scholarly journals Ωρίμανση ωοκυττάρων in vitro

2012 ◽  
Author(s):  
Ολυμπία Πικίου
Keyword(s):  
Protein Kinase ◽  
Tuberous Sclerosis ◽  
Adenosine Triphosphate ◽  
Tuberous Sclerosis Complex ◽  
Adenosine Monophosphate ◽  
Amp Activated Protein Kinase

Η μετφορμίνη, ένα παράγωγο της διγουανίδης, χρησιμοποιείται ως θεραπεία του σακχαρώδη διαβήτη τύπου 2 και στη θεραπεία του PCOS. Οι κύριες δράσεις της μετφορμίνης είναι η αναστολή της παραγωγής γλυκόζης από το ήπαρ και η μείωση της αντίστασης στην ινσουλίνη από περιφερικούς ιστούς, οδηγώντας σε αυξημένη πρόσληψη και χρήση της γλυκόζης από τους σκελετικούς μυς. Ο κύριος διαμεσολαβητής της δράσης της μετφορμίνης είναι η AMPK [AMP-activated protein kinase: πρωτεϊνική κινάση που ενεργοποιείται από την AMP (μονοφωσφορική αδενοσίνη)]. Η AMPK είναι ο κεντρικός αισθητήρας των επιπέδων ενέργειας στο κύτταρο, ο οποίος ανταποκρίνεται στην αύξηση του λόγου AMP/ATP (adenosine monophosphate/adenosine triphosphate: μονοφωσφορική/τριφωσφορική αδενοσίνη). Μελέτες σε ωοκύτταρα βοοειδών έχουν δείξει ότι η ενεργοποίηση της AMPK από τη μετφορμίνη σε υψηλές συγκεντρώσεις της τάξεως των mM ελέγχει την πυρηνική ωρίμανση. Το TSC2 (tuberous sclerosis complex 2: σύμπλεγμα οζώδους σκλήρυνσης 2) έχει αναγνωριστεί ως ο κατωφερής στόχος της AMPK. Σκοπός της παρούσης μελέτης ήταν η διερεύνηση της επίδρασης χαμηλών συγκεντρώσεων μετφορμίνης (1nM-10μΜ) (i) στη δημιουργία εμβρύων βοοειδών από συμπλέγματα ωοκυττάρου-ωοφόρου δίσκου, (ii) το ρυθμό διαίρεσης των εμβρύων και, (iii) την πιθανή ενεργοποίηση του TSC2 μέσω της AMPK. Τα συμπλέγματα ωοκυττάρου-ωοφόρου δίσκου ωρίμαζαν in vitro, γονιμοποιούνταν με αναβιωμένα σπερματοζωάρια ταύρου και τα ζυγωτά καλλιεργούνταν συνολικά για 72 ώρες μετά τη σπερματέγχυση. Η μετφορμίνη χορηγήθηκε σε όλα τα στάδια της παραγωγής των εμβρύων ή μόνο κατά το στάδιο της γονιμοποίησης. Προκειμένου να διερευνηθεί η παρουσία της TSC2 κατά τα πρώτα στάδια ανάπτυξης των εμβρύων και η πιθανή ενεργοποίηση του μορίου αυτού μέσω της AMPK πραγματοποιήθηκαν πειράματα ανοσοφθορισμού. Σύμφωνα με τα αποτελέσματα μας, η χορήγηση της μετφορμίνης είχε δοσο-εξαρτώμενη επίδραση στο ρυθμό διαίρεσης των εμβρύων. Συγκεκριμένα, παρουσία μετφορμίνης σε όλα τα στάδια της in vitro παραγωγής εμβρύων σε συγκέντρωση 1μΜ και 10μΜ ή μόνο στο στάδιο της in vitro γονιμοποίησης σε συγκέντρωση 0,1μΜ και 10μΜ, το ποσοστό των εμβρύων που έφτασαν στο στάδιο των ≥8-κυττάρων παρουσίασε στατιστικώς σημαντική μείωση, σε σχέση με αυτό της ομάδας ελέγχου. Η μείωση αυτή στο ποσοστό των εμβρύων ≥8-κυττάρων συνοδεύτηκε από αύξηση του ποσοστού των εμβρύων 2-κυττάρων. Η μετφορμίνη δεν είχε καμία επίδραση στο ποσοστό των ωοκυττάρων που εξελίχθηκαν σε έμβρυα. Σύμφωνα με τα αποτελέσματα μας, το TSC2 εκφράζεται κατά τα πρώτα στάδια ανάπτυξης των εμβρύων βοοειδών. Επιπλέον διαπιστώθηκε ότι, η χορήγηση 10μΜ μετφορμίνης είτε σε όλα τα στάδια της in vitro παραγωγής εμβρύων ή μόνο κατά το στάδιο της in vitro γονιμοποίησης είχε ως αποτέλεσμα την ενεργοποίηση του TSC2 μέσω της AMPK. Συγκεκριμένα διαπιστώθηκε ότι, τα επίπεδα του φωσφορυλιωμένου TSC2, μετά τη χορήγηση μετφορμίνης, αντιστοιχούν στην ολική ποσότητα TSC2 πρωτεΐνης στα κύτταρα γεγονός που προκύπτει τόσο από την αύξηση της PhosphoS1387-TSC2-ανοσοδραστικότητας όσο και από την αύξηση του λόγου PhosphoS1387-TSC2 : ολική TSC2 η οποία παρατηρήθηκε. Τα αποτελέσματα της παρούσης διατριβής υποδεικνύουν για πρώτη φορά ότι, η μετφορμίνη δεν έχει καμία επίδραση στο ποσοστό των ωοκυττάρων που εξελίσσονται σε έμβρυα και κατά συνέπεια δεν επηρεάζει την ωρίμανση των ωοκυττάρων όταν χορηγείται σε συγκεντρώσεις της τάξεως των μM. Εντούτοις, η μετφορμίνη σε αυτές τις συγκεντρώσεις έχει αρνητική δοσο-εξαρτώμενη επίδραση στο ρυθμό διαίρεσης των εμβρύων βοοειδών. Η δράση αυτή της μετφορμίνης στο ρυθμό διαίρεσης των εμβρύων είναι η ίδια είτε το φάρμακο χορηγείται καθ' όλη τη διάρκεια της in vitro παραγωγής των εμβρύων είτε μόνο κατά το στάδιο της in vitro γονιμοποίησης. Επιπλέον, δεδομένου ότι η μετφορμίνη είναι ενεργοποιητής της ΑΜΡΚ, τα αποτελέσματα μας σηματοδοτούν τη σπουδαιότητα της ρύθμισης της δραστηριότητας της ΑΜΡΚ κατά τα πρώτα στάδια ανάπτυξης των εμβρύων και δείχνουν ότι κάθε μεταβολή των επιπέδων δραστηριότητας του ενζύμου αυτού μπορεί να έχει αρνητική επίδραση στην ανάπτυξη των εμβρύων. Τέλος, από τα αποτελέσματα μας μπορεί να συναχθεί ότι το μοριακό μονοπάτι το οποίο εμπλέκεται στη μείωση του ρυθμού διαίρεσης των εμβρύων από τη μετφορμίνη περιλαμβάνει την ενεργοποίηση του TSC2 από την ΑΜΡΚ.

scholarly journals Role of AMP-activated protein kinase during postovulatory aging of mouse oocytes†

2020 ◽  
Vol 103 (3) ◽  
pp. 534-547
Author(s):  
Guang-Yi Sun ◽  
Shuai Gong ◽  
Qiao-Qiao Kong ◽  
Zhi-Bin Li ◽  
Jia Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Species Difference ◽  
Adenosine Monophosphate ◽  
Cytoplasmic Calcium ◽  
Ampk Activation ◽  
Mouse Oocytes ◽  
Compound C ◽  
Oocyte Aging ◽  
Amp Activated Protein Kinase

Abstract Studies suggested that postovulatory oocyte aging might be prevented by maintaining a high maturation-promoting factor (MPF) activity. Whether AMP-activated protein kinase (AMPK) plays any role in postovulatory oocyte aging is unknown. Furthermore, while activation of AMPK stimulates meiotic resumption in mouse oocytes, it inhibits meiotic resumption in pig and bovine oocytes. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies. This study showed that AMPK activation with metformin or 5-aminoimidazole- 4-carboxamide- 1-beta-d- ribofuranoside and inactivation with compound C significantly increased and decreased, respectively, the activation susceptibility (AS) and other aging parameters in aging mouse oocytes. While AMPK activity increased, MPF activity and cyclic adenosine monophosphate (cAMP) decreased significantly with time post ovulation. In vitro activation and inactivation of AMPK significantly decreased and increased the MPF activity, respectively. MPF upregulation with MG132 or downregulation with roscovitine completely abolished the effects of AMPK activation or inactivation on AS of aging oocytes, respectively. AMPK facilitated oocyte aging with increased reactive oxygen species (ROS) and cytoplasmic calcium. Furthermore, treatment with Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors significantly decreased AS and AMPK activation. Taken together, the results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

scholarly journals Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Jaspreet Singh ◽  
Hamid Suhail ◽  
Shailendra Giri
Keyword(s):  
Protein Kinase ◽  
Mitochondrial Dysfunction ◽  
Glial Cells ◽  
Adenosine Monophosphate ◽  
Proinflammatory Response ◽  
Inflammatory Protein ◽  
Lower Oxygen ◽  
Amp Activated Protein Kinase

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPKα1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice) mimics the human AMN phenotype and does not develop the cerebral inflammation characteristic of human ALD. In this study we document that AMPKα1 levelsin vivo(in brain cortex and spinal cord) andin vitroin Abcd1-KO mixed glial cells are similar to that of wild type mice. Deletion of AMPKα1 in the mixed glial cells of Abcd1-KO mice induced spontaneous mitochondrial dysfunction (lower oxygen consumption rate and ATP levels). Mitochondrial dysfunction in ALD patient-derived cells and in AMPKα1-deleted Abcd1-KO mice mixed glial cells was accompanied by lower levels of mitochondrial complex (1-V) subunits. More importantly, AMPKα1 deletion induced proinflammatory inducible nitric oxide synthase levels in the unstimulated Abcd1-KO mice mixed glial cells. Taken together, this study provides novel direct evidence for a causal role for AMPK loss in the development of mitochondrial dysfunction and proinflammatory response in X-ALD.

Activation of AMP-activated protein kinase may not be involved in AICAR- and metformin-mediated meiotic arrest in bovine denuded and cumulus-enclosed oocytes in vitro

Zygote ◽  
2010 ◽  
Vol 19 (2) ◽  
pp. 97-106 ◽  
Author(s):  
Sylvie Bilodeau-Goeseels ◽  
Paul L. Panich ◽  
John P. Kastelic
Keyword(s):  
Protein Kinase ◽  
Cumulus Cell ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Inhibitory Effect ◽  
Bovine Oocyte ◽  
Compound C ◽  
Oocyte Meiosis ◽  
Amp Activated Protein Kinase

SummaryThe adenosine monophosphate-activated protein kinase (AMPK) activators, 5′-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET), inhibit resumption of meiosis in bovine cumulus-enclosed oocytes (CEO) and denuded oocytes (DO). The objectives of this study were to: (1) examine the effects of AMPK inhibitors on bovine oocyte meiosis in vitro; and (2) determine if AICAR or MET activates oocyte and/or cumulus cell AMPK. The AMPK inhibitor compound C (CC; 0.5, 1, 5, and 10 μM) did not reverse the inhibitory effects of AICAR (1 mM) and MET (2 mM) on bovine oocyte meiosis. Additionally, CC (5 and 10 μM) inhibited meiosis (p < 0.05) in CEO and DO cultured for 7 h. Okadaic acid (1 μM) reversed the inhibitory effect of MET (2 mM) and CC (5 μM; p < 0.05) but not of AICAR (1 mM). Phosphorylation of the alpha subunit of AMPK on Thr172 is required for activation. Based on western blot analysis, AICAR, MET and CC did not affect Thr172 phosphorylation levels in DO and oocytes from complexes (p > 0.05). In cumulus cells, Thr172 phosphorylation decreased after 3 h of culture (p < 0.05), regardless of the presence of AMPK modulators in the culture medium. Higher concentrations of AICAR (2 mM) and MET (10 mM) did not affect Thr172 phosphorylation, but phosphorylation on Ser79 of ACC, a substrate of AMPK, was increased in response to MET (p < 0.05). In conclusion, we inferred that the inhibitory effect of AICAR and MET on bovine oocyte meiosis was probably not mediated through activation of AMPK. Moreover, these compounds probably inhibited meiosis through different pathways.

Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK

2001 ◽  
Vol 355 (2) ◽  
pp. 297-305 ◽  
Author(s):  
Diana L. LEFEBVRE ◽  
Yahong BAI ◽  
Nazanin SHAHMOLKY ◽  
Monika SHARMA ◽  
Raymond POON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cellular Response ◽  
Catalytic Domain ◽  
Metabolic Stress ◽  
Glucose Deprivation ◽  
Protein Band ◽  
Northern Blotting ◽  
Significant Similarity ◽  
Amp Activated Protein Kinase

Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress.

scholarly journals Interleukin-6 Increases Insulin-Stimulated Glucose Disposal in Humans and Glucose Uptake and Fatty Acid Oxidation In Vitro via AMP-Activated Protein Kinase

Diabetes ◽  
2006 ◽  
Vol 55 (10) ◽  
pp. 2688-2697 ◽  
Author(s):  
A. L. Carey ◽  
G. R. Steinberg ◽  
S. L. Macaulay ◽  
W. G. Thomas ◽  
A. G. Holmes ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Fatty Acid Oxidation ◽  
Interleukin 6 ◽  
Glucose Disposal ◽  
Acid Oxidation ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

2001 ◽  
Vol 280 (5) ◽  
pp. E677-E684 ◽  
Author(s):  
Nicolas Musi ◽  
Tatsuya Hayashi ◽  
Nobuharu Fujii ◽  
Michael F. Hirshman ◽  
Lee A. Witters ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Muscle Contractions ◽  
Treadmill Running ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-β-d-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPKα1 and AMPKα2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-β-d-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3- O-methyl-d-glucose (3-MG) uptake. There were dose-dependent increases in AMPKα2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPKα1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPKα2 activity and 3-MG uptake but had little effect on AMPKα1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPKα1 and -α2 activity and 3-MG uptake. Although the AMPKα1 and -α2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPKα2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.

scholarly journals Induction and properties of cytoplasmic factor(s) which enhance nuclear nonhistone protein phosphorylation in lymphocytes stimulated by anti-Ig.

1977 ◽  
Vol 146 (3) ◽  
pp. 653-664 ◽  
Author(s):  
Y Nishizawa ◽  
T Kishimoto ◽  
H Kikutani ◽  
Y Yamamura
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Adenosine Triphosphate ◽  
Active Substance ◽  
Nuclear Proteins ◽  
Concomitant Increase ◽  
Cytoplasmic Factor ◽  
Nonhistone Protein ◽  
Rabbit Lymphocytes

An increased in vitro phosphorylation of nonhistone nuclear proteins (NHP) was observed in the nuclei isolated from rabbit lymphocytes which had been stimulated with anti-Ig for 4 h. No concomitant increase of phosphorylation in histones or 0.14 M NaCl-soluble proteins was observed. The increase of in vitro phosphorylation of NHP was also observed in the nuclei isolated from nonstimulated cells when these nuclei were preincubated for 2 h with cell-free extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was maximally induced when lymphocytes were stimulated with anti-Ig for 2 h. The induction of an increased phosphorylation of NHP in nonstimulated nuclei with the cell-free extracts was not due to decrease of the adenosine triphosphate pool in the extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was not NHP-protein kinase itself, but it probably activated NHP-protein kinase in quiescent nuclei. The active substance was nondialyzable and probably protein. It was resistant against heating at 56 degrees C for 30 min, but the activity was completely destroyed by heating at 90 degrees C for 30 min. The active substance may be responsible for the transduction of the membrane-mediated signals given through Ig receptors to nuclei.

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