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During suboptimal growth conditions, Caenorhabditis elegans larvae undergo a global developmental arrest called "dauer." During this stage, the germline stem cells (GSCs) become quiescent in an AMP-activated Protein Kinase (AMPK)-dependent manner, and in the absence of AMPK, the GSCs overproliferate and lose their reproductive capacity, leading to sterility when mutant animals resume normal growth. These defects correlate with the altered abundance and distribution of a number of chromatin modifications, all of which can be corrected by disabling components of the endogenous small RNA pathway, suggesting that AMPK regulates germ cell integrity by targeting an RNA interference (RNAi)-like pathway during dauer. The expression of AMPK in somatic cells restores all the germline defects, potentially through the transmission of small RNAs. Our findings place AMPK at a pivotal position linking energy stress detected in the soma to a consequent endogenous small RNA-mediated adaptation in germline gene expression, thereby challenging the "permeability" of the Weismann barrier., Competing Interests: The authors have declared that no competing interests exist.

Elucidating the bioactive compound modes of action is crucial for increasing success rates in drug development. For anticancer drugs, defining effective drug combinations that overcome resistance improves therapeutic efficacy. Herein, by using a biologically annotated compound library, we performed a large-scale combination screening with Stearoyl-CoA desaturase-1 (SCD1) inhibitor, T-3764518, which partially inhibits colorectal cancer cell proliferation. T-3764518 induced phosphorylation and activation of AMPK in HCT-116 cells, which led to blockade of downstream fatty acid synthesis and acceleration of autophagy. Attenuation of fatty acid synthesis by small molecules suppressed the growth inhibitory effect of T-3764518. In contrast, combination of T-3764518 with autophagy flux inhibitors synergistically inhibited cellular proliferation. Experiments using SCD1 knock-out cells validated the results obtained with T-3764518. The results of our study indicated that activation of autophagy serves as a survival signal when SCD1 is inhibited in HCT-116 cells. Furthermore, these findings suggest that combining SCD1 inhibitor with autophagy inhibitors is a promising anticancer therapy.

Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction.

It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH) phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2) and 4 (PDK4). In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function.

The dietary fiber guar gum has beneficial effects on obesity, hyperglycemia and hypercholesterolemia in both humans and rodents. The major products of colonic fermentation of dietary fiber, the short-chain fatty acids (SCFAs), have been suggested to play an important role. Recently, we showed that SCFAs protect against the metabolic syndrome via a signaling cascade that involves peroxisome proliferator-activated receptor (PPAR) γ repression and AMP-activated protein kinase (AMPK) activation. In this study we investigated the molecular mechanism via which the dietary fiber guar gum protects against the metabolic syndrome. C57Bl/6J mice were fed a high-fat diet supplemented with 0% or 10% of the fiber guar gum for 12 weeks and effects on lipid and glucose metabolism were studied. We demonstrate that, like SCFAs, also guar gum protects against high-fat diet-induced metabolic abnormalities by PPARγ repression, subsequently increasing mitochondrial uncoupling protein 2 expression and AMP/ATP ratio, leading to the activation of AMPK and culminating in enhanced oxidative metabolism in both liver and adipose tissue. Moreover, guar gum markedly increased peripheral glucose clearance, possibly mediated by the SCFA-induced colonic hormone glucagon-like peptide-1. Overall, this study provides novel molecular insights into the beneficial effects of guar gum on the metabolic syndrome and strengthens the potential role of guar gum as a dietary-fiber intervention.

Background: AMP-activated protein kinase (Ampk) is a sensor of the cellular energy status and a powerful regulator of metabolism. Activation of Ampk was previously shown to participate in monocyte-to-fibroblast transition and matrix protein production in renal tissue. Thus, the present study explored whether the catalytic Ampkα1 isoform participates in the regulation of the renal fibrotic response following unilateral ureteral obstruction (UUO)., Methods: UUO was induced in gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and in corresponding wild-type mice (Ampkα1+/+). In the obstructed kidney and, for comparison, in the non-obstructed control kidney, quantitative RT-PCR, Western blotting and immunostaining were employed to determine transcript levels and protein abundance, respectively., Results: In Ampkα1+/+ mice, UUO significantly up-regulated the protein abundance of the Ampkα1 isoform, but significantly down-regulated the Ampkα2 isoform in renal tissue. Phosphorylated Ampkα protein levels were significantly increased in obstructed kidney tissue of Ampkα1+/+ mice but not of Ampkα1-/- mice. Renal expression of α-smooth muscle actin was increased following UUO, an effect again less pronounced in Ampkα1-/- mice than in Ampkα1+/+ mice. Histological analysis did not reveal a profound effect of Ampkα1 deficiency on collagen 1 protein deposition. UUO significantly increased phosphorylated and total Tgf-ß-activated kinase 1 (Tak1) protein, as well as transcript levels of Tak1-downstream targets c-Fos, Il6, Pai1 and Snai1 in Ampkα1+/+ mice, effects again significantly ameliorated in Ampkα1-/- mice. Moreover, Ampkα1 deficiency inhibited the UUO-induced mRNA expression of Cd206, a marker of M2 macrophages and of Cxcl16, a pro-fibrotic chemokine associated with myeloid fibroblast formation. The effects of Ampkα1 deficiency during UUO were, however, paralleled by increased tubular injury and apoptosis., Conclusions: Renal obstruction induces an isoform shift from Ampkα2 towards Ampkα1, which contributes to the signaling involved in cell survival and fibrosis.

Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.

The naturally occurring triterpenoid betulinic acid (BA) shows pronounced polypharmacology ranging from anti-inflammatory to anti-lipogenic activities. Recent evidence suggests that rather diverse cellular signaling events may be attributed to the same common upstream switch in cellular metabolism. In this study we therefore examined the metabolic changes induced by BA (10 µM) administration, with focus on cellular glucose metabolism. We demonstrate that BA elevates the rates of cellular glucose uptake and aerobic glycolysis in mouse embryonic fibroblasts with concomitant reduction of glucose oxidation. Without eliciting signs of obvious cell death BA leads to compromised mitochondrial function, increased expression of mitochondrial uncoupling proteins (UCP) 1 and 2, and liver kinase B1 (LKB1)-dependent activation AMP-activated protein kinase. AMPK activation accounts for the increased glucose uptake and glycolysis which in turn are indispensable for cell viability upon BA treatment. Overall, we show for the first time a significant impact of BA on cellular bioenergetics which may be a central mediator of the pleiotropic actions of BA.

Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway.

Objective: Intestinal barrier function is vital for homeostasis. Conditions where the mucosal barrier is compromised lead to increased plasma content of lipopolysaccharide (LPS). LPS acts on Toll-like receptor 4 (TLR4) and initiates cellular inflammatory responses. TLR4 receptors have been identified on enteric neurons and LPS exposure causes neuronal loss, counteracted by vasoactive intestinal peptide (VIP), by unknown mechanisms. In addition AMP activated protein kinase (AMPK) stimulation causes loss of enteric neurons. This study investigated a possible role of AMPK activation in LPS-induced neuronal loss., Design: Primary cultures of myenteric neurons isolated from rat small intestine were used. Cultures were treated with LPS (0.2-20 µg/mL) with and without TAK1-inhibitor (5Z)-7-Oxozeaenol (10-6 M) or AMPK inhibitor compound C (10-5 M). AMPK-induced neuronal loss was verified treating cultures with three different AMPK activators, AICAR (10-4-3×10-3 M), metformin (0.2-20 µg/mL) and A-769662 (10-5-3×10-4 M) with or without the presence of compound C (10-5 M). Upstream activation of AMPK-induced neuronal loss was tested by treating cultures with AICAR (10-3 M) in the presence of TAK1 inhibitor (5Z)-7-Oxozeaenol (10-6 M). Neuronal survival and relative numbers of neurons immunoreactive (IR) for VIP were evaluated using immunocytochemistry., Results: LPS caused a concentration dependent loss of neurons. All AMPK activators induced loss of myenteric neurons in a concentration dependent manner. LPS-, AICAR- and metformin-,but not A-769662-, induced neuronal losses were inhibited by presence of compound C. LPS, AICAR or metformin exposure increased the relative number of VIP-IR neurons; co-treatment with (5Z)-7-Oxozeaenol or compound C reversed the relative increase in VIP-IR neurons induced by LPS. (5Z)-7-Oxozeaenol, compound C or A-769662 did not per se change neuronal survival or relative numbers of VIP-IR neurons., Conclusion: AMPK activation mimics LPS-induced loss of cultured myenteric neurons and LPS-induced neuronal loss is counteracted by TAK1 and AMPK inhibition. This suggests enteric neuroimmune interactions involving AMPK regulation.

Macrophages play a key role in obesity-induced inflammation. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert anti-inflammatory functions in both humans and animal models, but the exact cellular signals mediating the beneficial effects are not completely understood. We previously found that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. Here we aim to determine whether ω-3 PUFAs antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway. Treatment of ω-3 PUFAs suppresses lipopolysaccharide (LPS)-induced cytokine expression in macrophages. Luciferase reporter assays, electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) assays show that treatment of macrophages with ω-3 PUFAs significantly inhibits LPS-induced NF-κB signaling. Interestingly, DHA also increases expression, phosphorylation and activity of the major isoform α1AMPK, which further leads to SIRT1 over-expression. More importantly, DHA mimics the effect of SIRT1 on deacetylation of the NF-κB subunit p65, and the ability of DHA to deacetylate p65 and inhibit its signaling and downstream cytokine expression require SIRT1. In conclusion, ω-3 PUFAs negatively regulate macrophage inflammation by deacetylating NF-κB, which acts through activation of AMPK/SIRT1 pathway. Our study defines AMPK/SIRT1 as a novel cellular mediator for the anti-inflammatory effects of ω-3 PUFAs.

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.

The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.