AMP-activated Protein Kinase Expression Research Articles

Occludin regulates glucose uptake and ATP production in pericytes by influencing AMP-activated protein kinase activity

Energetic regulation at the blood-brain barrier is critical for maintaining its integrity, transport capabilities, and brain demands for glucose. However, the underlying mechanisms that regulate these processes are still poorly explored. We recently characterized the protein occludin as a NADH oxidase and demonstrated its influence on the expression and activation of the histone deacetylase SIRT-1. Because SIRT-1 works in concert with AMP-activated protein kinase (AMPK) (AMPK), we investigated the impact of occludin on this metabolic switch. Here we show that in blood-brain barrier pericytes, occludin promotes AMPK expression and activation, influencing the expression of glucose transporters GLUT-1 and GLUT-4, glucose uptake, and ATP content. Furthermore, occludin expression, AMP-dependent protein kinase activity, and glucose uptake were altered under inflammatory (TNFα) and infectious (HIV) conditions. We also show that pericytes share glucose and mitochondria with astrocytes, and that occludin levels modify the ability of pericytes to share those energetic resources. In addition, we demonstrate that murine mitochondria can be transferred from live brain microvessels to energetically impaired human astrocytes, promoting their survival. Our findings demonstrate that occludin plays an important role in blood-brain barrier pericyte metabolism by influencing AMPK protein kinase activity, glucose uptake, ATP production, and by regulating the ability of pericytes to interact metabolically with astrocytes.

Mulberry granules protect against diabetic cardiomyopathy through the AMPK/Nrf2 pathway.

Mulberry granules(MLD) is a traditional Chinese medicine prescription that has been used in the treatment of diabetes for many years. Recently, we found that MLD protected the heart from diabetes-associated cardiomyopathy when it was used to treat diabetes. However, the beneficial effects and possible mechanism remain unknown. To elucidate these effects, an experimental myocardial ischemia/reperfusion(MI/R) injury model in diabetes rats was used in this study. Male C57BL/6mice were injected with streptozotocin to induce diabetes. The mice were pretreated with MLD for one month, and then exposed to 30min of ischemia followed by 24h of reperfusion. Infarct size, heart function and various cytokines in the heart were assessed. Expression of AMP-activated protein kinase(AMPK) and nuclear factor erythroid 2‑related factor2(Nrf2) were investigated by western blotting. Invitro, MLD significantly cleared oxygen-free radicals in DPPH and luminol chemiluminescence models. Invivo, fasting blood glucose, fasting blood insulin and lipids were significantly decreased by MLD. The results showed that MLD improved the cardiac function and decreased myocardial infarct size in the diabetic mice subjected to MI/R. Inaddition, upon pretreatment with MLD before MI/R treatment, GSH, SOD, CAT andGR were significantly increased in a dose-dependent manner. Pretreatment with MLD also significantly induced the expression of Nrf2, and the cardioprotective effects of MLD were abolished in Nrf2-knockoutmice. Furthermore, we also found that AMPK increase is upstream and was required for Nrf2 activation mediated by MLD. Inconclusion, MLD protects against diabetic-associated cardiomyopathy by suppressing oxidative stress induced by hyperglycemia and MI/R through the AMPK/Nrf2 signaling pathway.

Abstract 2323: The inhibitory role of Kaempferol-3-O-rutinoside induced AMPK activation on the growth of human breast cancer cell lines

Abstract Solidago virga-aurea, herbaceous perennial plant of the family Asteraceae, has astringent, diuretic, and antiseptic assets. In our current study, we found that kaempferol-3- O -rutinoside (KR) from Solidago virga-aurea extract prompted cellular apoptosis in breast cancer cells. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of inhibitory effect on cell viability against MDA-MB-231 and MCF-7 breast cancer cell lines. In addition, apoptotic protein, especially Bim, was also increased in KR treated MDA-MB-231 and MCF-7 cell line. However, an anti-apoptotic protein, Bcl-xL, was not changed by KR. To find out positive regulator of Bim expression, FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5' AMP-activated protein kinase) protein expressions were examined in KR treated cells. Immunoblot analysis showed dramatically increased expressions of FOXO3a and AMPK in KR treated MDA-MB-231 and MCF-7 cells. In addition, cell cycle analysis indicated that G0/G1 arrest was intensely increased in KR treated MDA-MB-231 and MCF-7 cells. Moreover, phospho-AMPK (pT172) and phospho-FOXO3a (S472) expression were also increased in KR treated MDA-MB-231 and MCF-7 cells. Our results suggest that KR induces apoptosis by AMPK-FOXO3a mediated Bim expression. The mechanisms of KR mediated AMPK and FOXO3a regulation is not fully elucidated yet, but further studies about these molecular mechanisms between two proteins will give some ideas for cancer therapies for certain breast cancer patients. Citation Format: Chea Ha Kim. The inhibitory role of Kaempferol-3-O-rutinoside induced AMPK activation on the growth of human breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2017-2323

TARGETING THE HDAC2/HNF-4A/MIR-101B/AMPK PATHWAY RESCUES TAUOPATHY AND DENDRITIC ABNORMALITIES IN ALZHEIMER’S DISEASE

Histone deacetylase 2 (HDAC2) plays a major role in the epigenetic regulation of gene expression. Previous studies have shown that HDAC2 expression is strongly increased in Alzheimer's disease (AD), a major neurodegenerative disorder and the most common form of dementia. Moreover, previous studies have linked HDAC2 to Ab overproduction in AD; however, its involvement in tau pathology and other memory-related functions remains unclear. Here, we first demonstrate that the HDAC2 levels are positively correlated with tau hyperphosphorylation in AD mice, and the overexpression of HDAC2, in contrast to HDAC1 or HDAC3, was sufficient to promote tau hyperphosphorylation, tau aggregation, and dendritic impairments in vivo. We then show that HDAC2 overexpression reduced the acetylation of hepatocyte nuclear factor 4α (HNF-4A), which inhibited its binding to the promoter region of miR-101b. The suppression of miR-101b leads to the upregulation of adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) expression at the post-transcriptional level. We subsequently antagonized this disease-modifying signal via the specific introduction of miR-101b mimics or silencing AMPK expression in cultured neurons and determined that tau hyperphosphorylation, aggregation and dendritic impairments induced by HDAC2 were rescued in these cells. A similar experimental strategy rescued the behavioral abnormalities in AD mice. We found that increased HDAC2 levels strongly correlate with phosphorylated tau in a mouse model of AD. HDAC2 overexpression induced AD-like tau hyperphosphorylation and aggregation, which were accompanied by a loss of dendritic complexity and spine density. The ectopic expression of HDAC2 resulted in the deacetylation of the hepatocyte nuclear factor 4a (HNF-4A) transcription factor, which disrupted its binding to the miR-101b promoter. The suppression of miR-101b caused an upregulation of its target, adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK). The introduction of miR-101b mimics or small interfering RNAs (siRNAs) against AMPK blocked HDAC2-induced tauopathy and dendritic impairments in vitro. Correspondingly, miR-101b mimics or AMPK siRNAs rescued tau pathology, dendritic abnormalities, and memory deficits in AD mice. Taken together, the current findings implicate the HDAC2/miR-101/AMPK pathway as a critical mediator of AD pathogenesis. These studies also highlight the importance of epigenetics in AD and provide novel therapeutic targets.

Melatonin Rescues Mesenchymal Stem Cells from Senescence Induced by the Uremic Toxin p-Cresol via Inhibiting mTOR-Dependent Autophagy.

p-Cresol, found at high concentrations in the serum of chronic kidney failure patients, is known to cause cell senescence and other complications in different parts of the body. p-Cresol is thought to mediate cytotoxic effects through the induction of autophagy response. However, toxic effects of p-cresol on mesenchymal stem cells have not been elucidated. Thus, we aimed to investigate whether p-cresol induces senescence of mesenchymal stem cells, and whether melatonin can ameliorate abnormal autophagy response caused by p-cresol. We found that p-cresol concentration-dependently reduced proliferation of mesenchymal stem cells. Pretreatment with melatonin prevented pro-senescence effects of p-cresol on mesenchymal stem cells. We found that by inducing phosphorylation of Akt and activating the Akt signaling pathway, melatonin enhanced catalase activity and thereby inhibited the accumulation of reactive oxygen species induced by p-cresol in mesenchymal stem cells, ultimately preventing abnormal activation of autophagy. Furthermore, preincubation with melatonin counteracted other pro-senescence changes caused by p-cresol, such as the increase in total 5′-AMP-activated protein kinase expression and decrease in the level of phosphorylated mechanistic target of rapamycin. Ultimately, we discovered that melatonin restored the expression of senescence marker protein 30, which is normally suppressed because of the induction of the autophagy pathway in chronic kidney failure patients by p-cresol. Our findings suggest that stem cell senescence in patients with chronic kidney failure could be potentially rescued by the administration of melatonin, which grants this hormone a novel therapeutic role.

Ulinastatin attenuates neuropathic pain via the ATP/P2Y2 receptor pathway in rat models

Ulinastatin, a serine protease inhibitor, which has anti-inflammatory properties and neuroprotective effects, is used to treat acute inflammatory disorders. Recent evidence indicates that administration of ulinastatin alleviates pain in rat model of neuropathic pain (NPP). However, its effect on NPP and the underlying mechanism requires further study. In this study, we evaluated the role of intrathecal administration of ulinastatin in rats with sciatic nerve ligation and observed the effect of ulinastatin on the ATP/P2Y2 receptor pathway. We performed mechanical and thermal sensitivity measurements, immunohistochemistry and double-label immunofluoresence studies to evaluate P2Y2 receptor and adenosine 5′-monophosphate-activated protein kinase (AMPK) expression in the dorsal horn of the lumbar enlargement region of the spinal cord, and a luciferase assay for the detection of ATP levels in the cerebrospinal fluid. The results showed that ulinastatin prevented the development of mechanical allodynia and thermal hypersensitivity in the rat sciatic nerve ligation model. Ulinastatin reduced the level of extracellular ATP, down-regulated P2Y2 receptor and AMPK expression in the spinal dorsal horn of the chronic constrictive injury model. We found that increased expression of P2Y2 receptor in microglia was likely involved in the activation of microglia after nerve injury, and ulinastatin inhibited the abnormal microglia activation in the dorsal horn after nerve injury. These findings demonstrated that ulinastatin might be a potential and effective drug for the treatment of NPP via the suppression of the ATP/P2Y2 receptor pathway.

AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia

Thymoquinone is a known inhibitor of neuroinflammation. However, the mechanism(s) involved in its action remain largely unknown. In this study, we investigated the roles of cellular reactive oxygen species (ROS), 5′ AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) in the anti-neuroinflammatory activity of thymoquinone. We investigated effects of the compound on ROS generation in LPS-activated microglia using the fluorescent 2′,7′-dichlorofluorescin diacetate (DCFDA)-cellular ROS detection. Immunoblotting was used to detect protein levels of p40phox, gp91phox, AMPK, LKB1 and SIRT1. Additionally, ELISA and immunofluorescence were used to detect nuclear accumulation of SIRT1. NAD+/NADH assay was also performed. The roles of AMPK and SIRT1 in anti-inflammatory activity of thymoquinone were investigated using RNAi and pharmacological inhibition. Our results show that thymoquinone reduced cellular ROS generation, possibly through inhibition of p40phox and gp91phox protein. Treatment of BV2 microglia with thymoquinone also resulted in elevation in the levels of LKB1 and phospho-AMPK proteins. We further observed that thymoquinone reduced cytoplasmic levels and increased nuclear accumulation of SIRT1 protein and increased levels of NAD+. Results also show that the anti-inflammatory activity of thymoquinone was abolished when the expressions of AMPK and SIRT1 were suppressed by RNAi or pharmacological antagonists. Pharmacological antagonism of AMPK reversed thymoquinone-induced increase in SIRT1. Taken together, we propose that thymoquinone inhibits cellular ROS generation in LPS-activated BV2 microglia. It is also suggested that activation of both AMPK and NAD+/SIRT1 may contribute to the anti-inflammatory, but not antioxidant activity of the compound in BV2 microglia.

AMPK expression patterns are significantly associated with poor prognosis in breast cancer patients.

Many investigators have examined the functions of AMP-activated protein kinase (AMPK) in cancer biology and its anti-neoplastic features in cancer models. The goal of this research is to assess the association of the immunohistochemical expression of AMPK in human mammary tumours with the clinical data of breast cancer patients. 449 cases of previously diagnosed breast cancer, and 27 tissue samples of fibroadenomas and normal breast were utilized for detection of AMPK expression using tissue microarrays and immunohistochemistry. Brownish nuclear and cytoplasmic staining were present in epithelial cells and stromal cells in 333 (74.16%) and 348 (77.5%) cancer cases respectively indicating AMPK expression. Twenty two (81.48%) control cases showed AMPK immunoexpression in both epithelial and stromal cells. Significant statistical association has been found between advanced stages of breast cancer and increased intensity of AMPK immunostaining only in epithelial cells (p-value=0.0001). Histotypes have been correlated with AMPK immunostaining in epithelial cells only (p-value=0.029). Low AMPK immunostaining scores were more dominant in DCIS, ductal and mixed type's ductal and mucinous histotypes, while high intense staining was more common in the lobular type. Furthermore, breast tumour cases with lymph node metastases showed significant AMPK expression in both epithelial and stromal cells (p-value=0.0001 and p-value=0.026). Low scores of AMPK immunostaining were common in breast cancer cases with positive vascular invasion (p-value=0.007) and disease recurrence (p-value=0.008). No significant differences in survival behavior distributions were observed for the different categories of AMPK immunostaining in epithelial and stromal cells. In conclusion, our results showed decreased AMPK expression in breast cancer in comparison with the control group. AMPK expression was significantly correlated with some clinicopathological factors like advanced stage, lymph node involvement, vascular invasion and disease recurrence which give indications for poor clinical outcomes. Immunohistochemical staining of AMPK protein is a valuable method which could predict cases of breast cancer with poor prognosis.

Effects of preventative moxibustion on AMPK and mTOR in myocardial tissue in rats with exhaustive exercise

To observe the effects of preventative moxibustion on adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in myocardial tissue in rats with exhaustive exercise, and to explore the action mechanism of preventative moxibustion for myocardial injury by exhaustive exercise. Eighteen SD male rats were randomly divided into a blank group, an exhaustive exercise group and a moxibustion group, 6 rats in each one. Rats in the blank group were treated with immobilization for 5 min per day, without any intervention. Rats in the exhaustive exercise group were treated with no intervention in the first 10 days. Rats in the moxibustion group were treated with moxibustion at "Zusanli" (ST 36) and "Guanyuan" (CV 4), 5 cones for each acupoint, for 10 days.On 11th day, rats in the exhaustive exercise group and moxibustion group were sacrificed to collect sample after exhaustive swimming, and time of exhaustive exercise was recorded. HE staining was used to observe the inflammatory changes of myocardial tissue; colorimetric method was used to measure lactic dehydrogenase (LDH), activity of superoxide dismutase (SOD) and malonaldehyde (MDA); immune suppression method was used to measure the content of creatine kinase isoenzyme (CK-MB) in serum; Elisa method was used to measure the content of troponinT (cTnT) in serum; western blot method was applied to measure the content of AMPKα2 and mTOR in myocardial tissue. The time of exhaustive exercise was (4 831.17±689.88) s in the moxibustion group, which was longer than (3 509.50±1 232.49) s in the exhaustive exercise group (P<0.05); HE staining indicated structure of the myocardium was clear and inflammatory cell infiltration was reduced in the moxibustion group. Compared with the blank group, the contents of serum LDH, CK-MB, cTnT were increased in the exhaustive exercise group (all P<0.05); MDA content was increased and SOD activity was decreased (both P<0.05); the AMPK α2 content in myocardial tissue was increased and the mTOR content was decreased (both P<0.05). Compared with the exhaustive exercise group, the contents of serum LDH, CK-MB, cTnT were reduced in the moxibustion group (all P<0.05); MDA content was decreased and SOD activity was increased (both P<0.05); the AMPKα2 content in myocardial tissue was increased, and the mTOR content was decreased (both P<0.05); AMPKα2 and mTOR were negatively correlated (r=-0.764, P<0.05). Preventative moxibustion is likely to regulate the expression of AMPK and mTOR to induce signaling pathway to recover myocardial injury by exhaustive exercise.

Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and carcinogenesis in tubular epithelial cells, which may be largely attenuated by renin-angiotensin system blockade, implying the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

The Role of O-GlcNAcylation in Perivascular Adipose Tissue Dysfunction of Offspring of High-Fat Diet-Fed Rats

Perivascular adipose tissue (PVAT), which reduces vascular contractility, is dysfunctional in the male offspring of rats fed a high-fat diet (HFD), partially due to a reduced NO bioavailability. O-GlcNAcylation of eNOS decreases its activity, thus we investigated the role of O-GlcNAcylation in the prenatal programming of PVAT dysfunction. Female Sprague-Dawley rats were fed either a control (10% fat) or an obesogenic HFD (45% fat) diet for 12 weeks prior to mating, and throughout pregnancy and lactation. Offspring were weaned onto the control diet and were killed at 12 and 24 weeks of age. Mesenteric arteries from the 12-week-old offspring of HFD dams (HFDO) contracted less to U46619; these effects were mimicked by glucosamine in control arteries. PVAT from 12- and 24-week-old controls, but not from HFDO, exerted an anticontractile effect. Glucosamine attenuated the anticontractile effect of PVAT in the vessels from controls but not from HFDO. AMP-activated protein kinase (AMPK) activation (with A769662) partially restored an anticontractile effect in glucosamine-treated controls and HFDO PVAT. Glucosamine decreased AMPK activity and expression in HFDO PVAT, although phosphorylated eNOS expression was only reduced in that from males. The loss of anticontractile effect of HFDO PVAT is likely to result from increased O-GlcNAcylation, which decreased AMPK activity and, in males, decreased NO bioavailability.

Dihydromyricetin prevents obesity-induced slow-twitch-fiber reduction partially via FLCN/FNIP1/AMPK pathway

Obesity is often accompanied by decreases in the proportion of skeletal muscle slow-twitch fibers and insulin sensitivity. Increased plasma non-esterified fatty acids (NEFA) levels are responsible for obesity-associated insulin resistance. Palmitate, one of the most elevated plasma NEFA in obesity, has been recognized as the principle inducer of insulin resistance. The present study showed that increased plasma NEFA levels were negatively linked to slow-twitch fiber proportion and insulin sensitivity, while slow-twitch fiber proportion was positively correlated to insulin sensitivity in high fat diet (HFD)-fed and ob/ob mice. Dihydromyricetin (DHM) intervention increased slow-twitch fiber proportion and improved insulin resistance. In cultured C2C12 myotubes, palmitate treatment resulted in decrease of slow-twitch fiber specific Myh7 expression and insulin resistance, concomitant with folliculin (FLCN) and folliculin-interacting protein 1 (FNIP1) expression increase, AMP-activated protein kinase (AMPK) inactivation and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression decrease. Those palmitate-induced effects could be blocked by knock-down of FLCN expression or DHM intervention. Meanwhile, the protective effects of DHM were alleviated by over-expression of FLCN. In addition, the changes in AMPK activity and expression of FLCN and FNIP1 in vivo were consistent with those occurring in vitro. These findings suggest that DHM treatment prevents palmitate-induced slow-twitch fibers decrease partially via FLCN-FNIP1-AMPK pathway thereby improving insulin resistance in obesity.

Protectin DX suppresses hepatic gluconeogenesis through AMPK-HO-1-mediated inhibition of ER stress

Several studies have shown that protectins, which are ω-3 fatty acid-derived proresolution mediators, may improve insulin resistance. Recently, protectin DX (PDX) was documented to attenuate insulin resistance by stimulating IL-6 expression in skeletal muscle, thereby regulating hepatic gluconeogenesis. These findings made us investigate the direct effects of PDX on hepatic glucose metabolism in the context of diabetes. In the current study, we show that PDX regulates hepatic gluconeogenesis in a manner distinct from its indirect glucoregulatory activity via IL-6. We found that PDX stimulated AMP-activated protein kinase (AMPK) phosphorylation, thereby inducing heme oxygenase 1 (HO-1) expression. This induction blocked hepatic gluconeogenesis by suppressing endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. These effects were significantly dampened by silencing AMPK or HO-1 expression with small interfering RNA (siRNA). We also demonstrated that administration of PDX to high fat diet (HFD)-fed mice resulted in increased hepatic AMPK phosphorylation and HO-1 expression, whereas hepatic ER stress was substantially attenuated. Furthermore, PDX treatment suppressed the expression of gluconeogenic genes, thereby decreasing blood glucose levels in HFD-fed mice. In conclusion, our findings suggest that PDX inhibits hepatic gluconeogenesis via AMPK-HO-1-dependent suppression of ER stress. Thus, PDX may be an effective therapeutic target for the treatment of insulin resistance and type 2 diabetes through the regulation of hepatic gluconeogenesis.

Upregulation of connexin 43 and apoptosis‑associated protein expression by high glucose in H9c2 cells was improved by resveratrol via the autophagy signaling pathway.

The expression of connexin43 (Cx43) protein and the apoptotic rate of cardiomyocytes may be regulated by autophagy and associated with diabetic cardiomyopathy. It is possible that the beneficial effect of resveratrol on diabetic cardiomyocytes occurs via the autophagy pathway. However, it remains to be elucidated whether resveratrol treatment may attenuate the hyperglycemia-induced remodeling of Cx43 and apoptosis through the regulation of autophagy. H9c2 cardiac cells were incubated with 5.5 and 25 mM glucose, 25 mM glucose with chloroquine (50 µM), and 25 mM glucose with or without resveratrol (10, 25 µM) for 24 h. H9c2 cells were also incubated with 25 µM resveratrol in the presence of chloroquine (50 µM). Cell viability was determined using an MTT cell survival assay. Cytotoxicity was determined by quantification of the release of lactate dehydrogenase. The expression of Cx43, autophagic maker proteins [Beclin-1, p62 and microtubule-associated protein 1 light chain 3 (LC3)], apoptosis maker proteins (B-cell lymphoma-2 and Bcl-2 associated X protein), AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were determined using western blotting. Resveratrol treatment led to reduced Cx43 expression levels compared with the 25 mM glucose treatment and significantly reduced the expression of apoptosis-associated proteins in H9c2 cells under hyperglycemic conditions. Autophagy was increased as indicated by the upregulation of Beclin-1 and p62 expression and the reduced LC3-II/LC3-I ratio. AMPK expression was increased, whereas mTOR expression was reduced in the resveratrol treatment groups. Treatment with chloroquine reversed effect of resveratrol. In conclusion, administration resveratrol may protect H9c2 cells against hyperglycemia-induced Cx43 upregulation and apoptosis, which may be mediated through the induction of the autophagy signaling pathway.

Grape seed procyanidin B2 ameliorates hepatic lipid metabolism disorders in db/db mice.

Diabetes is commonly associated with liver lipid metabolism disorders. AMP-activated protein kinase (AMPK) has a key role in regulating lipid metabolism. Grape seed procyanidin B2 (GSPB2), a natural polyphenol polymer, ameliorates mitochondrial dysfunction and inhibits oxidative stress or apoptosis via AMPK pathways. In the present study, the hypothesis that GSPB2 treatment may ameliorate liver lipid metabolic disorders by activating AMPK and downstream pathways was tested in diabetic mice. Db/m mice were used as controls, and diabetic db/db mice were randomly divided into 2 groups for treatment: Vehicle and GSPB2 (30mg/kg/day for 10weeks). Animals were weighed every week. Fasting blood was collected prior to sacrifice to measure fasting blood glucose (FBG), triglycerides (TG) and total cholesterol (TC). Hepatic TG and free fatty acid (FFA) levels were analyzed. Hepatic sections were examined by light microscopy following hematoxylin and eosin staining. The expression of hepatic AMPK, phosphorylated acetyl‑CoA carboxylase (ACC), carnitine palmitoyl transferase 1 (CPT1) and 4‑hydroxynonenal (4‑HNE) was measured by western blot analysis. Liver mitochondria were isolated to assess electron transport complex I (CI), complex II (CII) and complex IV by high-resolution respirometry. The results demonstrated that GSPB2 significantly decreased body weight and serum TG, TC and FFA levels, but not FBG levels in diabetic mice. GSPB2 visibly decreased lipid droplet accumulation in the liver and significantly reduced hepatic TG and FFA levels. In diabetic mice, GSPB2 restored liver AMPK and ACC phosphorylation, increased CPT1 protein expression, ameliorated lipid peroxidation damage, which was assessed by comparing 4‑HNE levels, and partially restored the damaged mitochondrial respiratory capacity of CI and CII in the liver. In conclusion, long‑term oral treatment with GSPB2 may benefit hepatic lipid metabolism disorders, potentially by decreasing hepatic lipid synthesis and increasing hepatic FFA β‑oxidation via the AMPK‑ACC pathway.

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.

AMP-activated protein kinase mediates the effects of lipoprotein-associated phospholipase A2 on endothelial dysfunction in atherosclerosis.

The present study aimed to investigate the effects of lipoprotein-associated phospholipase A2 (Lp-PLA2) on endothelial dysfunction in an in vitro cell model of atherosclerosis, and to determine whether AMP-activated protein kinase (AMPK) mediates the effects of Lp-PLA2 on endothelial dysfunction. A total of 392 patients with coronary artery disease (CAD), including various sub-conditions, were recruited, and the plasma concentrations of Lp-PLA2 were evaluated. In addition, an in vitro model of atherosclerosis was established by exposing human umbilical vein endothelial cells (HUVECs) to oxidized low-density lipoprotein (oxLDL). SB-435495 was used to inhibit Lp-PLA2, and compound C was used to suppress AMPK expression. Lp-PLA2, AMPKα and phosphorylated-AMPKα (T172) expression in HUVECs were evaluated using western blot analysis. The concentrations of nitric oxide (NO), endothelin 1 (ET-1), intercellular adhesion molecule 1 (ICAM-1) and platelet/endothelial cell adhesion molecule 1 (PECAM-1) in cell culture supernatant were determined using commercially available ELISA kits. MTT assays were employed to indicate changes in cell viability. The current study found the plasma Lp-PLA2 levels were elevated in the CAD patients with stable angina pectoris, unstable angina pectoris, acute coronary syndromes and acute myocardial infarction, compared with a healthy control population. In addition, the in vitro results showed that Lp-PLA2 expression levels were elevated in oxLDL-exposed HUVECs. Lp-PLA2 suppression could increase cell viability, induce the production of NO and decrease the secretion of ET-1, in addition to suppressing the expression of cell adhesion molecules, including ICAM-1 and PECAM-1 in oxLDL-exposed HUVECs. The expression of AMPKα and phosphorylated-AMPKα (T172) was regulated by Lp-PLA2, and AMPK suppression was able to reverse the effects of Lp-PLA2 with regard to cell viability, endothelial vasorelaxation capacity and the secretion of adhesion molecules in oxLDL-exposed HUVECs. In conclusion, the present study provides initial evidence that Lp-PLA2 is able to cause endothelial dysfunction in an in vitro model of atherosclerosis, and the effects of Lp-PLA2 on endothelial dysfunction was at least partially a result of the downregulation of AMPKα, thus contributing to the progression of atherosclerosis.

Abstract WP296: Short Term Acute Preconditioning Exercise Improves Stroke Outcomes and Reduces Neurovascular Injury via Increased Endothelial Nitric Oxide Synthase Activity

Physical exercise is becoming a prominent therapeutic strategy to improve stroke outcomes. It was previously shown that long term exercise reduces infarct volume, but this was never tested in acute short term preconditioning exercise in a thromboembolic model of stroke. Exercise induced shear stress improves vascular function through increased activity of endothelial nitric oxide synthase (eNOS) and its upstream principal activator, AMP-activated protein kinase (AMPK). However, this was not previously tested in a stroke setting. Accordingly, we tested the hypothesis that acute short term preconditioning exercise improves stroke outcomes through increased AMPK and eNOS activity. Methods: Male Wistar rats (300g) were subjected to treadmill exercise for four days (25 minutes/day), break for 2 days and then one acute bout for 30 minutes. Exercised animals were subjected to thromboembolic stroke at 1h, 6h or 24h after the last exercise session. At 24h, control (sedentary) and exercised rats were tested for neurological outcomes, infarct size and edema. Western Blotting was used to measure the expression of active eNOS (p-S1177-eNOS) and active AMPK (p-T172-AMPK). Results: Acute exercise significantly reduced infarct size and edema and improved functional outcomes compared to control. It also significantly increased the expression of peNOS and pAMPK in the brain, cerebral vessels and aorta (Table). Conclusion: Acute exercise preconditioning significantly reduced neurovascular injury and improved functional outcome after stroke through increased eNOS activity. Our findings are novel to point out the role of preconditioning exercise induced AMPK and eNOS activation in improving stroke outcomes.

Chronic metformin treatment facilitates seizure termination

The AMP-activated protein kinase (AMPK) is a key energy sensor. Its activator metformin could suppress epileptogenesis in the pentylenetetrazol (PTZ) kindling model. However, the effect of metformin on the acute and chronic seizures has not been studied. We first detected the expression of AMPK in the brain tissue of human and mice with chronic seizures, as well as in mice with acute seizures. Second, using behavioral assay and local filed potentials (LFPs) recording, we investigated the effect of chronic metformin treatment on seizures in a acute seizure model and a chronic seizure model. Our results showed that AMPK was expressed in neurons in the epileptic brain. The expression level was decreased in the brain tissue that experienced chronic and acute seizures. In PTZ-induced acute seizures model, behavioral assay showed that chronic metformin treatment decreased the mortality, and LFPs recording showed that chronic metformin treatment shortened the duration of generalized tonic-clonic seizures and prolonged the duration of postictal depression. Moreover, in kainic acid-induced chronic seizures model, LFPs recording showed that chronic metformin treatment shortened the duration of epileptic activity. Our study suggests that chronic metformin treatment could facilitate seizure termination.

α-Synuclein binds and sequesters PIKE-L into Lewy bodies, triggering dopaminergic cell death via AMPK hyperactivation

The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson's disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP+), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKE-L's neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.