Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.     

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas

TGF-β1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-β1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-β signaling, and through these pathways it is able to enhance TGF-β induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-β1 combination on cell growth and on TGF-β and mTOR signalling events in lymphoma cells.Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-β1 induced apoptosis and restored TGF-β1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-β sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin + TGF-β1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin + TGF-β1 induced apoptosis was not completely eliminated by a caspase inhibitor.These results suggest that high mTOR activity contributes to TGF-β resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-β1.     

AMPK-mediated inhibition of mTOR kinase is circumvented during immediate-early times of human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection increases synthetic rates in infected cells. The resulting increase in energy utilization could potentially increase the AMP:ATP ratio, causing activation of 5'-AMP-activated protein kinase (AMPK). Activated AMPK promotes inhibition of mammalian target of rapamycin (mTOR) kinase, which could be deleterious to the viral infection. Using the AMPK-activating drug 5-amino-4-imidazolecarboxamide ribose (AICAR), we showed that, by 12 h post-HCMV infection, inhibition of mTOR by AMPK is circumvented. However, growth curves showed that progeny virion production is inhibited when AICAR is added, suggesting other inhibitory effects of AICAR or activated AMPK.     

Liraglutide Improves Pancreatic Beta Cell Mass and Function in Alloxan-Induced Diabetic Mice

Glucagon-like peptide-1 (GLP-1) receptor agonists potentiate glucose-induced insulin secretion. In addition, they have been reported to increase pancreatic beta cell mass in diabetic rodents. However, the precise mode of action of GLP-1 receptor agonists still needs to be elucidated. Here we clarify the effects of the human GLP-1 analog liraglutide on beta cell fate and function by using an inducible Cre/loxP-based pancreatic beta cell tracing system and alloxan-induced diabetic mice. Liraglutide was subcutaneously administered once daily for 30 days. The changes in beta cell mass were examined as well as glucose tolerance and insulin secretion. We found that chronic liraglutide treatment improved glucose tolerance and insulin response to oral glucose load. Thirty-day treatment with liraglutide resulted in a 2-fold higher mass of pancreatic beta cells than that in vehicle group. Liraglutide increased proliferation rate of pancreatic beta cells and prevented beta cells from apoptotic cells death. However, the relative abundance of YFP-labeled beta cells to total beta cells was no different before and after liraglutide treatment, suggesting no or little contribution of neogenesis to the increase in beta cell mass. Liraglutide reduced oxidative stress in pancreatic islet cells of alloxan-induced diabetic mice. Furthermore, the beneficial effects of liraglutide in these mice were maintained two weeks after drug withdrawal. In conclusion, chronic liraglutide treatment improves hyperglycemia by ameliorating beta cell mass and function in alloxan-induced diabetic mice.     

Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside

The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), an activator of the AMP-activated protein kinase (AMPK), on the translational control of protein synthesis and signaling through the mammalian target of rapamycin (mTOR) in rat liver. Effects of AICAR observed in vivo were compared with those obtained in an in situ perfused liver preparation to investigate activation of AMPK in the absence of accompanying changes in hormones and nutrients. AMPK became hyperphosphorylated, as assessed by a gel-shift analysis, in response to AICAR both in vivo and in situ; however, increased relative phosphorylation at the Thr172 site on the kinase was observed only in perfused liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including ribosomal protein S6 kinase 1, eukaryotic initiation factor (eIF)4G, and eIF4E-binding protein (4E-BP)1. The phosphorylation changes in eIF4G and 4E-BP1 were accompanied by a reduction in the amount of eIF4E present in the active eIF4E.eIF4G complex and an increase in the amount present in the inactive eIF4E.4E-BP1 complex. Reduced insulin signaling as well as differences in nutrient availability may have contributed to the effects observed in vivo as AICAR caused a fall in the serum insulin concentration. Overall, however, the results from both experimental models support a scenario in which AICAR directly represses protein synthesis and mTOR signaling in the liver through an AMPK-dependent mechanism.     

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G₂M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.     

Modulatory effects of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24 h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -β, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development.     

5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.     

In vitro proliferation of adult human beta-cells

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells") population were plated on extracellular matrix from rat (804G) and human bladder carcinoma cells (HTB9) or bovine corneal endothelial ECM (BCEC). Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted) or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted), independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05).These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.     

Glucagon-Like Peptide-1 (GLP-1) Analog Liraglutide Inhibits Endothelial Cell Inflammation through a Calcium and AMPK Dependent Mechanism

Liraglutide is a glucagon-like peptide-1 (GLP-1) mimetic used for the treatment of Type 2 diabetes. Similar to the actions of endogenous GLP-1, liraglutide potentiates the post-prandial release of insulin, inhibits glucagon release and increases satiety. Recent epidemiological studies and clinical trials have suggested that treatment with GLP-1 mimetics may also diminish the risk of cardiovascular disease in diabetic patients. The mechanism responsible for this effect has yet to be determined; however, one possibility is that they might do so by a direct effect on vascular endothelium. Since low grade inflammation of the endothelium is an early event in the pathogenesis of atherosclerotic cardiovascular disease (ASCVD), we determined the effects of liraglutide on inflammation in cultured human aortic endothelial cells (HAECs). Liraglutide reduced the inflammatory responses to TNFα and LPS stimulation, as evidenced by both reduced protein expression of the adhesion molecules VCAM-1 and E-Selectin, and THP-1 monocyte adhesion. This was found to result from increased cell Ca²⁺ and several molecules sensitive to Ca²⁺ with known anti inflammatory actions in endothelial cells, including CaMKKβ, CaMKI, AMPK, eNOS and CREB. Treatment of the cells with STO-609, a CaMKK inhibitor, diminished both the activation of AMPK, CaMKI and the inhibition of TNFα and LPS-induced monocyte adhesion by liraglutide. Likewise, expression of an shRNA against AMPK nullified the anti-inflammatory effects of liraglutide. The results indicate that liraglutide exerts a strong anti-inflammatory effect on HAECs. They also demonstrate that this is due to its ability to increase intracellular Ca²⁺ and activate CAMKKβ, which in turn activates AMPK.     

AMPK activation attenuates S6K1, 4E-BP1, and eEF2 signaling responses to high-frequency electrically stimulated skeletal muscle contractions.

Regulation of protein translation through Akt and the downstream mammalian target of rapamycin (mTOR) pathway is an important component of the cellular response to hypertrophic stimuli. It has been proposed that 5'-AMP-activated protein kinase (AMPK) activation during muscle contraction may limit the hypertrophic response to resistance-type exercise by inhibiting translational signaling. However, experimental manipulation of AMPK activity during such a stimulus has not been attempted. Therefore, we investigated whether AMPK activation can attenuate the downstream signaling response of the Akt/mTOR pathway to electrically stimulated lengthening muscle contractions. Extensor digitorum longus muscles (n = 8/group) were subjected to a 22-min bout of lengthening contractions by high-frequency sciatic nerve electrical stimulation (STIM) in young adult (8 mo) Fischer 344 x Brown Norway male rats. Forty minutes before electrical stimulation, rats were subcutaneously injected with saline or 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR; 1 mg/g body wt), an AMPK activator. Stimulated and contralateral resting muscles were removed at 0, 20, and 40 min post-STIM, and AMPK, acetyl CoA carboxylase (ACC), Akt, eukaryotic initiation factor 4E-binding protein (4E-BP1), 70-kDa ribosomal protein S6 kinase (S6K1), and eukaryotic elongation factor 2 (eEF2) phosphorylations were assessed by Western blot. AICAR treatment increased (P < or = 0.05) post-STIM AMPK (Thr172) and ACC phosphorylation (Ser79/221), inhibited post-STIM S6K1 (Thr389) and 4E-BP1 (gel shift) phosphorylation, and elevated post-STIM eEF2 phosphorylation (Thr56). These findings suggest that translational signaling downstream of Akt/mTOR can be inhibited after lengthening contractions when preceded by AMPK activation and that energetic stress may be antagonistic to the hypertrophic translational signaling response to loaded muscle contractions.     

Lack of effect of prolonged treatment with liraglutide on cardiac remodeling in rats after acute myocardial infarction

Following the acute phase of a myocardial infarction, a set of structural and functional changes evolves in the myocardium, collectively referred to as cardiac remodeling. This complex set of processes, including interstitial fibrosis, inflammation, myocyte hypertrophy and apoptosis may progress to heart failure. Analogs of the incretin hormone glucagon-like peptide 1 (GLP-1) have shown some promise as cardioprotective agents. We hypothesized that a long-acting GLP-1 analog liraglutide would ameliorate cardiac remodeling over the course of 4 weeks in a rat model of non-reperfused myocardial infarction. In 134 male Sprague Dawley rats myocardial infarctions were induced by ligation of the left anterior descending coronary artery. Rats were randomized to either subcutaneous injection of placebo or 0.3 mg liraglutide once daily. Cardiac magnetic resonance imaging was performed after 4 weeks. Histology of the infarcted and remote non-infarcted myocardium, selected molecular remodeling markers and mitochondrial respiration in fibers of remote non-infarcted myocardium were analyzed. Left ventricular end diastolic volume increased in the infarcted hearts by 62% (from 0.58 ± 0.03 mL to 0.95 ± 0.07 mL, P < 0.05) compared to sham operated hearts and left ventricle ejection fraction decreased by 37% (63 ± 1%–40 ± 3%, P < 0.05). Increased interstitial fibrosis and phosphorylation of p38 Mitogen Activated Protein Kinase were observed in the non-infarct regions. Mitochondrial fatty acid oxidation was impaired. Liraglutide did not affect any of these alterations. Four-week treatment with liraglutide did not affect cardiac remodeling following a non-reperfused myocardial infarction, as assessed by cardiac magnetic resonance imaging, histological and molecular analysis and measurements of mitochondrial respiration.     

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.     

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways

Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H₂O₂ and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H₂O₂-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H₂O₂-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H₂O₂-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H₂O₂ induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.     

The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells

The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic beta-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of beta-cell function. Here, we show that inhibition of AMPK in beta-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the beta-cell.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Down-regulation of the ubiquitin�Cproteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes

The purpose of this work was to examine whether changes in dietary protein levels could elicit differential responses of tissue proteolysis and the pathway involved in this response. In rats fed with a high protein diet (55%) for 14?days, the liver was the main organ where adaptations occurred, characterized by an increased protein pool and a strong, meal-induced inhibition of the protein breakdown rate when compared to the normal protein diet (14%). This was associated with a decrease in the key-proteins involved in expression of the ubiquitin-proteasome and autophagy pathway gene and a reduction in the level of hepatic ubiquitinated protein. In hepatocytes, we demonstrated that the increase in amino acid (AA) levels was sufficient to down-regulate the ubiquitin proteasome pathway, but this inhibition was more potent in the presence of insulin. Interestingly, AICAR, an adenosine monophosphate-activated protein kinase (AMPK) activator, reversed the inhibition of protein ubiquination induced by insulin at high AA concentrations. Rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, reversed the inhibition of protein ubiquination induced by a rise in insulin levels with both high and low AA concentrations. Moreover, in both low and high AA concentrations in the presence of insulin, AICAR decreased the mTOR phosphorylation, and in the presence of both AICAR and rapamycin, AICAR reversed the effects of rapamycin. These results demonstrate that the inhibition of AMPK and the activation of mTOR transduction pathways, are required for the down-regulation of protein ubiquitination in response to high amino acid and insulin concentrations.     

AMPK activation suppresses mTOR/S6K1 phosphorylation and induces leucine resistance in rats with sepsis

Although it has been known that protein synthesis is suppressed in sepsis, which cannot be corrected by leucine supplement (also known as leucine resistance), the molecular signaling mechanism remains unclear. This study aimed to investigate the AMP‐activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway in sepsis‐induced leucine resistance and its upstream signals, and to seek a way to correct leucine resistance in sepsis. Sepsis was produced by cecal ligation and puncture (CLP) model in rat. Both septic rats and sham operation rat received total parenteral nutrition (TPN) with or without leucine for 24 h, and then protein synthesis and AMPK/mTOR and protein kinase B (PKB) were tested. In vitro C2C12 cells were treated with or without leucine, and we tested the AMPK/mTOR pathway and protein synthesis. We blocked AMPK by compound C and stimulated it by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) individually. The results showed that AMPK was highly phosphorylated and suppressed mTOR/S6K1 activation in CLP rats. In vitro when AMPK was activated by AICAR, protein synthesis was suppressed and leucine resistance was observed. High phosphorylation of AMPK was accompanied by PKB inactivation in CLP rats. When PKB was blocked, both AMPK activation and leucine resistance were observed. In CLP rats, nutrition support with intensive insulin therapy reversed leucine resistance by activating PKB and suppressing AMPK phosphorylation. These findings suggest that high phosphorylation of AMPK induced by PKB inactivation in sepsis suppresses mTOR, S6K1 phosphorylation, and protein synthesis and leads to leucine resistance. Intensive insulin treatment can reverse leucine resistance by suppressing AMPK activation through activation of PKB.     

mTOR integrates amino acid- and energy-sensing pathways

The AMP-activated protein kinase (AMPK) exists as a heterotrimetric complex comprising a catalytic alpha subunit and non-catalytic beta and gamma subunits. Under conditions of hypoxia, exercise, ischemia, heat shock, and low glucose, AMPK is activated allosterically by rising cellular AMP and by phosphorylation of the catalytic alpha subunit. The mammalian target of rapamycin (mTOR) controls cellular functions in response to amino acids and growth factors. Recent reports including our study have demonstrated the possible interplay between mTOR and AMPK signaling pathways, supporting a model in which mitochondrial dysfunction caused by the mitochondrial inhibitors or ATP depletion inhibits activation of p70 S6 kinase alpha (p70alpha), a downstream effector of mTOR, by activating AMPK. Leucine may stimulate p70alpha phosphorylation via mTOR pathway, in part, by serving both as a mitochondrial fuel through oxidative carboxylation and an allosteric activation of glutamate dehydrogenase. This hypothesis may support an idea in which leucine modulates mTOR function, in part by regulating mitochondrial function and AMPK. Further understanding of the role of mTOR in coordinating amino acid- and energy-sensing pathways would provide new insights into relationship between nutrients and cellular functions.