Elevation Of AMP Levels Research Articles

Protective effect of empagliflozin against palmitate-induced lipotoxicity through AMPK in H9c2 cells.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently emerged as novel cardioprotective agents. However, their direct impact on cardiomyocyte injury is yet to be studied. In this work, we investigate the underlying molecular mechanisms of empagliflozin (EMPA), an SGLT2 inhibitor, in mitigating palmitate (PA)-induced cardiomyocyte injury in H9c2 cells. We found that EMPA significantly attenuated PA-induced impairments in insulin sensitivity, ER stress, inflammatory cytokine gene expression, and cellular apoptosis. Additionally, EMPA elevated AMP levels, activated the AMPK pathway, and increased carnitine palmitoyl transferase1 (CPT1) gene expression, which collectively enhanced fatty acid oxidation and reduced stress signals. This study reveals a novel mechanism of EMPA's protective effects against PA-induced cardiomyocyte injury, providing new therapeutic insights into EMPA as a cardioprotective agent.

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics

NAD+ catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD+ catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD+ pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics.

Insertion of a knockout-first cassette in Ampd1 gene leads to neonatal death by disruption of neighboring genes expression.

AMPD1 is an adenosine monophosphate deaminase that catalyzes the deamination of AMP to IMP. To understand the physiological function of AMPD1, we obtained a strain of Ampd1 mutant mice from KOMP repository, which was generated by a knockout-first strategy. An elevated AMP level and almost complete lack of IMP was detected in the skeletal muscle of E18.5 Ampd1tm1a/tm1a mice. However, Ampd1tm1a/tm1a mice died in 2 days postnatally, which was contradicting to previous reports. After removal of the knockout-first cassette and critical exon, mice homozygous for the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d alleles survived to adulthood. RNA-seq analysis indicated that the expression of two neighboring genes, Man1a2 and Nras, were disrupted in the Ampd1tm1a/tm1a mice, but normal in the Ampd1tm1c/tm1c and Ampd1tm1d/tm1d mice. The neonatal lethality phenotype in the Ampd1tm1a/tm1a mice was consistent with the Man1a2-deficient mice. Our results indicated the knockout-first cassette may cause off-target effect by influence the expression of neighboring genes. This study, together with other reports, strongly suggests that removal of targeting cassette by site-specific recombinases is very important for the accurate phenotypic interpretation on mice generated by target mutations.

Role of AMP-activated protein kinase activators in antiproliferative multi-drug pituitary tumour therapies: effects of combined treatments with compounds affecting the mTOR-p70S6 kinase axis in cultured pituitary tumour cells.

AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)-secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down-regulation by specific small-interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR-p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF-4708671, with the effects of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor-induced p70S6K activity, as shown by the decrease of phospho-p70S6K levels. However, it was far less effective than rapamycin and PF-4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF-4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co-treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor-induced extracellular signal regulated kinase 1/2 phosphorylation. In conclusion, the results of the present study suggest the increased efficacy of combined antiproliferative therapies, including rapamycin analogues and AMPK activators in GH-secreting pituitary tumours, as a result of complementary and only partially overlapping mechanisms of action.

AMP-activated protein kinase regulates normal rat somatotroph cell function and growth of rat pituitary adenomatous cells

AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP and elevate AMP levels such as glucose deprivation and hypoxia. The AMPK system is primarily thought of as a regulator of metabolism and cell proliferation. Little is known about the regulation and the effects of AMPK in somatotroph cells. We present results from "in vitro" studies showing that AMPK activity has a role in regulating somatotroph function in normal rat pituitary and is a promising target for the development of new pharmacological treatments affecting cell proliferation and viability of pituitary adenomatous cells. In parallel, we show "in vivo" data obtained in the rat suggesting that AMPK is an intracellular transducer that may play a role in mediating the effects of the pharmacological treatment with dexamethasone on somatotrophs. In rat pituitary cell cultures, the AMP analog AICAR induced a rapid and clear-cut activation of AMPK. AICAR decreased GH release and total cellular GH content. An appropriate level of AMPK activation was essential for GH3 adenomatous cells. Remarkably, over-activation by AICAR induced apoptosis of GH3 whereas the AMPK inhibitor compound C was more effective at reducing cell proliferation. The role of endocrine or paracrine factors in regulating AMPK phosphorylation and activity in GH3 cells has been also studied. As to "in vivo" studies, western blot analysis revealed a significant decrease of phosphorylated AMPK alpha-subunit in pituitary homogenates of DEX-treated rats versus controls, suggesting reduced AMPK activity. In conclusion, our studies showed that AMPK has a role in regulating somatotroph function in normal rat pituitary and proliferation of pituitary adenomatous cells.

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP](2)-InsP(4)), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP](2)-InsP(4) synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP](2)-InsP(4) synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP](2)-InsP(4) levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP](2)-InsP(4) synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2'-amino-3'-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP](2)-InsP(4) is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.

Adenine nucleotides in snail muscles as one of biomarkers of fluoride toxicity

The aim of this work was to determine the extent of bioaccumulation of fluorides in tissues of Helix aspersa maxima. The toxicity of fluorides administered orally on the energy balance of the snail's foot was investigated based on measurements of concentrations of adenine nucleotides and their metabolism degradation products. Quantitation of fluoride levels was done in soft tissues (foot, hepatopancreas) and shells of mature snails. Qualitative and quantitative analysis of purine compounds was performed in slices of foot from mature snails. Fluoride concentrations in pulverized shells were measured using an ion-selective electrode. Gas chromatography was used to determine fluoride concentrations in soft tissues (hepatopancreas and foot). Purines were measured in foot muscle slices with high-performance liquid chromatography (HPLC). Fluoride levels in soft tissues of the snail cannot serve as an indicator for biomonitoring purposes as no significant accumulation was observed during exposure to maximum allowable concentrations of fluoride in drinking water. Contrary to this, levels of fluoride in the shell rose significantly with this concentration of fluoride in drinking water. The effect of fluorides on energy metabolism of foot muscle was evidenced by elevated AMP levels, increased adenine nucleotide pool and reduced conversion of ADP to ATP. Exposure to rising F(-) concentrations was accompanied by decreasing values of the adenylate energy charge AEC. Determination of AMP or AEC in foot muscle of exposed snails seems to be a useful indicator of fluoride effects on metabolic activity.

Effect of pentobarbital on fructose 2,6-bisphosphate metabolism in isolated rat hepatocytes.

Addition of the commonly used anesthetic pentobarbital to hepatocytes from fed rats resulted in a dose-dependent decrease in the level of fructose 2,6-bisphosphate. At a concentration of pentobarbital (0.4 mM) that lowered fructose 2,6-bisphosphate by 60%, there was no significant change in the level of fructose 6-phosphate, ATP, or L-glycerol 3-phosphate. Higher concentrations of pentobarbital (2 mM) enhanced both glycolysis and glycogenolysis and fructose 2,6-bisphosphate levels were reduced to less than 10% of the control. Concomitant with these changes there was a decrease in ATP, glucose 6-phosphate, and fructose 6-phosphate and a two- and fivefold increase in ADP and AMP, respectively. In hepatocytes from starved rats pentobarbital also lowered ATP levels and inhibited gluconeogenesis but had no effect on either lactate production or the already low level of sugar diphosphate. However, in the fasted case pentobarbital completely prevented the 10-fold elevation of fructose 2,6-bisphosphate brought about by 30 mM glucose. The anesthetic had no effect on cAMP-dependent protein kinase activity or on pyruvate kinase activity in hepatocytes from fed or starved rats but caused reciprocal changes in the activities of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Kinase activity was decreased and bisphosphatase activity was increased. These results suggest that the effects of pentobarbital on gluconeogenesis and glycolysis are due to inhibition of energy metabolism with elevated AMP levels causing activation of 6-phosphofructo-1-kinase and inhibition of fructose 1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes with time after capture in the metabolic activity of the carnivorous copepod Euchaeta norvegica Boeck

The rates of respiration and ammonium excretion of the carnivorous copepod Euchaeta norvegica Boeck decreased by 40 and 70%, respectively, during the first ≈10 h after capture. ETS (electron transport system) activity decreased in parallel with the rate of respiration. Increasing O/N and decreasing N/P atomic ratios during incubation indicated a change from protein to lipid metabolism, suggesting that starvation was the major factor responsible for the depression of the metabolic rates after capture. The adenylate energy charge (EC) ratio was significantly lower during the initial 13 h after capture than subsequently, caused mainly by an elevated AMP level. The total content of adenine nucleotides increased during this period of lowered EC. Starvation could also have been responsible for these changes in the adenine nucleotide levels through degradation of RNA. Our earlier interpretation that the lowered EC values after capture reflected capture stress has not been confirmed. The lactate level of E. norvegica just after capture was low (≈0.1 μg·mg dry wt −1) and thus not indicative of any oxygen debt.

Elevation of AMP Levels During Phagocytosis in Acanthamoeba castellanii

SUMMARY: Addition of particles to growing cultures of the amoeba, Acanthamoeba castellanii resulted in the production of large amounts of AMP, much of which was found extracellularly. At the time of maximal AMP production (13 h growth) adenylate energy charge values in conditioned and fresh medium were 0·58 and 0·1, respectively. Removal of particles from growth medium by filtration lowered the AMP level to 22% of the control value at the time of maximal AMP production; addition of particles to conditioned medium raised the level of AMP 3·6-fold. Uptake of killed yeast was accompanied by relatively small changes in levels of ATP and ADP, but large (5·7-fold) changes in AMP levels. Successive cycles of phagocytosis were paralleled by cyclic changes in AMP levels.