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1.
Mol Cell Endocrinol ; 476: 57-69, 2018 11 15.
Article in English | MEDLINE | ID: mdl-29715496

ABSTRACT

The effect of insulin priming on Il-10 expression, regulation of inflammatory cytokines and participation of intra-cellular signalling events, primarily ERK1/2 and PI3K/Akt, has been investigated in high glucose (HG) and/or lipopolysaccharide (LPS)-induced murine macrophages. Our results demonstrate that congruent with sharp increase in ERK1/2 and CREB phosphorylation, insulin stimulation in vitro promotes significant increase in Il-10 expression in mouse peritoneal macrophage and RAW 264.7 cells, both positive for anti-IRß. Pharmacological inhibition of MEK/MAPK, but not PI3K/Akt cascade, abrogates CREB phosphorylation and Il-10 synthesis indicating functional relevance of insulin action. Conversely, priming with PI3K inhibitor wortmannin prevents insulin attenuation of HG- and/or LPS-induced p38 MAPK and NF-κB activation, Tnf-α, Il-1ß expression as well as NO production. Congruent with reduced Il-10 expression, MEK inhibition abrogates insulin action allowing significant increase in Tlr4 expression and LPS response indicating insulin-induced Il-10 might have pivotal influence in regulation of chronic as well as acute inflammatory response.


Subject(s)
Insulin/metabolism , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/metabolism , Signal Transduction , Animals , Anti-Inflammatory Agents/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation/drug effects , Glucose/toxicity , Inflammation/pathology , Inflammation Mediators/metabolism , Insulin/pharmacology , Macrophages, Peritoneal/drug effects , Male , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , RAW 264.7 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Signal Transduction/drug effects
2.
Zygote ; 26(1): 62-75, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29229010

ABSTRACT

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2-M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30-120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.


Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Oocytes/physiology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Cyclic AMP/metabolism , Enzyme Inhibitors/pharmacology , Female , G2 Phase/physiology , In Vitro Oocyte Maturation Techniques , MAP Kinase Kinase 1/metabolism , Meiosis/physiology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Pregnenes/pharmacology , Signal Transduction/drug effects , Zebrafish , Zebrafish Proteins/metabolism
3.
Gen Comp Endocrinol ; 239: 21-31, 2016 12 01.
Article in English | MEDLINE | ID: mdl-26853486

ABSTRACT

Present study reports differential expression of the two insulin receptor (IR) subtypes in zebrafish ovary at various stages of follicular growth and potential involvement of IR in insulin-induced oocyte maturation. The results showed that mRNA expression for IR subtypes, insra and insrb, exhibited higher levels in mid-vitellogenic (MV) and full-grown (FG) rather than pre-vitellogenic (PV) oocytes. Interestingly, compared to the levels in denuded oocytes, mRNAs for both insra and insrb were expressed at much higher level in the follicle layer harvested from FG oocytes. Immunoprecipitation using IRß antibody could detect a protein band of desired size (∼95kDa) in FG oocyte lysates. Further, IRß immunoreactivity was detected in ovarian tissue sections, especially at the follicle layer and oocyte membrane of MV and FG, but not PV stage oocytes. While hCG (10IU/ml) stimulation was without effect, priming with insulin (5µM) could promote oocyte maturation of MV oocytes in a manner sensitive to de novo protein and steroid biosynthesis. Compared to hCG, in insulin pre-incubated MV oocytes, stimulation with maturation inducing steroid (MIS), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) elicited higher maturational response. Potential involvement of insulin-mediated action on acquisition of maturational competence and regulation of oocyte maturation was further manifested through up regulation of 20ß-hydroxysteroid dehydrogenase (20ß-hsd), MIS receptor (mPRα), insulin-like growth factor 3 (igf3) and IGF1 receptor (igf1rb), but not cyp19a expression in MV oocytes. Moreover, priming with anti-IRß attenuated insulin action on meiotic G2-M1 transition indicating the specificity of insulin action and physiological relevance of IR in zebrafish ovary.


Subject(s)
Insulin/pharmacology , Oogenesis/drug effects , Ovary/drug effects , Ovary/metabolism , Receptor, Insulin/genetics , Zebrafish/genetics , Animals , Female , Insulin/metabolism , Oocytes/drug effects , Oocytes/metabolism , Oogenesis/genetics , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , Ovary/physiology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptor, Insulin/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Somatomedins/metabolism , Zebrafish/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
4.
Zygote ; 24(2): 181-94, 2016 Apr.
Article in English | MEDLINE | ID: mdl-25707854

ABSTRACT

Regulation of insulin-mediated resumption of meiotic maturation in catfish oocytes was investigated. Insulin stimulation of post-vitellogenic oocytes promotes the synthesis of cyclin B, histone H1 kinase activation and a germinal vesicle breakdown (GVBD) response in a dose-dependent and duration-dependent manner. The PI3K inhibitor wortmannin abrogates recombinant human (rh)-insulin action on histone H1 kinase activation and meiotic G2-M1 transition in denuded and follicle-enclosed oocytes in vitro. While the translational inhibitor cycloheximide attenuates rh-insulin action, priming with transcriptional blocker actinomycin D prevents insulin-stimulated maturational response appreciably, albeit in low amounts. Compared with rh-insulin, human chorionic gonadotrophin (hCG) stimulation of follicle-enclosed oocytes in vitro triggers a sharp increase in 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) secreted in the incubation medium at 12 h. Interestingly, the insulin, but not the hCG-induced, maturational response shows less susceptibility to steroidogenesis inhibitors, trilostane or dl-aminoglutethimide. In addition, priming with phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) or cell-permeable dbcAMP or adenylyl cyclase activator forskolin reverses the action of insulin on meiotic G2-M1 transition. Conversely, the adenylyl cyclase inhibitor, SQ 22536, or PKA inhibitor H89 promotes the resumption of meiosis alone and further potentiates the GVBD response in the presence of rh-insulin. Furthermore, insulin-mediated meiotic maturation involves the down-regulation of endogenous protein kinase A (PKA) activity in a manner sensitive to PI3K activation, suggesting potential involvement of a cross-talk between cAMP/PKA and insulin-mediated signalling cascade in catfish oocytes in vitro. Taken together, these results suggest that rh-insulin regulation of the maturational response in C. batrachus oocytes involves down-regulation of PKA, synthesis of cyclin B, and histone H1 kinase activation and demonstrates reduced sensitivity to steroidogenesis and transcriptional inhibition.


Subject(s)
Cell Cycle/drug effects , Insulin/pharmacology , Meiosis/drug effects , Oocytes/drug effects , Animals , Catfishes , Cells, Cultured , Chorionic Gonadotropin/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclin B/metabolism , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Female , Hydroxyprogesterones/metabolism , Immunoblotting , Insulin/genetics , Oocytes/cytology , Oocytes/physiology , Ovarian Follicle/cytology , Protein Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Proteins/pharmacology , Time Factors
5.
Mol Cell Endocrinol ; 393(1-2): 109-19, 2014 Aug 05.
Article in English | MEDLINE | ID: mdl-24956082

ABSTRACT

High intra-cellular cyclic nucleotide (cAMP) ensures prophase-I arrest and prevent steroid-induced meiotic G2-M1 transition in full-grown oocytes; however, relatively less information is available for cAMP regulation of growth factor-stimulated signalling events in the oocyte model. Here using zebrafish oocytes, we show that priming with dibutyryl cAMP (dbcAMP) or cAMP modulators, e.g. adenylate cyclase activator, forskolin or phosphodiesterase inhibitors (IBMX/cilostamide) block insulin action on germinal vesicle breakdown (GVBD) and histone H1 kinase activation. Though high cAMP priming attenuates insulin-induced MAPK3/1 (ERK1/2) phosphorylation (activation), following 2h of insulin stimulation it fails to block MAPK activation and GVBD. Further, insulin stimulation promotes down regulation of phospho-PKAc (inactivation) and PKA inhibition by H89/PKI-(6-22)-amide overcomes negative regulation by cAMP and induces GVBD and MAPK activation. Moreover, MEK1/2 inhibitor U0126 has no influence on H89-induced GVBD; however, it delays GVBD response in insulin-stimulated oocytes. MAPK activation by okadaic acid (OA) promotes GVBD; however, high dbcAMP abrogates OA action suggesting cross-talk between cAMP/PKA and MAPK-mediated signalling pathways may contribute significantly in maturing zebrafish oocyte.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Meiosis , Mitogen-Activated Protein Kinase 3/chemistry , Oocytes/enzymology , Animals , Cells, Cultured , Electrophoresis , Immunoblotting , Insulin/pharmacology , Mitogen-Activated Protein Kinase 3/metabolism , Oocytes/drug effects , Oocytes/metabolism , Zebrafish
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