Orphan Nuclear Receptor Small Heterodimer Partner Negatively Regulates Growth Hormone-mediated Induction of Hepatic Gluconeogenesis through Inhibition of Signal Transducer and Activator of Transcription 5 (STAT5) Transactivation

Growth hormone (GH) is a key metabolic regulator mediating glucose and lipid metabolism. Ataxia telangiectasia mutated (ATM) is a member of the phosphatidylinositol 3-kinase superfamily and regulates cell cycle progression. The orphan nuclear receptor small heterodimer partner (SHP: NR0B2) plays a pivotal role in regulating metabolic processes. Here, we studied the role of ATM on GH-dependent regulation of hepatic gluconeogenesis in the liver. GH induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase gene expression in primary hepatocytes. GH treatment and adenovirus-mediated STAT5 overexpression in hepatocytes increased glucose production, which was blocked by a JAK2 inhibitor, AG490, dominant negative STAT5, and STAT5 knockdown. We identified a STAT5 binding site on the PEPCK gene promoter using reporter assays and point mutation analysis. Up-regulation of SHP by metformin-mediated activation of the ATM-AMP-activated protein kinase pathway led to inhibition of GH-mediated induction of hepatic gluconeogenesis, which was abolished by an ATM inhibitor, KU-55933. Immunoprecipitation studies showed that SHP physically interacted with STAT5 and inhibited STAT5 recruitment on the PEPCK gene promoter. GH-induced hepatic gluconeogenesis was decreased by either metformin or Ad-SHP, whereas the inhibition by metformin was abolished by SHP knockdown. Finally, the increase of hepatic gluconeogenesis following GH treatment was significantly higher in the liver of SHP null mice compared with that of wild-type mice. Overall, our results suggest that the ATM-AMP-activated protein kinase-SHP network, as a novel mechanism for regulating hepatic glucose homeostasis via a GH-dependent pathway, may be a potential therapeutic target for insulin resistance.     

ATM is involved in cell‐cycle control through the regulation of retinoblastoma protein phosphorylation

Ataxia telangiectasia mutated protein (ATM) is a member of the phosphatidylinositol‐3 kinase (PI3K) family, which has a role in the cellular response to DNA double‐strand breaks (DSBs). In the present study, we evaluated the role of ATM in cell‐cycle control in dopaminergic rat neuroblastoma B65 cells. For this purpose, ATM activity was either inhibited pharmacologically with the specific inhibitor KU‐55933, or the ATM gene was partially silenced by transfection with small interfering RNA (siRNA). Our data indicate that although ATM inhibition did not affect the cell cycle, both treatments specifically decreased the levels of cyclin A and retinoblastoma protein (pRb), phosphorylated at Ser780. Furthermore, ATM inhibition decreased the active form of p53, which is phosphorylated at Ser15, and also decreased Bax and p21 expression. Using H₂O₂ as a positive control of DSBs, caused a rapid pRb phosphorylation, this was prevented by KU‐55933 and siRNA treatment. Collectively, our data demonstrate how a new molecular network on ATM regulates the cell cycle through the control of pRb phosphorylation. These findings support a new target of ATM. J. Cell. Biochem. 110: 210–218, 2010. © 2010 Wiley‐Liss, Inc.     

AICAR induces phosphorylation of AMPK in an ATM-dependent, LKB1-independent manner

AMPK is an AMP-activated protein kinase that plays an important role in regulating cellular energy homeostasis. Metabolic stress, such as heat shock and glucose starvation, causes an 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 identified as an upstream kinase of AMPK. We found that in response to treatment with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), the LKB1 deficient cancer cell line, HeLa, exhibited AMPK-α phosphorylation. This indicates the existence of an LKB1-independent AMPK-α phosphorylation pathway. ATM is a protein that is deficient in the disease ataxia telangiectasia (A-T). We measured the activation of AMPK by AICAR in the normal mouse embryo fibroblast cell line, A29, and the mouse cell line lacking the ATM protein, A38. In A38 cells, the level of AICAR-induced AMPK-α phosphorylation was significantly 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. Thus, ATM may function as a potential AMPK kinase in response to AICAR treatment.     

The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via a γH2AX/p-p53/caspase-3-independent mechanism: Inhibition of calpain and cathepsin D

The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H₂O₂) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H₂O₂ or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H₂O₂-induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H₂O₂- and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H₂O₂-evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H₂O₂- and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal HT-22 cell line. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity.     

ATM protein kinase mediates full activation of Akt and regulates glucose transporter 4 translocation by insulin in muscle cells

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. Patients with A-T also have high incidences of type 2 diabetes mellitus. The gene mutated in this disease, ATM (A-T, mutated), encodes a protein kinase. Previous studies have demonstrated that cytoplasmic ATM is an insulin-responsive protein and a major upstream activator of Akt following insulin treatment. To further investigate the function of ATM in insulin signal transduction, insulin resistance was induced in rats by feeding them a high-fat diet. Muscle tissue of rats with insulin resistance had both dramatically reduced ATM levels and substantially decreased Akt phosphorylation at Ser473 in comparison to that of regular chow-fed controls. The decreased ATM expression suggests that ATM is involved in the development of insulin resistance through down-regulation of Akt activity. The role of ATM in activation of Akt was further confirmed in mouse embryonic fibroblast (MEF) A29 (ATM+/+) and A38 (ATM-/-) cells. In addition, insulin-mediated Akt phosphorylation in mouse L6 muscle cells was greatly reduced by KU-55933, a specific inhibitor of ATM. A 2-deoxyglucose incorporation assay showed that this inhibitor also caused a significant reduction in insulin-mediated glucose uptake in L6 cells. An immunofluorescence experiment demonstrated that in L6 cells transfected with wild-type (WT) ATM, insulin caused a dramatic increase of the cell surface glucose transporter 4 (GLUT4), while in cells transfected with kinase-dead (KD) ATM, translocation of GLUT4 to the cell surface in response to insulin was markedly inhibited.     

Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase

Chemotherapy that is used to treat human immunodeficiency virus type-1 (HIV-1) infection focuses primarily on targeting virally encoded proteins. However, the combination of a short retroviral life cycle and high mutation rate leads to the selection of drug-resistant HIV-1 variants. One way to address this problem is to inhibit non-essential host cell proteins that are required for viral replication. Here we show that the activity of HIV-1 integrase stimulates an ataxia-telangiectasia-mutated (ATM)-dependent DNA damage response, and that a deficiency of this ATM kinase sensitizes cells to retrovirus-induced cell death. Consistent with these observations, we demonstrate that a novel and specific small molecule inhibitor of ATM kinase activity, KU-55933, is capable of suppressing the replication of both wild-type and drug-resistant HIV-1.     

Deficiency of Ataxia Telangiectasia Mutated Kinase Modulates Cardiac Remodeling Following Myocardial Infarction: Involvement in Fibrosis and Apoptosis

Ataxia telangiectasia mutated kinase (ATM) is a cell cycle checkpoint protein activated in response to DNA damage. We recently reported that ATM plays a protective role in myocardial remodeling following β-adrenergic receptor stimulation. Here we investigated the role of ATM in cardiac remodeling using myocardial infarction (MI) as a model. Methods and Results: Left ventricular (LV) structure, function, apoptosis, fibrosis, and protein levels of apoptosis- and fibrosis-related proteins were examined in wild-type (WT) and ATM heterozygous knockout (hKO) mice 7 days post-MI. Infarct sizes were similar in both MI groups. However, infarct thickness was higher in hKO-MI group. Two dimensional M-mode echocardiography revealed decreased percent fractional shortening (%FS) and ejection fraction (EF) in both MI groups when compared to their respective sham groups. However, the decrease in %FS and EF was significantly greater in WT-MI vs hKO-MI. LV end systolic and diastolic diameters were greater in WT-MI vs hKO-MI. Fibrosis, apoptosis, and α-smooth muscle actin staining was significantly higher in hKO-MI vs WT-MI. MMP-2 protein levels and activity were increased to a similar extent in the infarct regions of both groups. MMP-9 protein levels were increased in the non-infarct region of WT-MI vs WT-sham. MMP-9 protein levels and activity were significantly lower in the infarct region of WT vs hKO. TIMP-2 protein levels similarly increased in both MI groups, whereas TIMP-4 protein levels were significantly lower in the infarct region of hKO group. Phosphorylation of p53 protein was higher, while protein levels of manganese superoxide dismutase were significantly lower in the infarct region of hKO vs WT. In vitro, inhibition of ATM using KU-55933 increased oxidative stress and apoptosis in cardiac myocytes.     

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.     

DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells

Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.     

Cadmium induced radioadaptive response via an ATM-independent H(2)S/cystathionine γ-lyase modulation

The combined exposure to environmental toxicants such as heavy metals and radiation is an important research area in health protection. Here we explored cadmium induced radioadaptive response (RAR) and investigated the role of hydrogen sulfide (H(2)S) and ATM kinase in this response. Our data showed that the cadmium ions with a sub-lethal concentration could induce RAR in Chang liver cells towards subsequent γ-irradiation and this response could be abrogated by DL-propargylglycine (PPG), the endogenous H(2)S synthetase inhibitor of cystathionine γ-lyase (CSE), but not by aminooxyacetic acid (AOAA), the inhibitor of cystathionine β-synthase (CBS). Moreover, the pretreatment of cells with NaHS also stimulated cellular adaptive response to radiation. Both cadmium treatment and irradiation up-regulated the expression of CSE protein in a time-dependent manner but had no influence on the expression of CBS protein. In the primed cells, the time course of CBS expression showed no significant difference with the cells treated with 2Gy irradiation alone, however, the CSE expression was easier to reach the maximum level, indicating a more efficient H(2)S production by CSE. Moreover, the cadmium-induced RAR was totally suppressed by KU-55933, a specific ATM inhibitor that did not change the CSE expression after radiation. However, exogenous H(2)S decreased the phosphorylation level of radiation-induced ATM. In conclusion, the present results demonstrate firstly that H(2)S is involved in the cadmium induced cross-adaptive response to challenging radiation. CSE, rather than CBS, may mainly responsible for the H(2)S production during this RAR which may also be mediated by ATM pathway. However, the activation of CSE is independent of ATM but could negatively regulate the phosphorylation of ATM.     

Metformin Increases Mitochondrial Energy Formation in L6 Muscle Cell Cultures

A popular hypothesis for the action of metformin, the widely used anti-diabetes drug, is the inhibition of mitochondrial respiration, specifically at complex I. This is consistent with metformin stimulation of glucose uptake by muscle and inhibition of gluconeogenesis by liver. Yet, mitochondrial inhibition is inconsistent with metformin stimulation of fatty acid oxidation in both tissues. In this study, we measured mitochondrial energy production in intact cells adapting an in vivo technique of phosphocreatine (PCr) formation following energy interruption (“PCr recovery”) to cell cultures. Metformin increased PCr recovery from either dinitrophenol (DNP) or azide in L6 cells. We found that metformin alone had no effect on cell viability as measured by total ATP concentration, trypan blue exclusion, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. However, treatments with low concentrations of DNP or azide reversibly decreased ATP concentration. Metformin increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction during recovery from either agent. Viability measured by trypan blue exclusion indicated that cells were intact under these conditions. We also found that metformin increased free AMP and, to a smaller extent, free ADP concentrations in cells, an action that was duplicated by a structurally unrelated AMP deaminase inhibitor. We conclude that, in intact cells, metformin can lead to a stimulation of energy formation, rather than an inhibition.     

Knockdown of cytoglobin expression sensitizes human glioma cells to radiation and oxidative stress

Cytoglobin is a recently identified vertebrate globin whose functions include scavenging reactive oxygen and nitrosative species. In tumor cells, CYGB may function as a tumor suppressor gene. Here we show that knockdown of cytoglobin expression can sensitize human glioma cells to oxidative stress induced by chemical inhibitors of the electron transport chain and as well can increase cellular radiosensitivity. When treated with antimycin A, an inhibitor of the mitochondrial electron transport chain, cytoglobin-deficient cells showed significantly higher H?O? levels, whereas H?O? levels were significantly reduced in cytoglobin-overexpressing cells. In addition, cytoglobin knockdown significantly decreased the doubling time of glioma cell lines, consistent with a putative tumor suppressor function. These finding suggest that modulating cytoglobin levels may be a promising treatment strategy for sensitizing human glioma cells to oxidative stress that is induced by ionizing radiation, certain chemotherapies and ischemia-reperfusion.     

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.     

C-terminal heat shock protein 90 inhibitor decreases hyperglycemia-induced oxidative stress and improves mitochondrial bioenergetics in sensory neurons

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70, and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress.     

Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells

Mitochondrial respiration, the key process behind cellular energy production, is critical for cell proliferation, growth and survival. However, the regulation of mitochondrial respiratory function in tumor cells is not well understood. In this study, we propose a model whereby tumor cells possess the capacity to fine-tune the balance between energy demands and mitochondrial reactive oxygen species (ROS) status, to maintain a milieu optimal for survival. This is achieved through the moderation of mitochondrial respiration, depending on the ROS context within the organelle, with the main players being Bcl-2 and cytochrome c oxidase (COX). We report a higher level of COX activity, oxygen consumption and mitochondrial respiration in tumor cells overexpressing Bcl-2. Transient overexpression, gene silencing and pharmacological inhibition of Bcl-2 corroborate these findings. Interestingly, Bcl-2 is also able to regulate mitochondrial respiration and COX activity in the face of mounting ROS levels, triggered by mitochondrial complex inhibitors. In this respect, it is plausible to suggest that Bcl-2 may be able to create an environment, most suited for survival by adjusting mitochondrial respiration accordingly to meet energy requirements, without incurring an overwhelming, detrimental increase in intracellular ROS.     

Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation

While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis. While AMP/ATP ratios were unaffected by metformin, phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase augmented. The inhibition of respiration provoked a rapid and sustained increase in superoxide levels, despite the increase in UCP2 and superoxide dismutase activity. The inhibition of respiration was rapidly reversed by fatty acids and thus respiration was lower in treated cells in the presence of pyruvate and glucose while rates were identical to control cells when palmitate was the substrate. We conclude that metformin reversibly inhibits mitochondrial respiration, it rapidly activates AMPK without altering the energy charge, and it inhibits fatty acid synthesis. Mitochondrial β-oxidation is facilitated by reversing the inhibition of complex I and, presumably, by releasing the inhibition of carnitine palmitoyltransferase. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).