AMP Levels Research Articles

Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes

Objective. In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism.Methods. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry.Results. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5’-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066).Conclusion. Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

A quantitative metabolomics peek into planarian regeneration.

The fresh water planarian species Schmidtea mediterranea is an emerging stem cell model because of its capability to regenerate a whole animal from a small piece of tissue. It is one of the best model systems to address the basic mechanisms essential for regeneration. Here, we are interested in studying the roles of various amines, thiols and nucleotides in planarian regeneration, stem cell function and growth. We developed mass spectrometry based quantitative methods and validated the differential enrichment of 35 amines, 7 thiol metabolites and 4 nucleotides from both intact and regenerating planarians. Among the amines, alanine in sexual and asparagine in asexual are the highest (>1000 ng/mg) in the intact planarians. The levels of thiols such as cysteine and GSH are 651 and 1107 ng mg(-1) in planarians. Among the nucleotides, the level of cGMP is the lowest (0.03 ng mg(-1)) and the level of AMP is the highest (187 ng mg(-1)) in both of the planarian strains. We also noticed increasing levels of amines in both anterior and posterior regenerating planarians. The blastema from day 3 regenerating planarians also showed higher amounts of many amines. Interestingly, the thiol (cysteine and GSH) levels are well maintained during planarian regeneration. This suggests an inherent and effective mechanism to control induced oxidative stress because of the robust regeneration and stem cell proliferation. Like in intact planarians, the level of cGMP is also very low in regenerating planarians. Surprisingly, the levels of amines and thiols in head regenerating blastemas are ∼3 times higher compared to those for tail regenerating blastemas. Thus our results strongly indicate the potential roles of amines, thiols and nucleotides in planarian regeneration.

Genetic introgression of ethylene-suppressed transgenic tomatoes with higher-polyamines trait overcomes many unintended effects due to reduced ethylene on the primary metabolome.

Ethylene regulates a myriad physiological and biochemical processes in ripening fruits and is accepted as the ripening hormone for the climacteric fruits. However, its effects on metabolome and resulting fruit quality are not yet fully understood, particularly when some of the ripening-associated biochemical changes are independent of ethylene action. We have generated a homozygous transgenic tomato genotype (2AS-AS) that exhibits reduced ethylene production as a result of impaired expression of 1-aminocyclopropane-1-carboxylate synthase 2 gene by its antisense RNA and had a longer shelf life. Double transgenic hybrid (2AS-AS × 579HO) developed through a genetic cross between 2AS-AS and 579HO (Mehta et al., 2002) lines resulted in significantly higher ethylene production than either the WT or 2AS-AS fruit. To determine the effects of reduced ethylene and introgression of higher polyamines’ trait, the metabolic profiles of ripening fruits from WT (556AZ), 2AS-AS, and 2AS-AS × 579HO lines were determined using 1H-NMR spectroscopy. The levels of Glu, Asp, AMP, Adenosine, Nucl1, and Nucl2 increased during ripening of the WT fruit. The increases in Glu, Asp, and AMP levels were attenuated in 2AS-AS fruit but recovered in the double hybrid with higher ethylene and polyamine levels. The ripening-associated decreases in Ala, Tyr, Val, Ile, Phe, malate, and myo-inositol levels in the 2AS-AS line were not reversed in the double hybrid line suggesting a developmental/ripening regulated accumulation of these metabolites independent of ethylene. Significant increases in the levels of fumarate, formate, choline, Nucl1, and Nucl2 at most stages of ripening fruit were found in the double transgenic line due to introgression with higher-polyamines trait. Taken together these results show that the ripening-associated metabolic changes are both ethylene dependent and independent, and that the fruit metabolome is under the control of multiple regulators, including ethylene and polyamines.

Inhibition of AMP Deaminase Activity Does Not Improve Glucose Control in Rodent Models of Insulin Resistance or Diabetes

Inhibition of AMP deaminase (AMPD) holds the potential to elevate intracellular adenosine and AMP levels and, therefore, to augment adenosine signaling and activation of AMP-activated protein kinase (AMPK). To test the latter hypothesis, novel AMPD pan inhibitors were synthesized and explored using a panel of in vitro, ex vivo, and in vivo models focusing on confirming AMPD inhibitory potency and the potential of AMPD inhibition to improve glucose control in vivo. Repeated dosing of selected inhibitors did not improve glucose control in insulin-resistant or diabetic rodent disease models. Mice with genetic deletion of the muscle-specific isoform Ampd1 did not showany favorable metabolic phenotype despite being challenged with high-fat diet feeding. Therefore, these results do not support the development of AMPD inhibitors for the treatment of type 2 diabetes.

Effects of Pharmacological AMP Deaminase Inhibition and Ampd1 Deletion on Nucleotide Levels and AMPK Activation in Contracting Skeletal Muscle

AMP-activated protein kinase (AMPK) plays a central role in regulating metabolism and energy homeostasis. It achieves its function by sensing fluctuations in the AMP:ATP ratio. AMP deaminase (AMPD) converts AMP into IMP, and the AMPD1 isoenzyme is expressed in skeletal muscles. Here, effects of pharmacological inhibition and genetic deletion of AMPD were examined in contracting skeletal muscles. Pharmacological AMPD inhibition potentiated rises in AMP, AMP:ATP ratio, AMPK Thr172, and acetyl-CoA carboxylase (ACC) Ser218 phosphorylation induced by electrical stimulation, without affecting glucose transport. In incubated extensor digitorum longus and soleus muscles from Ampd1 knockout mice, increases in AMP levels and AMP:ATP ratio by electrical stimulation were potentiated considerably compared with muscles from wild-type mice, whereas enhanced AMPK activation was moderate and only observed in soleus, suggesting control by factors other than changes in adenine nucleotides. AMPD inhibitors could be useful tools for enhancing AMPK activation in cells and tissues during ATP-depletion.

Increased Particulate Air Pollution and Markers of Platelet Aggregation

Background: Increased particulate air pollutant concentrations (PM) have previously been associated with platelet aggregation. Aspirin has been shown to protect against cardiovascular (CV) events. We hypothesized that increased ambient PM concentrations would be associated with increased markers of platelet aggregation, and that aspirin would lessen this response. Methods: We used data from a sequential therapy trial of aspirin and fish oil (n=30 subjects; 4 blood samples; 10/2010-3/2012) in type II diabetes patients. Using linear mixed models, we estimated changes in 3 platelet measures (aggregation induced by adenosine diphospate [ADP] or collagen, and thromboxane production) associated with interquartile range (IQR) increases in 1 to 96 hour mean concentrations of ambient PM2.5, black carbon, ultrafine particle (UFP; 10-100nm), and accumulation mode particle (AMP; 100-500nm) concentrations. Results: IQR increases in mean UFP and AMP concentrations were associated with significant decreases in platelet function, with the largest being a -1.7 ohm reduction in collagen-induced aggregation (95% CI= -3.1, -0.3) associated with each 2097 particles/cm3 increase in UFP, and a -0.43 decrease in log(thromboxane; pg/mL; 95% CI=-0.8, -0.1) associated with each 582 particles/cm3 increase in AMP, both in the previous 72 hours. We found no associations with PM2.5 or black carbon. Further, this UFP effect on thromboxane was greater when taking aspirin (-0.6; 95% CI = -1.0, -0.3) then when not (-0.0; 95% CI = -0.4, 0.3; interaction p=0.003). Conclusions: Inconsistent with our a priori hypothesis, we found that decreased platelet thromboxane production and collagen-induced aggregation were associated with increased UFP and AMP levels in the previous 3-4 days, with aspirin increasing this effect. The reason for this is unclear, but may be related to the disrupted lipid milieu and aspirin insensitivity in patients with type 2 diabetes.

Abstract 3735: Monitoring the level of the universal nucleotide AMP in diverse enzymatic reactions using bioluminescent homogenous assay platform

Abstract Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. Many biochemical reactions that generate AMP as a reaction product are pivotal players in a variety of signaling pathways and thus are considered validated drug targets. These include enzymes involved in protein ubiquitination (Ubiquitin ligases), protein synthesis (aminoacyl tRNA synthetases), second messenger signaling (cAMP-phosphodiesterases), and DNA synthesis and repair (prokaryotic and eukaryotic DNA ligases). Therefore, an assay that monitors the activity of these enzymes is desirable in the search for selective modulators and the development of novel therapeutics. As AMP is a common product of these enzymatic reactions, the development of an assay that monitors AMP in a homogenous and sensitive fashion will have significant impact in these diverse areas of research. In addition, enzyme reactions that generate products which can be converted to AMP can be also monitored using this assay. We demonstrate the utility of this assay platform in monitoring the activity of cAMP-PDE, aminoacyl tRNA synthetases, ubiquitin ligases, DNA ligases. Furthermore, enzyme reactions such as methyltransferases that produce product (SAH) which can be converted to AMP can be monitored using this assay platform Citation Format: Said A. Goueli, Kevin Hsiao, Hicham Zegzouti, Subhanjan Mondal. Monitoring the level of the universal nucleotide AMP in diverse enzymatic reactions using bioluminescent homogenous assay platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3735. doi:10.1158/1538-7445.AM2014-3735

Protective effect of an antithyroid compound against γ-radiation-induced damage in human colon cancer cells

We have previously reported the radioprotective effect of propylthiouracil (PTU) on thyroid cells. The aim of the present study was to analyze whether tumor cells and normal cells demonstrate the same response to PTU. Human colon carcinoma cells were irradiated with γ-irradiation with or without PTU. We evaluated the clonogenic survival, intracellular reactive oxygen species levels, catalase, superoxide dismutase and glutathione peroxidase activities, and apoptosis by nuclear cell morphology and caspase-3 activity assays. Cyclic AMP (cAMP) levels were measured by radioimmunoassay. PTU treatment increased surviving cell fraction at 2Gy (SF2) from 56.9±3.6 in controls to 75.0±3.5 (p<0.05) and diminished radiation-induced apoptosis. In addition, we observed that the level of antioxidant enzymes' activity was increased in cells treated with PTU. Moreover, pretreatment with PTU increased intracellular levels of cAMP. Forskolin (p<0.01) and dibutyryl cAMP (p<0.05) mimicked the effect of PTU on SF2. Co-treatment with H89, an inhibitor of protein kinase A, abolished the radioprotective effect of PTU. PTU reduces the toxicity of ionizing radiation by increasing cAMP levels and also possibly through a reduction in apoptosis levels and in radiation-induced oxidative stress damage. We therefore conclude that PTU protects both normal and cancer cells during exposure to radiation in conditions mimicking the radiotherapy.

Зміни енергетичного балансу печінки та підшлункової залози щурів при гіпоацидному стані шлунка та пошук підходів до його відновлення

It was established that the energy balance was disturbed in liver and pancreas of rats with long-term gastric hypochlorhydria, suggesting a decreased of ATP content while ATP and AMP levels were increasing. Multiprobiotic "Symbiter ®" restored adenine nucleotides balance in the analyzed organs of rats with gastric hypoacidity state.

The Effects of Nicotinic Acid on Lipid Profile of Albino Wistar Rats.

The effect of nicotinic acid on lipid profile of albino Wistar rats was investigated to assess its role in the management of lipid related disorder such as dyslipidemia, atherosclerosis and hypertension. Consequently, nicotinic acid at varying pharmacological doses was administered to different groups of albino Wistar rats. This was followed by the examination of the effect on serum total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). The nicotinic acid was administered to five groups of experimental animals (group B - F) for twenty-one days at the concentrations of 250mg, 500mg, 750, 1000mg and 1250mg per 70kg per day respectively while 'group A' served as the control. The results showed a reduction of TC in all groups when compared to the control. Interestingly, at a dose of 1000mg, there was a significant decrease in TG and VLDL, the percentage composition of HDL increased by 8% when compared to the control. The binding of nicotinic acid to its receptor (HM74/HM74A) is believed to decrease the level of intracellular cyclic AMP thereby inhibiting lipolysis in adipocytes.

Pre-diabetes alters testicular PGC1-α/SIRT3 axis modulating mitochondrial bioenergetics and oxidative stress

Pre-diabetes, a risk factor for type 2 diabetes development, leads to metabolic changes at testicular level. Peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) and Sirtuin 3 (Sirt3) are pivotal in mitochondrial function. We hypothesized that pre-diabetes disrupts testicular PGC-1α/Sirt3 axis, compromising testicular mitochondrial function. Using a high-energy-diet induced pre-diabetic rat model, we evaluated testicular levels of PGC-1α and its downstream targets, nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), mitochondrial transcription factor A (TFAM) and Sirt3. We also assessed mitochondrial DNA (mtDNA) content, mitochondrial function, energy levels and oxidative stress parameters. Protein levels were quantified by Western Blot, mtDNA content was determined by qPCR. Mitochondrial complex activity and oxidative stress parameters were spectrophotometrically evaluated. Adenine nucleotide levels, adenosine and its metabolites (inosine and hypoxanthine) were determined by reverse-phase HPLC.Pre-diabetic rats showed increased blood glucose levels and impaired glucose tolerance. Both testicular PGC-1α and Sirt3 levels were decreased. NRF-1, NRF-2 and TFAM were not altered. Testicular mtDNA content was decreased. Mitochondrial complex I activity was increased, whereas mitochondrial complex III activity was decreased. Adenylate energy charge was decreased in pre-diabetic rats, as were ATP and ADP levels. Conversely, AMP levels were increased, evidencing a decreased ATP/AMP ratio. Concerning to oxidative stress pre-diabetes decreased testicular antioxidant capacity and increased lipid and protein oxidation. In sum, pre-diabetes compromises testicular mitochondrial function by repressing PGC-1α/Sirt3 axis and mtDNA copy number, declining respiratory capacity and increasing oxidative stress. This study gives new insights into overall testicular bioenergetics at this prodromal stage of disease.

Abstract P2-09-15: Effect of [4-valine 5-glutamine] desmopressin, a novel vasopressin analog, on human breast cancer cell growth in two-dimensional (2D) and three-dimensional (3D) cultures

Abstract Background: Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in endothelium and also in some tumor cells. V2R stimulation is associated with increased levels of cyclic AMP and cyclic AMP-dependent protein kinase activation. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity. Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. After an extensive in vitro and in vivo characterization, novel peptide compound [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) exerted a remarkable and improved antitumor effect in comparison to parental peptide dDAVP. [V4Q5]dDAVP inhibited tumor growth in both syngeneic F3II and xenogeneic MDA-MB-231 breast cancer models, modulating tumor aggressiveness and vasculature. In this study, to further understand direct cytostatic action of this novel vasopressin analog on tumor cells, we assessed the effect of [V4Q5]dDAVP on clonogenic and three dimensional growth of V2R-expressing human breast carcinoma cell lines. Methods and results: We used MCF7 and triple negative MDA-MB-231 human breast cancer cell lines to conduct the experiments. Vasopressin V2 membrane receptor expression in tumor cell lines was assessed by RT-PCR and immunofluorescence. We first examined peptide ability to modify in vitro low density cell growth by the clonogenic growth assay. Incubation with [V4Q5]dDAVP (100-1500 nM) during one week significantly reduced the number of tumor cell colonies showing a concentration dependant manner and an IC50 value of 1135 nM. To assess the effect of novel analog on three dimensional growth of cancer cells we used two different assays. The first one evaluates the ability of basement membrane extract (BME)-embedded single cells to proliferate and form spheroids. The second assay evaluates growth of single fully established spheroids (Approx. 300 mm diameter) transplanted individually on a non-adherent surface. [V4Q5]dDAVP, at a concentration of 1000 nM, was able to significantly reduce the number of BME-embedded spheroids formed from single cells after 14 days of treatment. In a similar manner, incubation of established and isolated spheroids with [V4Q5]dDAVP using the same concentration for 9 days, caused a decrease in multicellular spheroids final diameter. Cellular metabolic activity analysis by the MTS assay showed no significant differences between treated and control groups. Conclusions: We report for the first time the inhibitory effect of [V4Q5]dDAVP on colony and spheroid formation and growth of human breast carcinoma cells. Our results using three dimensional models suggest that V2R stimulation in cancer cells may affect early stages of microtumor establishment. Further analysis of involved signaling pathways in treated 3D cultures is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-15.

Abstract 341: Expression of Leukocyte CD39 in Acute Coronary Syndrome

Background CD39 (ENTPD-1) is a membrane-spanning ectonucleotidase that is responsible for the phosphohydrolysis of ATP to ADP and ADP to AMP. As increased myocardial infarct sizes have been reported in murine CD39 knockout models when compared with controls, we hypothesized that CD39 might be expressed as part of the myocardial injury/repair response. CD39 levels have not been heretofore described in humans with acute coronary syndrome (ACS). Objectives To measure CD39 mRNA and protein levels in human study subjects with ACS compared with healthy controls. Methods Healthy subjects (n=24), defined as having no history of coronary or peripheral arterial disease, and subjects with ACS (n=13) were enrolled in the study. Blood was collected via venupuncture for healthy controls or at the time of cardiac catheterization for ACS subjects. Plasma and leukocytes were isolated from blood, and soluble CD39 protein from leukocytes was measured using ELISA. mRNA was extracted from leukocytes, and qPCR was used to measure differences in leukocyte CD39 mRNA. Mass spec was used to assess plasma ATP, ADP, AMP, and adenosine levels. Findings Increased CD39 mRNA expression was found in subjects with ACS when compared with healthy controls (2.39 vs. 1.66, p=0.021). There was a trend towards increased leukocyte CD39 protein concentrations in ACS patients (n=5) compared to controls (n=9) (7.50 ng/ml vs. 2.64 ng/ml, p=0.057) when measured by ELISA. There was a trend toward increased levels of soluble plasma CD39 levels in ACS patients (n=12) when compared with controls (n=24) (2.39 ng/ml vs. 1.67 ng/ml, p=0.59) when measured by ELISA. ACS patients (n=11) were found to have higher levels of circulating ADP as assessed by Mass spec when compared with healthy controls (n=17), 2.02 vs. 0.67, p=0.03. Leukocyte CD39 levels positively correlated with ATP (r 2 =.231) and ADP (r 2 =.155) levels (n=10). Conclusions This is the first report of increased CD39 gene expression in patients with ACS, when compared with healthy controls without known coronary disease. A trend toward increased plasma and leukocyte CD39 protein expression was present in those with ACS when compared with controls. CD39 could serve as a modulator of vascular homeostasis in ACS.

Forskolin induced increase in spontaneous activity of auditory brainstem neurons is comparable to acoustic stimulus evoked responses

Contemporary proposals for the pathophysiology of tinnitus due to cochlear damage underscore increased spontaneous activity of auditory brainstem neurons. One of the several consequences of the cochlear injury is the activation of the ERK pathway, suppression of phosphodiestase E activity, and putatively setting a long-term increase in intracellular levels of cyclic AMP at central auditory neurons. Local application of forskolin also increases intracellular cyclic AMP and spontaneous neural activity. We measured the effects of locally applied forskolin on spontaneous firing rate of isolated neurons in the peri-olivary region of the superior olive complex in anesthetized adult Long Evan rats. Forskolin induced increase in spontaneous neural activity was comparable to supra-threshold tone evoke neural responses. These results are viewed in context of hyperexcitability as a correlate of tinnitus.

The metabolism of 9-chloro-β-lapachone and its effects in isolated hepatocytes. The involvement of NAD(P)H:quinone oxidoreductase 1 (NQO1)

A β-lapachone analogue (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione) (9-chloro β-lapachone), named CGQ, with antitumoral, antiviral and antitrypanocidal activities was assayed for cytotoxic effects on isolated rat hepatocytes. The incubation of hepatocytes with this o-naphthoquinone showed (a) decreased adenylate energy charge, as a result of a decrease in ATP, and an increase in AMP levels; (b) increased NADP+ content, with a concomitant decrease of NADPH, NADH and NAD+ content; (c) decreased GSH content, accompanied by an increase in GSSG formation; (d) stimulated oxygen uptake as well as increased superoxide anion production and hydrogen peroxide formation; (e) inhibited lipid peroxidation; (f) hepatocyte viability was not reduced unless the NQO1 inhibitor dicoumarol was present. We hypothesize that the cytotoxicity of CGQ in dicoumarol-treated hepatocytes was the result of inhibition of the NQO1 detoxification pathway, thus allowing more quinone to be metabolized towards the one-electron pathway to form reactive semiquinones and/or reactive oxygen species. The results obtained indicate a protective role of NQO1 in preventing CGQ cytotoxicity in isolated rat hepatocytes.

Inverse correlation of intracellular calcium and cyclic AMP levels in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common renal tumour in adults. Altered levels of secondary messengers, that is, intracellular calcium and cyclic AMP (cAMP), have been implicated in the pathogenesis of various malignancies. In the present study, we measured levels of intracellular calcium and cAMP in RCC. The intracellular calcium level was significantly reduced, whereas the cAMP level was significantly augmented in RCC as compared with adjacent grossly normal renal parenchyma.

E-NTPDase and E-ADA activities are altered in lymphocytes of patients with indeterminate form of Chagas' disease

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P<0.05 and P<0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P<0.05) and both AMP (P<0.01) and adenosine (P<0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.

Mitochondrial DNA Variant for Complex I Reveals a Role in Diabetic Cardiac Remodeling

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.

Abstract 954: COX-2 inhibition reduces bone tumor growth in animal models:A role for celecoxib treatment in cAMP/protein kinase A-induced tumors

Abstract Background: Carney Complex (CNC) is a multiple neoplasia syndrome characterized by skin lesions, endocrine and other tumors, including osteochondromyxomas. It is caused by inactivating mutations in the PRKAR1A gene encoding the regulatory type 1A subunit of cAMP-dependent protein kinase A (PKA). This subunit is the main suppressor of activity of catalytic subunit (PRCKACA) of PKA which regulates cell proliferation and differentiation. Loss of PRKAR1A increases PKA activity and causes tumors in CNC-affected tissues. Mice with deletion of one prkar1a allele (prkar1a+/−) develop various CNC-like tumors and multiple tail osteotic lesions. Further deletion of a prkaca allele on the prkar1a+/− background unexpectedly increases the number and severity of bone lesions and reduces their age of onset. Recent data indicate that increased PKA activity stimulates the inflammasome without inflammation in mouse and human cells. The released prostaglandin E2 (PGE2) further increases cAMP, which in turn activates PKA and Wnt signaling and promotes the growth of these bony tumors. These data led us to investigate celecoxib, which is a selective inhibitor of cyclooxygenase-2 (COX-2) and therefore of PGE2's release, in prkar1α+/−/prkacα+/− mice. Methods: Eleven out of twenty-two mice 6-12 months old were exposed to celecoxib-containing diet (1,500 mg/kg of NIH-31 diet). The remaining mice were fed the standard NIH-31 diet. After a six-week treatment, we looked at the number, the PKA and phosphodiesterase (PDE) activities, c AMP levels and histology of bony lesions. Sixteen and seven days before sacrifice, mice were injected with calcein which labels bone forming surfaces. From calcein fluorescence observed under microscope, the rate of apposition of cortical bone at periosteal side was measured. This rate reflects tumor growth rate because growing tumors remain encapsulated by cortical bone forming at periosteal side and resorbing at endosteal side. Results: Celecoxib reduced the rate of tumor growth by two-fold as measured by calcein labeling (p=0.016). Histological examination revealed decreased cellularity in the lesions of treated animals due to treatment effect on the tumor cells. Treatment reduced PKA enzymatic activity in the tail tumors as measured by high-performance liquid chromatography (4.0 vs 3.0 CMP/ug protein, p=0.07) whereas cAMP levels and total PDE activity did not change. The expression of all inflammasome-related genes (ets-1, caspase-1, il1b and cox2) was down-regulated by at least 70% in the bone tumors from treated animals compared to controls. Conclusion: COX-2 inhibition reduces PKA activity and bone tumor growth in prkar1α+/−/prkacα+/− mice. Since local tumor control is clinically meaningful for patients with cAMP-induced tumors, celecoxib may merit consideration as the first molecular targeted therapy in this setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 954. doi:1538-7445.AM2012-954

Abstract LB-25: Activation of AMPK by ATM in reponse to AICAR and metformin

Abstract AMPK is an AMP-activated protein kinase that plays a key role in regulating cellular energy homeostasis. Metabolic stress, such as heat shock and glucose starvation, causes energy deficiency in the cell and leads to elevated levels of intracellular AMP. This results in the phosphorylation and activation of AMPK. LKB1, a tumor suppressor, has been known as an upstream kinase of AMPK. We found that in response to treatment with 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside (AICAR), a LKB1-null cancer cell line, HeLa, still exhibited AMPK phosphorylation. This indicates the existence of an LKB1-independent AMPK protein kinase. ATM is a protein kinase that is deficient in the disease ataxia telangiectasia (A-T). We measured the activation of AMPK by AICAR in the normal mouse embryo fibroblast (MEF) cell line, A29, and the MEF cell line lacking the ATM protein, A38. In A38 cells, the level of AICAR-induced AMPK phosphorylation was dramatically lower than that found in A29 cells. Furthermore, phosphorylation of AMPK in HeLa and A29 cells was inhibited by an ATM specific inhibitor, KU-55933. Our results demonstrate that AICAR treatment could lead to phosphorylation of AMPK in an ATM-dependent and LKB1-independent manner. Similar to the results of AICAR treatment, our work also revealed that the metformin-induced phosphorylation of AMPK in A38 cells is significantly lower than that in A29 cells. We also examined the effect of ATM on AMPK in rat hepatoma cell line H4IIE. Inhibition of ATM by KU-55933 dramatically reduced the phosphorylation of AMPK in response to metformin in this cell line. Thus, ATM may act as a potential AMPK kinase in response to both AICAR and metformin treatment. Metformin is the most commonly prescribed pharmaceutical drug for type 2 diabetes (T2D). In addition, patients with T2D or obesity/insulin resistance who take metformin have reduced rates of cancer occurrence, including breast cancer. However, the mechanism underlying metformin's effect on cancer prevention and/or treatment is unclear (Nature, New &amp; views, Feb. 2011; Science, News Focus, Jan. 2012). While ATM acts as a sensor of DNA damage by controlling cell cycle progression, it also has glucose regulatory function. Our results put ATM as a novel player in the AMPK signaling pathway in response to metformin. These findings provide new insights into the mechanisms by which metformin functions as a preventive and/or therapeutic agent for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-25. doi:1538-7445.AM2012-LB-25