Sex-specific regulation of AMP-activated protein kinase (AMPK) in the Pacific oyster Crassostrea gigas.

The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas.

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.

Metastasis suppressor NM23-H1 promotes repair of UV-induced DNA damage and suppresses UV-induced melanomagenesis.

Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3'-5' exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA-induced (290-400 nm) and UVC-induced (254 nm) DNA damage, whereas its 3'-5' exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data show a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.

In vivo RNA interference of a gonad-specific transforming growth factor-ß in the Pacific oyster Crassostrea gigas.

We investigated the role of oyster gonadal TGFß (og-TGFß) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFß, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFß dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFß appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.

Microarray-based identification of gonad transcripts differentially expressed between lines of Pacific oyster selected to be resistant or susceptible to summer mortality.

Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event, using a cDNA microarray that we designed. ANOVA analysis revealed that 34 genes were differentially expressed between R and S lines on four dates preceding the mortality event. Annotation of some of these genes highlights reproduction and its allocation and antioxidant defenses as the main pathways that operate differentially between R and S lines. This transcriptional analysis provides new indications to define markers for quantitative trait loci searches and functional studies and evaluate the potential role of each gene in the resistance to summer mortality.

Infrared spectroscopy: a reagent-free method to distinguish Alzheimer's disease patients from normal-aging subjects.

The physiopathogenesis of Alzheimer's disease (AD) is related to various biochemical mechanisms that may be reflected by changes in plasma components. In the current study, Fourier transform-infrared (FT-IR) spectroscopy was used to identify these biochemical variations by monitoring spectral differences in the plasma of 40 AD patients compared with those of 112 control subjects. A hierarchical classification in the whole mid-infrared region allowed a clear separation between AD and controls (C) that was optimized by using a restricted spectral range (1480-1428 cm(-1)). Spectral changes confirmed vibration differences between AD and C mostly related to modified lipid and nucleic acid structures involved in oxidative stress-dependent processes of AD. Moreover, the analysis of samples in the 1480-910-cm(-1) region allowed the distinction between C and AD with an accuracy of 98.4% and showed 2 subgroups C(1) and C(2) within the C group. Interestingly, the C(1) subgroup was located closer to the AD group than the C(2) subgroup, which suggests biochemical differences within the nondemented subjects. Biochemical studies revealed a significant increase in a specific marker of oxidative stress, F8-isoprostanes (8-epi-PGF2alpha) levels, in the plasma of AD patients as compared with total controls and subgroup C(2) but not subgroup C(1). Thus, these results suggest that use of FT-IR spectroscopy could be valuable to distinguish AD patients from normal-aging subjects.

Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg).

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.

Protein kinase CK2 acts as a signal molecule switching between the NDPK-A/AMPK alpha1 complex and NDPK-B.

Previously we elucidated the molecular interaction between the nucleoside diphosphate kinase A (NDPK-A)/AMP-activated protein kinase (AMPK) alpha1 complex, discovering a process we termed "substrate channeling." Here, we investigate the protein-protein interaction of the substrate channeling complex with the pleiotropic protein kinase, CK2 (formerly casein kinase 2). We show that CK2 is part of the NDPK-A/AMPK alpha1 complex under basal (background AMPK activity) conditions, binding directly to each of the complex components independently. We report that when S122 on NDPK-A is phosphorylated by AMPK alpha1 in vivo, (i.e., stimulation of AMPK using either metformin or phenformin) initiating the substrate channeling mechanism, the catalytic subunit of CK2 (CK2alpha) is expelled from the complex and translocates to bind NDPK-B, a closely related but independent isoform of NDPK. Thus, we find that the AMPK-dependent phospho-status of S122 on NDPK-A determines whether CK2alpha swaps partners between NDPK-A and NDPK-B. This is the first reported linkage between NDPK-A and NDPK-B via a phosphorylation pathway and could explain the complex biology of NDPK. This study also offers an explanation as to how CK2alpha exclusion mutations (S120A or S122D of NDPK-A) on NDPK-A might have implications in cancer biology and general cellular energy metabolism.

Understanding the molecular basis of the interaction between NDPK-A and AMPK alpha 1.

Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16 to 20 kDa) including two well-characterized isoforms (NDPK-A and -B). NDPK catalyzes the conversion of nucleoside diphosphates to nucleoside triphosphates, regulates a diverse array of cellular events, and can act as a protein histidine kinase. AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that responds to the cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of acetyl coenzyme A carboxylase 1 (ACC1), a regulator of cellular fatty acid synthesis. We recently reported that NDPK-A (but not NDPK-B) selectively regulates the alpha1 isoform of AMPK independently of the AMP concentration such that the manipulation of NDPK-A nucleotide trans-phosphorylation activity to generate ATP enhanced the activity of AMPK. This regulation occurred irrespective of the surrounding ATP concentration, suggesting that "substrate channeling" was occurring with the shielding of NDPK-generated ATP from the surrounding medium. We speculated that AMPK alpha1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK alpha1 in vivo. We find that NDPK-A S122 and S144 are phosphorylated by AMPK alpha1 and that the phosphorylation status of S122, but not S144, determines whether substrate channeling can occur. We report the cellular effects of the S122 mutation on ACC1 phosphorylation and demonstrate that the presence of E124 (absent in NDPK-B) is necessary and sufficient to permit both AMPK alpha1 binding and substrate channeling.

Acute oxidative stress is associated with cell proliferation in the mouse liver.

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.

NDPK-A (but not NDPK-B) and AMPK alpha1 (but not AMPK alpha2) bind the cystic fibrosis transmembrane conductance regulator in epithelial cell membranes.

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.

Expression patterns of nm23 genes during mouse organogenesis.

Nucleoside di-phosphate kinase enzyme (NDPK) isoforms, encoded by the nm23 family of genes, may be involved in various cellular differentiation and proliferation processes. We have therefore analyzed the expression of nm23-M1, -M2, -M3, and -M4 during embryonic mouse development. In situ hybridization data has revealed the differential expression of nm23 mRNA during organogenesis. Whereas nm23-M1 and -M3 are preferentially expressed in the nervous and sensory systems, nm23-M2 mRNA is found ubiquitously. Irrespective of the developmental state studied, nm23-M4 mRNA is only expressed at low levels in a few embryonic organs. In the cerebellum and cerebral cortex, nm23-M1, -M2, and -M3 are present in the neuronal differentiation layer, whereas nm23-M4 mRNA is distributed in the proliferating layer. Thus, nm23 mRNA is differentially expressed, and the diverse NDPK isoforms are sequentially involved in various developmental processes.

Increased lung metastasis in transgenic NM23-Null/SV40 mice with hepatocellular carcinoma.

BACKGROUND: The metastasis-suppressing role of the NM23 gene in the metastatic spread of solid tumors is still debated. We examined the role of NM23 in tumor development and metastatic dissemination by using transgenic mice that lack mouse NM23 (NM23-M1) in two mouse models of hepatocellular carcinoma (HCC) that recapitulate all steps of tumor progression. METHODS: We induced HCC in mice that contained (NM23-M1(+/+)) or lacked (NM23-M1(-/-)) NM23-M1 by diethylnitrosamine injection or by a crossing scheme that transferred a transgene that leads to liver expression of simian virus 40 large T antigen (ASV mice). We used microscopic examination and immunohistochemistry to analyze tumor progression. Expression of Nm23 protein isoforms (Nm23-M1 and Nm23-M2) and several tumor markers was analyzed in the primary tumor and in metastases by Western blotting. The statistical significance of differences in the incidence of Nm23-M2 overexpression in null mice relative to that in wild-type mice was tested by a one-sided Fisher's exact test. The statistical significance of differences in the incidence of metastases was examined using one-sided chi-square tests. All other statistical tests were two-sided. RESULTS: In both models, Nm23-M1 and/or Nm23-M2 were overexpressed in the primary liver tumors compared with nontumor liver tissue; however, the lack of the NM23-M1 gene had no effect on primary tumor formation in either model. ASV mice developed pulmonary metastases that were positive for the Hep-Par 1 antibody, which recognizes a specific hepatocyte antigen, whereas the few pulmonary nodules that developed in diethylnitrosamine-injected mice were negative for this antigen. Statistically significantly more ASV/NM23-M1(-/-) mice than ASV/NM23-M1(+/+) mice developed lung metastases (69.2% versus 37.5%; difference = 31.7%, 95% confidence interval = 13.1% to 50.3%; P<.001). In ASV/NM23-M1(+/+) mice, immunohistochemical staining for Nm23-M1 was highly heterogeneous among the primary liver tumors, but weak or negative among lung metastases. CONCLUSIONS: The lack of NM23-M1 expression promotes metastasis in the SV40 animal model of liver carcinogenesis.

Nm23-M2/NDP kinase B induces endogenous c-myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death.

The nm23 gene family encodes nucleoside diphosphate kinases (NDPKs) which supply the cell with (d)NTPs. The human NDPKB, also known as the PuF protein, binds the c-myc promoter and transactivates the c-myc protooncogene. We have now studied the effects of mouse NDPKA and NDPKB overexpression on endogenous c-myc transactivation in the mouse BAF3 and the rat PC12 cell lines. c-myc transcripts were found to be up-regulated by NDPKB only in the BAF3 line. This suggests that c-myc transcriptional control via NDPKB depends on the presence of cell-specific co-factors. Unexpectedly, NDPKB also induced NDPKA expression. This new effect was found in both cell lines, suggesting that NDPKB-dependent nm23-M1 gene transactivation requires cis and/or trans elements different from those involved in c-myc transactivation. Moreover, the BAF3 cell proliferation capacities were found to be independent of NDPKA or B cell contents. Interestingly, cell death induced by c-myc overexpression or H(2)O(2) exposure was decreased in nm23-transfected compared to control BAF3 cells. These data collectively suggest that NDPKs might improve cell survival by a mechanism coupling DNA repair and transcriptional regulation of genes involved in DNA damage response.

Oxidative and antioxidative status in pregnant women with either gestational or type 1 diabetes.

OBJECTIVES: To evaluate oxidative and antioxidative status in pregnant diabetic women between 26 and 32 weeks of gestation. DESIGN AND METHODS: Free and total malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX), and vitamins A and E were determined in plasma and erythrocytes of 54 pregnant women. Among these, 27 were diabetics with either gestational diabetes mellitus (GDM), sub-group I, or previous insulin-dependent diabetes mellitus (type 1 diabetes), sub-group II. The other 27 patients were controls. Fasting plasma glucose and HbA(1c) levels were determined in all women. RESULTS: HbA(1c) levels, plasma-, and erythrocyte-free MDA levels were significantly higher in all diabetic women and in both sub-groups than in controls. Plasma vitamin E and erythrocyte vitamin A levels were significantly lower in all diabetic women than in controls. Moreover, GPX and SOD activities were significantly reduced in all diabetic women, GPX in both sub-groups and SOD only in type 1 diabetes. CONCLUSIONS: The increased oxidative stress we demonstrated in pregnant women with previous type 1 diabetes or with GDM should be monitored by strictly controlling blood glucose during pregnancy with stringent recommendations and perhaps antioxidant supplementation.

Differential expression of nm23 genes in adult mouse dorsal root ganglia.

Nm23 has been identified as a gene family encoding different isoforms of nucleoside diphosphate kinase (NDPK). This protein is a key enzyme in nucleotide metabolism and has been shown to play important roles in various cellular functions. In the present study, we have investigated the expression of three isotypes in mouse dorsal root ganglia. In situ hybridization and reverse transcriptase-polymerase chain reaction analysis demonstrated high levels of nm23-M1, -M2, and -M3 mRNA expression in peripheral nervous tissue. Moreover, in situ hybridization also displayed a specific nuclear localization for nm23-M2 mRNA. Immunohistochemistry with light and electron microscopy on isoform-specific antibodies revealed a differential subcellular distribution of NDPK isoforms. Isoform A was mainly cytosolic, showing only partial association with organelles. In contrast, isoform B was also found in the nucleus, which is in agreement with its proposed role as a transcription factor. The results also indicate a preferential association of isoform C with endoplasmic reticulum and plasma membranes in neuronal cells. Furthermore, isoform C appeared to combine with other NDPK isoforms as demonstrated by double-labeling evidence by electron microscopy and might be responsible for binding NDPK oligomers to membranes. Thus, isoform C may be considered as a protein of importance for maintaining intracellular pools of GTP in the vicinity of membranes and, hence, for transmembrane signaling. The results indicate a high expression of NDPK isoforms, not only in the central but also in the peripheral nervous system. Their different subcellular compartmentalization suggests that they have isoform-specific roles in neuronal cell physiology.