Phosphorylation Levels Of AMPKα Research Articles

LEP inhibits intramuscular adipogenesis through the AMPK signaling pathway invitro.

Leptin can indirectly regulate fatty-acid metabolism and synthesis in muscle invivo and directly in incubated muscle exvivo. In addition, non-synonymous mutations in the bovine leptin gene (LEP) are associated with carcass intramuscular fat (IMF) content. However, the effects of LEP on lipid synthesis of adipocytes have not been clearly studied at the cellular level. Therefore, this study focused on bovine primary intramuscular preadipocytes to investigate the effects of LEP on the proliferation and differentiation of intramuscular preadipocytes, as well as its regulatory mechanism in lipid synthesis. The results showed that both the LEP and leptin receptor gene (LEPR) were highly expressed in IMF tissues, and their mRNA expression levels were positively correlated at different developmental stages of intramuscular preadipocytes. The overexpression of LEP inhibited the proliferation and differentiation of intramuscular preadipocytes, while interference with LEP had the opposite effect. Additionally, LEP significantly promoted the phosphorylation level of AMPKα by promoting the protein expression of CAMKK2. Meanwhile, rescue experiments showed that the increasing effect of AMPK inhibitors on the number of intramuscular preadipocytes was significantly weakened by the overexpression of LEP. Furthermore, the overexpression of LEP could weaken the promoting effect of AMPK inhibitor on triglyceride content and droplet accumulation, and prevent the upregulation of adipogenic protein expression (SREBF1, FABP4, FASN, and ACCα) caused by AMPK inhibitor. Taken together, LEP acted on the AMPK signaling pathway by regulating the protein expression of CAMKK2, thereby downregulating the expression of proliferation-related and adipogenic-related genes and proteins, ultimately reducing intramuscular adipogenesis.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKβ and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKβ induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

Antirheumatic drug leflunomide attenuates atherosclerosis by regulating lipid metabolism and endothelial dysfunction via DHODH/AMPK signaling pathway.

The probability of cardiovascular events has been reported lower in rheumatoid arthritis (RA) patients treated with leflunomide. However, the anti-atherosclerotic and cardiovascular protective effects and metabolism of leflunomide are not explored. In this study, we assessed the potential benefits of leflunomide on atherosclerosis and revealed the underlying mechanism. ApoE-/- mice were fed a western diet (WD) alone or supplemented with leflunomide (20 mg/kg, oral gavage, once per day) for 12 weeks. Samples of the aorta, heart, liver, serum, and macrophages were collected. We found that leflunomide significantly reduced lesion size in both en-face aortas and aortic root in WD-fed ApoE-/- mice. Leflunomide also obviously improved dyslipidemia, reduced hepatic lipid content, and improved disorders of glucose and lipid metabolism in vivo. RNA-Seq results showed that leflunomide effectively regulated the genes' expression involved in the lipid metabolism pathway. Importantly, leflunomide significantly increased the phosphorylation levels of AMPKα and acetyl-CoA carboxylase (ACC) in vivo. Furthermore, leflunomide and its active metabolite teriflunomide suppressed lipid accumulation in free fatty acid (FFA)-induced AML12 cells and improved endothelial dysfunction in palmitic acid (PA)-induced HUVECs through activating AMPK signaling and inhibiting dihydroorotate dehydrogenase (DHODH) signaling pathway. We present evidence that leflunomide and teriflunomide ameliorate atherosclerosis by regulating lipid metabolism and endothelial dysfunction. Our findings suggest a promising use of antirheumatic small-molecule drugs leflunomide and teriflunomide for the treatment of atherosclerosis and related cardiovascular diseases (CVDs).

AICAR Induces AMPK-Independent Cell Death in Adult T-Cell Leukemia/Lymphoma and Has Anti-Tumor Activity

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma caused by human T-cell lymphotropic virus type I (HTLV-1) and has a poor outcome. Recently, we reported that AMPKα, the catalytic unit of AMP-activated protein kinase (AMPK), is highly expressed in cells from patients with ATL and that an AMPK inhibitor had anti-tumor activity in ATL mice 1. On the other hand, acadesine or 5-aminoimidazole-4-carboxamide riboside (AICAR) is anAMPK activator, and was recently shown to have tumor suppressive effects on B cell chronic lymphocytic leukemia via the activation of AMPK. However, the effect of AICAR on the death of ATL-related cell lines is unknown. This study evaluated the effects of AICAR on death in ATL-related cell lines and its anti-tumor activity. Results: We demonstrated that AICAR inhibited the proliferation of ATL-related cell lines (S1T, MT-1 and MT-2) but not non-HTLV-1-infected Jurkat cells and peripheral blood mononuclear cells from healthy individuals. However, AICAR did not increase the phosphorylation levels of AMPKα. We found that AICAR increased annexin V-positive cell numbers in the sub-G0/G1 phase, and decreased the mitochondrial membrane potential and caspase-3/8/9 activation, which are hallmarks of apoptosis. However, AICAR did not increase the phosphorylation levels of AMPKα. On the other hand, HTLV-1 Tax, an HTLV-1-encoded oncogenic factor, expression did not affect AICAR-induced cell death. Moreover, AICAR induced the expression of death receptors (DR) DR4 and DR5 in vitro. Furthermore, AICAR inhibited the growth and infiltration of S1T cells transplanted subcutaneously in NOD/SCID/gamma mice. Conclusion: This is the first evidence to demonstrate the AMPK-independent effects of AICAR on death in ATL-related cell lines and its anti-tumor activity. Thus, AICAR might be a candidate for the treatment of ATL. Disclosures : No relevant conflicts of interest to declare. 1. Aikawa A, Kozako T, Uchida Y, et al. Cell death induced by dorsomorphin in adult T-cell leukemia/lymphoma is AMPK-independent. Febs j. 2020;287(18):4005-4015.

Anti-tumor activity of 5-aminoimidazole-4-carboxamide riboside with AMPK-independent cell death in human adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma caused by human T-cell lymphotropic virus type I (HTLV-1). Acadesine or 5-aminoimidazole-4-carboxamide riboside (AICAR) is an AMP-activated protein kinase (AMPK) activator that was recently shown to have tumor suppressive effects on B cell chronic lymphocytic leukemia, but not ATL. This study evaluated the cytotoxic effects of AICAR on ATL-related cell lines and its anti-tumor activity. Here, we demonstrated that AICAR induced cell death via apoptosis and the mitochondrial membrane depolarization of ATL-related cell lines (S1T, MT-1, and MT-2) but not non-HTLV-1-infected Jurkat cells. However, AICAR did not increase the phosphorylation levels of AMPKα. In addition, AICAR increased the expression of the death receptors (DR) DR4 and DR5, and necroptosis-related proteins including phosphorylated receptor-interacting protein family members and the mixed lineage kinase domain-like protein. Interestingly, HTLV-1 Tax, an HTLV-1-encoded oncogenic factor, did not affect AICAR-induced apoptosis. Furthermore, AICAR inhibited the growth of human ATL tumor xenografts in NOD/SCID/gamma mice in vivo. Together, these results suggest that AICAR induces AMPK-independent cell death in ATL-related cell lines and has anti-tumor activity, indicating that it might be a therapeutic agent for ATL.

Phosphoenolpyruvate induces endothelial dysfunction and cell senescence through stimulation of metabolic reprogramming

Endothelial dysfunction is a key early link in the pathogenesis of atherosclerosis, and the accumulation of senescent vascular endothelial cells causes endothelial dysfunction. Phosphoenolpyruvate (PEP), which is a high-energy glycolytic intermediate, protects against ischemia-reperfusion injury in isolated rat lung, heart, and liver tissue by quickly providing ATP. However, it was reported that serum PEP concentrations are 13-fold higher in healthy elderly compare to the young. Unlike that of other cell types, the energy required for the physiological function of endothelial cells is mainly derived from glycolysis. Recently, it is unclear whether circulating accumulation of PEP affects endothelial cell function. In this study, we found for the first time that 50-250μM of PEP significantly promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs) through increased expression of vascular endothelial adhesion factor 1 (VCAM1) and intercellular adhesion factor 1 (ICAM1) in HUVECs. Meanwhile, 50-250μM of PEP decreased the expression of endothelial nitric oxide synthase (eNOS) and cellular level of nitric oxide (NO) in HUVECs. Moreover, PEP increased levels of ROS, enhanced the numbers of SA-β-Gal-positive cells and upregulated the expression of cell cycle inhibitors such as p21, p16 and the phosphorylation level of p53 on Ser15, and the expression of proinflammatory factors including TNF-α, IL-1β, IL-6, IL-8, IL-18 and MCP-1 in HUVECs. Furthermore, PEP increased both oxygen consumption rate (OCR) and glycolysis rate, and was accompanied by reduced NAD+/NADH ratios and enhanced phosphorylation levels of AMPKα (Thr172), p38 MAPK (T180/Y182) and NF-κB p65 (Ser536) in HUVECs. Notably, PEP had no significant effect on hepG2 cells. In conclusion, these results demonstrated that PEP induced dysfunction and senescence in vascular endothelial cells through stimulation of metabolic reprogramming.

Arsenic induces autophagy-dependent apoptosis via Akt inactivation and AMPK activation signaling pathways leading to neuronal cell death

Inorganic arsenic (As3+), a well-known worldwide industrial and environmental pollutant, has been linked to neurodegenerative disorders (NDs). Autophagy plays an important role in controlling neuronal cell survival/death. However, limited information is available regarding the toxicological mechanism at the interplay between autophagy and As3+-induced neurotoxicity. The present study found that As3+ exposure induced a concomitant activation of apoptosis and autophagy in Neuro-2a cells, which was accompanied with the increase of phosphatidylserine exposure on outer membrane leaflets and apoptotic cell population, and the activation of caspase-3, -7, and PARP as well as the elevation of protein expressions of LC3-II, Atg-5, and Beclin-1, and the accumulation of autophagosome. Pretreatment of cells with autophagy inhibitor 3-MA, but not that of Z-VAD-FMK (a pan-caspase inhibitor), effectively prevented the As3+-induced autophagic and apoptotic responses, indicating that As3+-triggered autophagy was contributing to neuronal cell apoptosis. Furthermore, As3+ exposure evoked the dephosphorylation of Akt. Pretreatment with SC79, an Akt activator, could significantly attenuated As3+-induced Akt inactivation as well as autophagic and apoptotic events. Expectedly, inhibition of Akt signaling with LY294002 obviously enhanced As3+-triggered autophagy and apoptosis. Exposure to As3+ also dramatically increased the phosphorylation level of AMPKα. Pretreatment of AMPK inhibitor (Compound C) could markedly abrogate the As3+-induced phosphorylated AMPKα expression, and autophagy and apoptosis activation. Taken together, these results indicated that As3+ exerted its cytotoxicity in neuronal cells via the Akt inactivation/AMPK activation downstream-regulated autophagy-dependent apoptosis pathways, which ultimately lead to cell death. Our findings suggest that the regulation of Akt/AMPK signals may be a promising intervention to against As3+-induced neurotoxicity and NDs.

Choline and methionine regulate lipid metabolism via the AMPK signaling pathway in hepatocytes exposed to high concentrations of nonesterified fatty acids.

High concentrations of nonesterified fatty acids (NEFAs) and β-hydroxybutyric acid (BHBA) induce lipid peroxidation, resulting in liver damage. Choline and methionine (Met) can promote energy balance and benefit liver health in transition dairy cows; however, the regulating mechanism remains unclear. In the present study, we established the hepatocytedamage model by 1.5 mM NEFAs or BHBA treatment, and examined lipid metabolism in hepatocytes. The results showed that 1.5 mM NEFAs and 1.5 mM BHBA significantly decreased the messenger RNA (mRNA) expression of AMP-activated protein kinase (AMPK)-α as well as its target genes carnitine palmitoyltransferase-1α (CPT-1α), acetyl-CoA carboxylase, fatty acid synthetase, and Apolipoprotein B100 (ApoB100). Choline and Met upregulated the phosphorylation level of AMPK-α, which was blocked by BML (an AMPK-α inhibitor). The mRNA expression level of peroxisome proliferator-activated receptor-α (PPAR-α), CPT-1α, and ApoB100 showed a similar trend. The expressions of liver X recptoer α (LXR-α) and sterol regulatory element-binding protein 1c (SREBP-1c) were decreased by choline and Met, while only the decrease of LXR-α was blocked by BML. These findings indicate that the high-level NEFAs and BHBA weaken the lipid metabolism by impairing the fatty acid oxidation, synthesis, and transport proteins. Choline and Met regulate PPAR-α and LXR-α transcriptional activity through AMPK-α phosphorylation and regulate SREBP-1c independently of AMPK-α to promote lipid oxidation and transport in NEFAs-treated hepatocytes.

Molecular characteristics of AMPK and its role in regulating the phagocytosis of oyster hemocytes

Phagocytosis is one of the fundamental cellular immune defense parameter that helps in the elimination of the invading pathogens in both vertebrates and invertebrates, which require plenty of energy for functioning. In the present study, we identified the critical energy regulator AMP-activated protein kinase (AMPK) in Crassostrea hongkongensis which is composed of three subunits, named ChAMPK-α, ChAMPK-β, and ChAMPK-γ, and then analyzed the function of AMPK in regulating hemocyte phagocytosis. All the three ChAMPK subunits mRNA were detected to be expressed at various embryological stages, and also constitutively expressed in multiple tissues with high expression in gill and mantle. The phylogenetic tree showed that the three subunits of AMPK were correspondingly clustered with its orthologue branches. Furthermore Western Blot analysis revealed that the AMPK pharmacological inhibitors Compound C could effectively down-regulate the Thr172 phosphorylation level of AMPK-α, and the hemocyte phagocytosis was inhibited by Compound C (CC), which indicate its existence in the oyster. Our results showed that treatment of AMPK inhibitors significantly attenuated the capacity of hemocytes phagocytosis. Moreover, Compound C could also change the organization of actin cytoskeleton in the oyster hemocytes, demonstrating the crucial role of AMPK signaling in control of phagocytosis.

All-Trans-Retinoic Acid Suppresses Neointimal Hyperplasia and Inhibits Vascular Smooth Muscle Cell Proliferation and Migration via Activation of AMPK Signaling Pathway.

The proliferation and migration of vascular smooth muscle cells (VSMC) is extensively involved in pathogenesis of neointimal hyperplasia. All-trans-retinoic acid (ATRA) is a natural metabolite of vitamin A. Here, we investigated the involvement of AMP-activated protein kinase (AMPK) in the anti-neointimal hyperplasia effects of ATRA. We found that treatment with ATRA significantly reduced neointimal hyperplasia in the left common carotid artery ligation mouse model. ATRA reduced the proliferation and migration of VSMC, A7r5 and HASMC cell lines. Our results also demonstrated that ATRA altered the expression of proliferation-related proteins, including CyclinD1, CyclinD3, CyclinA2, CDK2, CDK4, and CDK6 in VSMC. ATRA dose-dependently enhanced the phosphorylation level of AMPKα (Thr172) in the left common carotid artery of experimental mice. Also, the phosphorylation level of AMPKα in A7r5 and HASMC was significantly increased. In addition, ATRA dose-dependently reduced the phosphorylation levels of mTOR and mTOR target proteins p70 S6 kinase (p70S6K) and 4E-binding protein 1 (4EBP1) in A7r5 and HASMC. Notably, the inhibition of AMPKα by AMPK inhibitor (compound C) negated the protective effect of ATRA on VSMC proliferation in A7r5. Also, knockdown of AMPKα by siRNA partly abolished the anti-proliferative and anti-migratory effects of ATRA in HASMC. Molecular docking analysis showed that ATRA could dock to the agonist binding site of AMPK, and the binding energy between AMPK and ATRA was -7.91 kcal/mol. Molecular dynamics simulations showed that the binding of AMPK-ATRA was stable. These data demonstrated that ATRA might inhibit neointimal hyperplasia and suppress VSMC proliferation and migration by direct activation of AMPK and inhibition of mTOR signaling.

Overexpression of CTRP3 protects against sepsis-induced myocardial dysfunction in mice.

C1q/tumor necrosis factor-related protein-3 (CTRP3) shows striking homologies of genomic structure to the adiponectin. In this study, we aimed to investigate the protective role of CTRP3 against sepsis-induced cardiomyopathy. Here, we overexpressed CTRP3 in myocardium by direct intramyocardial injection and constructed a model of lipopolysaccharide (LPS)-induced sepsis in mice. Our results demonstrated that cardiac-specific overexpression of CTRP3 remarkably attenuated myocardial dysfunction and increased the phosphorylation level of AMPKα during LPS-induced sepsis. The anti-inflammatory effects of CTRP3, as determined by decreased mRNA levels of TNF-α, IL-6 and a lower protein expression of phosphorylated NF-κB p65 and IκBα, was detected in mice following LPS treatment. Additionally, CTRP3 suppressed cardiac apoptosis induced by LPS in mice as indicated by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining and western blot for Cleaved-caspase3, Bax and Bcl-2. In conclusion, CTRP3 could protect against sepsis-induced myocardial dysfunction in mice. The cardioprotective effects of CTRP3 might be mediated by activating AMPKα signaling pathway and blunting inflammatory response and apoptosis.

Chronic Heat Stress Impairs the Quality of Breast-Muscle Meat in Broilers by Affecting Redox Status and Energy-Substance Metabolism.

We investigated the molecular mechanisms by which chronic heat stress impairs the breast-meat quality of broilers. Broilers were assigned to three groups: the normal control (NC) group, heat-stress (HS) group, and pair-fed (PF) group. After 7 days of heat exposure (32 °C), the high temperature had caused oxidative stress; elevated the activity of citrate synthase (CS), the mRNA expression of M-CPT1, and the phosphorylation level of AMPKα; and reduced the mRNA expression of avUCP. After 14 days of heat exposure, the heat stress had increased the lightness and drip loss and decreased the pH and shear force of the breast meat. Additionally, the heat exposure had increased the mRNA expressions of FAS, ACC, and PDK4; the content of lipids; and the activities of lactic dehydrogenase and pyruvate kinase, and it had decreased the mRNA expression of M-CPT1 and the activity of CS. In conclusion, chronic heat stress impairs meat quality by causing mitochondria to malfunction and affecting energy-substance aerobic metabolism, resulting in increased glycolysis and intramuscular fat deposition.

Green tea polyphenols alter lipid metabolism in the livers of broiler chickens through increased phosphorylation of AMP-activated protein kinase.

Our previous results showed that green tea polyphenols (GTPs) significantly altered the expression of lipid-metabolizing genes in the liver of chickens. However, the underlying mechanism was not elucidated. In this study, we further characterized how GTPs influence AMP-activated protein kinase (AMPK) in the regulation of hepatic fat metabolism. Thirty-six male chickens were fed GTPs at a daily dose of 0, 80 or 160 mg/kg of body weight for 4 weeks. The results demonstrated that oral administration of GTPs significantly reduced hepatic lipid content and abdominal fat mass, enhanced the phosphorylation levels of AMPKα and ACACA, and altered the mRNA levels and enzymatic activities of lipid-metabolizing enzymes in the liver. These results suggested that the activation of AMPK is a potential mechanism by which GTPs regulate hepatic lipid metabolism in such a way that lipid synthesis is reduced and fat oxidation is stimulated.

Antiaging Gene Klotho Deficiency Promoted High-Fat Diet-Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase.

Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-β1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-β1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity.

Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis.

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dismutase 1 mutant (SOD1G93A) and wild-type (SOD1WT) mouse models were exposed to H2O2. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenosine monophosphate-activated protein kinase (AMPK) α-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1WT cells, SOD1G93A embryonic neural stem cells were more likely to undergo H2O2-induced apoptosis. Phosphorylation of AMPKα in SOD1G93A cells was higher than that in SOD1WT cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKα. p53 protein levels were also correlated with AMPKα phosphorylation levels. Compound C, an inhibitor of AMPKα, attenuated the effects of H2O2. These results suggest that embryonic neural stem cells from SOD1G93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKα pathway.

Resveratrol Supplementation Does Not Improve Metabolic Function in Nonobese Women with Normal Glucose Tolerance

Resveratrol has been reported to improve metabolic function in metabolically abnormal rodents and humans, but it has not been studied in nonobese people with normal glucose tolerance. We conducted a randomized, double-blind, placebo-controlled trial to evaluate the metabolic effects of 12weeks of resveratrol supplementation (75mg/day) in nonobese, postmenopausal women with normal glucosetolerance. Although resveratrol supplementation increased plasma resveratrol concentration, it did not change body composition, resting metabolic rate, plasma lipids, or inflammatory markers. A two-stage hyperinsulinemic-euglycemic clamp procedure, in conjunction with stable isotopically labeled tracer infusions, demonstrated that resveratrol did not increase liver, skeletal muscle, or adipose tissue insulin sensitivity. Consistent with the absence of invivo metabolic effects, resveratrol did not affect its putative molecular targets, including AMPK, SIRT1, NAMPT, and PPARGC1A, in either skeletal muscle or adipose tissue. These findings demonstrate that resveratrol supplementation does not have beneficial metabolic effects innonobese, postmenopausal women with normal glucose tolerance.

Quercetin intake during lactation modulates the AMP-activated protein kinase pathway in the livers of adult male rat offspring programmed by maternal protein restriction

Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities including activation of adenosine-5’-monophosphate-activated protein kinase (AMPK). We investigated the effects of quercetin intake during lactation on the AMPK activation in the livers of adult offspring programmed by maternal protein restriction during gestation. Pregnant Wistar rats were fed control and low-protein diets during gestation. Following delivery, each dam received a control or 0.2% quercetin-containing control diet during lactation as follows: control on control (CC), control on restricted (LPC) and 0.2% quercetin-containing control on restricted (LPQ). At weaning (week 3), some of the pups from each dam were killed, and the remaining pups (CC, n=8; LPC, n=10; LPQ, n=13) continued to receive a standard laboratory diet and were killed at week 23. Blood chemistry and phosphorylation levels of AMPKα, acetyl-CoA carboxylase (ACC), endothelial nitric oxide synthase (eNOS) and mammalian target of rapamycin (mTOR) in the livers of male offspring were examined. At week 3, the level of phosphorylated AMPK protein in LPQ increased about 1.5- and 2.1-fold compared with LPC and CC, respectively, and the level in LPQ at week 23 increased about 1.9- and 2.9-fold, respectively. A significant increase in phosphorylated ACC and eNOS levels was found in LPQ. There was no significant difference among the three groups in the level of phosphorylated mTOR protein. In conclusion, quercetin intake during lactation up-regulates AMPK activation in the adult offspring of protein-restricted dams and modulates the AMPK pathway in the liver.

Abstract 4071: Sestrins modulate the expression of the metabolic stress sensor AMPK in breast cancer cells

Abstract Introduction: Sestrins (SESN) are stress response mediators that accumulate in cells exposed to genotoxic stresses, such as ultraviolet and ionizing radiation (IR). The three mammalian SESN (1-3) were recently shown to modulate signalling events. SESN1/2 are suggested to interact with and increasing the activity of the metabolic stress sensor AMP-activated protein kinase (AMPK). SESN1/2 have been postulated to act as scaffolding proteins that can bind targets of the AMPK metabolic pathway including AMPKα and TSC2. However, the direct mechanism in which SESN1/2 increase AMPK activity is still being elucidated. Furthermore, the ability of SESNs to modulate the expression of AMPK subunits in untreated or IR treated cells has not been examined. Methods: Immunoprecipitation of the AMPK subunits (α1-2, β1-2, γ1-3) with subunit-specific antibodies was preformed to determine the function AMPK heterotrimer complexes in MCF7 breast cancer cells. Overexpression of full length SESN2 cDNA was achieved in MCF7 cells using tetracycline inducible promoter system. SESN1/2 siRNA was introduced into cells using HiPerFect transfection reagent and the Qiagen protocol. Protein and mRNA levels were measured with western blotting and RT-PRC, respectively. Cells were treated with 0 – 8 Gy IR, and cell survival was measured using clonogenic assays. Results: Through immunoprecipitation we identified that the most prominent AMPK functional complex in MCF7 cells is the α1β1γ1 heterotrimer. SESN2 overexpression increases both mRNA and protein expression levels of AMPKα1 and AMPKβ1, and was co-immunoprecipitated with the AMPKβ1 subunit. Similarly, increasing SENS2 expression increased the phosphorylation levels of AMPKα (Thr172) and AMPKβ1 (Ser108), as well as phosphorylation of the AMPK substrate ACC. Conversely, siRNA against SESN2 decreased both AMPKα1 and AMPKβ1 mRNA and protein levels, while SESN1 siRNA blocked basal and IR-induced phosphorylation of AMPKα (Thr172). In addition, SESN2 overexpression sensitized MCF7 cells to the cytotoxic effects of IR. Conclusions: To date our work suggests that upregulation of SESNs in breast cancer cells modulates AMPK by stabilizing the AMPK (α1β1γ1) heterotrimer and by enhancing the subunit expression of AMPK. We observed that SESN2 was shown to directly interact with the AMPKβ1 subunit and also enhance its Ser108 phosphorylation, an additional marker of AMPK activity. Furthermore, silencing SESN1 expression blocks basal IR-induced AMPKα (Thr172) phosphorylation, while SESN2 overexpression acts as a radiation sensitizer in MCF7 cells. Taken together, this data indicates that SESNs not only regulate AMPK activity, but also play a role in stabilizing this enzymes basal and stress-induced expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2011-4071