Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling

The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD.     

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: a novel enzyme of the beta-lactamase fold family releasing anandamide and other N-acylethanolamines

N-acylethanolamines (NAEs) are a lipid class present in brain and other animal tissues and contains anandamide (an endocannabinoid) and other bioactive substances. NAEs are formed from N-acylphosphatidylethanolamines (NAPEs) by a phospholipase D (PLD)-type enzyme abbreviated to NAPE-PLD. Although this enzyme has been recognized for more than 20 years, its molecular cloning has only recently been achieved by us. We highly purified NAPE-PLD from the particulate fraction of rat heart, and on the basis of peptide sequences with the purified enzyme cloned its cDNA from mouse, rat and human. The deduced primary structures revealed no homology with any PLDs so far reported, but was suggested to belong to the beta-lactamase fold family. When overexpressed in COS-7 cells, the NAPE-PLD activity increased about 1000-fold in comparison with the endogenous activity. The recombinant enzyme generated various long-chain NAEs including anandamide from their corresponding NAPEs at similar rates. However, the enzyme was inactive with phosphatidylethanolamine and phosphatidylcholine and did not catalyze transphosphatidylation, a reaction characteristic of PLD. The enzyme was widely expressed in murine organs with higher levels in brain, testis and kidney. The existence of NAPE-PLD specifically hydrolyzing NAPEs to NAEs emphasizes physiological significance of NAEs including anandamide in brain and other tissues.     

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.     

Inhibition of phospholipase D alpha by N-acylethanolamines

N-Acylethanolamines (NAEs) are endogenous lipids in plants produced from the phospholipid precursor, N-acylphosphatidylethanolamine, by phospholipase D (PLD). Here, we show that seven types of plant NAEs differing in acyl chain length and degree of unsaturation were potent inhibitors of the well-characterized, plant-specific isoform of PLD-PLD alpha. It is notable that PLD alpha, unlike other PLD isoforms, has been shown not to catalyze the formation of NAEs from N-acylphosphatidylethanolamine. In general, inhibition of PLD alpha activity by NAEs increased with decreasing acyl chain length and decreasing degree of unsaturation, such that N-lauroylethanolamine and N-myristoylethanolamine were most potent with IC(50)s at submicromolar concentrations for the recombinant castor bean (Ricinus communis) PLD alpha expressed in Escherichia coli and for partially purified cabbage (Brassica oleracea) PLD alpha. NAEs did not inhibit PLD from Streptomyces chromofuscus, and exhibited only moderate, mixed effects for two other recombinant plant PLD isoforms. Consistent with the inhibitory biochemical effects on PLD alpha in vitro, N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) and Commelina communis at low micromolar concentrations. Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLD alpha physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.     

Viral activation of macrophages through TLR-dependent and -independent pathways

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.     

Regional distribution and age-dependent expression of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D in rat brain

The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.     

Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) inhibits myogenic differentiation via the activation of nuclear factor-kappaB (NF-kappaB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C(2)C(12) cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha-treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha-induced activation of NF-kappaB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappaB. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.     

Different modulation of phospholipase A2 activity by saturated and monounsaturated N-acylethanolamines

The physiological functions of N-acylethanolamines (NAEs) are poorly understood, although many functions were suggested for these naturally occurring membrane components of plants and animals. The binding with cannabinoid receptors CB1 and CB2 was demonstrated for some NAEs, such as anandamide. However, the chemical nature of these molecules suggests that some of their biological effects on biomembranes could be related, at least partially, to physical interactions with the lipid bilayer. The present work studies the effect of saturated and monounsaturated NAEs on phospholipase A2 (PLA2) activity, which is dependent on lipid bilayer features. The present study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene (Laurdan) fluorescence, demonstrates that the acyl chain length and the presence of a single double bond are crucial for the enzymatic activity modulation by NAEs. In fact, saturated NAEs with 10 carbon atoms don't affect the PLA2 activity, while NAEs with 12 and 16 carbon atoms largely activate the enzyme. On the other hand, an acyl chain length of 18 carbon atoms, with or without the presence of a double bond, only slightly affects the enzymatic activity. A structural model for NAE-lipid interactions is proposed in order to explain the differences in PLA2 activity modulation by these fatty acid derivatives.     

Serum lysophosphatidic acid is produced through diverse phospholipase pathways

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A(2) (sPLA(2)-IIA) and phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA(1), sPLA(2)-IIA, LCAT, and lysoPLD.     

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.     

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.     

Autotaxin induces lung epithelial cell migration through lysoPLD activity-dependent and -independent pathways

Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodelling and repair after lung injury. In the present paper we hypothesize that secreted ATX (autotaxin), which exhibits lysoPLD (lysophospholipase D) activity, stimulates lung epithelial cell migration through LPA (lysophosphatidic acid) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag overexpressed conditional medium had higher LPA levels compared with control medium and stimulated cell migration through G(αi)-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKC (protein kinase C) δ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to the LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of LPS (lipopolysaccharide) into the mouse airway induced ATX release and LPA production in BAL (bronchoalveolar lavage) fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodelling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated association of ATX with the epithelial cell-surface LPA receptor and integrin β4.     

Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.     

CD38 signaling regulates B lymphocyte activation via a phospholipase C (PLC)-gamma 2-independent, protein kinase C, phosphatidylcholine-PLC, and phospholipase D-dependent signaling cascade

The CD38 cell surface receptor is a potent activator for splenic, B lymphocytes. The molecular mechanisms regulating this response, however, remain incompletely characterized. Activation of the nonreceptor tyrosine kinase, Btk, is essential for CD38 downstream signaling function. The major Btk-dependent substrate in B cells, phospholipase C-gamma2 (PLC-gamma2), functions to generate the key secondary messengers, inositol-1,4,5 trisphosphate and diacylglycerol. Surprisingly, CD38 ligation results in no detectable increase in phosphoinositide metabolism and only a minimal increase in cytosolic calcium. We hypothesized that Btk functioned independently of PLC-gamma2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-gamma2 nor an increase in inositol-1,4,5 trisphosphate production. Furthermore, splenic B cells exhibit a normal CD38-mediated, proliferative response in the presence of the phosphoinositide-PLC inhibitor, U73122. Conversely, protein kinase C (PKC) beta-deficient mice, or PKC inhibitors, indicated the requirement for diacylglycerol-dependent PKC isoforms in this pathway. Loss of PKC activity blocked CD38-dependent, B cell proliferation, NF-kappaB activation, and subsequent expression of cyclin-D2. These results suggested that an alternate diacylglycerol-producing phospholipase must participate in CD38 signaling. Consistent with this idea, CD38 increased the enzymatic activity of the phosphatidylcholine (PC)-metabolizing enzymes, PC-PLC and phospholipase D. The PC-PLC inhibitor, D609, completely blocked CD38-dependent B cell proliferation, IkappaB-alpha degradation, and cyclin-D2 expression. Analysis of Btk mutant B cells demonstrated a partial requirement for Btk in the activation of both enzymes. Taken together, these data demonstrate that CD38 initiates a novel signaling cascade leading to Btk-, PC-PLC-, and phospholipase D-dependent, PLC-gamma2-independent, B lymphocyte activation.     

Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways

SCAR--also known as WAVE--is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization . In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed , but recent data suggest that it is essential for SCAR function . Here, we show that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA(-) cells move and chemotax surprisingly effectively. Our results show that the members of the complex have unexpectedly diverse biological roles.     

Enzymatic properties of phosphatidylinositol-glycan-specific phospholipase C from rat liver and phosphatidylinositol-glycan-specific phospholipase D from rat serum.

Using phosphatidylinositol-glycan (PtdIns-glycan) anchored acetylcholinesterase from bovine erythrocytes as substrate, we found PtdIns-glycan-anchor-degrading activity in rat liver and serum [corrected]. The hepatic enzyme was only soluble in detergents, whereas the serum enzyme occurs as soluble, slightly amphiphilic protein. Using 3-trifluoromethyl-3-(m- [125I]iodophenyl)diazirine-labelled acetylcholinesterase as substrate, we showed that the hepatic anchor-degrading enzyme had a cleavage specificity of a phospholipase C, whereas the serum enzyme was a phospholipase D. Both enzyme exhibited maximal activity in slightly acidic conditions and at low ionic strength. They had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 0.1 microM and 0.16 microM, respectively). Both hepatic PtdIns-glycan-specific phospholipase C and serum PtdIns-glycan-specific phospholipase D gave a large increase in activity between 0.1-10 microM Ca2+, indicating that PtdIns-glycan-specific phospholipases are only marginally active at physiological intracellular Ca2+ concentrations. The enzymes were inhibited by heavy metal chelating agents such as 1,10-phenanthroline and 2,2'-bipyridyl but not by the corresponding Fe2+ complexes or non-chelating analogues, indicating that they both require a heavy metal ion for the expression of catalytic activity in addition to Ca2+. Another interesting property of PtdIns-glycan-specific phospholipases is their inactivation by bicarbonate and cyanate. The inactivation was time- and pH-dependent and could be reversed by dialysis. These observations are in agreement with a covalent modification of the enzymes by carbamoylation.     

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation.