of hTERT mRNA and protein. We also demonstrated that compound C, a reversible AMPK inhibitor, suppressed the expression of hTERT. However, AMPK activators, including AICAR and metformin, did not increase the level of hTERT protein. Finally, we showed that tumor cells stably expressing hTERT are resistant to compound C treatment. These results indicate that AMPK activity is required for tumor progression.

Cathelicidin-WA attenuates LPS-induced inflammation and redox imbalance through activation of AMPK signaling

Dysregulated activation of inflammation is associated with the development and progression of many diseases. Generation of reactive oxygen species (ROS) has been shown to promote an inflammatory response. Cathelicidin peptides not only defend against the invasion of various microbes but also play an important role in regulating immune responses. The objective of this study was to investigate the effects and mechanisms of Cathelicidin-WA (CWA) on the inflammatory response and oxidative stress in macrophages. Our results showed that CWA efficiently attenuated lipopolysaccharide (LPS)-stimulated inflammation and oxidative stress both in vivo and in vitro. Mechanistically, we found that CWA significantly reduced the LPS-induced nuclear translocation of NF-κB, thus decreasing the production of the pro-inflammatory cytokines TNF-α and IL-6 in macrophages. On the other hand, CWA markedly promoted the nuclear translocation of Nrf2 via the AKT pathway and p38 signaling. This resulted in increased expression of the anti-oxidative genes NQO-1 and HO-1 and alleviated oxidative stress in LPS-stimulated macrophages. Interestingly, the effects of CWA were diminished when AMPK was knocked down. Consistently, we noticed that CWA failed to ameliorate the LPS-induced inflammatory response and oxidative stress in AMPK knockout mice. Furthermore, we discovered that LKB1 was essential for AMPK activation by CWA. These data demonstrated for the first time that CWA attenuated LPS-stimulated inflammation and redox imbalance through regulating LKB1-AMPK signaling. Such knowledge provides new insights into the mechanisms through which Cathelicidin peptides modulate immune responses.

Arhalofenate acid inhibits monosodium urate crystal-induced inflammatory responses through activation of AMP-activated protein kinase (AMPK) signaling

BackgroundArhalofenate acid, the active acid form of arhalofenate, is a non-agonist peroxisome proliferator-activated receptor γ (PPARγ) ligand, with uricosuric activity via URAT1 inhibition. Phase II studies revealed decreased acute arthritis flares in arhalofenate-treated gout compared with allopurinol alone. Hence, we investigated the anti-inflammatory effects and mechanisms of arhalofenate and its active acid form for responses to monosodium urate (MSU) crystals.MethodsWe assessed in-vivo responses to MSU crystals in murine subcutaneous air pouches and in-vitro responses in murine bone marrow-derived macrophages (BMDMs) by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE/Western blot, immunostaining, and transmission electron microscopy analyses.ResultsOral administration of arhalofenate (250 mg/kg) blunted total leukocyte ingress, neutrophil influx, and air pouch fluid interleukin (IL)-1β, IL-6, and CXCL1 in response to MSU crystal injection (p < 0.05 for each). Arhalofenate acid (100 μM) attenuated MSU crystal-induced IL-1β production in BMDMs via inhibition of NLRP3 inflammasome activation. In addition, arhalofenate acid dose-dependently increased activation (as assessed by phosphorylation) of AMP-activated protein kinase (AMPK). Studying AMPKα1 knockout mice, we elucidated that AMPK mediated the anti-inflammatory effects of arhalofenate acid. Moreover, arhalofenate acid attenuated the capacity of MSU crystals to suppress AMPK activity, regulated expression of multiple downstream AMPK targets that modulate mitochondrial function and oxidative stress, preserved intact mitochondrial cristae and volume density, and promoted anti-inflammatory autophagy flux in BMDMs.ConclusionsArhalofenate acid is anti-inflammatory and acts via AMPK activation and its downstream signaling in macrophages. These effects likely contribute to a reduction of gout flares.

MiR-1273g silences MAGEA3/6 to inhibit human colorectal cancer cell growth via activation of AMPK signaling

AMP-activated protein kinase (AMPK) is a metabolic regulator that acts to limit the growth of cancer cells. AMPK is downregulated by melanoma antigens A3/6 (MAGEA3/6), which are cancer-specific proteins that enhance the activity of specific E3 ubiquitin ligases to ubiquitinate and degrade AMP-activated protein kinase α1 (AMPKα1). Here, using a bioinformatic approach, we identified a microRNA, miR-1273g-3p, that is predicted to target the 3′ untranslated region (UTR) of MAGEA3/6. Analyzing miR-1273g-3p expression in human colon cancer tissues, we found a reduction in miR-1273g-3p expression correlating with increased MAGEA3/6 expression and AMPKα1 downregulation. Expression of miR-1273g in HT-29 cells and primary human colon cancer cells down-regulated MAGEA3/6, leading to AMPKα1 upregulation, inhibition of proliferation and cell apoptosis. The anti-CRC activity of miR-1273g was blocked by AMPKα1 knockout. MAGEA3/6 shRNA silencing mimicked and abolished miR-1273g-induced actions in HT-29 cells. In vivo, miR-1273g- or MAGEA3/6 shRNA-expressing HT-29 tumors grew significantly slower than control tumors. We propose a novel miRNA-based mechanism, whereby miR-1273g represses MAGEA3/6 expression in human CRC cells and tissues, which may provide a novel cancer-specific therapeutic.

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 in muscle is essential in ketone body utilization

BackgroundStarvation leads to a series of metabolic adaptations that enables continuous production and delivery of nutrients to critical organs, tissues and cells. Under conditions of nutrient withdrawal, ketone bodies provide an alternative source of energy for highly oxidative tissues. Although the AMP‐activated protein kinase (AMPK) is best known as a sensor of cellular energy status, its implication in ketone regulation remains unclear.ObjectiveThe aim of this study was to evaluate the effects of AMPKα2 on ketone body utilization, and to elucidate the underlying mechanisms.MethodsGlobal AMPKα2 knockout mice (AMPKα2−/−), muscle‐specific AMPKα2 knockout mice, and their age‐matched wild type (WT) littermates were subjected to either 3‐day fasting or received an intra‐peritoneal injection of β‐hydroxybutyrate salt (1.5 mg/kg body weight) to assess the β‐hydroxybutyrate consumption.ResultsFasting blood and urine ketone levels were significantly elevated in AMPKα2−/− mice when compared to their littermate WT mice. After an intra‐peritoneal bolus of β‐hydroxybutyrate, the main circulating ketone body, AMPKα2−/− mice presented a lower clearance of β‐hydroxybutyrate compared to their littermate WT mice. Further, fasting induced hyperketosis without any change in body weight or glycaemia in muscle‐specific AMPKα2 knockout mice. Consistently, these mice displayed impaired ketone utilization after β‐hydroxybutyrate injection. Furthermore, AMPKα2 deficiency in muscle significantly decreased locomotor activity, with a quicker switch from utilizing carbohydrate to fat as the predominant energy source compared to their littermate WT mice. Mechanistically, AMPKα2 deficiency in muscle presented a 50% reduced activity of succinyl‐CoA:3‐oxoacid CoA transferase compared to their littermate WT mice.ConclusionTaken together, these findings establish AMPKα2 in muscle as an essential mediator of ketolysis in fasting, through the regulation of the rate‐limiting ketolytic enzyme.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intestinal Epithelial AMPK Does Not Protect Against High Dose DSS‐Induced Colitis in Mice

AMP‐Activated Protein Kinase (AMPK) is a master regulator of cellular energy status whose activity requires α1 and α2 isoforms of the catalytic subunit. Recent evidence suggested AMPK protects against DSS‐induced colitis using villin‐Cre AMPKα1 knockout mice and nonspecific pharmacological activators. However, previous studies have not tested if both AMPKα isoforms are required for protection as α1/α2 double‐knockout is embryonic lethal in mice. Moreover, we previously showed AMPKα1 protein is increased in AMPKα2−/− mice, potentially confounding conclusions. Furthermore, the role of intestinal epithelial AMPK in inflammatory responses remains poorly understood. The goals of this study were to determine if AMPK is necessary for intestinal homeostasis and to confirm if intestinal epithelial AMPK is required for protection against colitis in a mouse model.MethodsIntestinal epithelial‐specific deletion of both AMPK catalytic isoforms (AMPKαΔIEC KO) was generated by crossing villin‐Cre recombinase mice with Prkaa1 and Prkaa2 floxed mice (AMPKαfl/fl). Barrier integrity and function were assessed histologically, by 40 kDa FITC‐dextran (FD4) intestinal permeability assay and by agonist stimulation of chloride secretion ex vivo in Ussing chambers. Colitis was induced with 5% dextran sulfate sodium (DSS) in drinking water for 5 days, followed by 3 days of water. Body weight, stool consistency and hemoccult were recorded daily to determine disease activity index scores. Intestinal permeability during colitis was determined by FD4 permeability. Mice were euthanized according to institutional IACUC protocols. Tissues were fixed in 4% paraformaldehyde for imaging or flash frozen for protein and RNA analysis.ResultsUnchallenged AMPKαΔIEC KO mice showed no histologic changes in intestinal integrity, nor any significant change in intestinal permeability or agonist‐stimulated chloride secretion. Mice administered 5% DSS developed severe colitis indicated by dramatic weight loss and increased disease activity index score compared to untreated controls. Colitis induced similar levels of colonic shortening and increased intestinal permeability in both floxed and AMPKαΔIEC KO mice, however AMPK‐regulated pro‐inflammatory gene expression was unchanged compared to untreated controls. Despite the lack of an overt phenotypic difference in AMPKαΔIEC KO mice compared to AMPKαfl/fl mice, we did observe significant differences in protein expression. The tight junction protein claudin 2 was significantly decreased in proximal colon of AMPKαΔIEC KO mice compared to AMPKαfl/fl mice without DSS challenge. Untreated and DSS‐treated AMPKαΔIEC KO mice had decreased levels of the apoptosis markers cleaved caspase 3 in ileum, and PARP in proximal colon respectively, while cleaved caspase 3 was increased in distal colon compared to matched AMPKαfl/fl mice.ConclusionLoss of AMPK alone altered levels of claudin 2 and apoptotic markers in intestinal epithelial cells but did not alter DSS‐induced colitis severity.Support or Funding InformationNIH 2R01DK091281 (DFM)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells.

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health.

High Fat Diet Attenuates the Anticontractile Activity of Aortic PVAT via a Mechanism Involving AMPK and Reduced Adiponectin Secretion.

Background and aim: Perivascular adipose tissue (PVAT) positively regulates vascular function through production of factors such as adiponectin but this effect is attenuated in obesity. The enzyme AMP-activated protein kinase (AMPK) is present in PVAT and is implicated in mediating the vascular effects of adiponectin. In this study, we investigated the effect of an obesogenic high fat diet (HFD) on aortic PVAT and whether any changes involved AMPK.Methods: Wild type Sv129 (WT) and AMPKα1 knockout (KO) mice aged 8 weeks were fed normal diet (ND) or HFD (42% kcal fat) for 12 weeks. Adiponectin production by PVAT was assessed by ELISA and AMPK expression studied using immunoblotting. Macrophages in PVAT were identified using immunohistochemistry and markers of M1 and M2 macrophage subtypes evaluated using real time-qPCR. Vascular responses were measured in endothelium-denuded aortic rings with or without attached PVAT. Carotid wire injury was performed and PVAT inflammation studied 7 days later.Key results: Aortic PVAT from KO and WT mice was morphologically indistinct but KO PVAT had more infiltrating macrophages. HFD caused an increased infiltration of macrophages in WT mice with increased expression of the M1 macrophage markers Nos2 and Il1b and the M2 marker Chil3. In WT mice, HFD reduced the anticontractile effect of PVAT as well as reducing adiponectin secretion and AMPK phosphorylation. PVAT from KO mice on ND had significantly reduced adiponectin secretion and no anticontractile effect and feeding HFD did not alter this. Wire injury induced macrophage infiltration of PVAT but did not cause further infiltration in KO mice.Conclusions: High-fat diet causes an inflammatory infiltrate, reduced AMPK phosphorylation and attenuates the anticontractile effect of murine aortic PVAT. Mice lacking AMPKα1 phenocopy many of the changes in wild-type aortic PVAT after HFD, suggesting that AMPK may protect the vessel against deleterious changes in response to HFD.

AntogomiR-451 protects human gastric epithelial cells from ethanol via activating AMPK signaling

The prevention and treatment efficiency of ethanol-induced gastric epithelial injury are not satisfied. We have previously shown that AMP-activated protein kinase (AMPK) activation exerts a pro-survival function in human gastric epithelial cells (GECs). miroRNA-451 (“miR-451”)’s inhibitor, antagomiR-451, can activate AMPK signaling. In the present study, we show that forced-expression of antagomiR-451 via a lentiviral vector depleted miR-451, leading to AMPK activation in established GES-1 cells and primary human GECs. AntagomiR-451 efficiently protected GES-1 cells and primary human GECs from ethanol-induced viability reduction and apoptosis. AMPK activation is required for antagomiR-451-induced GEC protection. AMPKα1 knockdown (by targeted-shRNAs) or knockout (by CRISPR-Cas-9 KO plasmid) blocked antagomiR-451-induced AMPK activation, and GEC protection against ethanol. Further experimental results show that antagomiR-451 significantly attenuated ethanol-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage. Collectively, antagomiR-451 protects human GECs from ethanol via activating AMPK signaling.

Rosmarinic acid attenuates cardiac fibrosis following long-term pressure overload via AMPK\u03b1/Smad3 signaling

Agonists of peroxisome proliferator-activated receptor gamma (PPAR-γ) can activate 5′ AMP-activated protein kinase alpha (AMPKα) and exert cardioprotective effects. A previous study has demonstrated that rosmarinic acid (RA) can activate PPAR-γ, but its effect on cardiac remodeling remains largely unknown. Our study aimed to investigate the effect of RA on cardiac remodeling and to clarify the underlying mechanism. Mice were subjected to aortic banding to generate pressure overload induced cardiac remodeling and then were orally administered RA (100 mg/kg/day) for 7 weeks beginning 1 week after surgery. The morphological examination, echocardiography, and molecular markers were used to evaluate the effects of RA. To ascertain whether the beneficial effect of RA on cardiac fibrosis was mediated by AMPKα, AMPKα2 knockout mice were used. Neonatal rat cardiomyocytes and fibroblasts were separated and cultured to validate the protective effect of RA in vitro. RA-treated mice exhibited a similar hypertrophic response as mice without RA treatment, but had an attenuated fibrotic response and improved cardiac function after pressure overload. Activated AMPKα was essential for the anti-fibrotic effect of RA via inhibiting the phosphorylation and nuclear translocation of Smad3 in vivo and in vitro, and AMPKα deficiency abolished RA-mediated protective effects. Small interfering RNA against Ppar-γ (siPpar-γ) and GW9662, a specific antagonist of PPAR-γ, abolished RA-mediated AMPKα phosphorylation and alleviation of fibrotic response in vitro. RA attenuated cardiac fibrosis following long-term pressure overload via AMPKα/Smad3 signaling and PPAR-γ was required for the activation of AMPKα. RA might be a promising therapeutic agent against cardiac fibrosis.

AMP-activated protein kinase agonist N6-(3-hydroxyphenyl)adenosine protects against fulminant hepatitis by suppressing inflammation and apoptosis

Both AMP-activated protein kinase (AMPK) agonist and inhibitor have been reported to protect against fulminant hepatitis, implying that AMPK may play a complicated role in the development of fulminant hepatitis. In this study, we exploited whether the novel AMPK agonist N6-(3-hydroxyphenyl)adenosine (named as M1) exerted protective effects on fulminant hepatitis and whether its beneficial effects were AMPK-dependent. Results showed that intraperitoneal injection of M1 improved liver function, ameliorated liver injury and finally raised the survival rate in d-galactosamine/lipopolysaccharide (GalN/LPS)-treated mice. These beneficial effects of M1 may attribute to the suppression of pro-inflammatory cytokines production and the prevention of hepatocyte apoptosis. Furthermore, M1 pretreatment mitigated LPS-stimulated TLR4 expression and NFκB activation in murine peritoneal macrophages and prevented actinomycin D (Act D)/tumor necrosis factor α (TNFα)-induced apoptosis by promoting protective autophagy in primary hepatocytes. Additionally, M1-induced AMPK activation was responsible both for its anti-inflammatory action in macrophages and for its anti-apoptotic action in hepatocytes. To our surprise, compared with the control AMPKα1lox/lox/AMPKα2lox/lox mice, liver-specific AMPKα1 knockout (AMPKα1LS−/−) mice were more sensitive to GalN/LPS administration but not AMPKα2LS−/−mice, and the beneficial effects of M1 on acute liver failure and the production of pro-inflammatory factors were dampened in AMPKα1LS−/− mice. Therefore, our study may prove that M1 could be a promising therapeutic agent for fulminant hepatitis, and targeting AMPK may be useful therapeutically in the control of LPS-induced hepatotoxicity.

Ezetimibe ameliorates steatohepatitis via AMP activated protein kinase-TFEB-mediated activation of autophagy and NLRP3 inflammasome inhibition

ABSTRACTImpairment in macroautophagy/autophagy flux and inflammasome activation are common characteristics of nonalcoholic steatohepatitis (NASH). Considering the lack of approved agents for treating NASH, drugs that can enhance autophagy and modulate inflammasome pathways may be beneficial. Here, we investigated the novel mechanism of ezetimibe, a widely prescribed drug for hypercholesterolemia, as a therapeutic option for ameliorating NASH. Human liver samples with steatosis and NASH were analyzed. For in vitro studies of autophagy and inflammasomes, primary mouse hepatocytes, human hepatoma cells, mouse embryonic fibroblasts with Ampk or Tsc2 knockout, and human or primary mouse macrophages were treated with ezetimibe and palmitate. Steatohepatitis and fibrosis were induced by feeding Atg7 wild-type, haploinsufficient, and knockout mice a methionine- and choline-deficient diet with ezetimibe (10 mg/kg) for 4 wk. Human livers with steatosis or NASH presented impaired autophagy with decreased nuclear TFEB and increased SQSTM1, MAP1LC3-II, and NLRP3 expression. Ezetimibe increased autophagy flux and concomitantly ameliorated lipid accumulation and apoptosis in palmitate-exposed hepatocytes. Ezetimibe induced AMPK phosphorylation and subsequent TFEB nuclear translocation, related to MAPK/ERK. In macrophages, ezetimibe blocked the NLRP3 inflammasome-IL1B pathway in an autophagy-dependent manner and modulated hepatocyte-macrophage interaction via extracellular vesicles. Ezetimibe attenuated lipid accumulation, inflammation, and fibrosis in liver-specific Atg7 wild-type and haploinsufficient mice, but not in knockout mice. Ezetimibe ameliorates steatohepatitis by autophagy induction through AMPK activation and TFEB nuclear translocation, related to an independent MTOR ameliorative effect and the MAPK/ERK pathway. Ezetimibe dampens NLRP3 inflammasome activation in macrophages by modulating autophagy and a hepatocyte-driven exosome pathway.