AMP-activated Protein Kinase Protein Expression Research Articles

AMPK activation attenuates central sensitization in a recurrent nitroglycerin-induced chronic migraine mouse model by promoting microglial M2-type polarization

BackgroundEnergy metabolism disorders and neurogenic inflammation play important roles in the central sensitization to chronic migraine (CM). AMP-activated protein kinase (AMPK) is an intracellular energy sensor, and its activation regulates inflammation and reduces neuropathic pain. However, studies on the involvement of AMPK in the regulation of CM are currently lacking. Therefore, this study aimed to explore the mechanism underlying the involvement of AMPK in the central sensitization to CM.MethodsMice with recurrent nitroglycerin (NTG)-induced CM were used to detect the expression of AMPK protein in the trigeminal nucleus caudalis (TNC). Following intraperitoneal injection of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound C, the mechanical pain threshold, activity level, and pain-like behaviors in the mice were measured. The expression of calcitonin gene-related peptide (CGRP) and cytokines, M1/M2 microglia, and NF-κB pathway activation were detected after the intervention.ResultsRepeated NTG injections resulted in a gradual decrease in AMPK protein expression, and the negative regulation of AMPK by increased ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression may counteract AMPK activation by increasing ADP/ATP. AICAR can reduce the hyperalgesia and pain-like behaviors of CM mice, improve the activity of mice, reduce the expression of CGRP, IL-1β, IL-6, and TNF-α in the TNC region, and increase the expression of IL-4 and IL-10. Moreover, AMPK in TNC was mainly located in microglia. AICAR could reduce the expression of inducible NO synthase (iNOS) in M1 microglia and increase the expression of Arginase 1 (Arg1) in M2 microglia by inhibiting the activation of NF-κB pathway.ConclusionsAMPK was involved in the central sensitization of CM, and the activation of AMPK reduced neuroinflammation in NTG-induced CM mice. AMPK may provide new insights into interventions for energy metabolism disorders and neurogenic inflammation in migraine.

Antidiabetic features of AdipoAI, a novel AdipoR agonist.

Adiponectin is an antidiabetic endogenous adipokine that plays a protective role against the unfavorable metabolic sequelae of obesity. Recent evidence suggests a sinister link between hypoadiponectinemia and development of insulin resistance/type 2 diabetes (T2D). Adiponectin's insulin-sensitizing property is mediated through the specific adiponectin receptors R1 and R2, which activate the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) α pathways. AdipoAI is a novel synthetic analogue of endogenous adiponectin with possibly similar pharmacological effects. Thus, there is a need of orally active small molecules that activate Adipoq subunits, and their downstream signaling, which could ameliorate obesity related type 2 diabetes. In the study we aim to investigate the effects of AdipoAI on obesity and T2D. Through in-vitro and in-vivo analyses, we investigated the antidiabetic potentials of AdipoAI and compared it with AdipoRON, another orally active adiponectin receptors agonist. Our results showed that in-vitro treatment of AdipoAI (0-5 µM) increased adiponectin receptor subunits AdipoR1/R2 with increase in AMPK and APPL1 protein expression in C2C12 myotubes. Similarly, in-vivo, oral administration of AdipoAI (25 mg/kg) observed similar effects as that of AdipoRON (50 mg/kg) with improved control of blood glucose and insulin sensitivity in diet-induced obesity (DIO) mice models. Further, AdipoAI significantly reduced epididymal fat content with decrease in inflammatory markers and increase in PPAR-α and AMPK levels and exhibited hepatoprotective effects in liver. Further, AdipoAI and AdipoRON also observed similar results in adipose tissue. Thus, our results suggest that low doses of orally active small molecule agonist of adiponectin AdipoAI can be a promising therapeutic target for obesity and T2D.

Activated AMPK Protects Against Chronic Cerebral Ischemia in Bilateral Carotid Artery Stenosis Mice.

AMP-activated protein kinase (AMPK) is a regulator of cellular energy metabolism. Long-term use of metformin, an AMPK activator, was previously reported to be neuroprotective, as it promotes behavioral improvement and angiogenesis following an acute ischemic injury of the brain. However, only a few studies have demonstrated the role of AMPK in alleviating chronic cerebral ischemia (CCI) in mice models in the long-term (over 3 months). Therefore, we established a mouse model of CCI via bilateral carotid artery stenosis (BCAS) to explore the effect of AMPK on CCI. We used four groups of 3-month-old male C57BL/6 mice labeled as Sham, BCAS, BCAS + metformin treatment (BCAS + Met) and BCAS + AMPKα2 gene knockout (BCAS + KO). Three months after BCAS, we measured the AMPK protein expression, spatial learning and memory, Nissl bodies, cell apoptosis, astrocyte activation, and oligodendrocyte maturation. Additionally, we observed the brain tissues for changes in cell morphology. We observed that mice in the BCAS group had impaired spatial learning and memory compared with those in the sham group. The brain tissues of mice with CCI injury showed altered cell morphology, fewer Nissl bodies, cerebral cells apoptosis, and astrocyte activation. Interestingly, compared with mice from the BCAS group, the brains of mice from BCAS + Met group suffered lesser damage, whereas those of mice from the BCAS + KO group suffered more damage. The activation of AMPK, especially AMPKα2, plays a neuroprotective role during CCI in a mouse model of BCAS.

Dietary supplementation with metformin improves testis function and semen quality and increases antioxidants and autophagy capacity in goats

ATP is essential for mammalian sperm to maintain fertilizing capacity. Metformin (Met) can activate 5′-AMP-activated protein kinase (AMPK) to maintain energy homeostasis. Thus, the aim of the present study was to investigate whether Met can improve testis function, semen quality, antioxidant and autophagy capacity through AMPK mediation of energy metabolism in goats. Twelve adult goats were randomly divided into three dietary treatments. All goats were fed a basal diet for 3 weeks and then assigned to a Met supplementation diet containing 0, 150, or 300 mg/kg for 8 weeks. The results showed that sperm viability, sperm membranal functional integrity, and acrosome integrity increased (P < 0.05) relative to the other treatments in the 300 mg/kg Met group. Growth hormone (GH) and insulin-like growth factor (IGF-1) in the 300 mg/kg Met group significantly decreased (P < 0.05) relative to the control group. Estrogen levels (E2) in the 300 mg/kg Met group remarkably improved (P < 0.05) compared with the control group. The activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GSH-px), and superoxide dismutase (SOD) significantly increased (P < 0.05) in the 300 mg/kg Met group relative to the control group. A significant increase in AMPK and p-AMPK protein expression in the 300 mg/kg Met group was observed relative to the control group (P < 0.05). Belicin-1 and LC3II/I protein expression was significantly increased by adding Met to the diet (P < 0.05) and reached a maximum in the 300 mg/kg Met group. In addition, differentially expressed genes (DEGs) of goat testis were confirmed by RNA-seq. GO enrichment analysis revealed that DEGs were enriched in testicular metabolism and sperm development-related functional pathways. Overall, the results indicate that Met may play an important role in the regulation of testis function, semen quality, antioxidant, and autophagy capacity. These findings will help elucidate the role of Met in goat testis development.

Differentiated modulation of signaling molecules AMPK and SIRT1 in experimentally drug-induced hepatocyte injury.

Currently available medicines have little to offer in terms of supporting the regeneration of injured hepatic cells. Previous experimental studies have shown that resveratrol and metformin, less specific activators of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), can effectively attenuate acute liver injury. The aim of this experimental study was to elucidate whether modulation of AMPK and SIRT1 activity can modify drug/paracetamol (APAP)-induced hepatocyte damage in vitro. Primary rat hepatocytes were pretreated with mutual combinations of specific synthetic activators and inhibitors of SIRT1 and AMPK and followed by a toxic dose of APAP. At the end of cultivation, medium samples were collected for biochemical analysis of alanine-aminotransferase and nitrite levels. Hepatocyte viability, thiobarbituric reactive substances, SIRT1 and AMPK activity and protein expression were also assessed. The harmful effect of APAP was associated with decreased AMPK and SIRT1 activity and protein expression alongside enhanced oxidative stress in hepatocytes. The addition of AMPK activator (AICAR) or SIRT1 activator (CAY10591) significantly attenuated the deleterious effects of AMPK inhibitor (Compound C) on the hepatotoxicity of APAP. Furthermore, CAY10591 but not AICAR markedly decreased the deleterious effect of APAP in combination with SIRT1 inhibitor (EX-527). Our findings demonstrate that decreased AMPK activity is associated with the hepatotoxic effect of APAP which can be significantly attenuated by the administration of a SIRT1 activator. These findings suggest that differentiated modulation of AMPK and SIRT1 activity could therefore provide an interesting and novel therapeutic opportunity in the future to combat hepatocyte injury.

Tissue-Specific Warburg Effect in Breast Cancer and Cancer-Associated Adipose Tissue-Relationship between AMPK and Glycolysis.

Simple SummarySpecific metabolic phenotypes of breast cancer result from local interactions such as cancer-adipocyte cross-talk and systemic metabolic influences such as obesity. Here we examined key regulatory enzymes involved in glucose metabolism in breast cancer tissue and cancer-associated adipose tissue of normal-weight and overweight/obese premenopausal women in comparison to benign breast tumor tissue and adipose tissue of weight-matched women. We show a simultaneous increase in 5′-AMP-activated protein kinase (AMPK) protein expression with glucose utilization favoring glycolysis and pentose phosphate pathway in breast cancer tissue. In parallel, we show an increased AMPK protein expression with glucose utilization favoring the pentose phosphate pathway in cancer-associated adipose tissue. Moreover, specific features of cancer tissue glycolysis and glycogen metabolism differ between normal-weight and overweight/obese women. The results suggest context-dependent induction of tissue-specific Warburg effect in breast cancer and cancer-associated adipose tissue.Typical features of the breast malignant phenotype rely on metabolic reprogramming of cancer cells and their interaction with surrounding adipocytes. Obesity is strongly associated with breast cancer mortality, yet the effects of obesity on metabolic reprogramming of cancer and cancer-associated adipose tissue remain largely unknown. Paired biopsies of breast tumor tissue and adipose tissue from premenopausal women were divided according to pathohistological analyses and body mass index on normal-weight and overweight/obese with benign or malignant tumors. We investigated the protein expression of key regulatory enzymes of glycolysis, pentose phosphate pathway (PPP), and glycogen synthesis. Breast cancer tissue showed a simultaneous increase in 5′-AMP-activated protein kinase (AMPK) protein expression with typical features of the Warburg effect, including hexokinase 2 (HK 2) overexpression and its association with mitochondrial voltage-dependent anion-selective channel protein 1, associated with an overexpression of rate-limiting enzymes of glycolysis (phosphofructokinase 1—PFK-1) and pentose phosphate pathway (glucose-6-phosphate dehydrogenase—G6PDH). In parallel, cancer-associated adipose tissue showed increased AMPK protein expression with overexpression of HK 2 and G6PDH in line with increased PPP activity. Moreover, important obesity-associated differences in glucose metabolism were observed in breast cancer tissue showing prominent glycogen deposition and higher glycogen synthase kinase-3 protein expression in normal-weight women and higher PFK-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression in overweight/obese women. In conclusion, metabolic reprogramming of glycolysis contributes to tissue-specific Warburg effect in breast cancer and cancer-associated adipose tissue.

Prion peptide-mediated calcium level alteration governs neuronal cell damage through AMPK-autophagy flux

BackgroundThe distinctive molecular structure of the prion protein, PrPsc, is established only in mammals with infectious prion diseases. Prion protein characterizes either the transmissible pathogen itself or a primary constituent of the disease. Our report suggested that prion protein-mediated neuronal cell death is triggered by the autophagy flux. However, the alteration of intracellular calcium levels, AMPK activity in prion models has not been described. This study is focused on the effect of the changes in intracellular calcium levels on AMPK/autophagy flux pathway and PrP (106–126)-induced neurotoxicity.MethodsWestern blot and Immunocytochemistry was used to detect AMPK and autophagy-related protein expression. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells. Calcium measurement was employed using fluo-4 by confocal microscope.ResultsWe examined the effect of calcium homeostasis alterations induced by human prion peptide on the autophagy flux in neuronal cells. Treatment with human prion peptide increased the intracellular calcium concentration and induced cell death in primary neurons as well as in a neuronal cell line. Using pharmacological inhibitors, we showed that the L-type calcium channel is involved in the cellular entry of calcium ions. Inhibition of calcium uptake prevented autophagic cell death and reduction in AMP-activated protein kinase (AMPK) activity induced by human prion peptide.ConclusionOur data demonstrated that prion peptide-mediated calcium inflow plays a pivotal role in prion peptide-induced autophagic cell death, and reduction in AMPK activity in neurons. Altogether, our results suggest that calcium influx might play a critical role in neurodegenerative diseases, including prion diseases.3p5PCBHnn1RWkUoaQCoeATVideo

Novel Method To Visualize AMPK Protein Localization In Single Human Muscle Fibers Via Confocal Microscopy

PURPOSE: AMP-activated protein kinase (AMPK) is the energy regulator of skeletal muscle cells. Current methods can identify the magnitude of AMPK expression in skeletal muscle cells via Western blotting and Capillary Nano-Immunoassay (CNIA); however, these methods lack the ability to visually identify AMPK localization within single muscle fibers. Identifying AMPK in human muscle is important because it is involved in various exercise training adaptations such as mitochondrial biogenesis and glucose transport. Therefore, we aimed to develop a novel confocal microscopy method to identify AMPK protein expression (relative intensity) and localization within human single muscle fibers. METHODS: A vastus lateralis muscle biopsy was obtained from a healthy male and immediately fixed (4% PFA). Twenty fibers were isolated, placed on microscope slides, incubated in 0.1% Triton (15min), then incubated in 5% normal goat serum (blocking solution; 4h). This was followed by exposure to a 1 antibody (Ab) (anti-AMPKα2) in 5% bovine serum albumin (14h at 4°C). Fibers were then exposed to a 2 Ab (anti-rabbit IgG conjugated w/AlexaFluor 488) and phalloidin (AlexaFluor 568) to label actin (2h). Finally, fibers were mounted under coverslips with AntiFade Gold w/DAPI for myonuclei detection. Confocal microscopy imaging was conducted using a Zeiss LSM 710 with 63x plan apochromatic objective (oil emersion). Images were processed via ImageJ. RESULTS: Muscle fiber contractile proteins (actin; red), myonuclei (blue), and AMPK proteins (green) were successfully visually identified at rest (AMPK fluorescence intensity = 1199.64 + 630 AU). To ensure that no auto-fluorescence or non-specific binding was observed, images were compared to control slides: 1) DAPI only, 2) 1 Ab only, 3) 2 Ab only, and 4) no staining. CONCLUSION: These methods allow for the successful visualization (relative intensity) and localization of AMPK proteins within single human muscle fibers. This method could be used in future research to investigate the response and myonuclear co-localization of AMPK following exercise in human skeletal muscle to elucidate how they may play a role in these physiological processes.

Anomalous AMPK-regulated angiotensin AT1R expression and SIRT1-mediated mitochondrial biogenesis at RVLM in hypertension programming of offspring to maternal high fructose exposure

BackgroundTissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high-fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP-activated protein kinase (AMPK)-regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)-dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring.MethodsPregnant female rats were randomly assigned to receive normal diet (ND) or HFD (60% fructose) chow during pregnancy and lactation. Both ND and HFD offspring returned to ND chow after weaning, and blood pressure (BP) was monitored from age 6 to 12 weeks. At age of 8 weeks, ND and HFD offspring received oral administration of simvastatin or metformin; or brain microinfusion of losartan. BP was monitored under conscious condition by the tail-cuff method. Nutrient sensing molecules, AT1R, subunits of NADPH oxidase, mitochondrial biogenesis markers in rostral ventrolateral medulla (RVLM) were measured by Western blot analyses. RVLM oxidative stress was measured by fluorescent probe dihydroethidium and lipid peroxidation by malondialdehyde assay. Mitochondrial DNA copy number was determined by quantitative real-time polymerase chain reaction.ResultsIncreased systolic BP, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in RVLM where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG-CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD-programmed increase in AT1R and decreases in SIRT1, PGC-1α and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension.ConclusionDysfunction of AMPK-regulated AT1R expression and SIRT1-mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and BP in young offspring programmed by excessive maternal fructose consumption.

Anomalous AMPK‐Regulated Angiotensin AT1R Expression and SIRT1‐Mediated Mitochondrial Biogenesis at RVLM in Hypertension Programming of Offspring to Maternal High Fructose Exposure

Tissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high‐fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP‐activated protein kinase (AMPK)‐regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)‐dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring. Increased systolic and diastolic blood pressure, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in rostral ventrolateral medulla (RVLM) where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator‐activated receptor g co‐activator a (PGC‐1a) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG‐CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD‐programmed increase in AT1R and decreases in SIRT1, PGC‐1a and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension. Together these results suggest that dysfunction of AMPK‐regulated AT1R expression and SIRT1‐mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and blood pressure in young offspring programmed by excessive maternal fructose consumption.Support or Funding InformationThis work was supported by research grants from the Chang Gung Medical Foundation to J.Y.H. Chan (CMRPG8C0053‐33 and OMRPG8G0011). Y.M. Chao was supported by a Postdoctoral Fellowship (MOST107‐2811‐B‐182A‐501) from the Ministry of Science and Technology, Taiwan.

Spilanthol from Traditionally Used Spilanthes acmella Enhances AMPK and Ameliorates Obesity in Mice Fed High-Fat Diet.

Spilanthol (SP) is a bioactive compound found in Spilanthes acmella, giving the flowers and leaves a spicy taste. Studies found that phyto-ingredients stored in spice plants act against obesity-related diseases. SP has antimicrobial, anti-inflammatory, and analgesic properties, but the effects on obesity are not yet known. We investigated the effects of SP in differentiated adipocytes (3T3-L1 cells) and mice fed a high-fat diet (HFD). SP significantly inhibited intracellular lipid accumulation and significantly reduced the expression of lipogenesis-related proteins, including acetyl-CoA carboxylase (ACC) and fatty-acid synthase (FAS). In contrast, SP increased the expression of carnitine palmitoyltransferase (CPT)1 and AMP-activated protein kinase (AMPK) in adipocytes. However, SP suppressed the levels of cyclooxygenase-2 (COX-2), phospho-p38 (pp38), and phospho-JNK (c-Jun N-terminal kinase) (pJNK) in LPS (lipopolysaccharide)-stimulated murine pre-adipocytes. SP administered to HFD-induced obese mice via intraperitoneal injections twice a week for 10 weeks decreased body weight gain, visceral adipose tissue weight, and adipocyte size. SP inhibited lipogenic proteins FAS and ACC, and suppressed adipogenic transcription factors, enhancing lipolysis and AMPK protein expression in the liver. SP has anti-obesity effects, upregulating AMPK to attenuate lipogenic and adipogenic transcription factors.

Cell Type‐Specific Differential Effects of Nitric Oxide on PDE3 Expression in Pulmonary Vasculature

Persistent pulmonary hypertension of the newborn (PPHN) is an important cause of morbidity and mortality in neonates. Often used in combination with hyperoxia, the pulmonary vasodilator, inhaled nitric oxide (iNO), is the only FDA‐approved therapy for PPHN, although up to 40% of patients are non‐responders. In the pulmonary vasculature, NO increases cGMP levels, promoting vasodilation. cGMP competitively inhibits phosphodiesterase 3 (PDE3), thereby decreasing cAMP hydrolysis and further enhancing vasodilation. However, in animal models, NO treatment has been shown to increase PDE3 expression and/or activity and has been postulated to be the reason for rebound PH upon iNO withdrawal. Furthermore, it has been reported that that addition of a PDE3 inhibitor to iNO enhances pulmonary vasorelaxation. AMP‐activated protein kinase (AMPK), a critical regulator of energy homeostasis with downstream effects on NO synthase (NOS), has been shown to be regulated by PDE3 isoforms in other cell types and implicated in PH pathogenesis. We hypothesize that NO regulates the AMPK pathway via modulating PDE3 activity in a cell‐type specific manner in the pulmonary vasculature. Human PASMC (hPASMC) were treated with the NO donor DETA NONOate (1 mM) or DMSO and incubated in 21% or 85% O2 for 48 h. PDE3A, PDE3B, phosphorylated (p) and total (T)‐AMPK, p‐ and T‐ACC, and β‐actin protein expression were quantified by Western blot analyses. A similar experiment was performed using hPMVEC. Additionally, p‐ and T‐endothelial NOS (eNOS) protein expression were quantified. Our results show that hPASMC PDE3A protein expression was increased following Deta NONOate treatment in normoxia and hyperoxia (5.5±0.4 ‐fold, p=0.0004; 7.5±0.8 ‐fold, p&lt;0.0001) relative to 21% O2 vehicle. Additionally, p/T‐AMPK protein expression was increased by 3‐fold after Deta NONOate treatment (p&lt;0.0001). Consistent with these results, p/T‐ACC (a downstream target of AMPK), was also increased with the addition of NO donor in both conditions. In hPMVEC, the addition of NO donor increased PDE3A protein expression (2.4±0.1 ‐fold, p&lt;0.0001). The combination of hyperoxia + NO donor, however, blunted the increase in PDE3A to a level similar to that in 21% O2, vehicle‐treated cells. Similarly, NO donor alone increased p/T‐AMPK (4.1±0.1 ‐fold, p&lt;0.0001), whereas the combination of hyperoxia + NO blunted this increase. Moreover, p‐eNOS/β‐actin and T‐eNOS/β‐actin were both increased with the NO donor (1.8±0.03 ‐fold, p&lt;0.0001; 2.9‐fold±0.20 SEM, p=0.0068), whereas the combination of hyperoxia + NO blunted the increase in p‐eNOS levels. In conclusion, exogenous NO donor increased PDE3A expression and AMPK activation in hPASMCs in both normoxia and hyperoxia. Alternatively, in hPMVEC, the increase in PDE3A expression, AMPK and eNOS activation after the addition of NO donor was blunted by hyperoxia. We speculate that these cell‐type specific differences in regulation of PDE3 and AMPK may be the reason for the variability in response of the pulmonary vasculature to PDE3 inhibition and AMPK modulators in PH.Support or Funding InformationNIH, NHLBI, R01, 1R01HL136963‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metformin attenuates lipid accumulation in hepatocytes of blunt snout bream (Megalobrama amblycephala) via activation of AMP-activated protein kinase

Currently, there is a trend to use high-fat diets in intensive aquaculture that is accompanied with incidence of fatty liver when dietary lipid level surpasses an upper limit. So, it is necessary to develop appropriate strategies to reduce the risk of fatty liver in commercial fish farming. Studies in mammals have revealed a correlation between fatty liver and AMP-activated protein kinase (AMPK) activity, which has been recognized as a key modulator of lipid metabolism. Considering the frequent occurrence of fatty liver in blunt snout bream farming, an in-vitro study was designed to evaluate the efficiency of metformin, as a stimulator of AMPK, in activation of AMPK and its subsequent effects on lipid metabolism in primary hepatocytes. Fish hepatocytes were seeded at a density of 1 × 106 ml−1 in 6-well tissue culture plates and treated with three different media including: 1) Leibovitz's L-15 medium [L15] as control, 2) high-fat medium [L15 + 400 μM oleic acid], and 3) metformin medium [L15 + 400 μM oleic acid+200 μM metformin]. After 48 h of culture, the cells and supernatant were collected for analysis. The results showed significant (P < .05) enhancement of cell triglyceride and total cholesterol concentrations in the high-fat group over control, and metformin addition significantly reduced the values. Also, the high-fat group exhibited significantly higher aspartate aminotransferase activity than both control and metformin groups. The lowest AMPK and phospho-AMPK protein expression was found in the high-fat group while metformin addition significantly up-regulated their expression levels. Mitochondrial and peroxisomal oxidation rates in the high-fat group were significantly lower than control while similar oxidation rates were observed for metformin treated and control groups. The high-fat group showed significantly lower CPT I activity than control, and metformin inclusion increased the activity. Expression of genes associated with lipid metabolism such as PPARα, CPT I, AOX, PGC-1α and TFAM was suppressed in the high-fat group, and metformin supplementation up-regulated their expression levels. The opposite trend was true for the expression of ACC2 gene. Also, the results showed down-regulation of FAS and SREBP-1C genes in the high-fat medium group, and metformin addition resulted in further reduction of their expression level. The lowest activities of mitochondrial complexes (I-III) were found in the high-fat group and metformin prevented high-fat-induced reduction of mitochondrial complexes activity. Notably increased concentrations of reactive oxygen species and malondialdehyde were found in the high-fat group, and metformin treatment reduced their concentrations. Moreover, metformin group exhibited higher glutathione peroxidase activity than the high-fat group. The findings in this study showed clearly that metformin activated AMPK in blunt snout bream hepatocytes, which contributed to enhanced lipid metabolism and attenuated lipid deposition in the cells incubated with high-fat medium.

Protein Expression Differences in Hypertrophic Cardiomyopathy Transgenic Mice

Hypertrophic Cardiomyopathy (HCM) is the most common genetic cardiac disease, occurring in 1 out of every 500 individuals. The pathologic hallmarks of HCM are myocyte hypertrophy, myocyte disarray, fibrosis and arteriolar remodeling. Majority of HCM patients have mutations in genes coding for sarcomeric proteins. However, the mechanisms whereby the mutations lead to myocyte hypertrophy are not fully elucidated. Despite successes in animal models, there is no known therapy that can prevent the cardiac phenotype in HCM patients. We hypothesized that differences in pathophysiology of the cardiac phenotype in HCM could underlie failure of clinical trials in HCM patients. Hence, in this study, we investigated 2 transgenic mouse models of HCM, namely the R403Q mutation in the alpha Myosin Heavy Chain gene (R403Q‐MHC) and the R92W mutation in the Troponin T gene (R92W‐TnT). Since energetic deficits and abnormalities in calcium handling are 2 postulated mechanisms underlying the cardiac phenotypes in HCM, we investigated expression levels of AMP‐Activated Protein Kinase (AMPK), Ca2+/Calmodulin‐Dependent Protein Kinase II (CamKII) and the mitochondrial calcium uniporter (MCU) in these 2 mouse models using western immunoblotting.MethodsTransgenic mice of age 5 weeks and 24 weeks containing the R403Q‐MHC and R92W‐TnT HCM mutations were euthanized and each heart was harvested and homogenized in 2% Triton‐Ripa buffer. Protein from whole heart homogenate was isolated by centrifugation and subjected to Western Immunoblotting.ResultsIn vitro studies have shown protein expression differences in all three transgenic mouse models, R403Q‐MHC, R92W‐TnT, and wild type, at both age 5 weeks and 24 weeks. Total target protein, AMPK, CamKII, and MCU were equally expressed in all three models; however, there was a 25% increase in phosphorylated AMPK protein expression in R403Q‐MHC transgenic mice than R92W‐TnT transgenic mice of age 5 weeks and 24 weeks. Phosphorylated CamKII (pCamKII) had 38% increase in protein expression in R92W‐TnT transgenic mice than R403Q‐MHC transgenic mice of age 5 weeks. Phosphorylated MCU had 13% increase in protein expression in R92W‐TnT transgenic mice of age 5 weeks and 26% increase in R92W‐TnT transgenic mice of age 24 weeks than R403Q‐MHC transgenic mice of age 5 weeks and 24 weeks. Although pCamKII lacked expression in transgenic mice of age 24 weeks, this result can lead us to further investigations testing other protein expression differences to gain additional insight of underlying mechanisms how mutations lead to myocyte hypertrophy.ConclusionR403Q‐MHC results in abnormal energy caused by conditions of low ATP, while R92W‐TnT results in high calcium sensitivity by an increase of calcium ions. Results confirm that there is disruption in energy and calcium handling due to the respective mutations compared to that of control. At 5 weeks, in the early development of disease, phosphorylated AMPK protein was expressed 15% lower than at 24 weeks, showing more energy stress at a later stage of disease. However, the opposite holds true for pCamKII, as it was expressed 100% higher at age of 5 weeks, as it lacked expression at age of 24 weeks. This shows that there may be abnormal calcium handling at an early developing stage of disease, but not when the disease is well developed.Support or Funding InformationThis research was supported by the JTB Foundation to Roselle Abraham.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3520: Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression

Abstract Solidago virga-aurea (European goldenrod or woundwort) is an herbaceous perennial plant of the family Asteraceae. It is used for medical system with astringent, diuretic, antiseptic and homeopathic properties. And the studies about Solidago virga-aurea are in progress for the other functions. Recently, we found that Solidago virga-aurea extract induces apoptosis of breast cancer cells. MTT assay showed that cellular viability was decreased in Solidago virga-aurea extract treated MDA-MB-231 and MCF-7 breast cancer cell lines by dose and time dependent manner. And apoptotic protein, especially bim, was increased in Solidago virga-aurea extract treated MCF-7 cell line. However, anti-apoptotic protein, Bcl-xL, does not changed by Solidago virga-aurea extract. In order to find out positive regulator of bim expression, we tested whether FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5’ AMP-activated protein kinase) protein expression. In immunoblot analysis showed that FOXO3a expression level was dramatically increased in Solidago virga-aurea treated MCF-7 cells without changes of AMPK expression. Moreover, immunostaining showed that Solidago virga-aurea induces co-localization of FOXO3a and bim proteins in MCF-7 and MB-231 cells. Taken together, Solidago virga-aurea induces apoptosis by FOXO3a mediated up-regulation of bim protein. The mechanisms of Solidago virga-aurea mediated FOXO3a regulation is not fully elucidated yet, but further studies about this molecular mechanism between two proteins will give some ideas for cancer therapies for certain breast cancer patients. Citation Format: Haesung Kim, Chea Ha Kim, Jeong-Hyeon Kim, Jeong-Min Lee, Jeong-Ho Jeon, Jae-Yong Lee. Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3520.

Abstract B57: Sucrose nonfermenting 1-related kinase (SNRK) expression and function in ovarian cancer.

Abstract Purpose of study: SNRK is a member of the sucrose nonfermenting (SNF)-related kinase family of serine/threonine kinases with sequence similarity to AMP-activated protein kinase (AMPK). AMPK proteins play a central role in cellular energy regulation. Active AMPK restricts cellular proliferation, growth, and metabolic substrate synthesis, and increases ATP-generating processes, such as glucose and fatty acid oxidation. SNKR has been previously shown to regulate metabolism and to suppress adipocyte inflammation via mTORC1, which is a pathway known to be aberrant in ovarian cancer. SNRK has also been shown to reduce colon cancer cell proliferation and stable knockdown of SNRK increases in vitro colon cancer cell tumorigenicity indicating that SNRK is a regulator of cell proliferation. The goal of this study is to describe the expression and function of SNRK in ovarian cancer and benign cell lines. Experimental Procedures: The ovarian cancer cell line A2780 was used as well as the benign surface epithelial cell line, IOSE, for comparison. Quantitative PCR and western blot analysis were used to compare SNRK mRNA and protein expression between cell lines. SNRK was knocked down in each cell line using lentiviral transduction. Proliferation, migration, and invasion assays were performed to determine the effects of SNRK knockdown on tumorigenicity. Alterations in metabolism as a result of SNRK knockdown were assessed using the XF Extracellular Flux Analyzer (Seahorse Bioscience). Western blot analysis was used to determine changes in active phosphorylated AMPK (pAMPK) and mTOR protein expression. Summary of data: IOSE and A2780 cells have similar levels of SNRK mRNA and protein expression. We observed increased proliferation, migration, and invasion in SNRK knockdown cells compared to controls with significant differences seen in the IOSE cells. A2780 and IOSE SNRK knockdown cells have decreased oxygen consumption rates and decreased spare respiratory capacity in the presence of fatty acids as a substrate compared to controls as well as decreased pAMPK and mTOR expression. Conclusions: Our results show that decreased SNRK levels lead to increased tumorigenicity and impairment in fatty acid oxidation particularly in the benign IOSE cells. Along with these functional changes, a decrease in pAMPK expression is also seen. Our results suggest that SNRK plays a role in regulating growth and metabolism and that this regulation appears to be required in benign ovarian cells, while malignant cells may have the ability to control growth independent of SNRK. Citation Format: Erin A. Bishop, Stephanie M. Cossette, Ramani Ramchandran. Sucrose nonfermenting 1-related kinase (SNRK) expression and function in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B57.

Metformin attenuates palmitic acid-induced insulin resistance in L6 cells through the AMP-activated protein kinase/sterol regulatory element-binding protein-1c pathway

AMP-activated protein kinase (AMPK) is an important effector of metformin action on glucose uptake in skeletal muscle cells. We recently reported that metformin improved insulin receptor substrate-1 (IRS-1)-associated insulin signaling by downregulating sterol regulatory element-binding protein-1c (SREBP-1c) expression. In this study, we investigated whether AMPK activation and SREBP-1c inhibition contribute to the beneficial effects of metformin on IRS-1-associated insulin signaling in L6 myotubes. L6 muscle cells were incubated with palmitic acid (PA) to induce insulin resistance and then treated with metformin and/or the AMPK inhibitor, compound C. AMPK, SREBP-1c, IRS-1 and Akt protein expression levels were determined by western blot analysis. The effects of metformin on SREBP-1c gene transcription were determined by a luciferase reporter assay. Glucose uptake was evaluated using the 2-NBDG method. In the PA-treated L6 cells, metformin treatment enhanced AMPK phosphorylation, reduced SREBP-1c expression and increased IRS-1 and Akt protein expression, whereas treatment with compound C blocked the effects of metformin on SREBP-1c expression and the IRS-1 and Akt levels. Moreover, metformin suppressed SREBP-1c promoter activity and promoted glucose uptake through AMPK. The results from this study demonstrate that metformin ameliorates PA-induced insulin resistance through the activation of AMPK and the suppression of SREBP-1c in skeletal muscle cells.

Effect of exhaustive ultra-endurance exercise in muscular glycogen and both Alpha1 and Alpha2 Ampk protein expression in trained rats

Glycogen is the main store of readily energy in skeletal muscle and plays a key role in muscle function, demonstrated by the inability to sustain prolonged high-intensity exercise upon depletion of these glycogen stores. With prolonged exercise, glycogen depletion occurs and 5'-AMP-activated protein kinase (AMPK), a potent regulator of muscle metabolism and gene expression, is activated promoting molecular signalling that increases glucose uptake by muscular skeletal cells. The aim of this study was primarily to determine the effect of ultra-endurance exercise on muscle glycogen reserves and secondly to verify the influence of this type of exercise on AMPK protein expression. Twenty-four male Wistar rats, 60 days old, were divided into four experimental groups: sedentary, sedentary exhausted (SE), endurance trained (T) and endurance trained exhausted (TE). The animals ran for 10 to 90 min/day, 5 days/week, for 12 weeks to attain trained status. Rats were killed immediately after the exhaustion protocol, which consisted of running on a treadmill (at approximately 60% Vmax until exhaustion). Optical density of periodic acid-Schiff was detected and glycogen depletion observed predominantly in type I muscle fibres of the TE group and in both type I and II muscle fibres in the SE group. Plasma glucose decreased only in the TE group. Hepatic glycogen was increased in T group and significantly depleted in TE group. AMPK protein expression was significantly elevated in TE and T groups. In conclusion, acute exhaustive ultra-endurance exercise promoted muscle glycogen depletion. It seems that total AMPK protein and gene expression is more influenced by status training.

Chronic caffeine intake reverses age-induced insulin resistance in the rat: effect on skeletal muscle Glut4 transporters and AMPK activity

The role of caffeine consumption on insulin action is still under debate. The hypothesis that chronic caffeine intake reverses aging-induced insulin resistance in the rat was tested in this work. The mechanism by which caffeine restores insulin sensitivity was also investigated. Six groups of rats were used: 3 months old (3 M), 3 months old caffeine-treated (3MCaf), 12 months old (12 M), 12 months old caffeine-treated (12MCaf), 24 months old (24 M), and 24 months old caffeine-treated (24MCaf). Caffeine was administered in drinking water (1 g/l) during 15 days. Insulin sensitivity was assessed by means of the insulin tolerance test. Blood pressure, body weight, visceral and total fat, fasting glycemia and insulinemia, plasma nonesterified fatty acids (NEFA), total antioxidant capacity (TAC), cortisol, nitric oxide, and catecholamines were monitored. Skeletal muscle Glut4 and 5'-AMP activated protein kinase (AMPK) protein expression and activity were also assessed. Aged rats exhibited diminished insulin sensitivity accompanied by hyperinsulinemia and normoglycemia, increased visceral and total fat, decreased TAC and plasma catecholamines, and also decreased skeletal muscle Glut4 and AMPK protein expression. Chronic caffeine intake restored insulin sensitivity and regularized circulating insulin and NEFA in both aging models. Caffeine neither modified skeletal muscle AMPK expression nor activity in aged rats; however, it decreased visceral and total fat in 12 M rats and it restored skeletal muscle Glut4 expression to control values in 24 M rats. We concluded that chronic caffeine intake reverses aging-induced insulin resistance in rats by decreasing NEFA production and also by increasing Glut4 expression in skeletal muscle.

Nutrient sensor–mediated programmed nonalcoholic fatty liver disease in low birthweight offspring

We hypothesized that gestationally programmed nonalcoholic fatty liver disease in low-birthweight offspring is mediated through nutrient sensors nicotinamide adenine dinucleotide+-dependent histone deacetylase (SIRT1) and AMP-activated protein kinase (AMPK). Pregnant dams received ad libitum food or were 50% food restricted from pregnancy days 10-21 to produce control and low-birthweight newborn offspring, respectively. All pups were nursed by control dams and weaned to ad libitum feed. We determined hepatic SIRT1 and AMPK activities and protein expression of lipid targets in low-birthweight and control fetuses, newborns, and adult offspring (3 months). Low-birthweight fetuses demonstrated increased prenatal hepatic SIRT1 activity, although with increased lipogenesis. After birth, low-birthweight newborn offspring undergo postnatal suppression of hepatic SIRT1 and AMPK activities in conjunction with increased lipogenesis, decreased lipolysis, and increased fat stores. These findings suggest that undernutrition stress in utero may program hepatic nutrient sensors to perceive normal postnatal nutrition as a state of nutrient excess with the induction of hepatic lipid storage.