AMP-activated Protein Kinase Α1 Research Articles

Blocking of tripartite motif 8 protects against lipopolysaccharide (LPS)-induced acute lung injury by regulating AMPKα activity

Acute lung injury (ALI) and its more serious form, respiratory distress syndrome (ARDS), are considered as an acute and severe inflammatory process existing in lungs, and still remain high mortality rates. Tripartite motif 8 (TRIM8) contains an N-terminal RING finger, which is followed by two B-boxes and a coiled-coil domain, belonging to the TRIM/RBCC family and playing significant role in meditating inflammation, oxidative stress and apoptosis. In the study, we investigated the role of TRIM8 in ALI induced by lipopolysaccharide (LPS) and the underlying molecular mechanisms. The in vitro results indicated that LPS time-dependently enhanced TRIM8 expression in lung epithelial cells. Suppressing TRIM8 markedly ameliorated LPS-elicited inflammatory response, as evidenced by the down-regulated mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in cells mainly through inactivating nuclear factor-kappa B (NF-κB) signaling pathway; however, over-expressing TRIM8 markedly promoted inflammation in LPS-challenged cells. In addition, LPS-induced oxidative stress was accelerated by TRIM8 over-expression, while being alleviated by TRIM8 knockdown by regulating Nrf2 signaling. Importantly, TRIM8 could negatively meditate AMP-activated protein kinase-α (AMPKα) activation to modulate LPS-triggered inflammatory response and ROS generation in vitro. Additionally, our in vivo findings suggested that TRIM8 knockdown effectively attenuated LPS-induced lung injury nu decrease of lung wet/dry (W/T) ratio, protein concentrations, neutrophil infiltration, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) production and superoxide dismutase (SOD) depletion. Meanwhile, the loss of TRIM8 markedly lessened IL-1β, IL-6 and TNF-α expression in lung tissues of LPS-challenged mice, and reduced NF-κB phosphorylation. Furthermore, TRIM8 knockdown evidently improved nuclear factor-erythroid 2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions in lung of LPS-treated mice. The anti-inflammation and anti-oxidant role of TRIM8-silence might be associated with AMPKα phosphorylation. Together, our study firstly provided a support that TRIM8 knockdown effectively protected LPS-induced ALI against inflammation and oxidative stress largely dependent on the promotion of AMPKα pathway.

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

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

Differential Role of Hypothalamic AMPKα Isoforms in Fish: an Evolutive Perspective.

In mammals, hypothalamic AMP-activated protein kinase (AMPK) α1 and α2 isoforms mainly relate to regulation of thermogenesis/liver metabolism and food intake, respectively. Since both isoforms are present in fish, which do not thermoregulate, we assessed their role(s) in hypothalamus regarding control of food intake and energy homeostasis. Since many fish species are carnivorous and mostly mammals are omnivorous, assessing if the role of hypothalamic AMPK is different is also an open question. Using the rainbow trout as a fish model, we first observed that food deprivation for 5days did not significantly increase phosphorylation status of AMPKα in hypothalamus. Then, we administered adenoviral vectors that express dominant negative (DN) AMPKα1 or AMPKα2 isoforms. The inhibition of AMPKα2 (but not AMPKα1) led to decreased food intake. The central inhibition of AMPKα2 resulted in liver with decreased capacity of use and synthesis of glucose, lipids, and amino acids suggesting that a signal of nutrient abundance flows from hypothalamus to the liver, thus suggesting a role for central AMPKα2 in the regulation of peripheral metabolism in fishes. The central inhibition of AMPKα1 induced comparable changes in liver metabolism though at a lower extent. From an evolutionary point of view, it is of interest that the function of central AMPKα2 remained similar throughout the vertebrate lineage. In contrast, the function of central AMPKα1 in fish relates to modulation of liver metabolism whereas in mammals modulates not only liver metabolism but also brown adipose tissue and thermogenesis.

Insulin signaling in LepR cells modulates fat and glucose homeostasis independent of leptin.

Hypothalamic neurons detect changes in circulating hormones such as leptin and insulin and put forward outputs to sustain energy and glucose homeostasis. Because leptin and insulin receptors colocalize in ~40-60% of neurons in the hypothalamus, we characterized the metabolic phenotype of mice with selective deletion of the insulin receptor (InsR) in LepR cells. LRΔInsR mice presented no difference in body weight and insulin levels but increased fat mass. In the light phase, LRΔInsR mice exhibited increased food intake, locomotor activity, carbon dioxide production, and respiratory exchange rate. These mice showed reduced fat oxidation and reduced expression of cluster of differentiation 36 and AMP-activated protein kinase-α1 in the liver, increased glucose oxidation in the light phase, and overall reduced basal glucose levels. To verify the impact of InsR deletion in LepR cells in obesity, we generated ob/ ob InsRfl, ob/ ob LRcre, and ob/ ob LRΔInsR mice. The ob/ ob LRΔInsR mice had higher body weight, fat mass, and expression of genes related to fat metabolism in the liver. No difference in food intake despite increased neuropeptide Y and agouti-related peptide expression, and no difference in energy expenditure, fat, or glucose oxidation was found in ob/ ob LRΔInsR compared with LRcre or LRΔInsR controls. Remarkably, basal glucose levels were reduced, and the expression of genes associated with glucose metabolism in the liver was higher. Insulin signaling in LepR cells is required for the proper fat and glucose oxidation. These effects are independent of leptin given that the leptin-deficient ob/ ob LRΔInsR mice also presented reduced glycemia and higher adiposity. The mechanisms underlying these responses remain to be unveiled.

Control of hepatic gluconeogenesis by Argonaute2

ObjectiveThe liver performs a central role in regulating energy homeostasis by increasing glucose output during fasting. Recent studies on Argonaute2 (Ago2), a key RNA-binding protein mediating the microRNA pathway, have illustrated its role in adaptive mechanisms according to changes in metabolic demand. Here we sought to characterize the functional role of Ago2 in the liver in the maintenance of systemic glucose homeostasis. MethodsWe first analyzed Ago2 expression in mouse primary hepatocyte cultures after modulating extracellular glucose concentrations and in the presence of activators or inhibitors of glucokinase activity. We then characterized a conditional loss-of-function mouse model of Ago2 in liver for alterations in systemic energy metabolism. ResultsHere we show that Ago2 expression in liver is directly correlated to extracellular glucose concentrations and that modulating glucokinase activity is adequate to affect hepatic Ago2 levels. Conditional deletion of Ago2 in liver resulted in decreased fasting glucose levels in addition to reducing hepatic glucose production. Moreover, loss of Ago2 promoted hepatic expression of AMP-activated protein kinase α1 (AMPKα1) by de-repressing its targeting by miR-148a, an abundant microRNA in the liver. Deletion of Ago2 from hyperglycemic, obese, and insulin-resistant Lepob/ob mice reduced both random and fasted blood glucose levels and body weight and improved insulin sensitivity. ConclusionsThese data illustrate a central role for Ago2 in the adaptive response of the liver to fasting. Ago2 mediates the suppression of AMPKα1 by miR-148a, thereby identifying a regulatory link between non-coding RNAs and a key stress regulator in the hepatocyte.

Fenofibrate improves vascular endothelial function and contractility in diabetic mice

Fenofibrate, a peroxisome proliferator-activated receptors α (PPARα) agonist, reduces vascular complications of diabetic patients but its protective mechanisms are not fully understood. Here we tested the hypothesis that fenofibrate improves vascular endothelial dysfunction by balancing endothelium-dependent relaxation and contractility of the aorta in diabetes mellitus (DM). In streptozotocin-induced diabetic mice, eight weeks of fenofibrate treatment (100 mg/Kg/d) improved endothelium dependent relaxation in the macro- and microvessels, increased nitric oxide (NO) levels, reduced renal damage markers and effects of the vasoconstrictor prostaglandin. Levels of superoxide dismutase and catalase were both reduced and hydrogen peroxide was increased in vehicle-treated DM, but these changes were reversed by fenofibrate treatment. Vasodilation of the aorta after fenofibrate treatment was reversed by PPARα or AMPKα inhibitors. Western blots showed that fenofibrate treatment elevated PPARα expression, induced liver kinase B1 (LKB1) translocation from the nucleus to the cytoplasm and activated AMP-activated protein kinase-α (AMPKα), thus activating endothelial NO synthase (eNOS). Also, fenofibrate treatment decreased NF-κB p65 and cyclooxygenase 2 proteins in aortas. Finally, incubation with indomethacin in vitro improved aortic contractility in diabetic mice. Overall, our results show that fenofibrate treatment in diabetic mice normalizes endothelial function by balancing vascular reactivity via increasing NO production and suppressing the vasoconstrictor prostaglandin, suggesting mechanism of action of fenofibrate in mediating diabetic vascular complications.

Regulation of mitochondrial biosynthesis and function by dietary carbohydrate levels and lipid sources in juvenile blunt snout bream Megalobrama amblycephala

This study aimed to investigate the effects of dietary non-protein energy adjustments on the mitochondrial biosynthesis and function of juvenile Megalobrama amblycephala. Fish (average weight: 37.98 ± 0.07 g) were fed eight diets containing two dietary carbohydrate levels (30% and 43%) and four lipid sources (fish oil, soybean oil, palm oil and the mixed oil) for 11 weeks. Liver mitochondrial respiratory chain complex V activity and ATP (adenosine triphosphate) content both increased significantly with increasing dietary carbohydrate levels, whereas the opposite was true for the AMP (adenosine 5′-monophosphate)/ATP ratio, hepatic transcripts of AMP-activated protein kinase α1 (AMPKα1), AMPKα2, peroxisome proliferators γ-activated receptor coativator-1α (PGC-1α), NADH dehydrogenase 1 and cytochrome c oxidase 1 (COX1) as well as the activities of Na+-K+-ATPase, succinate dehydrogenase (SDH), citrate synthase (CS) and mitochondrial respiratory chain complex I, III and IV. Additionally, hepatic ATP content, the transcripts of AMPKα, COX1 and ATP6 and the activities of Na+-K+-ATPase, SDH, CS and mitochondrial respiratory chain complex III were all significantly affected by lipid sources. Furthermore, an interaction between dietary carbohydrate levels and lipid sources was also observed in the activities of liver mitochondrial Na+-K+-ATPase and respiratory chain complex III as well as the transcripts of ATP6 and PGC-1α. Overall, these findings suggested that dietary carbohydrate levels and lipid sources remarkably affected the mitochondrial biosynthesis and function of M. amblycephala. A diet containing 30% carbohydrate and FO could boost its mitochondrial biosynthesis, while that of 30% carbohydrate and SO could enhance the mitochondrial function.

Abstract P196: Fenofibrate Improves Vascular Contractility and Endothelial Function in Diabetic Mice

Aim: To exam the hypothesis that fenofibrate improves vascular endothelial dysfunction via balancing endothelium-dependent relaxation and constriction of aorta in diabetic mice. Methods: The streptozotocin-induced diabetic mice were treated with fenofibrate (100 mg/Kg/d, 8 weeks). The responses were assessed from serum and aortic parameters, vascular reactivity by wire myograph and aortic protein expression of endothelial nitric oxide synthase (eNOS), peroxisome proliferator-activated receptor α (PPARα), AMP-activated protein kinase-α (AMPKα) and liver kinase B1 (LKB1) and cyclooxygenase-2 (COX-2) in control and diabetic mice given placebo or fenofibrate. Results: Comparing with the control mice (12.2±2.0 μmol/L), serum creatinine increased by 114.8% in diabetic mice (26.2±2.0 μmol/L) and by 66.2% in fenofibrate-treated diabetic mice (21.6±2.8 μmol/L, p<0.01). NO level in aorta was 49.5±8.2 (μmol/g) in diabetic mice and 71.7±5.2 (μmol/g) in fenofibrate-treated diabetic mice (p<0.01). Compared with placebo-treated diabetic group, phosphorylated-eNOS protein in aorta was 3.6±1.0 fold higher (p<0.01) and endothelium-dependent relaxation was increased in fenofibrate-treated diabetic mice (p<0.01). The aorta vasodilation by fenofibrate treatment was reversed with PPARα inhibitor or AMPKα inhibitor. Fenofibrate treatment elevated PPARα expression, subsequently induced LKB1 translocation from nucleus to cytoplasm to activate AMPKα, thus activated eNOS in diabetic aorta (p<0.01). Fenofibrate treatment reduced vascular contractility and expression of COX-2 in aorta from diabetic mice(p<0.01). Prostaglandin E2 and thromboxane A2 were 174.9±16.4 and 128.9±4.4 (pg/ml) in diabetic mice, and reduced to 142.7±14.1 and 105.9±5.7 (pg/ml) in fenofibrate-treated mice (p<0.01). Conclusion: Fenofibrate treatment in diabetic mice normalizes endothelial function by balancing vascular reactivity via increasing NO production and suppressing vasoconstrictor prostaglandin. Our results suggest that implication of fenofibrate as a putative mediator of diabetic vascular complications.

AMPK contributes to autophagosome maturation and lysosomal fusion

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK α1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK α1 knockout cells. The transient expression of AMPK α1 can rescue autophagosome maturation. These results indicate that AMPK α1 is required for efficient autophagosome maturation and lysosomal fusion.

Effect of microRNA-26a on vascular endothelial cell injury caused by lower extremity ischemia-reperfusion injury through the AMPK pathway by targeting PFKFB3.

Vascular endothelial cell (VEC) dysfunction plays an important role in the ischemia-reperfusion injury (IRI)-related diseases, and microRNAs (miRNAs) are key factors during this process. We conducted this study to investigate whether miRNA-26a (miR-26a) has effect on the IRI-induced VEC injury via the AMPK pathway by targeting 6-phosphofructo-2-kinase-fructose-2,6-biphosphatase 3 (PFKFB3). IRI rat models were successfully constructed by an abdominal incision. Additionally, the cultured VECs were further treated with miR-26a mimic or inhibitor, and si-PFKFB3. Both the reverse-transcription quantitative polymerase chain reaction and the western blot assay method were carried out to examine the expressions of PFKFB3, endothelial nitric oxide synthase (eNOS), and 5'-adenosine monophosphate-activated protein kinase (AMPK) α1, as well as the extent of the AMPK α1 phosphorylation levels in vascular tissues. Circulating endothelial cell (CEC), von Willebrand factor (VWF), thrombomodulin (TM), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), and endothelin (ET) were all measured. In the rat model of an IRI, a poorly expressed miR-26a and contrarily highly expressed PFKFB3 were identified in vascular tissues. In response to an overexpression of miR-26a or to the PFKFB3 gene silencing, decreased CEC number, TM, VWF, MDA, and ET contents, increased AMPK α1, and eNOS levels, as well as the extent of AMPK α1 phosphorylation coordinate with both increased SOD and NO contents based on the restoration of the AMPK pathway. Overexpression of the miR-26a or si-PFKFB3 provides an elevation in cell proliferation. Our study suggests that the miR-26a RNA alleviates lower extremity IRI-induced VEC injury in rats through the activation of the AMPK pathway by inhibiting PFKFB3.

Herb-partitioned moxibustion alleviates colon injuries in ulcerative colitis rats.

AIMTo observe the effect of herb-partitioned moxibustion (HPM) on expression of colonic cytokines in ulcerative colitis (UC) rats.METHODSA UC rat model was established by protein immunization in combination with topical chemical stimulation. Rats in the HPM group (n = 8) received HPM at bilateral Tianshu (ST25) points. The gross injury and pathological scores of the colon were recorded. The expression profile of colonic cytokines was assayed using the protein microarray technique. Specific differential cytokines were selected and verified by ELISA. The corresponding UniProt Accessions of the differentially expressed cytokines were retrieved in the UniProt database. The pathways involved were analyzed with the help of the KEGG PATHWAY database. The DAVID database was used for functional cluster and pathway analysis.RESULTSHPM improved colon injuries in UC rats, manifested by accelerated repair of ulcers and alleviation of inflammation, and the gross injury and pathological scores both significantly decreased (P < 0.01). Fold change > 1.3 or < 0.77 was taken as the screening standard. There were 77 down-regulated and 9 up-regulated differentially expressed colonic cytokines in the HPM group compared with the model group, and expression of 20 differed significantly (P < 0.05). Twelve of the 20 significantly differentially expressed cytokines [β-catenin, interleukin-1 receptor 6 (IL-1R6), IL-1β, B7-1, nerve growth factor receptor, AMP-activated protein kinase-α1, neuropilin-2, orexin A, adipocyte differentiation-related protein, IL-2, Fas and FasL] were up-regulated in the model group (n = 3, compared with the normal group) but down-regulated in the HPM group (n = 3, compared with the model group). Functional cluster analysis showed that the differentially expressed colonic cytokines in the HPM group regulated apoptosis and protein phosphorylation. KEGG pathway analysis showed that 52 down-regulated and 7 up-regulated differentially expressed colonic cytokines in the HPM group had pathways. The pathways that interacted between the cytokines and their receptors accounted for the largest proportion (28 of the down-regulated and 5 of the up-regulated cytokines).CONCLUSIONHPM promotes the repair of colon injuries in UC rats, which is related to the regulation of several abnormally expressed cytokines.

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

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

Pinosylvin enhances leukemia cell death via down-regulation of AMPKα expression.

Resveratrol at high concentrations (50-100μmol/L) is known to induce cell death in leukemia cells. Here, we investigated whether pinosylvin, a resveratrol analogue, induced cell death in leukemia cells. Cell death was found to be markedly elevated by 50- to 100-μmol/L pinosylvin in THP-1 and U937 cells. It was also shown that pinosylvin induced caspase-3 activation, flip-flop of phosphatidylserine, LC3-II accumulation, LC3 puncta, and p62 degradation in both THP-1 and U937 cells. These data indicate that pinosylvin-induced cell death may occur through apoptosis and autophagy. In addition, we showed that pinosylvin down-regulates AMP-activated protein kinase α1 (AMPKα1) in leukemia cells. Therefore, we correlated AMPKα1 down-regulation and leukemia cell death. AMPKα1 inhibition appeared to decrease pinosylvin-induced apoptosis and autophagy in leukemia cells, implying that AMPK is a key regulator of leukemia cell death. Moreover, we found that both pinosylvin-induced autophagy and apoptotic progress were reduced in AMPKα1-overexpressed leukemia cells, when compared with vector-transfected cells. Cell death was elevated by AMPKα1 overexpression, whereas pinosylvin-induced cell death was markedly decreased by caspase-3 inhibitors or autophagy inhibitors. These results suggest that pinosylvin-induced depletion of AMPKα1 enhances cell death via apoptosis and autophagy in leukemia cells.

The changes of HIF-1α and AMPKα2 expression levels during skeletal muscle blunt injury recovery

To explore the possible biological mechanisms of skeletal muscle injury recovery by observing the changes of AMP-activated protein kinase α2 (AMPKα2) and hypoxia inducible factor-1α (HIF-1α) expression levels of rats in the skeletal muscle blunt injury recovery process. Six of the 48 male Wistar rats was randomly selected as the normal control group. The blunt injury model was set up by injuring the hind legs of remaining 42 rats with heavy objects. Then they were divided into 7 groups (n=6). The changes of AMPKα2 and HIF-1α expression levels were tested in the hind limb triceps surae of the model rats from each of the 7 groups at 12 hours, 2 days, 5 days, 7 days, 10 days, 15 days and 30 days respectively following the injury. The expression levels of AMPKα2 and HIF-1α all increased significantly at 12 hours following the injury and fell close to normal levels at 15 days following the injury. The values of AMPKα2 and HIF-1α expression peaked within 2 days after injury and the expression levels began to decline at 5 days after injury. Except the peak, the changes of the mRNA expressions of the two proteins were basically consistent with those of protein expression at the other time points. HIF-1α and AMPKα2 might play roles in mediating hypoxia adaptation, muscle cell regeneration, and energy compensation to promote recovery after injury.

AMP-activated protein kinase α1 in Megalobrama amblycephala: Molecular characterization and the transcriptional modulation by nutrient restriction and glucose and insulin loadings

This study aimed to characterize the full-length cDNA of AMP-activated protein kinase α1 (AMPKα1) from Megalobrama amblycephala and investigate the transcriptional response of this kinase to nutrient restriction and glucose and insulin loadings. The cDNA obtained was 3545-bp long with an open reading frame of 1710 bp encoding 570 amino acids. Multiple alignments and phylogenetic analyses revealed a high degree of conservation (80–100%) among most fish, retaining one kinase domain (KD), one auto-inhibitory domain (AID), one C-terminal domain (α-CTD), one regulatory-subunit-interacting motif (α-RIM), one serine/threonine-rich loop (ST loop), one α-hook, and several phosphorylation sites. AMPKα1 mRNA was predominantly expressed in white muscle, gill, and brain tissues, whereas little was expressed in the intestines. After a fasting-refeeding trial, phosphorylation and mRNA levels of AMPKα1 were significantly greater in fish fasted for 10 days, while in re-fed fish at 1 h after re-feeding, the levels of this kinase were intermediate between those of the fish in the fed and fasted groups. Further, AMPKα1 mRNA levels were quantified in the liver and muscle tissues of fish injected intraperitoneally with 1.67 g glucose per kg body weight and 0.052 mg insulin per kg body weight, respectively. Glucose and insulin administration resulted in a significant decrease in AMPKα1 expression in both tissues with minimum values attained at 2 h and 4 h after injection, respectively. Thereafter, the expression increased significantly to the basal value at 24 h after injection, except in the liver in which the maximum value was obtained at 12 h post-glucose injection. Overall, AMPKα1 of M. amblycephala was similar to that of other vertebrates, and nutrient restriction modified its phosphorylation and mRNA levels in liver and muscle tissues. Furthermore, substantial expression of this kinase was induced in both liver and muscle tissues by glucose and insulin administration.

Hypocholesterolaemic effect of whole-grain highland hull-less barley in rats fed a high-fat diet.

Whole-grain highland hull-less barley (WHLB) contains high amounts of bioactive compounds that potentially exhibit cholesterol-lowering effects. This study investigated the hypocholesterolaemic effect of WHLB. A total of seventy-two male Sprague-Dawley rats were divided into four groups and were fed with the normal control diet, high-fat diet (HFD) and HFD containing low or high dose (10 or 48·95 %) of WHLB. High dose of WHLB significantly decreased the organ indexes of liver and abdominal fat and lipid levels of plasma and liver in HFD rats. The lipid regulation effect of WHLB, which was reconfirmed through hepatocyte morphologic observation, was accompanied by a large excretion of bile acids in the small intestinal contents and the faeces. Real-time PCR analyses, which were further reconfirmed through Western blot analyses, revealed that a high dose of WHLB significantly enhanced the hepatic expressions of AMP-activated protein kinase α, cholesterol 7α-hydroxylase, LDL receptor, liver X receptor, and PPARα and decreased the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase. It also enhanced the ileal expression of farnesoid X receptor and resulted in the decrease of expression of apical sodium-dependent bile acid transporter. WHLB exhibited hypocholesterolaemic effects mainly by inhibiting cholesterol synthesis, cholesterol accumulation in peripheral tissue, and bile acid reabsorption and by stimulating bile acid synthesis.

Reactivation of visual cortical plasticity by caloric restriction mediated by AMPK-SIRT1 pathway from monocular deprivation in adult mice

Objective This study was to investigate the effect of caloric restriction on the plasticity of visual cortex in adult monocular deprivation (MD) amblyopic mice, as well as to promote the treatment of amblyopia, and to explore the possible molecular mechanism of this benefical effect. Methods Fifty healthy newborn Kunming mice of clean grade were randomly divided into 3 groups using a random number table method: normal control group (n=14), MD+ ad libitum group (n=18) and MD+ caloric restriction group (n=18). A mouse model of adult MD amblyopia was established, and caloric restriction intervention and ad libitum were performed on MD+ caloric restriction group and MD+ ad libitum group, respectively.The visual acuity and flash visual evoked potential (F-VEP) of each group were detected.The synaptic structure of visual cortex neurons was observed by transmission electron microscope, and the expression of phosphorylated AMP-activated protein kinase-α (p-AMPKα) and silent information regulator 1 (SIRT1) in visual cortex were detected by Western blot.The animal feeding and use was in accordance with the standards set by the ARVO. Results The weight of mice in MD+ caloric restriction group increased from the beginning of the first week, and was significantly lower than that in the MD+ ad libitum group (P<0.05). Compared with the MD+ ad libitum group, the visual acuity was restored, the latency was shortened, and the amplitude of F-VEP was increased in the deprived eyes of MD+ caloric restriction group (all at P<0.05). Transmission electron microscope observation showed that the width of synaptic gap of visual cortical neurons was significantly narrower, and the thickness of postsynaptic density was significantly thicker in MD+ caloric restriction group than that in the MD+ ad libitum group (both at P<0.05); compared with the normal control group, the synaptic gap was widened and the postsynaptic density was significantly thicker than that in the MD+ ad libitum group (both at P<0.05). Western blot showed that the expression of p-AMPKα in visual cortex in the normal control group, MD+ caloric restriction group and MD+ ad libitum group was 0.89±0.03, 0.94±0.02 and 0.74±0.02, and the expression of SIRT1 was 0.97±0.11, 0.95±0.14 and 0.58±0.13, respectively, showing significant differences among the three groups (F=14.57, P=0.00; F=23.91, P=0.00), the expressions of p-AMPKα and SIRT1 in visual cortex were increased in MD+ caloric restriction group than those in MD+ ad libitum group (both at P<0.05). Conclusions Caloric restriction can restore the ultrastructure of synapses and improve the visual cortical plasticity in adult MD mice, so that help to improve visual function.Its mechanism may be related to the activation of AMPK-SIRT1 pathway. Key words: Caloric restriction; Adult mice; Monocular deprivation; Visual cortical; Plasticity

Gallotannin derivatives from mango (Mangifera indica L.) suppress adipogenesis and increase thermogenesis in 3T3-L1 adipocytes in part through the AMPK pathway

Expansion of adipose tissue in obesity is associated with dysregulation of adipokines, which can lead to long-term metabolic disorders. Gallotannin derivatives from mango possess anti-inflammatory activities, but their potential role in lipid metabolism is not well investigated. In this study, 3T3-L1 preadipocytes were differentiated into adipocytes and treated with mango polyphenols (MG), or pyrogallol (PG) for 6 days. The anti-adipogenic activity of PG was demonstrated by reduced number of lipid droplets and expressions of adipogenic markers, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). In mature adipocytes, PG promoted adipocyte browning and increased the expressions of uncoupling protein 1 (UCP1) and sirtuin 1 (Sirt1). Knockdown of AMP-activated protein kinase (AMPK) α1 with siRNA partially abolished the effect of PG on adipogenesis. Results indicate that gallotannin derivatives modulate lipid metabolism, at least in part, though the AMPK pathway, and possess potential to prevent obesity-related conditions.

Abstract 460: Modulation of the SUMOylation of Fish Oil Receptor G-protein Coupled Receptor (GPR) 120 by AMP-activated Protein Kinase α2 Controls the Anti-atherosclerotic Effects of Fish Oils in vivo

Background: Fish oils including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have cardiovascular benefits in general population but fail to show therapeutic effects in patients with cardiovascular diseases (CVD). The aim of this study was to determine how fish oils are ineffective in CVD patients. Methods: The effects of fish oils on atherosclerosis, the expression of AMP-activated protein kinase (AMPK), fish oil receptor G-protein coupled receptor (GPR) 120, and inflammation were assessed in CVD patients, atherosclerosis prone LDLR -/- mice with or without AMPKα2, lipid reversal atherosclerotic mouse model (LDLR -/- ApoB100 +/+ ) and cultured cells. Results: Healthy individuals had significant higher levels of EPA and DHA in blood than CVD patients. The levels of EPA or DHA were negatively correlated with SYTNAX scores in CVD patients with high AMPKα2 expression but had no correlation in those patients with low AMPKα2 expression. Administration of fish oils (5%, 12 weeks) significantly reduced aortic lesions and inflammation in both LDLR -/- mice and LDLR -/- ApoB100 +/+ but not in LDLR -/- /AMPKα2 -/- mice. In both human and mouse arteries, AMPKα2 was inversely correlated with GPR120. Further, AMPKα2 suppresses GPR120 SUMOylation resulting in an increase of GPR120 internalization. Consistently, GPR120 inhibition abolished the anti-atherosclerotic effects of fish oils in cultured cells. Moreover, selective inhibition of AMPKα2 but not AMPKα1 increased both SUMOylated protein UBC9 and its transcriptional factor c-myc expression. Furthermore, inhibition of c-myc ablated UBC9 transcription and GPR120 SUMOylation. Mechanistically, we found that AMPKα2 controls c-myc stability by phosphorylating c-myc at serine 67, a key site for its degradation. Conclusion: We conclude that AMPK α2 mediates the anti-atherosclerotic effects of fish oils by modulating GPR120 SUMOylation and internalization.

AMP‐Activated Protein Kinase (AMPK) Modulates Myoendothelial Junctions (MEJ) in Murine Microvessels

Myoendothelial junctions (MEJ) in microvessels are not only the site of direct cell communication via gap junctions but also represent a signaling microdomain critical for regulating vascular processes such as endothelium dependent hyperpolarization (EDH). The 5′‐adenosine monophosphate‐activated protein kinase (AMPK) has been shown to influence vascular tone. It has not yet been studied, however, whether AMPK could also affect MEJ. Thus, the aim of this project was to elucidate the impact of AMPK on MEJ dynamics and its potential mechanism of action.We studied isolated small mesenteric and skeletal muscle resistance arteries of mice using a pressure myograph system developed in our laboratory. Using confocal and two photon excitation fluorescence microscopy we identified the internal elastic lamina, which in these vessels exhibits small holes that allow direct contact between endothelial and smooth muscle cells. On average, we found that in about 35% of the holes, structures with a bright actin core as assessed by using F‐actin fluorescence in LifeAct mice or by staining with phalloidin which we could define as sites of MEJ. These structures showed expression of Cx37 and Cx43 suggesting that gap junctions (MEGJs) were also localized in these structures. Over observation times of up to 2 hours, we found that the number and localization of these MEJ varied, while they always positioned in the area of holes of IEL only. Thus, for the first time we could investigate MEJ dynamics. AMPK negatively regulates MEJ formation, since in AMPK α1, but not α2 knockout (KO) mice, the number of MEJ was significantly increased. In accordance, in wild type mice, incubation with the AMPK inhibitory compound C (CC) significantly augmented the number of MEJ. Furthermore, KO of AMPK α1, in intact mice as well as in cultured human and porcine smooth muscle cells, went along with an increase in PAI‐1 expression. Accordingly, incubation of arteries with exogenous PAI‐1 also increased the number of MEJ. The negative effect of AMPK on PAI‐1 could be explained by enhanced expression of the silencer of the PAI‐1 gene, small heterodimer partner (SHP). Since MEJ are also involved in EDH, we studied whether an enhanced formation of MEJ correlated with the magnitude of EDH induced vasodilation. Indeed, a higher amount of MEJs in α1 KO mice went along with a left shift of acetylcholine (ACh) ‐induced dilation dose effect curve.This work describes for the first time a role of the α1 subunit of AMPK as a potent modulator of MEJ dynamics which is probably mediated by controlling PAI‐1 expression. Our data also for the first time demonstrate a dynamic regulation of MEJ expression in intact blood vessels.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.