Stimulation of Lactate Production in Human Granulosa Cells by Metformin and Potential Involvement of Adenosine 5′ Monophosphate-Activated Protein Kinase

Abstract Context: Production of 3-carbon units (as lactate) by granulosa cells (GCs) is important in follicular and oocyte development and may be modulated by metformin. Objective: The aim of the study was to examine the action of metformin on GC lactate production and potential mediation via AMP-activated protein kinase (AMPK). Design: GCs were prepared from follicular aspirates. After exposure to metformin and other potential modulators of AMPK in culture, aspects of cellular function were examined. Setting: The study was conducted in a private fertility clinic/university academic center. Patients: Women undergoing routine in vitro fertilization participated in the study. Interventions: All agents were added in culture. Main Outcome Measures: Lactate output of GCs was measured. Cell extracts were prepared after culture, and phosphorylated forms of AMPK and acetyl CoA carboxylase (ACC) were assayed using Western analysis. Results: Metformin led to a rapid increase in lactate production by GCs [minimum effective dose, 250 μm; maximum dose studied, 1 mm (1.22-fold; P < 0.01)]. This dose range of metformin was similar to that required for stimulation of phospho-AMPK in GCs [minimum effective dose, 250 μm; maximum effect, 500 μm (2.01-fold; P < 0.001)]. Increasing phospho-ACC, as a representative downstream target regulated by AMPK, was apparent over a lower range (minimum effective dose, 31 μm; maximum effect, 250 μm; P < 0.001). A level of metformin (125 μm) insufficient for the stimulation of lactate output when used alone potentiated the effects of suboptimal doses of insulin on lactate production. Adiponectin (2.5 μg/ml) had a small but significant effect on lactate output. Conclusions: Metformin activates AMPK in GCs, stimulating lactate production and increasing phospho-ACC. Metformin also enhances the action of suboptimal insulin concentrations to stimulate lactate production.

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CXADR-like membrane protein (CLMP) in the rat ovary: stimulation by human chorionic gonadotrophin during the periovulatory period

Reproduction Fertility and Development ◽

10.1071/rd14201 ◽

2016 ◽

Vol 28 (6) ◽

pp. 742

Author(s):

Feixue Li ◽

Xiaoping Miao ◽

Yonglong Chen ◽

Thomas E. Curry

Keyword(s):

Cell Adhesion ◽

Protein Kinase ◽

Membrane Protein ◽

Mrna Expression ◽

Granulosa Cells ◽

Receptor Activation ◽

Chorionic Gonadotrophin ◽

Human Chorionic Gonadotrophin ◽

Rat Ovary ◽

Stimulation Of

CXADR-like membrane protein (CLMP) is a novel cell–cell adhesion molecule. The present study investigated the spatiotemporal expression pattern of CLMP and its regulation in the rat ovary during the periovulatory period. Real-time polymerase chain reaction analysis revealed that Clmp mRNA was rapidly stimulated in intact ovaries by 4 h after human chorionic gonadotrophin (hCG) treatment. In situ hybridisation analysis demonstrated that Clmp mRNA expression was stimulated in theca cells at 4 h after hCG and remained elevated until 12 h. Clmp mRNA was also upregulated in granulosa cells and was present in forming corpora lutea. Our data indicate that the protein kinase A but not the protein kinase C pathway regulates the expression of Clmp mRNA in granulosa cells. Phosphatidylinositol 3 kinase and p38 kinase are also involved in regulating Clmp mRNA expression. The stimulation of Clmp mRNA by hCG requires new protein synthesis. Furthermore, inhibition of epidermal growth factor receptor activation significantly inhibited Clmp mRNA expression, whereas inhibition of prostaglandin synthesis or progesterone action had no effect. The stimulation of CLMP in the rat ovary may be important in cell adhesion events during ovulation and luteal formation such as maintaining the structure and communication of ovarian follicular and luteal cells.

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Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.577 ◽

1996 ◽

Vol 16 (2) ◽

pp. 577-583 ◽

Cited By ~ 53

Author(s):

K H Holt ◽

B G Kasson ◽

J E Pessin

Keyword(s):

Protein Kinase ◽

Mitogen Activated Protein Kinase ◽

Insulin Stimulation ◽

Anion Exchange Column ◽

Cell Extracts ◽

Erk Phosphorylation ◽

Extracellular Signal ◽

Stimulation Of

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

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Regulation of rat Sertoli cell function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B pathway

Journal of Endocrinology ◽

10.1677/joe.0.1740195 ◽

2002 ◽

Vol 174 (2) ◽

pp. 195-204 ◽

Cited By ~ 81

Author(s):

SB Meroni ◽

MF Riera ◽

EH Pellizzari ◽

SB Cigorraga

Keyword(s):

Protein Kinase ◽

Sertoli Cell ◽

Mechanism Of Action ◽

Sertoli Cells ◽

Cell Function ◽

Protein Kinase B ◽

Lactate Production ◽

Phosphatidylinositol 3 Kinase ◽

Stimulation Of

The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway via activation of a G protein. Recently, links between cAMP, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) signaling pathways in thyroid and granulosa cells have been observed. The aim of this study was to investigate the possible role of the PI3K/PKB pathway in FSH regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. An increase in phosphorylated PKB (P-PKB) levels in response to FSH and dibutyryl-cAMP was observed. These increments in P-PKB levels were not observed in the presence of two PI3K inhibitors, wortmannin and Ly 294002. Inhibition of protein kinase A (PKA) by H89 did not decrease FSH stimulation of P-PKB levels. Taken together, these results indicate that FSH increases P-PKB levels in a PI3K-dependent and PKA-independent manner in rat Sertoli cells. In addition, wortmannin partially inhibited the ability of FSH to stimulate two well-known parameters of Sertoli cell function - transferrin secretion and lactate production - at doses equal to or lower than 0.1 microM. Related to lactate production, a decrease in FSH stimulation of lactate dehydrogenase activity and of basal and FSH-stimulated glucose uptake was observed in the presence of wortmannin. These metabolic changes were in most cases accompanied by changes in the levels of P-PKB. Altogether, these results suggest a meaningful role of the PI3K/PKB pathway in the mechanism of action of FSH in rat Sertoli cells.

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Participation of phosphatidyl inositol 3-kinase/protein kinase B and ERK1/2 pathways in interleukin-1β stimulation of lactate production in Sertoli cells

Reproduction ◽

10.1530/rep.1.01091 ◽

2007 ◽

Vol 133 (4) ◽

pp. 763-773 ◽

Cited By ~ 12

Author(s):

María Fernanda Riera ◽

María Noel Galardo ◽

Eliana Herminia Pellizzari ◽

Silvina Beatriz Meroni ◽

Selva Beatriz Cigorraga

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Sertoli Cell ◽

Sertoli Cells ◽

Protein Kinase B ◽

Lactate Production ◽

Phosphatidyl Inositol ◽

Interleukin 1Β ◽

Mrna Levels ◽

Stimulation Of

Interleukin-1β (IL1β ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1β regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1β showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1β . PD and W, but not R, decreased IL1β-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1β was also analyzed. It was observed that W decreased IL1β-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1β , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1β stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1β utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1β utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.

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Activation of phospholipase D in rat type II pneumocytes by ATP and other surfactant secretagogues

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.2.l133 ◽

1993 ◽

Vol 264 (2) ◽

pp. L133-L140 ◽

Cited By ~ 3

Author(s):

S. A. Rooney ◽

L. I. Gobran

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase D ◽

Primary Cultures ◽

Maximum Effect ◽

Type Ii ◽

Kinase C ◽

Atp Concentration ◽

Surfactant Secretion ◽

Stimulation Of

Surfactant phospholipid secretion can be stimulated by a variety of agonists acting via a number of signal-transduction mechanisms. To determine whether phospholipase D has a role in surfactant secretion, we examined phosphatidylethanol formation in response to surfactant secretagogues in primary cultures of rat type II cells. Phosphatidylethanol formation was stimulated by ATP, 12-O-tetradecanoylphorbol-13 acetate (TPA), and dioctanoylglycerol, surfactant secretagogues that also activate protein kinase C. Surfactant secretagogues that act via other signaling mechanisms had no effect on phosphatidylethanol formation. The effect of ATP on phosphatidylethanol formation was dependent on time, with the maximum stimulation being achieved in approximately 10 min. It was also dependent on ATP concentration. The ATP concentration eliciting 50% of the maximum effect (EC50) was 2.45 x 10(-6) M. This was similar to the EC50 reported for ATP stimulation of surfactant secretion. ATP analogues also stimulated phosphatidylethanol formation with a potency order generally similar to that reported for surfactant secretion. The effects of ATP, TPA, and dioctanoylglycerol were antagonized by protein kinase C inhibitors. We speculate that activation of protein kinase C either directly by TPA and dioctanoylglycerol or indirectly subsequent to phosphoinositide-specific phospholipase C activation by ATP leads to initial stimulation of surfactant secretion as well as activation of phospholipase D. The action of phospholipase D on cellular phospholipids then leads to further generation of diacylglycerols, continued activation of protein kinase C, and sustained surfactant secretion.

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Follicle-Stimulating Hormone and Insulin-Like Growth Factor I Synergistically Induce Up-Regulation of Cartilage Link Protein (Crtl1) via Activation of Phosphatidylinositol-Dependent Kinase/Akt in Rat Granulosa Cells

Endocrinology ◽

10.1210/en.2002-220900 ◽

2003 ◽

Vol 144 (3) ◽

pp. 793-801 ◽

Cited By ~ 20

Author(s):

Guang Wei Sun ◽

Hiroshi Kobayashi ◽

Mika Suzuki ◽

Naohiro Kanayama ◽

Toshihiko Terao

Keyword(s):

Protein Kinase ◽

Granulosa Cells ◽

Kinase Inhibitor ◽

Primary Cultures ◽

Cumulus Cell ◽

Mitogen Activated Protein Kinase ◽

Link Protein ◽

Cell Extracts ◽

Igf I ◽

Mitogen Activated Protein

FSH and IGF-I are both important determinants of follicle development and the process of cumulus cell-oocyte complex expansion. FSH stimulates the phosphorylation of Akt by mechanisms involving phosphatidylinositol 3-kinase (PI3-K), a pattern of response mimicking that of IGF-I. Cartilage link protein (Crtl1) is confined to the cartilaginous lineage and is assembled into a macroaggregate complex essential for hyaluronan-rich matrix stabilization. The present studies were performed to determine the actions of FSH and IGF-I on Crtl1 production in rat granulosa cells. Primary cultures of granulosa cells were prepared from 24-d-old rats. After treatments, cell extracts and media were prepared, and the Crtl1 level was determined by immunoblotting analysis using anti-Crtl1 antibodies. Here we showed that 1) treatment with FSH (≥25 ng/ml) or IGF-I (≥25 ng/ml) for 4 h increased Crtl1 production; 2) maximal stimulatory effects of FSH or IGF-I were observed at 100 or 50 ng/ml, respectively; 3) FSH caused a concentration-dependent increase in IGF-I-induced Crtl1 production and vice versa; 4) FSH and IGF-I also up-regulate the expression of Crtl1 mRNA; 5) FSH- and IGF-I-dependent Crtl1 production were abrogated by PI3-K inhibitors (LY294002 and wortmannin), and inhibition of Crtl1 production by p38 mitogen-activated protein kinase inhibitor (SB202190) was partial (∼30%), suggesting that PI3-K and, to a lesser extent, p38 mitogen-activated protein kinase are critical for the response. Our study represents the first report that FSH amplifies IGF-I-mediated Crtl1 production, possibly via PI3-K-Akt signaling cascades in rat granulosa cells.

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Assessment of the roles of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase/protein kinase B pathways in the basic fibroblast growth factor regulation of Sertoli cell function

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310279 ◽

2003 ◽

Vol 31 (2) ◽

pp. 279-289 ◽

Cited By ~ 16

Author(s):

MF Riera ◽

SB Meroni ◽

EH Pellizzari ◽

SB Cigorraga

Keyword(s):

Protein Kinase ◽

Sertoli Cell ◽

Kinase Inhibitors ◽

Cell Function ◽

Lactate Production ◽

Phosphatidyl Inositol ◽

Mitogen Activated Protein Kinase ◽

Ldh Activity ◽

Mitogen Activated Protein ◽

Stimulation Of

Basic fibroblast growth factor (bFGF) belongs to the large set of intratesticular regulators that provide the fine tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to determine the participation of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase/protein kinase B (PI3K/PKB) pathways in bFGF regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. Stimulation of the cultures with bFGF showed a time-dependent increment in phosphorylated MAPK and PKB levels that reached maximal values in 5-min incubations. MAPK kinase inhibitors U0126 (U) and PD98059 (PD) and a PI3K inhibitor wortmannin (W) were able to block the stimulatory effects of bFGF on phosphorylated MAPK and PKB levels respectively. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of two well-known Sertoli cell-differentiated functions, lactate and transferrin production, was next explored. As for lactate production, PD and W did not modify the ability of bFGF to stimulate lactate production. However, a combination of PD and W partially impaired the increase in lactate production elicited by bFGF. The participation of MAPK- and PI3K/PKB-signaling pathways in the regulation by bFGF of glucose uptake and lactate dehydrogenase (LDH) activity was also analysed. In this respect, it was observed that W markedly decreased basal and bFGF-stimulated glucose uptake and that U and PD did not modify it. On the other hand, U and PD decreased the stimulation of LDH activity by bFGF whereas W did not modify it. As for transferrin production, while both MAPK kinase inhibitors partially decreased the ability of bFGF to stimulate transferrin secretion, the PI3K inhibitor did not modify it. In summary, the results demonstrated that bFGF stimulates MAPK- and PI3K/PKB-dependent pathways in rat Sertoli cells. Moreover, these results showed that while bFGF utilizes the MAPK pathway to regulate transferrin production and LDH activity, it uses the PI3K/PKB pathway to regulate glucose transport into the cell.

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Follicle Stimulating Hormone-Regulated Expression of Serum/Glucocorticoid-Inducible Kinase in Rat Ovarian Granulosa Cells: A Functional Role for the Sp1 Family in Promoter Activity

Molecular Endocrinology ◽

10.1210/mend.11.13.0033 ◽

1997 ◽

Vol 11 (13) ◽

pp. 1934-1949 ◽

Cited By ~ 51

Author(s):

Tamara N. Alliston ◽

Anita C. Maiyar ◽

Patricia Buse ◽

Gary L. Firestone ◽

JoAnne S. Richards

Keyword(s):

Protein Kinase ◽

Granulosa Cell ◽

Granulosa Cells ◽

Binding Activity ◽

Mrna Levels ◽

Protein Levels ◽

Dna Binding Activity ◽

Cell Extracts ◽

Cell Progression ◽

Inducible Protein

Abstract Recently, a family of novel, serine/threonine protein kinases has been identified. One of these transcriptionally inducible, immediate-early genes encodes serum/glucocorticoid inducible-protein kinase, sgk. By in situ hybridization, we show that sgk expression in the rat ovary is selectively localized to granulosa cells. In culture, FSH or forskolin, activators of the protein kinase A (PKA) pathway, rapidly (2 h) and transiently increased sgk mRNA levels in undifferentiated granulosa cells. Sgk mRNA exhibited a biphasic expression pattern, with maximal levels observed at 48 h of FSH/forskolin as granulosa cells differentiate to the preovulatory phenotype. Deletion analyses using sgk promoter-reporter constructs (−4.0 kb to −35 bp) identified a region between −63 and− 43 bp that mediated FSH and forskolin-responsive transcription in undifferentiated and differentiated granulosa cells. This G/C-rich region 1) conferred both basal and inducible transcription to the minimal −35 sgk promoter chloramphenicol acetyltransferase reporter construct, 2) specifically bound Sp1 and Sp3 present in granulosa cell extracts, and 3) bound recombinant Sp1. Mutation of 2 bp in this region not only prevented Sp1 and Sp3 binding, but also abolished the PKA-mediated transactivation observed when using the wild type construct. Sp1 and Sp3 DNA-binding activity and protein levels did not change significantly during sgk induction. Collectively, these data indicate that Sp1/Sp3 transactivation of the sgk promoter likely involves regulated, phosphorylation-dependent interaction with other factors. Thus the novel, biphasic induction of sgk that correlates with granulosa cell progression from proliferation to differentiation appears to involve sequential, coordinated actions of FSH, PKA, and transcription factors, including Sp1 and Sp3.

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Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.5.c1605 ◽

1997 ◽

Vol 273 (5) ◽

pp. C1605-C1612 ◽

Cited By ~ 8

Author(s):

Chris R. J. Kennedy ◽

Richard L. Hébert ◽

Minh T. Do ◽

Pierre R. Proulx

Keyword(s):

Arachidonic Acid ◽

Protein Kinase C ◽

Protein Kinase ◽

Kinase Inhibitor ◽

Active Form ◽

Mitogen Activated Protein Kinase ◽

Cell Extracts ◽

Kinase C ◽

A 23187 ◽

Stimulation Of

Bradykinin (BK)-induced release of arachidonic acid (AA) from Madin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol 12-myristate 13-acetate (PMA) caused a synergistic increase in BK- and A-23187-induced release of AA but alone had no effect on this release. Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS) abolished the synergistic effects of PMA but did not affect AA release caused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMA resulted in a reduction of AA release induced by BK or A-23187 addition, which corresponded to a decrease in cytoplasmic phospholipase A2(cPLA2) activity as measured in cell extracts. Although Western blotting revealed no differences in cPLA2 expression as a result of PMA treatment, phosphorylation of the enzyme, as assessed by phosphoserine content, was significantly reduced in PKC-depleted cells. These results imply that, with PKC downregulation, subsequent BK stimulation results in a Ca2+-dependent translocation of a less phosphorylated, less active form of cPLA2. Any stimulation of PKC by BK addition did not appear as a significant event in onset reponses leading to AA release. On the other hand, inhibition of the mitogen-activated protein kinase (MAPK) cascade with the MAPK kinase inhibitor, PD-98059, significantly decreased BK-induced release of AA, a finding that, with our other results, points to the existence of a PKC-independent route for stimulation of MAPK and the propagation of onset responses.

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Effect of a GnRH analogue on rat granulosa cell lactate production in vitro

Acta Endocrinologica ◽

10.1530/acta.0.1050112 ◽

1984 ◽

Vol 105 (1) ◽

pp. 112-118 ◽

Cited By ~ 2

Author(s):

Håkan Billig ◽

C. S. Sheela Rani ◽

Carl Ekholm ◽

Claes Magnusson ◽

Torbjörn Hillensjö

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Culture Medium ◽

Cumulus Cell ◽

Lactate Production ◽

Gnrh Analogue ◽

Minimal Essential Medium ◽

Complex Culture ◽

Stimulation Of

Abstract. The effect of a GnRH analogue ((D-Ala6, des-Gly10-NH2)-GnRH-ethylamide,GnRHa) on granulosa and cumulus cell glycolysis in presence or absence of FSH was studied. Cumulus complexes and granulosa cells from PMSG-treated rats were cultured in Eagle's minimal essential medium (MEM) for a period of 72 h. Media were changed at 24 and 48 h and lactate content was assayed by fluorimetry. GnRHa alone stimulated lactate production in granulosa cells. GnRH combined with FSH increased lactate production in granulosa cells during the 0–24 h period and decreased it during the 48–72 h period as compared to FSH alone. GnRHa did not stimulate lactate production in cumulus complexes during 72 h culture in MEM, while FSH did. In a less complex culture medium, BMOC, GnRHa caused a small increase in lactate production and slightly enhanced the FSH effect. In conclusion, GnRHa has a direct stimulatory effect on granulosa cell glycolysis. GnRHa also modulates the FSH stimulation of granulosa cells biphasically, i.e. early enhancement (0–24 h) and late inhibition (48–72 h). GnRHa has no consistent direct effects on cumulus cell glycolysis.

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