AMPK-activated Protein Kinase Research Articles

Deletion of AMPK Reduces Oxidative Metabolism in Gvhd-Causing Human T Cells

BACKGROUND Allogeneic hematopoietic stem cell transplantation (AlloHSCT) is a curative therapy for many hematological disorders including primary immunodeficiencies and relapsed/refractory leukemia. However, the therapeutic application of AlloHSCT is limited by acute graft-versus-host-disease (GVHD), where donor T cells recognize and destroy recipient tissues in the skin, liver, and gastrointestinal tract. GVHD occurs following 30-50% of allogeneic transplants and is a leading cause of non-relapse morbidity and mortality. Recent efforts to treat GVHD have improved but attempts to prevent GVHD remain an important area of ongoing investigation. Following transplantation, alloreactive T cells exhibit heightened energy requirements that present a promising area for therapeutic intervention. Previously, we have shown that murine T cells upregulate activity of the cellular energy sensor AMPK-activated protein kinase (AMPK) during GVHD initiation and furthermore, that transfer of AMPK-deficient T cells increased recipient survival while preserving leukemia clearance and immune reconstitution. Whether these findings translate to human cells is unknown. Here, we report successful targeting of AMPK in primary human T cells and demonstrate clear similarities in phenotype between murine and human T cells lacking AMPK. METHODS To delete AMPK, primary human T cells were isolated from healthy donors and electroporated with Cas 9 ribonuclear protein complexes targeting the AMPKa1 locus. Deletion of AMPK was assessed via DNA sequencing (TIDE analysis) and phosphorylation of downstream target Unc-51-like kinase (ULK1) by immunoblot. Oxidative metabolism and spare respiratory capacity (SRC) were determined via extracellular flux analysis (Agilent Seahorse). Mitochondrial transmembrane potential (TMRM) and cell phenotype (mitochondrial density, apoptosis) were evaluated by flow cytometry after cells stimulation. Intracellular cytokine expression was assessed following 72 hours in a mixed lymphocyte reaction (MLR), followed by re-stimulation with PMA/Ionomycin in the presence of Brefeldin A. AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients for 7 days for comparative analysis. RESULTS CRISPR treatment of primary human T cells efficiently deleted AMPK as measured by both DNA sequencing (92.3% insertion/deletion frequency by TIDE analysis) and immunoblot analysis of downstream ULK1 phosphorylation. When re-stimulated in physiological glucose (5.5 mM), AMPK-deficient human T cells significantly decreased mitochondrial respiration (Fig. 1A) as measured by both maximal oxygen consumption (174.6 ± 27.73 vs 277.3 ± 22.88, p<0.0001) and spare respiratory capacity (SRC, 126.9 ± 22.78 vs 207.1 ± 19.68, p<0.0001). This result was strikingly similar to the metabolism of post-transplant murine T cells lacking AMPK, which exhibited a proportional decrease in SRC (-6.499 ± 5.156 vs 29.11 ± 7.064, p<0.0001) and maximal oxygen consumption (46.82 ± 10.11 vs 80.01 ± 6.946, p<0.0001). AMPK-deficient human T cells also experienced decreased cellular viability (43.4% vs 51.8%) as well as limited mitochondrial membrane potential (Fig. 1B); however, no differences in mitochondrial density were observed. Upon co-culture with allogeneic antigen presenting cells, AMPK-deficient human CD4+ T cells produced lower levels of the pro-inflammatory cytokines TNFa (97.7 MFI vs 209 MFI) and IL-2 ( 391 vs 454 MFI). DISCUSSION Here, we demonstrate a method to effectively delete AMPK in human T cells, a modification which decreases oxidative metabolism identical to what is observed in AMPK-deficient murine T cells. Specifically, human T cells lacking AMPK decreased their maximal mitochondrial respiration and spare respiratory capacity, a result we attribute to decreased mitochondrial transmembrane potential without changes in mitochondrial mass. Together with decreased pro-inflammatory cytokine production, we predict these changes will limit the viability and function of alloreactive T cells in vivo. As such, future studies will assess the disease potential of AMPK-deficient human T cells in a modified model of xenogeneic GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity

G6PD (glucose-6-phosphate dehydrogenase) is the rate-limiting enzyme in the oxidative pentose phosphate pathway that can generate cytosolic NADPH for biosynthesis and oxidative defense. Since cytosolic NADPH can be compensatively produced by other sources, the enzymatic activity deficiency alleles of G6PD are well tolerated in somatic cells but the effect of null mutations is unclear. Herein, we show that G6PD KO sensitizes cells to the stresses induced by hydrogen peroxide, superoxide, hypoxia, and the inhibition of the electron transport chain. This effect can be completely reversed by the expressions of natural mutants associated with G6PD deficiency, even without dehydrogenase activity, exactly like the WT G6PD. Furthermore, we demonstrate that G6PD can physically interact with AMPK (AMPK-activated protein kinase) to facilitate its activity and directly bind to NAMPT (nicotinamide phosphoribosyltransferase) to promote its activity and maintain the NAD(P)H/NAD(P)+ homeostasis. These functions are necessary to the antistress ability of cells but independent of the dehydrogenase activity of G6PD. In addition, the WT G6PD and naturally inactive mutant also can similarly regulate the metabolism of glucose, glutamine, fatty acid synthesis, and GSH and interact with the involved enzymes. Therefore, our findings reveal the previously unidentified functions of G6PD that can act as the important physiological neutralizer of stresses independently of its enzymatic activity.

Chronic AMPK Activation Reduces the Expression and Alters Distribution of Synaptic Proteins in Neuronal SH-SY5Y Cells.

Neuronal growth and synaptic function are dependent on precise protein production and turnover at the synapse. AMPK-activated protein kinase (AMPK) represents a metabolic node involved in energy sensing and in regulating synaptic protein homeostasis. However, there is ambiguity surrounding the role of AMPK in regulating neuronal growth and health. This study examined the effect of chronic AMPK activation on markers of synaptic function and growth. Retinoic-acid-differentiated SH-SY5Y human neuroblastoma cells were treated with A-769662 (100 nM) or Compound C (30 nM) for 1, 3, or 5 days before AMPK, mTORC1, and markers for synapse function were examined. Cell morphology, neuronal marker content, and location were quantified after 5 days of treatment. AMPK phosphorylation was maintained throughout all 5 days of treatment with A-769662 and resulted in chronic mTORC1 inhibition. Lower total, soma, and neuritic neuronal marker contents were observed following 5 d of AMPK activation. Neurite protein abundance and distribution was lower following 5 days of A-769662 treatment. Our data suggest that chronic AMPK activation impacts synaptic protein content and reduces neurite protein abundance and distribution. These results highlight a distinct role that metabolism plays on markers of synapse health and function.

Extracellular Nicotinamide Phosphoribosyltransferase Is a Component of the Senescence-Associated Secretory Phenotype.

Cellular senescence is a stress or damage response by which a cell adopts of state of essentially permanent proliferative arrest, coupled to the secretion of a number of biologically active molecules. This senescence-associated secretory phenotype (SASP) underlies many of the degenerative and regenerative aspects of cellular senescence - including promoting wound healing and development, but also driving diabetes and multiple age-associated diseases. We find that nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in nicotinamide adenine dinucleotide (NAD) biosynthesis, is elevated in senescent cells without a commensurate increase in NAD levels. This elevation is distinct from the acute DNA damage response, in which NAD is depleted, and recovery of NAD by NAMPT elevation is AMPK-activated protein kinase (AMPK)-dependent. Instead, we find that senescent cells release extracellular NAMPT (eNAMPT) as part of the SASP. eNAMPT has been reported to be released as a catalytically active extracellular vesicle-contained dimer that promotes NAD increases in other cells and extends lifespan, and also as free monomer that acts as a damage-associated molecular pattern and promotes conditions such as diabetes and fibrosis. Senescent cells released eNAMPT as dimer, but surprisingly eNAMPT appeared in the soluble secretome while being depleted from exosomes. Finally, diabetic mice showed elevated levels of eNAMPT, and this was lowered by treatment with the senolytic drug, ABT-263. Together, these data reveal a new SASP factor with implications for NAD metabolism.

Transcriptome analysis reveals the underlying mechanism of heptanal against Aspergillus flavus spore germination.

Methods of controlling Aspergillus flavus contamination in agro-products have attracted attention because of its impact on global food security. We previously reported that the natural cereal volatile heptanal could effectively inhibit A. flavus growth and showed great potential as a bio-preservative agent. In this study, the minimum inhibitory concentration and minimum fungicide concentration of heptanal could change the surface morphology of A. flavus spores, causing them to wrinkle and collapse. Transcriptomic analysis showed that heptanal treatment significantly changed the expression of several genes involved in cell wall and plasma damage, reactive oxygen species (ROS) accumulation, energy metabolism, AMPK-activated protein kinase, biosynthesis of unsaturated fatty acids, RNA degradation, and DNA replication. Heptanal-induced early apoptosis of A. flavus spores was characterized by decreased mitochondrial membrane potential, increased intracellular ROS production, and DNA fragmentation. This study provides new insight into the inhibitory mechanism of heptanal against A. flavus and points to its potential application as a bio-preservative. KEY POINTS: • Heptanal can effectively inhibit A. flavus growth in cereal grains. • The transcriptional changes in A. flavus spores exposed to heptanal were analyzed. • The antifungal mechanism of heptanal against A. flavus was elucidated.

Abstract 93: AMPK alpha1 and AMPK alpha 2 control TNBC proliferation through cell cycle regulation

Abstract Introduction. Triple negative breast cancer (TNBC) is very aggressive, has a higher metastatic rate than other breast cancer subtypes, and lacks traditional targetable factors. AMPK-activated protein kinase (AMPK) is an important energy regulator within the cell that also impacts cancer progression. AMPK's catalytic subunit is composed of α1 and α2 isoforms, but little is known about their individual roles in TNBC. The purpose of this study was to establish the specific function of each isoform in TNBC with respect to expression, cell proliferation, cell cycle progression, and metabolism. Methods. (i) Immunohistochemistry, immunofluorescence, and immunoblotting were used to determine the expression and localization of AMPKα1 and AMPKα2 in TNBC PDX samples or cell lines. (ii) To study the role of each isoform individually, TNBC cells were transfected with siRNA to NTC, AMPKα1, or AMPKα2 (50 nM) and were either harvested for immunoblotting or subseeded for cell cycle analysis, proliferation assays, or Seahorse analysis. Results. (i) AMPKα1 expression was localized in the cytoplasm of all 9 TNBC PDX cell lines, whereas AMPKα2 was expressed predominantly in the nucleus in 6 of the 9 of TNBC samples. IF analysis of MDA-MB-231 cells confirmed expression findings in the PDX cell lines with AMPKα1 expression noted in the cytoplasm and AMPKα2 expression in both the cytoplasm and nucleus. (ii) siRNA knockdown of AMPKα1 or AMPKα2 induced G1 cell cycle arrest and decreased proliferation of MDA-MB-231 cells. Moreover, knockdown of AMPKα1 decreased levels of cyclin D1 in MDA-MB-231, MDA-MB-468, and BT-20 cells. siRNA knockdown of AMPKα1, but not AMPKα2, led to decreased basal and compensatory glycolysis in MDA-MB-231 cells. Conclusions. Our findings indicate that both AMPKα1 and AMPKα2 can impact cell cycle progression and proliferation in TNBC; both isoforms promote progression through the cell cycle, thus supporting the proliferative capacity of TNBC. Importantly, AMPKα1 may also affect TNBC proliferation by increasing energy availability through glycolysis. Citation Format: Jeremy Johnson, Piotr Richahou, B. Mark Evers. AMPK alpha1 and AMPK alpha 2 control TNBC proliferation through cell cycle regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 93.

Garlic Scape Extract Decreases Adipogenesis and Lipogenesis by Activating AMK-Activated Protein Kinase in 3T3-L1 Adipocytes

Abstract Objectives Excessive fat accumulation in the body, indicated as obesity causes development of other metabolic diseases, such as diabetes and cardiovascular diseases. Due to limitations of current strategies such as long-term engagement and high cost, new strategies to reduce the prevalence of obesity are needed. Garlic scape (Allium sativum L.) is the flower bud of the garlic plant that is removed to promote the growth of the bulb. Although most of garlic scape are trashed as a byproduct of garlic in the farm, it is still edible and includes various phytochemicals to improve human health. The purpose of this study was to investigate anti-obesity effect of garlic scape and its mechanism using 3T3-L1 cells. Methods Garlic scape extract (GSE) was prepared by solvent extraction using 80% methanol (v/v). 3T3-L1 cells, mouse embryonic fibroblast cells that mimic preadipocytes, were incubated with GSE during the differentiation. Total RNA and proteins from the cells were extracted. The expression of genes related to lipid metabolism such as peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding protein (C\EBP) α and β, adipocyte protein (aP) 2, acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), sterol regulated element binding protein (SREBP) 1c, and carnitine palmitoyltransferase (CPT) 1α was determined using a quantitative PCR. The expression of proteins such as hormone sensitive lipase (HSL), ACC, and AMPK-activated protein kinase (AMPK) was measured using a western blot. Effect of GSE on lipid accumulation were visualized and quantified using Oil Red O staining. Results GSE decreased the expression of genes related to adipogenesis (PPARγ, C\EBPα, and C\EBPβ) and lipogenesis (ACC, FAS, and SREBP1c) whereas HSL and CPT1α genes and proteins related to lipolysis and fatty acid oxidation were not changed. Phosphorylation on both AMPK and ACC were increased. Adipocytes that were treated with GSE showed reduced lipid accumulation. Conclusions GSE inhibits fat accumulation in white adipocytes by decreasing adipogenesis and lipogenesis through possibly a AMPK pathway. Garlic scape may be a potential candidate to improve obese conditions. Funding Sources This work was supported by USDA.

Naja atra Cardiotoxin 3 Elicits Autophagy and Apoptosis in U937 Human Leukemia Cells through the Ca2+/PP2A/AMPK Axis.

Cardiotoxins (CTXs) are suggested to exert their cytotoxicity through cell membrane damage. Other studies show that penetration of CTXs into cells elicits mitochondrial fragmentation or lysosome disruption, leading to cell death. Considering the role of AMPK-activated protein kinase (AMPK) in mitochondrial biogenesis and lysosomal biogenesis, we aimed to investigate whether the AMPK-mediated pathway modulated Naja atra (Taiwan cobra) CTX3 cytotoxicity in U937 human leukemia cells. Our results showed that CTX3 induced autophagy and apoptosis in U937 cells, whereas autophagic inhibitors suppressed CTX3-induced apoptosis. CTX3 treatment elicited Ca2+-dependent degradation of the protein phosphatase 2A (PP2A) catalytic subunit (PP2Acα) and phosphorylation of AMPKα. Overexpression of PP2Acα mitigated the CTX3-induced AMPKα phosphorylation. CTX3-induced autophagy was via AMPK-mediated suppression of the Akt/mTOR pathway. Removal of Ca2+ or suppression of AMPKα phosphorylation inhibited the CTX3-induced cell death. CTX3 was unable to induce autophagy and apoptosis in U937 cells expressing constitutively active Akt. Met-modified CTX3 retained its membrane-perturbing activity, however, it did not induce AMPK activation and death of U937 cells. These results conclusively indicate that CTX3 induces autophagy and apoptosis in U937 cells via the Ca2+/PP2A/AMPK axis, and suggest that the membrane-perturbing activity of CTX3 is not crucial for the cell death signaling pathway induction.

Lipopolysaccharide-Induced Dephosphorylation of AMPK-Activated Protein Kinase Potentiates Inflammatory Injury via Repression of ULK1-Dependent Autophagy.

AMP-activated protein kinase (AMPK) is a crucial metabolic regulator with profound modulatory activities on inflammation. Although the anti-inflammatory benefits of AMPK activators were well documented in experimental studies, the pathological significance of endogenous AMPK in inflammatory disorders largely remains unknown. This study investigated the phosphorylation status of endogenous AMPK and the potential roles of AMPK in mice with lipopolysaccharide (LPS)-induced lethal inflammation. The results indicated that LPS dose-dependently decreased the phosphorylation level of AMPK and its target protein acetyl-CoA carboxylase (ACC). Reactivation of AMPK with the AMPK activator A-769662 suppressed LPS-induced elevation of interleukin 6, alleviated histological abnormalities in lung and improved the survival of LPS-challenged mice. Treatment with A-769662 restored LPS-induced suppression of autophagy, inhibition of autophagy by 3-MA reversed the beneficial effects of A-769662. Treatment with A-769662 suppressed LPS-induced activation of mammalian target of rapamycin (mTOR), co-administration of mTOR activator abolished the beneficial effects of A-769662, and the suppressive effects of A-769662 on uncoordinated-51-like kinase 1 (ULK1) phosphorylation. Inhibition of ULK1 removed the beneficial effects of A-769662. These data indicated that LPS-induced dephosphorylation of AMPK could result in weakened inhibition of mTOR and repression of ULK1-dependent autophagy, which might potentiate the development of LPS-induced inflammatory injury. These data suggest that pharmacological restoration of AMPK activation might be a beneficial approach for the intervention of inflammatory disorders.

PAN-AMPK activator O304 improves glucose homeostasis and microvascular perfusion in mice and type 2 diabetes patients.

AMPK activated protein kinase (AMPK), a master regulator of energy homeostasis, is activated in response to an energy shortage imposed by physical activity and caloric restriction. We here report on the identification of PAN-AMPK activator O304, which - in diet-induced obese mice - increased glucose uptake in skeletal muscle, reduced β cell stress, and promoted β cell rest. Accordingly, O304 reduced fasting plasma glucose levels and homeostasis model assessment of insulin resistance (HOMA-IR) in a proof-of-concept phase IIa clinical trial in type 2 diabetes (T2D) patients on Metformin. T2D is associated with devastating micro- and macrovascular complications, and O304 improved peripheral microvascular perfusion and reduced blood pressure both in animals and T2D patients. Moreover, like exercise, O304 activated AMPK in the heart, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved left ventricular stroke volume in mice, but it did not increase heart weight in mice or rats. Thus, O304 exhibits a great potential as a novel drug to treat T2D and associated cardiovascular complications.

Skeletal Muscle Gene Expression in High Fat Diet‐fed LKB1 Knockout Mice.

The development of obesity leads to many alterations in skeletal muscle metabolism. Liver kinase B1 (LKB1) is well‐known to regulate skeletal muscle function and metabolism through its phosphorylation and regulation of AMPK activated protein kinase (AMPK) family members. LKB1 and AMPK signaling is known to affect gene expression in skeletal muscle through regulation of transcription factors and epigenetic regulation. The purpose of this study was to determine how the lack of LKB1 affects gene expression in skeletal muscle under high fat diet‐induced obesity (DIO). To test this, skeletal muscle specific LKB1 knockout (skmLKB1‐KO) and littermate LKB1‐floxed (WT) mice were fed high fat diet (HFD) or a standard chow diet (SCD) for 14 weeks. RNA sequencing and RT‐PCR was performed using RNA that was isolated from quadriceps muscles. Western blotting was performed using quadriceps homogenates. Gene ontology (GO) analysis showed 549 terms that were significantly affected by genotype in high fat fed mice. When ranked by p‐value, those GO terms with the highest level of significance were mitochondria/respiration‐related and oxidative stress‐related genes in the skmLKB1‐KO vs. WT muscles. Sixty‐nine genes were differentially expressed when comparing skmLKB1‐KO and WT muscles from HFD fed mice. The most under‐expressed of these in the skmLKB1‐KO vs. WT mice was dickkopf‐related protein 3 (DKK3), a regulator of the Wnt pathway. RT‐PCR confirmed the low level of DKK3 expression in skmLKB1‐KO muscles, but also suggested a potential upregulation by HFD feeding. Western blot analysis of DKK3 likewise showed increased expression in muscles from HFD vs. SCD mice. Intriguingly, when the mice were separated into HFD‐induced obesity resistant and obesity‐prone subgroups, DKK3 was only upregulated by HFD in the obesity resistant animals, suggesting a potential protective role against the development of obesity. In conclusion, skmLKB1‐KO under high‐fat diet conditions is associated with altered mitochondrial and oxidation‐reduction related gene expression, and DKK3 expression is greatly reduced in skmLKB1‐KO muscles.Support or Funding InformationThis work was funded by a BYU Mentoring Environment Grant and Graduate Research Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Moderate treadmill exercise ameliorates amyloid-β-induced learning and memory impairment, possibly via increasing AMPK activity and up-regulation of the PGC-1α/FNDC5/BDNF pathway

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with loss of memory and cognitive abilities. Previous evidence suggested that exercise ameliorates learning and memory deficits by increasing brain derived neurotrophic factor (BDNF) and activating downstream pathways in AD animal models. However, upstream pathways related to increase BDNF induced by exercise in AD animal models are not well known. We investigated the effects of moderate treadmill exercise on Aβ-induced learning and memory impairment as well as the upstream pathway responsible for increasing hippocampal BDNF in an animal model of AD. Animals were divided into five groups: Intact, Sham, Aβ1−42, Sham-exercise (Sham-exe) and Aβ1−42-exercise (Aβ-exe). Aβ was microinjected into the CA1 area of the hippocampus and then animals in the exercise groups were subjected to moderate treadmill exercise (for 4 weeks with 5 sessions per week) 7 days after microinjection. In the present study the Morris water maze (MWM) test was used to assess spatial learning and memory. Hippocampal mRNA levels of BDNF, peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), fibronectin type III domain-containing 5 (FNDC5) as well as protein levels of AMPK-activated protein kinase (AMPK), PGC-1α, BDNF, phosphorylation of AMPK were measured. Our results showed that intra-hippocampal injection of Aβ1−42 impaired spatial learning and memory which was accompanied by reduced AMPK activity (p-AMPK/total-AMPK ratio) and suppression of the PGC-1α/FNDC5/BDNF pathway in the hippocampus of rats. In contrast, moderate treadmill exercise ameliorated the Aβ1−42-induced spatial learning and memory deficit, which was accompanied by restored AMPK activity and PGC-1α/FNDC5/BDNF levels. Our results suggest that the increased AMPK activity and up-regulation of the PGC-1α/FNDC5/BDNF pathway by exercise are likely involved in mediating the beneficial effects of exercise on Aβ-induced learning and memory impairment.

Manipulation and Measurement of AMPK Activity in Pancreatic Islets.

The role of the energy sensor AMPK-activated protein kinase (AMPK) in the insulin-secreting β-cell remains unclear and a subject of intense research. With this chapter, we aim to provide a detailed description of the methods that our group routinely applies to the study of AMPK function in mouse and human pancreatic islets. Thus, we provide detailed protocols to isolate and/or culture mouse and human islets, to modulate and measure AMPK activity in isolated islets, and to evaluate its impact on islet function.

Hypoglycemic effect of &lt;i&gt;Berberis microphylla&lt;/i&gt; G Forst root extract

Purpose: To evaluate the effect of the root extract of Berberis microphylla on glucose uptake and AMPK-activated protein kinase (AMPK) activity in non-resistant and insulin-resistant HepG2 cells. Methods: B. microphylla root was extracted with absolute ethanol, filtered, concentrated and lyophilized. Subsequently, liver cells, HepG2 (resistant and non-insulin resistant), were exposed for 24 h to different concentrations of the extract (10, 5, 2.5 and 1.25 x 10 -3 μg/μL) to determine the stimulation of glucose uptake and phosphorylation of AMPK. Results: In HepG2 cells without resistance exposed to B. microphylla root extract, glucose uptake varied from 34 to 59 % of the available glucose while AMPK phosphorylation was 1.9 to 3.6 times the phosphorylation of the control. In insulin-resistant HepG2 cells, glucose uptake varied from 68 to 95 % of available glucose while AMPK phosphorylation was 1.8 to 3.3 times the phosphorylation of the control. Conclusion: The root extract o f B. microphylla possesses hypoglycemic effects and stimulates glucose uptake in HepG2 cells with and without resistance by activating AMPK protein. Keywords: Calafate, Diabetes, Antihyperglycemic effect, Phytomedicine, Berberis, Insulin resistance

Apoptosis triggered by isoquercitrin in bladder cancer cells by activating the AMPK-activated protein kinase pathway.

Cancer cells are well known to require a constant supply of protein, lipid, RNA, and DNA via altered metabolism for accelerated cell proliferation. Targeting metabolic pathways is, therefore, a promising therapeutic strategy for cancers. Isoquercitrin (ISO) is widely distributed in dietary and medicinal plants and displays selective cytotoxicity to cancer cells, primarily by inducing apoptosis and cell cycle arrest. The aims of this study were to find out whether ISO could stabilize in a bladder-like acidic environment and inhibit bladder cancer cell proliferation by affecting their metabolism, and to investigate its molecular mechanism. In this study, the exposure of T24 bladder cancer cells to ISO (20-80 μM) decreased cell viability by causing ROS overproduction. This ROS change regulated the AMPK signaling pathway, and caused Caspase-dependent apoptosis as well as metabolism dysfunction. Metabolic alterations elevated metabolic pathway variation, which in turn destabilized lipid synthesis and altered anaerobic glycolysis. This linkage was proved by immunoblotting assay, and metabolomics as identified by UHPLC-QTOF-MS. Our findings provide comprehensive evidence that ISO influenced T24 bladder cancer cell metabolism, and that this process was mainly involved in activating the AMPK pathway. This study could lead to an understanding of how ISO suppresses bladder cancer cell growth, and whether the affected cancer metabolism is a common mechanism by which nutritional compounds suppress cancers.

The soluble epoxide hydrolase determines cholesterol homeostasis by regulating AMPK and SREBP activity

Inhibition or deletion of the soluble epoxide hydrolase (sEH) has been linked to reduced cholesterol and protection against atherosclerosis. This study set out to identify sEH substrate(s) or product(s), altered in livers from sEH−/− mice that contribute to these beneficial effects.In livers and isolated hepatocytes, deletion of sEH decreased expression of HMG CoA reductase, fatty acid synthase and low density lipoprotein receptor. Sterol regulatory element binding proteins (SREBPs) regulate the expression of all three enzymes and SREBP activation was attenuated in the absence of sEH. The effect was attributed to the AMPK-activated protein kinase (AMPK) which was activated in the absence of sEH. Livers from wild-type versus sEH−/− littermates contained significantly higher levels of the sEH substrate 12,13-epoxyoctadecenoic acid, which elicited AMPK activation, while the corresponding sEH product was inactive. Thus, AMPK activation and subsequent inhibition of SREBP can account for the altered expression of lipid metabolizing enzymes in sEH−/− mice.

AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages.

C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway.

Estrogen Receptor‐Alpha Activation of AMPK in Vascular Smooth Muscle Cells and Vascular Tone

The cardioprotective effects of estrogen have long been known to result, in part, from its influence on vascular tone. The classical ERs, ERalpha (ERα) and ERbeta (ERβ), can localize within the cell and function as ligand‐activated transcription factors, modulating gene expression in response to estrogen. ERα and ERβ can also localize to the plasma membrane and participate in rapid cellular activation by interacting with cellular signaling pathways. Estrogen has been shown to activate AMPK‐activated protein kinase (AMPK), suggesting that AMPK may mediate some of the effects of estrogen, including its influence on vascular tone. In this study, the selective ERα agonist 4,4′,4′‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT), AMPK activators 5‐aminoimidazole‐4‐carboxamide (AICAR) and metformin, and the AMPK inhibitor Compound C were used to examine the importance of ERα‐AMPK signaling in the relaxation of aortic rings from male and female C57BL/6 mice. The results suggest that activation of AMPK represents a novel pathway through which ERα affects vasomotor tone. (Supported by 5K12HD043451).

AMPK-activated protein kinase activation by Impatiens balsamina L. is related to apoptosis in HSC-2 human oral cancer cells.

Objective:In the present study, we investigated the efficacy of a methanol extract from Impatiens balsamina L. (MEIB) against HSC-2 human oral cancer cells.Materials and Methods:The anti-cancer efficacies of MEIB were performed by methanethiosulfonate assay, phospho-kinase array, Western blot, 4’-6-diamidino-2-phenylindole staining, trypan blue exclusion assay and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide assay.Results:MEIB decreased the cell viability of HSC-2 cells. According to phospho-kinase arrays, MEIB markedly activated AMP-activated protein kinase (AMPK) signaling, but inactivated mammalian target of rapamycin signaling. MEIB induced apoptosis as evidenced by activation of caspase-3, poly (ADP-ribose) polymerase cleavage and nuclear condensation. In addition, AMPK activation by two known activators (5-aminoimidazole-4-carboxamide-1-β-ribofuranoside and metformin) decreased cell viability and induced apoptosis. Moreover, MEIB increased the expression levels of mitochondria-related proteins (t-Bid, Bak and Bad), which contributed to the disruption of mitochondrial membrane potential, cytochrome C release and activation of caspase-9. Metformin also increased t-Bid expression and the subsequent release of cytochrome C into the cytosol.Conclusion:These results suggest that MEIB may be of therapeutic value for treating oral cancer and that its mechanism of action occurs through AMPK and t-Bid.

Arginase inhibition ameliorates hepatic metabolic abnormalities in obese mice.

ObjectivesWe examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity.Methods and ResultsAfter obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells.ConclusionsArginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function.