Monophosphate-activated Protein Kinase Pathway Research Articles

Liquorice flavonoid oil suppresses hyperglycaemia accompanied by skeletal muscle myocellular GLUT4 recruitment to the plasma membrane in KK-Ay mice

For over 4000 years, liquorice has been one of the most frequently employed botanicals as a traditional herbal medicine. Although previous reports have found that liquorice flavonoids possess various health beneficial effects, the underlying mechanism responsible for the anti-diabetic effect of liquorice flavonoids remains unclear. The present study demonstrates that liquorice flavonoid oil (LFO) improves type 2 diabetes mellitus through GLUT4 translocation to the plasma membrane by activating both the adenosine monophosphate-activated protein kinase (AMPK) pathway and Akt pathway in muscle of KK-Ay mice. Furthermore, LFO lowered postprandial hyperglycaemia in a human study. These results indicate that LFO may exert a therapeutic effect on metabolic disorders, such as diabetes and hyperglycaemia, by modulating glucose metabolism through AMPK- and insulin-dependent pathways in skeletal muscle.

Maternal perinatal calorie restriction temporally regulates the hepatic autophagy and redox status in male rat

Intrauterine growth restriction (IUGR) leads to adult obesity, cardiovascular disease, and non-alcoholic fatty liver disease/steatohepatitis. Animal models have shown that combined intrauterine and early postnatal calorie restriction (IPCR) ameliorates these sequelae in adult life. The mechanism by which IPCR protects against adult onset disease is not understood. Autophagy, a lysosomal degradative process, recycles cellular constituents and eliminates damaged organelles, proteins, and oxidants. In this study, we hypothesized that IPCR could regulate autophagy in the liver of male rat offspring. At birth (d1) of male IUGR rat offspring and on day 21 (p21) of life, IPCR male rat offspring had a profound decrease in hepatic autophagy in all three stages of development: initiation, elongation, and maturation. However, upon receiving a normal diet ad-lib throughout adulthood, aged IPCR rats (day 450 of life (p450)), had increased hepatic autophagy, in direct contrast to what was seen in early life. The decreased autophagy at d21 led to the accumulation of ubiquitinated proteins and lipid oxidative products, whereas the increased autophagy in late life had the opposite effect. Oxidized lipids were unchanged at d1 by IUGR treatment indicating that decreased autophagy precedes oxidative stress in early life. When cellular signaling pathways regulating autophagy were examined, the 5′ adenosine monophosphate-activated protein kinase pathway (AMPK), and not endoplasmic stress pathways, was found to be altered, suggesting that autophagy is regulated through AMPK signaling pathway in IPCR rats. Taken together, this study reveals that the perinatal nutritional status establishes a nutritionally sensitive memory that enhances hepatic autophagy in late life, a process that perhaps acts as a protective mechanism to limited nutrition.

Sirtuin 3-dependent mitochondrial redox homeostasis protects against AGEs-induced intervertebral disc degeneration

Intervertebral disc (IVD) degeneration contributes largely to pathoanatomical and degenerative changes of spinal structure that increase the risk of low back pain. Apoptosis in nucleus pulposus (NP) can aggravate IVD degeneration, and increasing studies have shown that interventions targeting NP cell apoptosis can ameliorate IVD degeneration, exhibiting their potential for use as therapeutic strategies. Recent data have shown that advanced glycation end products (AGEs) accumulate in NP tissues in parallel with the progression of IVD degeneration and form a microenvironment of oxidative stress. This study examined whether AGEs accumulation aggravates NP cell apoptosis and IVD degeneration, and explored the mechanisms underlying these effects. We observed that the viability and proliferation of human NP cells were significantly suppressed by AGEs treatment, mainly due to apoptosis. Furthermore, activation of the mitochondrial apoptosis pathway was detected after AGEs treatment. In addition, the molecular data showed that AGEs could significantly aggravate the generation of mitochondrial reactive oxygen species and prolonged activation of the mitochondrial permeability transition pore, as well as the increased level of Bax protein and decreased level of Bcl-2 protein in mitochondria. These effects could be reduced by antioxidant (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) and Visomitin (SKQ1). Importantly, we identified that impairment of Sirtuin3 (SIRT3) function and the mitochondrial antioxidant network were vital mechanisms in AGEs-induced oxidative stress and secondary human NP cell apoptosis. Finally, based on findings that nicotinamide mononucleotide (NMN) could restore SIRT3 function and rescue human NP cell apoptosis through adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor-γ coactivator 1α (AMPK-PGC-1α) pathway in vitro, we confirmed its protective effect on AGEs-induced IVD degeneration in vivo. In conclusion, our data demonstrate that SIRT3 protects against AGEs-induced human NP cell apoptosis and IVD degeneration. Targeting SIRT3 to improve mitochondrial redox homeostasis may represent a potential therapeutic strategy for attenuating AGEs-associated IVD degeneration.

Proteomic Profiling Analysis of Male Infertility in Spodoptera Litura Larvae Challenged with Azadirachtin and its Potential-Regulated Pathways in the Following Stages.

Biopesticides are considered as an alternative to synthetic pesticide with a focus on increasing agricultural productivity as well as maintaining the ecosystem. Prior to application, its potential mechanism should be clearly addressed. Here, the effects of azadirachtin on the reproductive behavior in male Spodoptera litura (Fabricius) are determined. To further explore its molecular mechanism, an iTRAQ (isobaric tags for relative and absolute quantitation) based approach is applied to identify the differentially expressed proteins regulated by azadirachtin at two developmental stages. The results demonstrate that many proteins in the pathway of focal adhesion are regulated to exert influences in detachment of cell attachment, the loss of cell-cell interactions and inducing apoptosis at pupal stage, and many proteins in adenosine monophosphate-activated protein kinase pathway are also changed at the adult stage after azadirachtin-treatment as larvae. Moreover, based on their important roles, it is suggested that some proteins, such as ACTB-G1, ste20-related adaptor protein alpha, and regulatory-associated protein of mTOR (mTORC1) could serve as potential target proteins of azadirachtin to induce male infertility. The results of this study could provide evidence to illuminate the mechanism of male infertility induced by azadirachtin and potential targets for the development of environmentally friendly pesticides.

Metformin inhibits visceral allodynia and increased gut permeability induced by stress in rats.

Metformin has been shown to have anti-cytokine property. Lipopolysaccharide (LPS)-induced or repeated water avoidance stress (WAS)-induced visceral allodynia and increased gut permeability were pro-inflammatory cytokine-dependent responses, which were considered to be animal models of irritable bowel syndrome (IBS). We hypothesized that metformin improves symptoms in the patients with IBS by attenuating these visceral changes and tested the hypothesis in rats. The threshold of the visceromotor response induced by colonic balloon distention was measured. Colonic permeability was determined in vivo by quantifying the absorbed Evans blue for 15min spectrophotometrically. Subcutaneously injected LPS (1mg/kg) reduced the threshold of visceromotor response, and metformin (5-50mg/kg for 3days) intraperitoneally attenuated this response in a dose-dependent manner. Repeated WAS (1h daily for 3days) induced visceral allodynia, which was also blocked by metformin. The antinociceptive effect of metformin on the LPS-induced allodynia was reversed by compound C, an adenosine monophosphate-activated protein kinase inhibitor or NG -nitro-L-arginine methyl ester, a nitric oxide synthesis inhibitor but not modified by naloxone. Additionally, it was blocked by sulpiride, a dopamine D2 receptor antagonist, but domperidone, a peripheral dopamine D2 receptor antagonist, did not alter it. Metformin also blocked the LPS-induced or repeated WAS-induced increased colonic permeability. Metformin attenuated the visceral allodynia and increased gut permeability in animal IBS models. These actions may be evoked via activation of adenosine monophosphate-activated protein kinase, nitric oxide, and central dopamine D2 pathways. These results indicate the possibility that metformin can be useful for treating IBS.

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway.

To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway. Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 μmol/L CoCl2), Gs-Rb1 group (200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group (500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group (500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot. Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A (P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A (P<0.01) and was not affected by AICAR =0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B II, the LC3B II/I ratio and cathepsin B activity of hypoxia cardiomyocytes (P<0.05), each of these protein levels was significantly enhanced by Ara A (all P<0.01), but was not affected by AICAR (all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871). Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.

Dihydromyricetin Inhibits Lead-Induced Cognitive Impairments and Inflammation by the Adenosine 5'-Monophosphate-Activated Protein Kinase Pathway in Mice.

Dihydromyricetin (DHM), a natural flavonoid derived from the medicinal and edible plant Ampelopsis grossedentata, exhibits antioxidant, antiapoptosis, antitumor, and anti-inflammatory bioactivities. This study evaluated the effects of DHM on Pb-induced neurotoxicity and explored the underlying mechanisms. DHM significantly ameliorated behavioral impairments of Pb-induced mice. It decreased the levels of lipid peroxidation and protein carbonyl and increased the activities of superoxide dismutase and catalase in the brains. DHM suppressed Pb-induced apoptosis, as indicated by the decreased levels of Bax and cleaved caspase-3. DHM also decreased inflammatory cytokines in the brains of Pb-treated mice. DHM decreased amyloid-beta (Aβ) level and nuclear factor-κB nuclear translocation. Moreover, DHM induced the adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation and inhibited the activation of p38, Toll-like receptor 4, myeloid differentiation factor 88, and glycogen synthase kinase-3. Collectively, this is the first report indicating that DHM could improve Pb-induced cognitive functional impairment by preventing oxidative stress, apoptosis, and inflammation and that the protective effect was mediated partly through the AMPK pathway.

Chronic Heat Stress Damages Small Intestinal Epithelium Cells Associated with the Adenosine 5'-Monophosphate-Activated Protein Kinase Pathway in Broilers.

Heat-stressed broilers usually reduce their feed intake, leading to energy imbalance and disturbing the homeostasis in the small intestine. This study was aimed to explore heat-stress-mediated physiological features that may be ascribed to impairments in the intestinal tract of broilers. The results revealed that heat exposure increased the activities of trypsin and Na+/K+-ATPase, while it decreased the activities of amylase, lipase, and maltase as well as the proliferating cell nuclear antigen cells in the jejunum after 14 days of heat exposure. Meanwhile, heat stress upregulated the mRNA expressions of AMPKα1, LKB1, and HIF-1α and protein expressions of p-AMPKαThr172 and p-LKB1Thr189 in the small intestine after 7 or 14 days of heat exposure. In conclusion, chronic heat exposure impeded the development of digestive organs, disordered the activities of intestinal digestive enzymes, and impaired the intestinal epithelial cells by increasing the cell apoptosis and declining cell proliferation, which might be correlated with the adenosine 5'-monophosphate-activated protein kinase signaling pathway. Additionally, heat stress upregulated the gene expression of HIF-1α, which indicated that heat stress may disturb the homeostasis in the intestine.

Differentially expressed novel alternatively spliced transcript variant of tumor suppressor Stk11 gene in mouse

Serine/threonine kinase 11 (STK11) is a protein kinase that is encoded by Stk11 gene located on chromosome 19 and 10 in humans and mouse respectively. It acts as a master kinase of adenine monophosphate-activated protein kinase (AMPK) pathway that coordinates the regulation of cellular energy metabolism and cell division. STK11 exerts effect by activating more than 14 kinases including AMPK and AMPK-related kinases. It is also known to regulate cell polarity and acts as tumor suppressor. Alternative splicing of pre-mRNA is a mechanism which results in multiple transcript variants of a single gene. In human, two STK11 isoforms have been reported, an alternatively spliced isoform which has variation at its C-terminal and mostly expressed in testis (LKB1S). Another isoform exhibiting oncogenic properties lacks few residues at its N-terminal (ΔN-LKB1). In the present study, we report the identification of a new transcript variant Stk11N which is generated through alternative splicing. The new variant was found to have differential and tissue specific expression at Postnatal-7 and adult stages of mouse. As compared to the known variant Stk11C, the conceptually translated amino acid sequences of the new variant differ from exon-E2 onwards. In silico post translational studies of the new and published variant show similarity in some of the properties while differ in properties like nuclear export signals, phosphorylation, glycosylation, etc. Thus, alternative splicing of Stk11 gene generating new variant with heterogeneous properties suggests for complex regulation of these variants in controlling the AMPK pathway and other functions.

An in vitro comparative study of the antioxidant activity and SIRT1 modulation of natural compounds

Oxidative stress arises from an imbalance between the production of free radicals and antioxidant defences. Several studies have suggested that dietary antioxidants (such as polyphenols and berberine) may counteract oxidative stress through the involvement of the Sirtuin 1/Adenosine Monophosphate-Activated Protein Kinase (SIRT1/AMPK) pathway. The aim of this study was to evaluate the direct and specific antioxidant activity of some natural compounds, as well as their ability to modulate the expression of SIRT1 and the activation of AMPK.Quercetin, tyrosol, ferulic acid, catechin, berberine and curcumin were evaluated for their specific and direct antioxidant activity with TOSC assay. Their ability to modulate SIRT1 and AMPK was assessed by immunoblotting assay, while their cytotoxicity by CellTiter-Blue Cell Viability Assay.No statistically significant decrease (p > 0.05) in the number of viable cells was found upon challenging with the natural compounds. Quercetin exhibited the highest antioxidant activity against peroxyl radical and peroxinitrate derivates, while curcumin showed the best anti-hydroxyl activity with respect to the other compounds and, most importantly, respect to the reference antioxidants. Finally, all the tested compounds significantly increased the SIRT1 expression and the activation of AMPK.Our results clearly disclose the specific antioxidant activity of these natural compounds and their ability to increase SIRT1 expression and AMPK activation.

Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti

Gut microbes positively affect the physiology of many animals, but the molecular mechanisms underlying these benefits remain poorly understood. We recently reported that bacteria-induced gut hypoxia functions as a signal for growth and molting of the mosquito Aedes aegypti In this study, we tested the hypothesis that transduction of a gut hypoxia signal requires hypoxia-induced transcription factors (HIFs). Expression studies showed that HIF-α was stabilized in larvae containing bacteria that induce gut hypoxia but was destabilized in larvae that exhibit normoxia. However, we could rescue growth of larvae exhibiting gut normoxia by treating them with a prolyl hydroxylase inhibitor, FG-4592, that stabilized HIF-α, and inhibit growth of larvae exhibiting gut hypoxia by treating them with an inhibitor, PX-478, that destabilized HIF-α. Using these tools, we determined that HIF signaling activated the insulin/insulin growth factor pathway plus select mitogen-activated kinases and inhibited the adenosine monophosphate-activated protein kinase pathway. HIF signaling was also required for growth of the larval midgut and storage of neutral lipids by the fat body. Altogether, our results indicate that gut hypoxia and HIF signaling activate multiple processes in A. aegypti larvae, with conserved functions in growth and metabolism.

Anti-insulin resistant effect of ferulic acid on high fat diet-induced obese mice

Objective: To evaluate the insulin sensitivity action of ferulic acid (FA) in skeletal muscle and hypothalamus of high-fat diet (HFD)-induced obese mice. Methods: Obese mouse model was induced by HFD (45 kcal% lard fat) for 16 weeks. After 8 weeks of HFD feeding, these obese mice were orally treated with FA at doses of 25 and 50 mg/kg/day for 8 weeks. At the end of all treatments, the epididymal fat, pancreas, skeletal muscle and hypothalamus were removed for biochemical parameter and protein expression examinations. Results: FA treatment significantly decreased leptin level in fat tissue and insulin level in pancreas (P < 0.05). Interestingly, obese mice treated with FA increased the protein expressions of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and phosphorylated-protein kinase B in both muscle and brain (P < 0.05). The phosphorylations of adenosine monophosphate-activated protein kinase and acetyl-CoA carboxylase in muscle, and leptin receptor protein in hypothalamus were also increased (P < 0.05). The pancreatic islets histology showed smaller size in obese mice treated with FA compared to untreated obese mice. Conclusions: These findings indicate the beneficial effect of FA in improving insulin resistance in HFD-induced obese mice. These effects are probably mediated via modulating the insulin receptor substrate/phosphatidylinositol 3-kinase/protein kinase B or adenosine monophosphate-activated protein kinase pathways.

Antidiabetic Potential of Monoterpenes: A Case of Small Molecules Punching above Their Weight.

Monoterpenes belong to the terpenoids class of natural products and are bio-synthesized through the mevalonic acid pathway. Their small molecular weight coupled with high non-polar nature make them the most abundant components of essential oils which are often considered to have some general antioxidant and antimicrobial effects at fairly high concentrations. These compounds are however reported to have antidiabetic effects in recent years. Thanks to the ingenious biosynthetic machinery of nature, they also display a fair degree of structural complexity/diversity for further consideration in structure-activity studies. In the present communication, the merit of monoterpenes as antidiabetic agents is scrutinized by assessing recent in vitro and in vivo studies reported in the scientific literature. Both the aglycones and glycosides of these compounds of rather small structural size appear to display antidiabetic along with antiobesity and lipid lowering effects. The diversity of these effects vis-à-vis their structures and mechanisms of actions are discussed. Some key pharmacological targets include the insulin signaling pathways and/or the associated PI3K-AKT (protein kinase B), peroxisome proliferator activated receptor-γ (PPARγ), glucose transporter-4 (GLUT4) and adenosine monophosphate-activated protein kinase (AMPK) pathways; proinflammatory cytokines and the NF-κB pathway; glycogenolysis and gluconeogenesis in the liver; glucagon-like-1 receptor (GLP-1R); among others.

Caloric restriction can improve learning and memory in C57/BL mice probably via regulation of the AMPK signaling pathway

Caloric restriction (CR) is effective in slowing aging and delaying aging-related diseases in many species, but the mechanism is complex and not fully elucidated. This study aimed to evaluate the beneficial effects of a caloric restriction diet on learning and memory, and further to elucidate the mechanisms. Thirty-six week-old male C57/BL mice were randomly divided into three groups: normal control (NC group), high-energy (HE group) and CR group. After 44weeks, the Morris water maze was used to examine learning and memory abilities. Western blotting and immunohistochemistry were used to detect changes in proteins involved in the adenosine monophosphate-activated protein kinase (AMPK) pathway in the mouse hippocampus. Compared with NC group, the swimming distance and escape latency were shorter in the CR group. The protein and mRNA expression of AMPK and glucose transporter type 4 (GLUT4) in the CR group were significantly higher than that in HE group. CR increased serum insulin-like growth factor, adiponectin and vaspin, decreased blood glucose and serum malondialdehyde, and improved insulin sensitivity. Our findings demonstrate that a CR diet may improve hippocampus-dependent spatial learning ability of C57/BL mice, accompanied with an increase in AMPK and GLUT4 expression, which indicates AMPK pathway was associated with the neuroprotective effect of CR in mice.

Hempseed Peptides Exert Hypocholesterolemic Effects with a Statin-Like Mechanism

This study had the objective of preparing a hempseed protein hydrolysate and investigating its hypocholesterolemic properties. The hydrolysate was prepared treating a total protein extract with pepsin. Nano HPLC-ESI-MS/MS analysis permitted identifying in total 90 peptides belonging to 33 proteins. In the range 0.1-1.0 mg/mL, it inhibited the catalytic activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) in a dose-dependent manner. HepG2 cells were treated with 0.25, 0.5, and 1.0 mg/mL of the hydrolysate. Immunoblotting detection showed increments in the protein levels of regulatory element binding proteins 2 (SREBP2), low-density lipoprotein receptor (LDLR), and HMGCoAR. However, the parallel activation of the phospho-5'-adenosine monophosphate-activated protein kinase (AMPK) pathway, produced an inactivation of HMGCoAR by phosphorylation. The functional ability of HepG2 cells to uptake extracellular LDL was raised by 50.5 ± 2.7%, 221.5 ± 1.6%, and 109 ± 3.5%, respectively, versus the control at 0.25, 0.5, and 1.0 mg/mL concentrations. Finally, also a raise of the protein level of proprotein convertase subtilisin/kexintype 9 was observed. All of these data suggest that the mechanism of action has some similarity with that of statins.

Differential expression of microRNAs in luteinising hormone-treated mouse TM3 Leydig cells.

Testosterone is primarily produced by Leydig cells of the mammalian male gonads. The cellular functions of Leydig cells are regulated by the hypothalamus-pituitary-gonad axis, whereas the microRNA (miRNA) changes of LH-treated Leydig cells are unknown. Mouse TM3 Leydig cells were treated with LH, and deep sequencing showed that 29 miRNAs were significantly different between two groups (fold change of >1.5 or <0.5, p<.05), of which 27 were upregulated and two were downregulated. The differential expression of miR-29b-3p, miR-378b, miR-193b and miR-3695 was confirmed by quantitative real-time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs were involved in the Wingless and INT-1, adenosine 5'-monophosphate-activated protein kinase, NF-kappa B and Toll-like receptor signalling pathways. Results showed that miRNAs might be involved in the regulation of LH to Leydig cells.

Proposed mechanisms of the effects of proanthocyanidins on glucose homeostasis.

Proanthocyanidins are a major group of flavonoids in the human diet, known for their strong antioxidant properties. Emerging evidence from clinical studies indicates a role of proanthocyanidins in modulating glucose homeostasis, and higher proanthocyanidin intake has been associated with reduced risk of diabetes. On the other hand, recent studies report limited bioavailability of proanthocyanidins. At relatively low concentrations in the systemic circulation, proanthocyanidins may act as cell-signaling molecules to modulate glucose homeostasis. For example, they affect hepatic glucose production via adenosine monophosphate-activated protein kinase and/or insulin-signaling pathways. There is also evidence for a direct role of proanthocyanidins in modulating several pancreatic β-cell functions: prevention of oxidative stress, enhancement of insulin secretion, and promotion of β-cell survival. Therefore, greater understanding of the potentially beneficial effects of proanthocyanidins on cell-signaling pathways implicated in glucose homeostasis is needed. In addition, further investigation to address the in vivo metabolism of proanthocyanidins and the comparative effectiveness of proanthocyanidin-derived metabolites is warranted. The dosage and the experimental model should be given special attention when results from mechanistic studies using proanthocyanidins are interpreted.

Abstract 4855: Loss of USP18 represses invasion and metastasis of lung cancer

Abstract Metastasis is a major cause of human lung cancer mortality. Uncovering novel targets and mechanisms involved in regulating metastasis is critical for developing effective ways to improve lung cancer survival. It is reported that ubiquitin and ubiquitin-like pathways can regulate invasion and metastasis. We previously found that USP18 (Ubiquitin Specific Peptidase 18) is substantially upregulated in several cancers, including lung cancer. Engineered repression of the deubiquitinase USP18 decreased growth, increased apoptosis and augmented chemotherapeutic agent response of lung cancer cells. Our prior work showed that loss of USP18 can destabilize specific oncogenic proteins and this results in the marked reduction of lung cancer cell growth and in vivo tumorigenicity. This study sought to explore the precise role of USP18 expression in invasion, migration and metastasis of lung cancer. In murine (KC2 and 344SQ) and human (A549 and H1299) lung cancer cells with varying degrees of metastatic potential, knock-down of USP18 by different short hairpin RNAs (shRNAs) decreased cell growth and increased cell death as compared to vector control transfectants. The findings from wound-healing migration and Transwell invasion assays established that USP18 knock-down in all studied lung cancer cell lines conferred substantial reduction of migration and invasion versus vector control transfected cell lines (p &amp;lt; 0.001). In mouse models, USP18 knock-down of lung cancer cell lines displayed a lower number of lung cancer metastasis in vivo. To discern engaged mechanisms, Reverse Phase Protein Arrays (RPPAs) were performed to interrogate over 300 growth-regulatory proteins in murine (344SQ and KC2) and human (A549 and H1299) lung cancer cells following USP18 knock-down. Using RPPAs, potential targets identified were differentially expressed between USP18 knock-down and vector control transfected lung cancer cells. Highlighted species included Myc, eEF2K (Eukaryotic Elongation Factor 2 Kinase), Programmed Cell Death 4 (PDCD4), Hes Family BHLH Transcription Factor 1 (Hes) and Polo Like Kinase 1 (PLK1), among other species. In addition, phosphorylated Acetyl-CoA Carboxylase (ACC), a key component of the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway, was significantly affected in lung cancer cell lines following USP18 knock-down (p &amp;lt; 0.05). Ingenuity Pathway Analysis and functional validation are now underway to uncover specific pathways directly involved in the observed reduction of lung cancer metastases. Taken together, this study implicates the deubiquitinase USP18 as a molecular target to combat lung cancer metastases. Citation Format: Lin Zheng, Lisa Mustachio, Yulong Chen, Xi Liu, Jason Roszik, Jonathan Kurie, Ethan Dmitrovsky. Loss of USP18 represses invasion and metastasis of lung cancer [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 4855. doi:10.1158/1538-7445.AM2017-4855

Sestrin 2 suppresses cells proliferation through AMPK/mTORC1 pathway activation in colorectal cancer.

Sestrin 2 is a conserved antioxidant protein that reduces reactive oxygen species (ROS) and inhibits mammalian target of rapamycin complex 1 (mTORC1). We previously showed that sestrin 2 is abnormally decreased in colorectal cancer (CRC). To elucidate the molecular mechanism behind the potential contribution of sestrin 2 to CRC, we used a lentiviral expression vector system to determine the effects of sestrin 2 overexpression on human CRC cells. We found that sestrin 2 overexpression decreased ROS production, inhibited cell growth, and stimulated apoptosis in two CRC cell lines. In parallel, expression of the proliferation marker PCNA was decreased, proapoptotic caspase 3, 7, and 9 levels were increased, and expression of the anti-apoptotic protein survivin was reduced. Sestrin 2 overexpression also activated the adenosine monophosphate-activated protein kinase (AMPK) pathway, and suppressed mTORC1 signaling. Treating CRC cells with compound C, an AMPK inhibitor, reversed or attenuated changes in proliferation, apoptosis, and signaling proteins of the AMPK/mTORC1 axis. In a xenograft mouse model, CRC growth was attenuated by sestrin 2 overexpression. These results suggest that sestrin 2 suppresses CRC cell growth through activation of the AMPK/mTORC1 pathway and induction of apoptosis, and could be a novel pharmacological target for the treatment of CRC.

Specific Inhibition of Acyl-CoA Oxidase-1 by an Acetylenic Acid Improves Hepatic Lipid and Reactive Oxygen Species (ROS) Metabolism in Rats Fed a High Fat Diet

A chronic high fat diet results in hepatic mitochondrial dysfunction and induction of peroxisomal fatty acid oxidation (FAO); whether specific inhibition of peroxisomal FAO benefits mitochondrial FAO and reactive oxygen species (ROS) metabolism remains unclear. In this study a specific inhibitor for the rate-limiting enzyme involved in peroxisomal FAO, acyl-CoA oxidase-1 (ACOX1) was developed and used for the investigation of peroxisomal FAO inhibition upon mitochondrial FAO and ROS metabolism. Specific inhibition of ACOX1 by 10,12-tricosadiynoic acid increased hepatic mitochondrial FAO via activation of the SIRT1-AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway and proliferator activator receptor α and reduced hydrogen peroxide accumulation in high fat diet-fed rats, which significantly decreased hepatic lipid and ROS contents, reduced body weight gain, and decreased serum triglyceride and insulin levels. Inhibition of ACOX1 is a novel and effective approach for the treatment of high fat diet- or obesity-induced metabolic diseases by improving mitochondrial lipid and ROS metabolism.