Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor.Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.     

Evidence that tyrphostins AG10 and AG18 are mitochondrial uncouplers that alter phosphorylation-dependent cell signaling

Receptor agonists that initiate fluid secretion in salivary gland epithelial cells also increase protein phosphorylation. To assess contributions of tyrosine phosphorylation to secretion, changes in muscarinic receptor-initiated secretion (estimated from sodium pump-dependent increases in oxygen consumption) were measured in parotid acinar cells exposed to tyrosine kinase inhibitors. However, like the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, tyrphostins AG10 and AG18 increased the rate of oxygen consumption and reduced cellular ATP by approximately 90% in the absence of the muscarinic agonist carbachol, indicating that these tyrphostins uncouple mitochondria. Exposure of isolated mitochondria to five structurally related tyrphostins demonstrated that their relative potencies as uncouplers differed from their in vitro kinase-inhibitory potencies due to different molecular requirements for the two effects. AG10 and AG18 blocked parotid phosphorylation events only at concentrations that reduced ATP content. The tyrosine kinase inhibitor genistein reduced ATP content by 15-20% and weakly uncoupled isolated mitochondria, but its inhibition of carbachol-mediated protein kinase Cdelta tyrosine phosphorylation and ERK1/2 activation appeared attributable to blocking tyrosine kinases directly. Carbachol itself rapidly reduced ATP content by 15-20%. Carbachol, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (P2X(7) receptor agonist), AG10, AG18, and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone rapidly activated the fuel sensor AMP-activated protein kinase (AMPK); however, only AMPK activation by carbachol and BzATP was due to sodium pump stimulation. AG10 and AG18 also activated AMPK and/or uncoupled mitochondria in PC12, HeLa, and HEK293 cells. These studies demonstrate that some tyrosine kinase inhibitors produce cellular effects that are mechanistically different from their primary in vitro characterizations and, as do salivary secretory stimuli, promote rapid metabolic alterations that initiate secondary signaling events.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

Regulation of neurotensin receptor function by the arachidonic acid-lipoxygenase pathway in prostate cancer PC3 cells

Neurotensin (NT) elevates leukotriene levels in animals and stimulates 5-HETE formation in prostate cancer PC3 cells. PC3 cell growth is stimulated by NT and inhibited by lipoxygenase (LOX) blockers. This led us to test LOX blockers (NDGA, MK886, ETYA, Rev5901, AA861 and others) for effects on NT binding and signaling. LOX blockers dramatically enhanced 125I-neurotensin binding to NT receptor NTR1 in PC3 cells, whereas they inhibited NT-induced inositol phosphate formation. These effects were indirect (binding to isolated membranes was unaffected), receptor-specific (binding to beta2-adrenergic, V1a-vasopressin, EGF and bombesin receptor was unaffected) and pathway-specific (cyclooxygenase inhibitors were inactive). NT receptor affinity was increased but receptor number and % internalization were unchanged. Also supporting the involvement of arachidonic acid metabolism in NTR1 regulation was the finding that inhibitors of PLA2 and DAG lipase enhanced NT binding. These findings suggest that NTR1 is regulated by specific feedback mechanism(s) involving lipid peroxidation and/or LOX-dependent processes.     

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.     

Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells

The mechanism by which neurotensin (NT) promotes the growth of prostate cancer epithelial cells is not yet defined. Here, androgen-independent PC3 cells, which express high levels of the type 1 NT-receptor (NTR1), are used to examine the involvement of epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (ERK, SAPK/JNK and p38), PI3 kinase and PKC in the mitogenic effect of NT. NT dose dependently (0.1–30 nM) enhanced phosphorylation of EGFR, ERK and Akt, reaching maximal levels within 3 min as measured by Western blotting. These effects were associated with an accumulation of EGF-like substance(s) in the medium (assayed by EGFR binding) and a 2-fold increase in DNA synthesis (assayed by [³H]thymidine incorporation). The DNA synthesis enhancement by NT was non-additive with that of EGF. The NT-induced stimulation of EGFR/ERK/Akt phosphorylation and DNA synthesis was inhibited by EGFR-tyrosine kinase inhibitors (AG1478, PD153035), metallo-endopeptidase inhibitor phosphoramidon and by heparin, but not by neutralizing anti-EGF antibody. Thus, transactivation of EGFR by NT involved heparin-binding EGF (HB-EGF or amphiregulin) rather than EGF. The effects of NT on EGFR/ERK/Akt activation and DNA synthesis were attenuated by PLC-inhibitor (U73122), PKC-inhibitors (bisindolylmaleimide, staurosporine, rottlerin), MEK inhibitor (U0126) and PI3 kinase inhibitors (wortmannin, LY 294002). We conclude that NT stimulated mitogenesis in PC3 cells by a PKC-dependent ligand-mediated transactivation of EGFR, which led to stimulation of the Raf–MEK–ERK pathway in a PI3 kinase-dependent manner.     

Molecular and cellular regulation of neurotensin receptor under acute and chronic agonist stimulation

Neurotensin is a tridecapteptide acting mostly in the brain and gastrointestinal tract. NT binds two G protein coupled receptors (GPCR), NTS1 and NTS2, and a single transmembrane domain receptor, NTS3/gp95/sortilin receptor. NTS1 mediates the majority of NT action in neurons and the periphery. Like many other GPCRs, upon agonist stimulation, NTS1 is internalized, endocytosed, and the cells are desensitized. It is tacitly acknowledged that the intensity and the lasting of cellular responses to NT are dependent on free and functional NTS1 at the cell surface. Understanding how NTS1 expression is regulated at the membrane should provide a better comprehension towards its function. This review analyzes and discusses the current cellular and molecular mechanisms affecting the expression of NTS1 at the cellular membrane upon acute and chronic NT stimulation.     

Distinct regions of C-terminus of the high affinity neurotensin receptor mediate the functional coupling with pertussis toxin sensitive and insensitive G-proteins

The functional coupling of C-terminally truncated mutants of the high affinity rat neurotensin (NT) receptor (NTS1) was characterized in transfected Chinese hamster ovary cells. On cells expressing NTRDelta372 (truncated NTS1 lacking the entire 52 amino acid C-terminus), NT failed to promote [(35)S]guanosine 5'-[gamma-(35)S]triphosphate binding whereas a robust pertussis toxin (PTx) sensitive response was observed in cells expressing a partially truncated receptor (NTRDelta401 lacking the last 23 residues). Similar results were obtained when measuring the ability of NT to induce the production of arachidonic acid. Since neither deletions impaired the NT-induced phosphoinositide hydrolysis, these results indicate that the membrane proximal region of the C-terminus is specifically involved in the functional coupling of the receptor with PTx sensitive G-proteins. This region was also found to be involved in the control of receptor internalization. However, PTx failed to impair internalization, indicating that these two properties are not directly related.     

Insulin-like growth factor-1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells

Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.     

Intracellular signals mediating the food intake-suppressive effects of hindbrain glucagon-like peptide-1 receptor activation

Glucagon-like peptide-1 receptor (GLP-1R) activation within the nucleus tractus solitarius (NTS) suppresses food intake and body weight (BW), but the intracellular signals mediating these effects are unknown. Here, hindbrain (fourth i.c.v.) GLP-1R activation by Exendin-4 (Ex-4) increased PKA and MAPK activity and decreased phosphorylation of AMPK in NTS. PKA and MAPK signaling contribute to food intake and BW suppression by Ex-4, as inhibitors RpcAMP and U0126 (fourth i.c.v.), respectively, attenuated Ex-4's effects. Hindbrain GLP-1R activation inhibited feeding by reducing meal number, not meal size. This effect was attenuated with stimulation of AMPK activity by AICAR (fourth i.c.v.). The PKA, MAPK, and AMPK signaling responses by Ex-4 were present in immortalized GLP-1R-expressing neurons (GT1-7). In conclusion, hindbrain GLP-1R activation suppresses food intake and BW through coordinated PKA-mediated suppression of AMPK and activation of MAPK. Pharmacotherapies targeting these signaling pathways, which mediate intake-suppressive effects of CNS GLP-1R activation, may prove efficacious in treating obesity.     

NTS2 modulates the intracellular distribution and trafficking of NTS1 via heterodimerization

Neurotensin (NT) receptors NTS1 and NTS2 are known to display considerable distributional overlap in mammalian central nervous system (CNS). Using co-immunoprecipitation approaches, we demonstrated here that NTS1 forms constitutive heterodimers with NTS2 in transfected COS-7 cells. We also showed that co-expression of NTS2 with NTS1 markedly decreases the cell surface density of NTS1 without affecting ERK1/2 MAPK activity or NT-induced NTS1 internalization. However, radioligand-binding studies indicated that upon prolonged NT stimulation, cell surface NTS1 receptors are more resistant to down-regulation in cells co-expressing NTS1 and NTS2 than in cells expressing NTS1 alone. Taken together, these data suggest that NTS1/NTS2 heterodimerization affects the intracellular distribution and trafficking of NTS1 by making it more similar to that of NTS2 as witnessed in cells expressing NTS2 alone. NTS1/NTS2 heterodimerization might therefore represent an additional mechanism in the regulation of NT-triggered responses mediated by NTS1 and NTS2 receptors.     

Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.     

Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK,DNA Synthesis and Proliferation in Pancreatic Cancer Cells

Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser⁷⁹. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr³⁸⁹ and S6 at Ser^240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α₁ and α₂ catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.     

Activation of AMPK attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury

AMP-activated protein kinase (AMPK) is activated by increases in the intracellular AMP-to-ATP ratio and plays a central role in cellular responses to metabolic stress. Although activation of AMPK has been shown to have anti-inflammatory effects, there is little information concerning the role that AMPK may play in modulating neutrophil function and neutrophil-dependent inflammatory events, such as acute lung injury. To examine these issues, we determined the effects of pharmacological activators of AMPK, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) and barberine, on Toll-like receptor 4 (TLR4)-induced neutrophil activation. AICAR and barberine dose-dependently activated AMPK in murine bone marrow neutrophils. Exposure of LPS-stimulated neutrophils to AICAR or barberine inhibited release of TNF-alpha and IL-6, as well as degradation of IkappaBalpha and nuclear translocation of NF-kappaB, compared with findings in neutrophil cultures that contained LPS without AICAR or barberine. Administration of AICAR to mice resulted in activation of AMPK in the lungs and was associated with decreased severity of LPS-induced lung injury, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-alpha and IL-6 in bronchoalveolar lavage fluid. These results suggest that AMPK activation reduces TLR4-induced neutrophil activation and diminishes the severity of neutrophil-driven proinflammatory processes, including acute lung injury.     

AMP-Activated protein kinase is activated by the stimulations of G(q)-coupled receptors

The AMP-activated protein kinase (AMPK) functions as a metabolic sensor that monitors cellular AMP and ATP levels. Platelet-activating factor (PAF) activates endogeneous AMPKalpha1 in Chinese hamster ovary cells expressing the PAF receptor coupled with both G(i) and G(q), but its activity was not inhibited after treatment with islet-activating protein. Norepinephrine and bradykinin also activated AMPKalpha1 in cells expressing the G(q)-coupled alpha(1b)-adrenergic receptor and bradykinin receptor, respectively. Stimulations of the G(i)-coupled alpha(2A)-adrenergic receptor, fMet-Leu-Phe receptor, prostaglandin EP3alpha receptor, and G(s)-coupled beta(2)-adrenergic receptor did not activate AMPKalpha1. AMPKalpha1 thus is activated specifically by stimulation of G(q)-coupled receptors. G(q)-coupled receptors transmit the signal for GLUT4 translocation and glucose uptake through an insulin-independent pathway. However, direct activation of AMPKalpha1 with treatment of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside did not trigger GLUT4 translocation nor stimulate glucose uptake in our cells. Thus, activation of AMPKalpha1 via G(q) is not sufficient to trigger GLUT4 translocation or stimulate glucose uptake.     

Biochemical regulation of mammalian AMP-activated protein kinase activity by NAD and NADH

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of beta-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by beta-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.     

Expanding roles for AMP‐activated protein kinase in neuronal survival and autophagy

AMP‐activated protein kinase (AMPK) is an evolutionarily conserved cellular switch that activates catabolic pathways and turns off anabolic processes. In this way, AMPK activation can restore the perturbation of cellular energy levels. In physiological situations, AMPK senses energy deficiency (in the form of an increased AMP/ATP ratio), but it is also activated by metabolic insults, such as glucose or oxygen deprivation. Metformin, one of the most widely prescribed anti‐diabetic drugs, exerts its actions by AMPK activation. However, while the functions of AMPK as a metabolic regulator are fairly well understood, its actions in neuronal cells only recently gained attention. This review will discuss newly emerged functions of AMPK in neuroprotection and neurodegeneration. Additionally, recent views on the role of AMPK in autophagy, an important catabolic process that is also involved in neurodegeneration and cancer, will be highlighted.     

The human glucagon-like peptide-1 analogue liraglutide regulates pancreatic beta-cell proliferation and apoptosis via an AMPK/mTOR/P70S6K signaling pathway

Glucagon-like peptide-1 (GLP-1), an effective therapeutic agent for the treatment of diabetes, has been proven to protect pancreatic beta cells through many pathways. Recent evidence demonstrates that AMP-activated protein kinase (AMPK), as a metabolic regulator, coordinates beta-cell protein synthesis through regulation of the mammalian target of rapamycin (mTOR) signaling pathway. The purpose of the present study was to explore whether liraglutide, a human GLP-1 analogue, protects beta cells via AMPK/mTOR signaling. We evaluated INS-1 beta-cell line proliferation using the Cell Counting Kit-8, and examined the effect of GLP-1 on cellular ATP levels using an ATP assay kit. mTOR pathway protein expression levels were tested by Western blotting and glucolipotoxicity-induced cell apoptosis was evaluated by flow cytometry. Liraglutide increased beta-cell viability at an optimum concentration of 100 nmol/L in the presence of 11.1 or 30 mmol/L glucose. Liraglutide (100 nmol/L) activated mTOR and its downstream effectors, 70-kDa ribosomal protein S6 kinase and eIF4E-binding protein-1, in INS-1 cells. This effect was abated by pathway blockers: the AMPK activator AICAR and the mTOR inhibitor rapamycin. Furthermore, the effect of liraglutide on beta-cell proliferation was inhibited by AICAR and rapamycin. Liraglutide increased cellular ATP levels. In addition, liraglutide protected beta cells from glucolipotoxicity-induced apoptosis. This response was also prevented by rapamycin treatment. These results suggest that the enhancement of beta-cell proliferation by that GLP-1 receptor agonist liraglutide is mediated, at least in part, by AMPK/mTOR signaling. Liraglutide also prevents beta-cell glucolipotoxicity by activating mTOR.     

AMP-activated protein kinase induces a p53-dependent metabolic checkpoint

Replicative cell division is an energetically demanding process that can be executed only if cells have sufficient metabolic resources to support a doubling of cell mass. Here we show that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G(1)/S boundary and is regulated by AMP-activated protein kinase (AMPK). This cell-cycle arrest occurs despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53 on serine 15, and this phosphorylation is required to initiate AMPK-dependent cell-cycle arrest. AMPK-induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence. Thus, AMPK is a cell-intrinsic regulator of the cell cycle that coordinates cellular proliferation with carbon source availability.     

Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues

Background

Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.

Methodology/Principal Findings

A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.

Conclusions/Significance

Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.