AMP-activated Protein Kinase Knockout Research Articles

AMPK Deficiency Increases DNA Methylation and Aggravates Colorectal Tumorigenesis in AOM/DSS Mice

The incidence of colorectal cancer (CRC) is closely linked to metabolic diseases. Accumulating evidence suggests the regulatory role of AMP-activated protein kinase (AMPK) in cancer metabolic reprogramming. In this study, wild-type and AMPK knockout mice were subjected to azoxymethane-induced and dextran sulfate sodium (AOM/DSS)-promoted colitis-associated CRC induction. A stable AMPK-deficient Caco-2 cell line was also established for the mechanistic studies. The data showed that AMPK deficiency accelerated CRC development, characterized by increased tumor number, tumor size, and hyperplasia in AOM/DSS-treated mice. The aggravated colorectal tumorigenesis resulting from AMPK ablation was associated with reduced α-ketoglutarate production and ten-eleven translocation hydroxylase 2 (TET2) transcription, correlated with the reduced mismatch repair protein mutL homolog 1 (MLH1) protein. Furthermore, in AMPK-deficient Caco-2 cells, the mRNA expression of mismatch repair and tumor suppressor genes, intracellular α-ketoglutarate, and the protein level of TET2 were also downregulated. AMPK deficiency also increased hypermethylation in the CpG islands of Mlh1 in both colonic tissues and Caco-2 cells. In conclusion, AMPK deficiency leads to reduced α-ketoglutarate concentration and elevates the suppressive epigenetic modifications of tumor suppressor genes in gut epithelial cells, thereby increasing the risk of colorectal tumorigenesis. Given the modifiable nature of AMPK activity, it holds promise as a prospective molecular target for the prevention and treatment of CRC.

Frequent loss-of-function mutations in the AMPK-α2 catalytic subunit suggest a tumour suppressor role in human skin cancers.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status activated by increases in AMP or ADP relative to ATP. Once activated, it phosphorylates targets that promote ATP-generating catabolic pathways or inhibit ATP-consuming anabolic pathways, helping to restore cellular energy balance. Analysis of human cancer genome studies reveals that the PRKAA2 gene (encoding the α2 isoform of the catalytic subunit) is often subject to mis-sense mutations in cancer, particularly in melanoma and non-melanoma skin cancers, where up to 70 mis-sense mutations have been documented, often accompanied by loss of the tumour suppressor NF1. Recently it has been reported that knockout of PRKAA2 in NF1-deficient melanoma cells promoted anchorage-independent growth in vitro, as well as growth as xenografts in immunodeficient mice in vivo, suggesting that AMPK-α2 can act as a tumour suppressor in that context. However, very few of the mis-sense mutations in PRKAA2 that occur in human skin cancer and melanoma have been tested to see whether they cause loss-of-function. We have addressed this by making most of the reported mutations and testing their activity when expressed in AMPK knockout cells. Of 55 different mis-sense mutations (representing 75 cases), 9 (12%) appeared to cause a total loss of activity, 18 (24%) a partial loss, 11 (15%) an increase in phenformin-stimulated kinase activity, while just 37 (49%) had no clear effect on kinase activity. This supports the idea that AMPK-α2 acts as a tumour suppressor in the context of human skin cancer.

AMPK Amplifies IL2-STAT5 Signaling to Maintain Stability of Regulatory T Cells in Aged Mice.

AMP-activated protein kinase (AMPK), an important regulator of the aging process, is expressed in various immune cells. However, its role in regulatory T cell (Treg) stability during aging is poorly understood. Here, we addressed the role of AMPK in Treg function and stability during aging by generating Treg-specific AMPKα1 knockout mice. In this study, we found that AMPKα1-deficient Tregs failed to control inflammation as effectively as normal Tregs did during aging. AMPK knockout from Tregs reduces STAT5 phosphorylation in response to interleukin (IL)-2 stimulation, thereby destabilizing Tregs by decreasing CD25 expression. Thus, our study addressed the role of AMPK in Tregs in sensing IL-2 signaling to amplify STAT5 phosphorylation, which, in turn, supports Treg stability by maintaining CD25 expression and controlling inflamm-aging.

Cardiac AMPK is not required for cardioprotection by metformin in pulmonary hypertension induced right heart failure

Right ventricle (RV) dysfunction dictates survival in pulmonary hypertension (PH) and other clinical conditions. Metformin, a clinically relevant AMP activated protein kinase (AMPK) activator, has been proposed as a therapy for PH induced right heart failure (PH‐RHF) due to its well‐reported cardioprotective properties. Indeed, our group has recently shown that metformin is protective in PH‐RHF, but the mechanisms of protection in the RV remain unknown. The objective of this study was to use determine whether the protective properties of metformin require cardiac AMPK. We used Cre‐Lox recombination to create a cardiomyocyte specific AMPK deleted with temporal regulation of expression. AMPK knockout (KO) and wild type (WT) control mice were placed in a hypobaric hypoxia (HH) chamber (~17,000ft) for 4 weeks to induce PH‐RHF, with or without metformin (200mg/kg/day) in drinking water. Cardiomyocyte specific AMPK deletion exacerbated RV and LV remodeling in response to HH compared to WT, with significant sex differences evidenced by males undergoing worse remodeling than females. AMPK deletion exacerbated cardiac output and RV stroke volume compared to WT mice. Metformin rescued cardiac remodeling in male KO mice but did not attenuate remodeling in female mice. We suggest that metformin is a potential therapeutic target to attenuate maladaptive cardiac remodeling induced by PH‐RHF, but only in male mice. Metformin does not require cardiac AMPK for cardioprotection. The mechanisms by which metformin affords protection in PH‐RHF are still unclear, but may include AMPK activity in non‐myocyte cell populations. Ongoing work will elucidate the role of AMPK activation in fibroblasts as well as other systemic and cardiac mechanisms which may explain how metformin protects against PH‐RHF.

Maternal exercise intergenerationally drives muscle-based thermogenesis via activation of apelin-AMPK signaling.

SummaryBackgroundSarcolipin and uncoupling protein 3 (UCP3) mediate muscle-based non-shivering thermogenesis (NST) to improve metabolic homeostasis. The impacts of maternal obesity (MO) and maternal exercise (ME) on NST in offspring muscle remain unexamined.MethodsFemale mice were fed with a control diet or high fat diet to induce obesity. Then, obese mice were further separated into two groups: obesity only (OB) and OB plus daily exercise (OB/Ex). Fetal muscle was collected at embryonic day 18.5 and offspring mice at 3-month-old. Apelin administration during pregnancy and apelin receptor (APJ) knockout mouse were further used for investigating the mediatory role of APJ on muscle-based thermogenesis. To explore the direct effects of exercise on AMP-activated protein kinase (AMPK) downstream targets, AMPK knockout mouse was used.FindingsMO inhibited while ME activated AMPK and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in fetal muscle. AMPK activation increased sarcolipin expression, which inhibited the uptake of calcium ions into sarcoplasmic reticulum, thereby activating CaMKK2. Consistently, the expression of UCP3 and sarcolipin was suppressed due to MO but activated in ME fetal muscle. Importantly, changes of UCP3 and sarcolipin maintained in offspring muscle, showing the transgenerational effects. Furthermore, apelin administration during pregnancy mimicked the effects of ME on AMPK and CaMKK2 activation, and UCP3 and sarcolipin expression, underscoring the mediatory roles of apelin-AMPK signaling in improving fetal muscle development.InterpretationME, via activation of apelin signaling-AMPK axis, enhances NST gene expression in fetal and offspring muscle impaired due to MO, which intergenerationally protects offspring from diet-induced obesity and metabolic disorders.FundingThis work was supported by National Institutes of Health Grant R01-HD067449.

Dexmedetomidine Protects Human Cardiomyocytes Against Ischemia-Reperfusion Injury Through α2-Adrenergic Receptor/AMPK-Dependent Autophagy.

Background: Ischemia-reperfusion injury (I/R) strongly affects the prognosis of children with complicated congenital heart diseases (CHDs) who undergo long-term cardiac surgical processes. Recently, the α2-adrenergic receptor agonist Dexmedetomidine (Dex) has been reported to protect cardiomyocytes (CMs) from I/R in cellular models and adult rodent models. However, whether and how Dex may protect human CMs in young children remains largely unknown. Methods and Results: Human ventricular tissue from tetralogy of Fallot (TOF) patients and CMs derived from human-induced pluripotent stem cells (iPSC-CMs) were used to assess whether and how Dex protects human CMs from I/R. The results showed that when pretreated with Dex, the apoptosis marker-TUNEL and cleaved caspase 3 in the ventricular tissue were significantly reduced. In addition, the autophagy marker LC3II was significantly increased compared with that of the control group. When exposed to the hypoxia/reoxygenation process, iPSC-CMs pretreated with Dex also showed reduced TUNEL and cleaved caspase 3 and increased LC3II. When the autophagy inhibitor (3-methyladenine, 3-MA) was applied to the iPSC-CMs, the protective effect of Dex on the CMs was largely blocked. In addition, when the fusion of autophagosomes with lysosomes was blocked by Bafilomycin A1, the degradation of p62 induced by Dex during the autophagy process was suspended. Moreover, when pretreated with Dex, both the human ventricle and the iPSC-CMs expressed more AMP-activated protein kinase (AMPK) and phospho AMPK (pAMPK) during the I/R process. After AMPK knockout or the use of an α2-adrenergic receptor antagonist-yohimbine, the protection of Dex and its enhancement of autophagy were inhibited. Conclusion: Dex protects young human CMs from I/R injury, and α2-adrenergic receptor/AMPK-dependent autophagy plays an important role during this process. Dex may have a therapeutic effect for children with CHD who undergo long-term cardiac surgical processes.

The AMP-Activated Protein Kinase Plays a Role in Antioxidant Defense and Regulation of Vascular Inflammation.

Cardiovascular diseases represent the leading cause of global deaths and life years spent with a severe disability. Endothelial dysfunction and vascular oxidative stress are early precursors of atherosclerotic processes in the vascular wall, all of which are hallmarks in the development of cardiovascular diseases and predictors of future cardiovascular events. There is growing evidence that inflammatory processes represent a major trigger for endothelial dysfunction, vascular oxidative stress and atherosclerosis and clinical data identified inflammation as a cardiovascular risk factor on its own. AMP-activated protein kinase (AMPK) is a central enzyme of cellular energy balance and metabolism that has been shown to confer cardio-protection and antioxidant defense which thereby contributes to vascular health. Interestingly, AMPK is also redox-regulated itself. We have previously shown that AMPK largely contributes to a healthy endothelium, confers potent antioxidant effects and prevents arterial hypertension. Recently, we provided deep mechanistic insights into the role of AMPK in cardiovascular protection and redox homeostasis by studies on arterial hypertension in endothelial and myelomonocytic cell-specific AMPK knockout (Cadh5CrexAMPKfl/fl and LysMCrexAMPKfl/fl) mice. Using these cell-specific knockout mice, we revealed the potent anti-inflammatory properties of AMPK representing the molecular basis of the antihypertensive effects of AMPK. Here, we discuss our own findings in the context of literature data with respect to the anti-inflammatory and antioxidant effects of AMPK in the specific setting of arterial hypertension as well as cardiovascular diseases in general.

1831-P: Activating AMPK Mediates FGF1 Protection from NAFLD in Diabetic Mice

Fibroblast growth factor 1 (FGF1) demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic (T2D) and obese mice by uncertain mechanism. This study investigated the therapeutic activity and mechanism of a non-mitagenic FGF1 variant (FGF1ΔHBS) against NAFLD. FGF1ΔHBS administration was effective in 9-month old db/db mice with established NAFLD; liver weight, lipid deposition and inflammation declined and liver function improved. FGF1ΔHBS reduced oxidative stress by nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1ΔHBS also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of AMP-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1ΔHBS. In palmitate treated hepatic cells, siRNA knockdown of Nrf2 abolished only FGF1ΔHBS anti-oxidative actions but not lipid metabolism improvement. In contrast, AMPK inhibition by pharmacological agents or siRNA abolished both FGF1ΔHBS benefits on oxidative stress and lipid metabolism that was FGF receptor 4 (FGFR4) dependent. Further support of these in vitro findings is that liver specific AMPK knockout abolished therapeutic effects of FGF1ΔHBS against high-fat-high-sugar diet-induced hepatic steatosis. These findings indicate that FGF1ΔHBS is effective in late stage NAFLD and acts by activation of AMPK via hepatocyte FGFR4. Disclosure Q. Lin: None. G. Cai: None. Z. Liu: None. J. Li: None. J. Li: None. K.A. Wintergerst: None. P.N. Epstein: None. L. Cai: None. Y. Li: None. Y. Tan: None. Funding American Diabetes Association (1-13-JF-53 to Y.T.), (1-18-IBS-082 to L.C.)

AMPK Deletion in the Intestinal Epithelium Reveals Region‐Specific Roles in Health and Disease

AMP‐Activated Protein Kinase (AMPK) is a conserved energy sensor that has been suggested to modulate intestinal barrier function. Intestinal barrier function contributes to partitioning of the gut lumen and lamina propria and is primarily mediated by intestinal epithelial cell (IEC) control of permeability, ion transport, and regulation of cell turnover. Dysfunction of the intestinal barrier is a feature of pathologies such as Inflammatory Bowel Disease (IBD) and diarrhea. The aim of this study was to identify the impact of AMPK activity on intestinal barrier function by utilizing a tissue‐specific knockout mouse model lacking both AMPK‐α catalytic isoforms.MethodsIntestinal epithelial‐specific AMPK knockout mice (AMPKαΔIEC KO) were created by crossing Prkaa1 and Prkaa2 floxed mice (AMPKαfl/fl) with Villin‐Cre mice. Large intestine (cecum, proximal, and distal colon) was stripped of the seromuscular layer and mounted in Ussing chambers containing Kreb’s Ringer’s buffer. Transepithelial electrical resistance (TER), 4 kDa FITC‐dextran (FD4) permeability and ion transport responses to Forskolin (20 μM, bilateral) and Carbachol (300 μM, serosal) were measured. Protein expression was analyzed in isolated IECs or whole tissue by immunoblotting or by immunostaining of whole intestinal tissue. Colitis was induced by 5% Dextran Sodium Sulfate (DSS) in drinking water (ad libitum for 5 days) followed by 3 days of water.ResultsAMPKαΔIEC KO mice had decreased TER only in the proximal colon vs. AMPKαfl/fl mice (p = 0.057, n = 6–7), with no change in FD4 permeability. Decreased TER in the proximal colon was associated with reduced Claudin‐4 expression. Claudin‐4 in the distal colon was significantly decreased in AMPKαΔIEC KO mice (48% decrease, p = 0.0136, n = 5). Ion transport responses to Carbachol were increased in the cecum of AMPKαΔIEC KO mice vs. AMPKαfl/fl mice (p = 0.012, n = 3), while Forskolin responses were unchanged. Expression of the AMPK‐regulated ion transport protein, Na+‐K+‐Cl− Cotransporter 1 (NKCC1) was increased in the cecum and distal colon of AMPKαΔIEC KOvs. AMPKαfl/fl mice, thus indicating that AMPK repressed NKCC1 expression in these regions (p<0.05 ‐ p<0.001). Inflammatory challenge (DSS colitis) caused increased cell death (TUNEL staining) in cecum and distal colon but TUNEL staining was reduced in the cecum of AMPKαΔIEC KO mice vs. AMPKαfl/fl mice (6.2‐fold, p = 0.0241, n = 2–3). Consistent with unaltered TUNEL staining in the proximal colon, the apoptosis markers cleaved caspase‐3 and PARP‐1 were also reduced in both mouse genotypes. However, despite increased TUNEL staining, these cleavage events were not increased in the cecum or distal colon of DSS‐treated mice suggesting cell death occurred by a mechanism independent of caspase‐3 and PARP cleavage.ConclusionsEpithelial AMPK regulation of intestinal barrier permeability, ion transport, and inflammation‐induced cell death displays regional heterogeneity along the large intestine.Support or Funding InformationNIH 2R01DK091281 (DFM)

Adipose tissue-specific knockout of AMPKα1/α2 results in normal AICAR tolerance and glucose metabolism

AMP-activated protein kinase (AMPK) is a member of Ser/Thr kinases that has been shown to regulate energy balance. Although recent studies have demonstrated the function of AMPK in adipose tissue using different fat-specific AMPK knockout mouse models, the results were somewhat inconsistent. For this study, we tested the hypothesis that AMPK in adipose tissue regulates whole body glucose metabolism. To determine the role of adipose tissue AMPK in vivo, we generated fat-specific AMPKα1/α2 knockout mice (AMPKFKO) using the Cre-loxP system. Body weights of AMPKFKO mice were not different between 8 and 27 weeks of age. Furthermore, tissue weights (liver, kidney, muscle, heart and white and brown adipose tissue) were similar to wild type littermates and DEXA scan analysis revealed no differences in percentages of body fat and lean mass. Knockout of AMPKα1/α2 in adipose tissue abolished basal and AICAR-stimulated phosphorylation of AMPK and Acetyl-CoA Carboxylase, a downstream of AMPK. Despite of the ablation of AICAR-stimulated AMPK phosphorylation, the blood glucose-lowering effect of AICAR injection (i.p.) was normal in AMPKFKO mice. In addition, AMPKFKO displayed normal fasting blood glucose concentration, glucose tolerance, insulin tolerance, and insulin signaling, indicating normal whole body glucose metabolism. These data demonstrate that adipose tissue AMPK plays a minimum role in whole body glucose metabolism on a chow diet.

Sulforaphane ameliorates high-fat diet-induced spermatogenic deficiency in mice†.

Sulforaphane (SFN), a dietary isothiocyanate that is mainly found in cruciferous vegetables, possesses anti-oxidative and anticancer activity and modulates inflammation. However, little is known about the role of SFN in obesity-related male reproductive defects. The present study aimed to investigate the effects of SFN on high-fat diet (HFD)-induced male spermatogenic impairment and further clarify the possible underlying mechanisms. In this study, 8-week-old mice were randomly divided into four groups. Mice were fed a normal diet or an HFD with or without SFN supplementation. Sulforaphane was subcutaneously injected at a dose of 0.5 mg/kg 5 days/week for 4 weeks beginning 8 weeks after initiation of the HFD. The results demonstrated that SFN could protect against HFD-induced reproductive dysfunction in male mice. Moreover, SFN also improved reproductive ability, as demonstrated by an increased pregnancy rate and decreased embryo resorption rate in comparison to the corresponding HFD group. We also observed a decrease in apoptosis and an attenuation of endoplasmic reticulum (ER) stress after SFN treatment. In vitro studies of mouse and human sperm samples also revealed that SFN protects against the palmitic acid-induced reduction in sperm viability and motility by inhibiting ER stress in an AMP-activated protein kinase (AMPK)-dependent manner. AMPK-dependent ER stress attenuation by SFN was further confirmed using AMPK knockout mice. Taken together, these data show that SFN protects against HFD-induced male reproductive dysfunction by inhibiting ER stress and apoptosis. These findings may be helpful for identifying new therapeutic methods to treat male infertility.

Loss of Intestinal Epithelial AMPK Does Not Alter Susceptibility to Acute Chemical Colitis or Bacterial Infection

AMP‐Activated Protein Kinase (AMPK) is a master regulator of cellular energy status. Its primary actions are mediated by its alpha catalytic subunit, which has two isoforms. Previous evidence in mice has suggested that loss of an individual AMPK‐α isoform compromises critical functions of the intestinal epithelium such as tight junction permeability while intact AMPK activity is protective against mucosal inflammation. These processes are essential to maintain the intestinal barrier and prevent chronic inflammation. However, AMPK‐dependent effects under cellular stress conditions have not been consistent and appear context‐dependent. Additionally, published studies have not confirmed whether AMPK is necessary and sufficient for these effects. The goals of this study were to determine if AMPK is required to protect from mouse models of acute colitis.MethodsDeletion of AMPKα specifically in intestinal epithelial cells was accomplished by crossing Prkaa1/2 floxed mice with Villin‐Cre mice. Dextran sulfate sodium (DSS) colitis was induced in conventionally housed intestinal epithelial cell‐specific AMPK knockout (AMPKαΔIEC KO) and AMPK floxed (AMPKαfl/fl) mice. 40kDa DSS was administered at 2.5% in drinking water ad libitum for 5 days followed by 3 days of water. Disease activity index was calculated from daily measures of body weight, stool consistency, and presence of hemaoccult. On the day of sacrifice mice were orally gavaged with 4kDa FITC‐dextran and blood was collected after 4 hours to determine intestinal permeability. Mice were sacrificed and intestinal tissues were flash frozen. Citrobacter rodentium infection was performed in mice housed under specific pathogen‐free (SPF) conditions following overnight treatment of 50ng/mL streptomycin. Mice were orally gavaged with ~108 CFU of C. rodentium (DBS100 streptomycin‐resistant strain). Fecal bacterial load was determined regularly until sacrifice on day 10 post‐infection. Colon and extra‐intestinal tissues (mesenteric lymph nodes, spleen, and liver) were excised to determine bacterial burden.ResultsAMPKαΔIEC KO mice did not exhibit increased susceptibility to 2.5% DSS compared to AMPKαfl/fl mice as determined by similar changes in body weight and disease activity index throughout the course of disease. In addition, there were no significant differences in colon weight or length after DSS (n = 4 – 15). AMPKαΔIEC KO mice do not appear to have significantly increased intestinal permeability after DSS treatment. To test if AMPK is important for responses to enteric infection, mice were infected with Citrobacter rodentium, a mouse model of enteropathogenic (EPEC) and enterohemorrhagic E. coli (EHEC) infection in humans. Infected mice had identical increases in fecal bacterial burden over time (~107 CFU/g, n = 3–6) and loss of AMPK did not affect disease progression. In addition, AMPK deficiency did not increase bacterial invasion of extra‐intestinal tissues (n = 3–4).ConclusionsThese data suggest that AMPK plays only a minor role in intestinal epithelial function and suggests that protection from colitis is mediated by AMPK‐independent pathways.Support or Funding InformationNIH 2R01DK091281 (DFM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autoantibody against \u03b21-adrenoceptor promotes the differentiation of natural regulatory T cells from activated CD4+ T cells by up-regulating AMPK-mediated fatty acid oxidation

Therapeutic adoptive transfer of natural regulatory T cells (nTreg, CD4+ CD25+ Foxp3+ T cells) or in vivo selective expansion of nTreg cells has been demonstrated to improve the cardiac function in various cardiovascular disease models. The differentiation of nTreg cells is mediated by catecholamines via β1-adrenergic receptor (β1-AR) activation. Autoantibody against β1-adrenoceptor (β1-AA) as a β1-AR agonist is closely associated with the occurrence and deterioration of cardiac dysfunction. However, whether β1-AA has any impact on nTreg cells has not been reported. The aim of the present study was intended to assess the potential impact of β1-AA on nTreg cell differentiation and explore the underlying mechanism. It was found that the expression of multiple proteins involved in nTreg cell differentiation, immunosuppressive function, and migration was up-regulated in mice after β1-AA administration, suggesting that β1-AA may promote nTreg cell activation. In vitro, β1-AA promoted nTreg cell differentiation by up-regulating mitochondrial fatty acid oxidation (FAO) in activated CD4+ T cells via AMP-activated protein kinase (AMPK) activation and mitochondrial membrane potential reduction. In addition, the AMPK agonist facilitated β1-AA-mediated FAO and nTreg cell differentiation. To further confirm the role of AMPK in β1-AA-mediated nTreg cell differentiation, β1-AA was acted on the CD4+ T cells isolated from AMPK-deficient (AMPK−/−) mice. The result showed that the effect of β1-AA on nTreg cell differentiation was attenuated markedly after AMPK knockout. In conclusion, AMPK-mediated metabolic regulation targeting for nTreg cell restoration may be a promising therapeutic target for β1-AA-positive patients with cardiac dysfunction.

Increased Innate Lymphoid Cells in Periodontal Tissue of the Murine Model of Periodontitis: The Role of AMP-Activated Protein Kinase and Relevance for the Human Condition.

Innate lymphoid cells (ILCs) are master regulators of immune and inflammatory responses, but their own regulatory mechanisms and functional roles of their subtypes (i.e., ILC1s–ILC3s) remain largely unresolved. Interestingly, AMP-activated protein kinase (AMPK), influences inflammatory responses, but its role in modulation of ILCs is not known. Periodontitis is a prevalent disorder with impairment of immune and inflammatory responses contributing importantly to its pathogenesis; however, neither the role of ILCs nor AMPK has been explored in this condition. We tested the hypotheses that (a) periodontitis increases ILCs and expression of relevant cytokines thereby contributing to inflammation and (b) knockdown of AMPK worsens indices of periodontitis in association with further increases in subtypes of ILCs and cytokine expression. The studies utilized wild-type (WT) and AMPK knockout (KO) mice, subjected to ligature-induced periodontitis or sham operation, in association with the use of micro-CT for assessment of bone loss, immunogold electron microscopy to show presence of ILCs in periodontal tissues, flow cytometry for quantitative assessment of subtypes of ILCs and RT-polymerase chain reaction analyses to measure mRNA expression of several relevant cytokines. The results for the first time show (a) presence of each subtype of ILCs in periodontal tissues of sham control and periodontitis animals, (b) that periodontitis is associated with increased frequencies of ILC1s–ILC3s with the effect more marked for ILC2s and differential phenotypic marker expression for ILC3s, (c) that AMPK KO mice display exacerbation of indices of periodontitis in association with further increases in the frequency of subtypes of ILCs with persistence of ILC2s effect, and (d) that periodontitis increased mRNA for interleukin (IL)-33, but not IL-5 or IL-13, in WT mice but expression of these cytokines was markedly increased in AMPK KO mice with periodontitis. Subsequently, we showed that human periodontitis is associated with increases in each ILCs subtype with the effect more marked for ILC2s and that mRNA expressions for IL-33 and IL-5 are markedly greater for sites affected by periodontitis than healthy sites. Collectively, these novel observations indicate a pivotal role for ILCs in pathogenesis of periodontitis and that AMPK is a regulator of their phenotype expression in this condition.

Examining the Role of the AMP‐activated Protein Kinase in Stress‐Dependent O‐GlcNAc Signaling

Acute enhancement of O‐linked‐beta‐N‐acetylglucosamine (O‐GlcNAc) modification on intracellular proteins activates an endogenous protective program in numerous models of injury, including cardiac ischemia reperfusion injury (IR/I). The O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA) enzymes are responsible for adding and removing the O‐GlcNAc moiety from proteins, respectively. This project aims to understand how cells regulate OGT and OGA in response to oxidative stress to promote cell survival and cardioprotection. AMP‐activated protein kinase (AMPK) integrates cellular stress signals with energy balance to affect cellular function, and thus plays a critical role in the cellular stress response. Our data and other studies suggest a complex interplay between OGT, OGA, AMPK, and downstream kinases that may impact stress‐induced O‐GlcNAcylation. These data include: 1) mTOR interacts with OGA in response to oxidative stress; 2) OGT and AMPK interact in response to oxidative stress, and AMPK is O‐ GlcNAc modified; and 3) ULK‐1 interacts with OGT in response to oxidative stress. To explore the significance of these interactions, we are taking two approaches. Firstly, we are examining stress‐induced glycosylation in AMPK knockout MEFs. Secondly, we are generating tagged constructs of OGT and AMPK to test the impact of the interaction between these enzymes on their function, as well as the impact of O‐GlcNAcylation on AMPK function. These experiments will further elucidate the molecular mechanisms by which OGT is regulated during injury.

AMPK improves gut epithelial differentiation and barrier function via regulating Cdx2 expression.

Impairment in gut epithelial integrity and barrier function is associated with many diseases. The homeostasis of intestinal barrier is based on a delicate regulation of epithelial proliferation and differentiation. AMP-activated protein kinase (AMPK) is a master regulator of energy metabolism, and cellular metabolites are intrinsically involved in epigenetic modifications governing cell differentiation. We aimed to evaluate the regulatory role of AMPK on intestinal epithelial development and barrier function. In this study, AMPK activator (AICAR) improved the barrier function of Caco-2 cells as indicated by increased transepithelial electrical resistance and reduced paracellular FITC-dextran permeability; consistently, AICAR enhanced epithelial differentiation and tight junction formation. Transfection of Caco-2 cells with AMPK WT plasmid, which enhances AMPK activity, improved epithelial barrier function and epithelial differentiation, while K45R (AMPK dominant negative mutant) impaired; these changes were correlated with the expression of caudal type homeobox 2 (CDX2), the key transcription factor committing cells to intestinal epithelial lineage. CDX2 deficiency abolished intestinal differentiation promoted by AMPK activation. Mechanistically, AMPK inactivation was associated with polycomb repressive complex 2 regulated enrichment of H3K27me3, the inhibitory histone modification, and lysine-specific histone demethylase-1-mediated reduction of H3K4me3, a permissive histone modification. Those histone modifications provide a mechanistic link between AMPK and CDX2 expression. Consistently, epithelial AMPK knockout in vivo reduced CDX2 expression, impaired intestinal barrier function, integrity and ultrastructure of tight junction, and epithelial cell migration, promoted intestinal proliferation and exaggerated dextran sulfate sodium-induced colitis. In summary, AMPK enhances intestinal barrier function and epithelial differentiation via promoting CDX2 expression, which is partially mediated by altered histone modifications in the Cdx2 promoter.

Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms

People with Type 2 diabetes mellitus (T2DM) have reduced bone mineral density and an increased risk of fractures due to altered mesenchymal stem cell (MSC) differentiation in the bone marrow. This leads to a shift in the balance of differentiation away from bone formation (osteogenesis) in favour of fat cell development (adipogenesis). The commonly used anti-diabetic drug, metformin, activates the osteogenic transcription factor Runt-related transcription factor 2 (Runx2), which may suppress adipogenesis, leading to improved bone health. Here we investigate the involvement of the metabolic enzyme, AMP-activated protein kinase (AMPK), in these protective actions of metformin. The anti-adipogenic actions of metformin were observed in multipotent C3H10T1/2 MSCs, in which metformin exerted reciprocal control over the activities of Runx2 and the adipogenic transcription factor, PPARγ, leading to suppression of adipogenesis. These effects appeared to be independent of AMPK activation but rather through the suppression of the mTOR/p70S6K signalling pathway. Basal AMPK and mTOR/p70S6K activity did appear to be required for adipogenesis, as demonstrated by the use of the AMPK inhibitor, compound C. This observation was further supported by using AMPK knockout mouse embryo fibroblasts (MEFs) where adipogenesis, as assessed by reduced lipid accumulation and expression of the adipogeneic transcription factor, C/EBPβ, was found to display an absolute requirement for AMPK. Further activation of AMPK in wild type MEFS, with either metformin or the AMPK-specific activator, A769662, was also associated with suppression of adipogenesis. It appears, therefore, that basal AMPK activity is required for adipogenesis and that metformin can inhibit adipogenesis through AMPK-dependent or -independent mechanisms, depending on the cellular context.

IGF-I and IGFBP-2 Stimulate AMPK Activation and Autophagy, Which Are Required for Osteoblast Differentiation.

IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3-6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.

AMPK a 1 subunit is required for proper intestinal epithelial differentiation

Intestinal epithelial cells renew every 2-4 days, which involves rapid proliferation and differentiation. AMP-activated protein Kinase (AMPK) is a key sensor and regulator of energy catabolism, and recent studies suggest that it is also an important regulator of cell differentiation. We hypothesized that AMPKa1 subunit is required for the differentiation of intestinal epithelial cells and the formation of epithelial tight junctions. To test, AMPKa1flox/flox – CreER conditional knock out (KO) mice were used, and the deletion of AMPK was induced by 4-hydrotamoxifen. Fetal intestines were sampled from WT and AMPKa1 KO E13.4 embryos, which were cultured in vitro to establish a “mini-gut” system. The differentiation was monitored by immunofluoresent staining of intestinal differentiation markers. The mini-gut from WT forms a clearly defined, well-organized epithelial layer, which was absent in that from AMPK KO. In addition, the formation of enterocytes were also decreased and disorganized in KO mini-guts compared to WT, accompanied by the disordered formation of epithelial tight junctions. In addition to impaired epithelial differentiation, the supporting mesenchymal cells, including smooth muscle cells, were also disarranged in mini-guts from KO. Taken together, these data clearly show that AMPKa1 is required for proper differentiation and development of gut epithelium (NIH R15HD073864).

AMP-Activated Protein Kinase α1 but Not α2 Catalytic Subunit Potentiates Myogenin Expression and Myogenesis

The link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic α subunits, α1 and α2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the α1 but not the α2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPKα1 and AMPKα2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPKα1-specific effect on myogenesis was likely due to the dominant expression of α1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPKα1 subunit for myogenesis in vivo, we prepared both DsRed AMPKα1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wild-type myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPKα1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPKα1.