Liver Kinase B1 Research Articles

IQGAP1 binds AMPK and is required for maximum AMPK activation

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

Metformin in cardiovascular diabetology: a focused review of its impact on endothelial function.

As a first-line treatment for diabetes, the insulin-sensitizing biguanide, metformin, regulates glucose levels and positively affects cardiovascular function in patients with diabetes and cardiovascular complications. Endothelial dysfunction (ED) represents the primary pathological change of multiple vascular diseases, because it causes decreased arterial plasticity, increased vascular resistance, reduced tissue perfusion and atherosclerosis. Caused by “biochemical injury”, ED is also an independent predictor of cardiovascular events. Accumulating evidence shows that metformin improves ED through liver kinase B1 (LKB1)/5'-adenosine monophosphat-activated protein kinase (AMPK) and AMPK-independent targets, including nuclear factor-kappa B (NF-κB), phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), forkhead box O1 (FOXO1), krüppel-like factor 4 (KLF4) and krüppel-like factor 2 (KLF2). Evaluating the effects of metformin on endothelial cell functions would facilitate our understanding of the therapeutic potential of metformin in cardiovascular diabetology (including diabetes and its cardiovascular complications). This article reviews the physiological and pathological functions of endothelial cells and the intact endothelium, reviews the latest research of metformin in the treatment of diabetes and related cardiovascular complications, and focuses on the mechanism of action of metformin in regulating endothelial cell functions.

Hepatoprotective effect of galangin on carbon tetrachloride-induced hepatotoxicity via the LKB1/AMPK pathway

To investigate the protective effects of galangin on liver toxicity induced by carbon tetrachloride (CCl4) in mice. Mouse hepatotoxicity model was established by intraperitoneal injection (i.p.) of 10 ml/kg body weight CCl4 that diluted with corn oil to a proportion of 1:500 on Kunming mice. The mice were randomly divided into five groups named control group, model group, and 1, 5, and 10 mg/kg galangin group. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed by ELISA. Liver histopathological examination was observed via optical microscopy. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and glutathion (GSSG) were analyzed to assess oxidative stress. Finally, western blot assay was carried out to analyse the expression levels of total AMP-activated protein kinase (AMPK), phospho-AMPK (p-AMPK), total liver kinase B1 (LKB1), and phospho-LKB1 (p-LKB1). Compared with the control group, in the model group, the levels of AST, ALT, MDA, and GSSG increased significantly ( p < 0.01); the activity of SOD and GSH decreased significantly ( p < 0.01); and the histopathological examination revealed liver necrosis. However, treatment with galangin (5 and 10 mg/kg) significantly reversed these CCl4-induced liver damage indicators. Furthermore, treatment with galangin (10 mg/kg) significantly increased the p-AMPK and p-LKB1 expression levels ( p < 0.01). This study supports the hepatoprotective effect of galangin against hepatotoxicity, perhaps occurring mainly through the LKB1/AMPK-mediated pathway.

Sodium Butyrate Supplementation Inhibits Hepatic Steatosis by Stimulating Liver Kinase B1 and Insulin-Induced Gene.

Background and AimsButyric acid is an intestinal microbiota-produced short-chain fatty acid, which exerts salutary effects on alleviating nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism of butyrate on regulating hepatic lipid metabolism is largely unexplored.MethodsA mouse model of NAFLD was induced with high-fat diet feeding, and sodium butyrate (NaB) intervention was initiated at the eighth week and lasted for 8 weeks. Hepatic steatosis was evaluated and metabolic pathways concerning lipid homeostasis were analyzed.ResultsHere, we report that administration of NaB by gavage once daily for 8 weeks causes an augmentation of insulin-induced gene (Insig) activity and inhibition of lipogenic gene in mice fed with high-fat diet. Mechanistically, NaB is sufficient to enhance the interaction between Insig and its upstream kinase AMP-activated protein kinase (AMPK). The stimulatory effects of NaB on Insig-1 activity are abolished in AMPKα1/α2 double knockout (AMPK−/−) mouse primary hepatocytes. Moreover, AMPK activation by NaB is mediated by LKB1, as evidenced by the observations showing NaB-mediated induction of phosphorylation of AMPK, and its downstream target acetyl-CoA carboxylase is diminished in LKB1–/– mouse embryonic fibroblasts.ConclusionsThese studies indicate that NaB serves as a negative regulator of hepatic lipogenesis in NAFLD and that NaB attenuates hepatic steatosis and improves lipid profile and liver function largely through the activation of LKB1-AMPK-Insig signaling pathway. Therefore, NaB has therapeutic potential for treating NAFLD and related metabolic diseases.

LSR promotes epithelial ovarian cancer cell survival under energy stress through the LKB1-AMPK pathway

Lipolysis-stimulated lipoprotein receptor (LSR), also known as a component of tricellular tight junctions, is highly expressing in epithelial ovarian cancer (EOC). However, the biological role of LSR in EOC cells remains unclear. In this study, we evaluated liver kinase B1 (LKB1) mediated AMP-activated protein kinase (AMPK) activity and investigated the effect of LSR on EOC cell survival under energy stress. LSR increased the levels of phospho-AMPKα at Thr172 and phospho-acetyl-CoA carboxylase (ACC) at Ser79 via LKB1-AMPK pathway in glucose deprivation in vitro. The increase of P-AMPKα (Thr172) and P-ACC (Ser79) was also detected in tumor microenvironment in vivo. Meanwhile, LSR promoted LKB1 localization at the cell membrane of EOC cells. By cell survival analysis, LSR attenuated glucose deprivation-induced cell death in EOC cells in vitro. Our results suggest that LSR promotes EOC cell survival and tumor growth through the LKB1-AMPK pathway.

Cloning and prokaryotic expression of the chicken liver kinase B1 (LKB1) and its localization in liver, heart and hypothalamus

Liver kinase B1 (LKB1) is a member of the serine/threonine kinase family, which plays an indispensable role in the organism of animals. In the current study, the chicken LKB1 protein gene was amplified by PCR based on the primers and cDNA templates. Then, the cloning vector was constructed and the target gene was cloned. After that, the target gene was inserted into the expression vector to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) host cells and the LKB1 recombinant proteins were successfully expressed by using Isopropyl-β-D-thiogalactopyranoside (IPTG). Finally, purified LKB1 proteins were used as antigen and the rabbit-derived antiserums were collected. The antiserum titer determined by ELISA was not less than 1:128000. The results of Western blot suggested that the polyclonal antibody is highly specific to chicken LKB1 protein. Immunofluorescence indicated that the LKB1 protein is mainly expressed in the cytoplasm of liver, heart and hypothalamus cells of chicken. Our study showed that the LKB1 polyclonal antibodies produced by this method are effective and can be used to further study the role of LKB1 in the pathogenesis of chicken disease.

Cigarette smoke-induced LKB1/AMPK pathway deficiency reduces EGFR TKI sensitivity in NSCLC

Smoker patients with non-small cell lung cancer (NSCLC) have poorer prognosis and survival than those without smoking history. However, the mechanisms underlying the low response rate of those patients to EGFR tyrosine kinase inhibitors (TKIs) are not well understood. Here we report that exposure to cigarette smoke extract enhances glycolysis and attenuates AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR; this in turn reduces the sensitivity of NSCLC cells with wild-type EGFR (EGFRWT) to EGFR TKI by repressing expression of liver kinase B1 (LKB1), a master kinase of the AMPK subfamily, via CpG island methylation. In addition, LKB1 expression is correlated positively with sensitivity to TKI in patients with NSCLC. Moreover, combined treatment of EGFR TKI with AMPK activators synergistically increases EGFR TKI sensitivity. Collectively, the current study suggests that LKB1 may serve as a marker to predict EGFR TKI sensitivity in smokers with NSCLC carrying EGFRWT and that the combination of EGFR TKI and AMPK activator may be a potentially effective therapeutic strategy against NSCLC with EGFRWT.

(-)-Epicatechin induces mitochondrial biogenesis and markers of muscle regeneration in adults with Becker muscular dystrophy.

IntroductionWe conducted an open‐label study to examine the effects of the flavonoid (−)‐epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD).MethodsSeven participants received (−)‐epicatechin 50 mg twice per day for 8 weeks. Pre‐ and postprocedures included biceps brachii biopsy to assess muscle structure and growth‐relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests.ResultsWestern blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5′‐adenosine monophosphate–activated protein kinase). Peroxisome proliferator‐activated receptor gamma coactivator‐1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae‐associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor‐5, myoblast determination protein 1, myocyte enhancer factor‐2, and structure‐associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states.Discussion(−)‐Epicatechin, an exercise mimetic, appears to have short‐term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.

Loss of cellular identity in common pre-clinical models of serine‑threonine kinase 11 (Liver kinase B1) loss

Nearly 1/3 of lung adenocarcinomas have loss of STK11 (LKB1) function. Herein, a bioinformatics approach was used to determine how accurately preclinical model systems reflect the in vivo biology of STK11 loss in human patients. Hierarchical and K-mean clustering, principle component, and gene set enrichment analyses were employed to model gene expression due to STK11 loss in patient cohorts representing nearly 1000 lung adenocarcinoma patients. K-means clustering classified STK11 loss patient tumors into three distinct sub-groups; positive (54%), neuroendocrine (NE) (35%) and negative (11%). The positive and NE groups are both defined by the expression of NKX2–1. In addition to NKX2–1, NE patients express neuroendocrine markers such as ASCL1 and CALCA. In contrast, the negative group does not express NKX2–1 (or neuroendocrine markers) and is characterized by significantly reduced survival relative to the two other groups. Two gene expression signatures were derived to explain both neuroendocrine features and differentiation (NKX2–1 loss) and were validated through two public datasets involving chemical differentiation (DCI) and NKX2–1 reconstitution. Patients results were then compared with established cell lines, transgenic mice, and patient-derived xenograft models of STK11 loss. Interestingly, all cell line and PDX models cluster and show expression patterns similar with the NKX2–1 negative subset of STK11-loss human tumors. Surprisingly, even mouse models of STK11 loss do not resemble patient tumors based on gene expression patterns. Results suggest pre-clinical models of STK11 loss are pronounced by marked elimination of type II pneumocyte identity, opposite of most in vivo human tumors.

Liver kinase B1 inhibits smooth muscle calcification via high mobility group box 1

Vascular calcification is a common pathological feature of atherosclerosis, chronic kidney disease, vascular injury, and aging. Liver kinase B1 (LKB1) plays pivotal roles in cellular processes such as apoptosis, metabolism, and cell cycle regulation. In addition, growing evidence has indicated that LKB1 functions as a tumor suppressor gene. However, its role in vascular calcification has not been reported. LKB1flox/flox mice were hybridized with SM22-CreERT2 transgenic mice and adult mice received tamoxifen to obtain smooth muscle-specific LKB1-knockout (LKB1SMKO) mice. LKB1 expression was decreased under calcifying conditions, and LKB1 overexpression had a protective effect on vascular calcification. However, high mobility group box 1 (HMGB1) overexpression partially counteracted the promotion of vascular calcification induced by LKB1 overexpression. Mechanically, LKB1 could bind to HMGB1 to promote HMGB1 degradation. Furthermore, LKB1SMKO mice showed intensified vascular calcification, which was alleviated by treatment with the HMGB1 inhibitor glycyrrhizic acid. Based on our results, LKB1 may inhibit vascular calcification via inhibiting HMGB1 expression.

Optimization of WZ4003 as NUAK inhibitors against human colorectal cancer.

NUAK, the member of AMPK (AMP-activated protein kinase) family of protein kinases, is phosphorylated and activated by the LKB1 (liver kinase B1) tumor suppressor protein kinase. Recent work has indicated that NUAK1 is a key component of the antioxidant stress response pathway, and the inhibition of NUAK1 will suppress the growth and survival of colorectal tumors. As a promising target for anticancer drugs, few inhibitors of NUAK were developed. With this goal in mind, based on NUAK inhibitor WZ4003, a series of derivatives has been synthesized and evaluated for anticancer activity. Compound 9q, a derivative of WZ4003 by removing a methoxy group, was found to be the most potential one with stronger inhibitory against NUAK1/2 enzyme activity, tumor cell proliferation and inducing apoptosis of tumor cells. By invivo efficacy evaluations of colorectal SW480 xenografts, 9q suppresses tumor growth more effectively with an excellent safety profile invivo and is therefore seen as a suitable candidate for further investigation.

PSPH induces cell autophagy and promotes cell proliferation and invasion in the hepatocellular carcinoma cell line Huh7 via the AMPK/mTOR/ULK1 signaling pathway.

Phosphoserine phosphatase (PSPH), a key enzyme of the l-serine synthesis pathway, has been involved in cancer progression and survival. However, limited evidence revealed the PSPH influence on hepatocellular carcinoma (HCC). Herein, we observed that PSPH expression was upregulated in both HCC tissues and cell lines, which was determined by western blotting. TCGA database showed that the PSPH protein levels were significantly upregulated and affected patient survival rates in HCC. Then gain- and loss-of-function manipulations were performed by transfection with a pcDNA-PSPH expression vector or a specific short interfering RNA against PSPH in Huh7 cells. Huh7 cell proliferation, stemness, invasion, and apoptosis were assessed by using CCK-8 test, colony formation assay, Transwell assay, and Flow cytometry analysis, respectively, and levels of autophagy-related proteins were detected by using western blotting. The results showed that PSPH could induce Huh7 cell autophagy, promote cell proliferation and invasion, and inhibit apoptosis. The knockdown of PSPH could inhibit Huh7 cell proliferation, invasion, and autophagy. Furthermore, PSPH activated Liver kinase B1 (LKB1) and TGF beta-activated kinase 1 (TAK1), affected the adenosine 5'-monophosphate-activated protein kinase (AMPK)/mTOR/ULK1 signaling pathway, but could not activate calcium/calmodulin-dependent protein kinase kinase (CaMKK) in Huh7 cells. Inhibition of either LKB1, TAK1, or AMPK could eliminate the effect of PSPH overexpression on Huh7 cell behaviors. However, inhibition of CaMKK could not influence the effect of PSPH overexpression on Huh7 cell behaviors. In conclusion, PSPH could induce autophagy, promote proliferation and invasion, and inhibit apoptosis in HCC cells via the AMPK/mTOR/ULK1 signaling pathway.

Lkb1 suppresses amino acid-driven gluconeogenesis in the liver

Excessive glucose production by the liver is a key factor in the hyperglycemia observed in type 2 diabetes mellitus (T2DM). Here, we highlight a novel role of liver kinase B1 (Lkb1) in this regulation. We show that mice with a hepatocyte-specific deletion of Lkb1 have higher levels of hepatic amino acid catabolism, driving gluconeogenesis. This effect is observed during both fasting and the postprandial period, identifying Lkb1 as a critical suppressor of postprandial hepatic gluconeogenesis. Hepatic Lkb1 deletion is associated with major changes in whole-body metabolism, leading to a lower lean body mass and, in the longer term, sarcopenia and cachexia, as a consequence of the diversion of amino acids to liver metabolism at the expense of muscle. Using genetic, proteomic and pharmacological approaches, we identify the aminotransferases and specifically Agxt as effectors of the suppressor function of Lkb1 in amino acid-driven gluconeogenesis.

Autophagy compensates for Lkb1 loss to maintain adult mice homeostasis and survival.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11) is the major energy sensor for cells to respond to metabolic stress. Autophagy degrades and recycles proteins, macromolecules, and organelles for cells to survive starvation. To assess the role and cross-talk between autophagy and Lkb1 in normal tissue homeostasis, we generated genetically engineered mouse models where we can conditionally delete Stk11 and autophagy essential gene, Atg7, respectively or simultaneously, throughout the adult mice. We found that Lkb1 was essential for the survival of adult mice, and autophagy activation could temporarily compensate for the acute loss of Lkb1 and extend mouse life span. We further found that acute deletion of Lkb1 in adult mice led to impaired intestinal barrier function, hypoglycemia, and abnormal serum metabolism, which was partly rescued by the Lkb1 loss-induced autophagy upregulation via inhibiting p53 induction. Taken together, we demonstrated that autophagy and Lkb1 work synergistically to maintain adult mouse homeostasis and survival.

Naringenin alleviates nonalcoholic steatohepatitis in middle-aged Apoe−/−mice: role of SIRT1

BackgroundNaringenin is naturally isolated from citrus fruits possessing many pharmacological activities. However, little is known about the effect of naringenin on nonalcoholic steatohepatitis (NASH) in the model of metabolic syndrome. PurposeThe present study is aimed to investigate the effect of naringenin on NASH in 12-mo-old male ApoE−/− mice and its possible underlying mechanism. MethodsIn vivo, 12-mo-old male ApoE−/− mice were administrated with naringenin by intragastric gavage for 12 weeks. At the end of experiment, the blood samples and liver tissues were collected. Metabolic parameters in serum, levels of triglyceride, cholesterol and hydroxyproline, activities of antioxidant enzymes, and content of inflammatory cytokines (TNF-α and IL-6) in liver were examined by corresponding assay kits. Pathological changes in liver were observed by hematoxylin-eosin, oil red O, masson's trichrome, picro-sirius red and senescence β-galactosidase staining. Dihydroethidium was used for detection of reactive oxygen species (ROS). In vitro, AML-12 cells were treated with oleic acid in the presence or absence of naringenin for 24 h. Transfection of SIRT1 siRNA was also conducted in vitro. Lipid accumulation, cellular ROS generation, malondialdehyde content, antioxidant enzyme activities and secretion levels of TNF-α and IL-6 were examined. Both in vivo and in vitro, gene expressions were detected by real-time PCR or western blot. ResultsNaringenin administration improved metabolic parameters, suppressed hepatic steatosis, regulated expression of genes involved in lipid metabolism (FASN, SCD1, PPARα and CPT1α), reduced hepatic fibrosis and cell senescence, inhibited hepatic inflammation as evidenced by the decreased macrophage recruitment and content of TNF-α and IL-6, and reduced hepatic oxidative stress by suppressing ROS generation and normalizing activities of antioxidant enzymes. Notably, naringenin administration increased hepatic SIRT1 protein expression and activity along with the increased deacetylation of liver kinase B1 (LKB1), PGC1α and NF-κB. In vitro study, the benefits of naringenin on lipid accumulation, oxidative stress and inflammation were diminished by SIRT1 siRNA transfection. ConclusionsThese results indicate that naringenin administration may be a potential curative therapy for NASH treatment and the activation of hepatic SIRT1-mediated signaling cascades is involved in its beneficial effects.

Kaempferol Promotes Glucose Uptake in Myotubes through a JAK2-Dependent Pathway.

Kaempferol possesses various health-promoting functions including antihyperglycemic activity, but its underlying molecular mechanism is poorly understood. Glucose transporter 4 (GLUT4) plays an important role in the uptake of blood glucose into muscle cells after its translocation to the plasma membrane. In this study, we demonstrated that kaempferol at 1.0 nM or more significantly increased the uptake of 2-[3H]- deoxy-d-glucose by 1.3-1.4-fold in L6 myotubes. Kaempferol at 10 pM or more also significantly increased GLUT4 translocation by 1.3-1.6-fold. Kaempferol at 1.0 nM significantly increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) by 2.9-fold, liver kinase B1 and Janus kinase 2 (JAK2) by 1.9-fold, and signal transducer and activator of transcription 3 by 3.7-fold. In addition, kaempferol increased phosphorylation of phosphoinositide 3-kinase (PI3K) by 1.8-fold but not the insulin receptor. Small interfering RNA (siRNA) for AMPK, JAK2, or PI3K canceled kaempferol-induced glucose uptake and GLUT4 translocation. Furthermore, siRNA for JAK2 canceled kaempferol-induced phosphorylation of AMPK and PI3K. These results indicate that a JAK2-depdendent pathway regulates kaempferol-induced glucose uptake and GLUT4 translocation in L6 myotubes and that kaempferol may be an effective compound for the prevention of hyperglycemia.

Current Status and Application of Metformin for Prostate Cancer: A Comprehensive Review.

Metformin, an oral biguanide used for first-line treatment of type 2 diabetes mellitus, has attracted attention for its anti-proliferative and anti-cancer effects in several solid tumors, including prostate cancer (PCa). Liver kinase B1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK) activation, inhibition of the mammalian target of rapamycin (mTOR) activity and protein synthesis, induction of apoptosis and autophagy by p53 and p21, and decreased blood insulin level have been suggested as direct anti-cancer mechanisms of metformin. Research has shown that PCa development and progression are associated with metabolic syndrome and its components. Therefore, reduction in the risk of PCa and improvement in survival in metformin users may be the results of the direct anti-cancer mechanisms of the drug or the secondary effects from improvement of metabolic syndrome. In contrast, some research has suggested that there is no association between metformin use and PCa incidence or survival. In this comprehensive review, we summarize updated evidence on the relationship between metformin use and oncological effects in patients with PCa. We also highlight ongoing clinical trials evaluating metformin as an adjuvant therapy in novel drug combinations in various disease settings.

Curcumin Prevents Neuroinflammation by Inducing Microglia to Transform into the M2-phenotype via CaMKKβ-dependent Activation of the AMP-Activated Protein Kinase Signal Pathway.

Neuroinflammation plays an important role in the pathophysiological process of various neurodegenerative diseases. It is well known that curcumin has obvious anti-inflammatory effects in various neuroinflammation models. However, its effect on the modulation of microglial polarization is largely unknown. This study aimed to investigate whether curcumin changed microglia to an anti-inflammatory M2-phenotype by activating the AMP-activated protein kinase (AMPK) signaling pathway. LPS treatment was used to establish BV2 cells and primary microglia neuroinflammation models. The neuroinflammation mouse model was established by an intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) in the lateral septal complex region of the brain. TNF-α was measured by ELISA, and cell viability was measured by Cell Counting Kit-8 (CCK-8). The expression of proinflammatory and anti-inflammatory cytokines was examined by Q-PCR and Western blot analysis. Phenotypic polarization of BV2 microglia was detected by immunofluorescence. Curcumin enhanced AMPK activation in BV2 microglial cells in the presence and absence of LPS. Upon LPS stimulation, the addition of curcumin promoted M2 polarization of BV2 cells, as evidenced by suppressed M1 and the elevated M2 signature protein and gene expression. The effects of curcumin were inhibited by an AMPK inhibitor or AMPK knockdown. Calmodulin-dependent protein kinase kinase β (CaMKKβ) and liver kinase B1 (LKB1) are upstream kinases that activate AMPK. Curcumin can activate AMPK in Hela cells, which do not express LKB1. However, both the CaMKKβ inhibitor and siRNA blocked curcumin activation of AMPK in LPS-stimulated BV2 cells. Moreover, the CaMKKβ inhibitor and siRNA weaken the effect of curcumin suppression on M1 and enhancement of M2 protein and gene expression in LPS-stimulated BV2 cells. Finally, curcumin enhanced AMPK activation in the brain area where microglia were over-activated upon LPS stimulation in an in vivo neuroinflammation model. Moreover, curcumin also suppressed M1 and promoted M2 signature protein and gene expression in this in vivo model. Curcumin enhances microglia M2 polarization via the CaMKKβ-dependent AMPK signaling pathway. Additionally, curcumin treatment was found to be neuroprotective and thus might be considered as a novel therapeutic agent to treat the neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, etc.

Modeling Marginal Zone Lymphomagenesis

Introduction: Indolent B-Cell Non-Hodgkin's Lymphomas (NHL) represent a heterogeneous group of lymphoproliferative malignancies, that remain largely incurable. Marginal zone lymphomas (MZL) are the second most common subtype of indolent NHL, and lack a unique cytogenetic identifying abnormality. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K) pathway. Furthermore, novel PI3K inhibitors, such as copanlisib and parsaclisib, have shown impressive clinical activity in several indolent lymphomas including MZL. This further supports the important role of the PI3K pathway in the pathogenesis of this tumor. Therefore, we hypothesized that the PI3K-mTOR pathway is sufficient for driving the pathogenesis of MZL. Methods: In order to test our hypothesis, we generated a genetically engineered mouse model carrying heterozygous global knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1). This led to over-activation of the PI3K-mTOR pathway in all mouse tissues. We closely monitored these mice for tumor formation via weekly physical examinations for several months. Upon tumor detection, the mouse was sacrificed, and tumors were sectioned for histological characterization. In order to generate a more specific model of B cells, and more accurately mimic the underlying human disease, we used the Cre-LoxP system to create the CD19-Cre-PTENfl/fl-LKB1fl/fl. Results: Thirty mice of global KO PTEN+/- LKB1 +/- died or were sacrificed due to disease progression, defined as either lymph node enlargement and/or splenomegaly. All mice showed either abnormal lymphadenopathy or splenomegaly. By Kaplan-Meier analysis, we saw a steady decrease in both tumor-free and overall survival after 3 months of age. Utilizing the product limit method, the median survival time was 6 months (95% CI: 6, 8). A total of 51 lymph nodes were sent for immunohistochemistry and pathological characterization. Of the 51 nodes, 61.5% (N=32) showed indolent Non-Hodgkin's Lymphoma, 25% (N=13) were atypical, and 11.5% (N=6) were reactive. All lymph nodes with indolent NHL were of MZL subtype. Compared to wild type (n=3), the new CD19-Cre-PTENfl/fl -LKB1fl/fl (n=3) showed an overall increase in spleen mass (120 vs 196 mg, p=0.0564), % B1 cells (4% vs 59%, p= 0.0075), % MZ cells (5% vs 30%, p=0.0547), % plasma cells (1% vs 12%, p=0.0729), and decrease in % FO cells (80% vs 12%, p=0.0003) by flowcytometry. Further characterization of the new model is currently underway. Conclusion: Marginal zone lymphoma remains an incurable lymphoma that lacks reliable preclinical models. Our data provides, for the first time, a proof of concept on the role of the PI3K-mTOR pathway in the pathogenesis of marginal zone lymphoma and paves the way for future studies understanding the biology of this disease, and developing rational therapies for this incurable malignancy. Disclosures Yazbeck: Celgene: Consultancy; AstraZeneca: Consultancy; Gilead: Research Funding; Seattle Genetics: Consultancy; Verastem: Speakers Bureau.

Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly tothe risk of strokein humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.