Activating AMPK by targeting the ADaM site in β2 complexes induces skeletal muscle glucose uptake and thus lowers blood glucose.
The potential of AMPK activation for treatment of T2DM was reinforced by findings that it was activated by metformin (5). Metformin is the frontline drug treatment in T2DM, prescribed to more than 150 million patients worldwide. It activates AMPK by inhibiting mitochondrial ATP synthesis, thus increasing cellular AMP and ADP concentrations.
http://science.sciencemag.org/content/357/6350/455.full
//////////////AMPK exists as complexes comprising three subunits—a catalytic α subunit and regulatory β and γ subunits, with multiple isoforms (α1, α2, β1, β2, γ1, γ2, and γ3)—giving rise to 12 possible complexes that have tissue-specific expression patterns////////
The mammalian 5-prime-AMP-activated protein kinase (AMPK) appears to play a role in protecting cells from stresses that
cause ATP depletion by switching off ATP-consuming biosynthetic pathways. AMPK is a heterotrimeric protein composed
of 1 alpha subunit, 1 beta subunit (e. g., PRKAB1; 602740), and 1 gamma subunit (e. g., PRKAG1; 602742). The catalytic
alpha subunit requires phosphorylation for full activity Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. Mo JS et al. (2015) YAP (Yes-associated protein) is a transcription co-activator in the Hippo tumour suppressor pathway and controls cell growth, tissue homeostasis and organ size. YAP is inhibited by the kinase Lats, which phosphorylates YAP to induce its cytoplasmic localization and proteasomal degradation. YAP induces gene expression by binding to the TEAD family transcription factors. Dysregulation of the Hippo-YAP pathway is frequently observed in human cancers. Here we show that cellular energy stress induces YAP phosphorylation, in part due to AMPK-dependent Lats activation, thereby inhibiting YAP activity. Moreover, AMPK directly phosphorylates YAP Ser 94, a residue essential for the interaction with TEAD, thus disrupting the YAP-TEAD interaction. AMPK-induced YAP inhibition can suppress oncogenic transformation of Lats-null cells with high YAP activity. Our study establishes a molecular mechanism and functional significance of AMPK in linking cellular energy status to the Hippo-YAP pathway.////////////////// AMPK modulates Hippo pathway activity to regulate energy homeostasis. Wang W et al. (2015) The Hippo pathway was discovered as a conserved tumour suppressor pathway restricting cell proliferation and apoptosis. However, the upstream signals that regulate the Hippo pathway in the context of organ size control and cancer prevention are largely unknown. Here, we report that glucose, the ubiquitous energy source used for ATP generation, regulates the Hippo pathway downstream effector YAP. We show that both the Hippo pathway and AMP-activated protein kinase (AMPK) were activated during glucose starvation, resulting in phosphorylation of YAP and contributing to its inactivation. We also identified glucose-transporter 3 (GLUT3) as a YAP-regulated gene involved in glucose metabolism. Together, these results demonstrate that glucose-mediated energy homeostasis is an upstream event involved in regulation of the Hippo pathway and, potentially, an oncogenic function of YAP in promoting glycolysis, thereby providing an exciting link between glucose metabolism and the Hippo pathway in tissue maintenance and cancer prevention.//////////////////
NCBI Summary:
The protein encoded by this gene belongs to the ser/thr protein kinase family. It is the catalytic subunit of the 5'-prime-AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensor conserved in all eukaryotic cells. The kinase activity of AMPK is activated by the stimuli that increase the cellular AMP/ATP ratio. AMPK regulates the activities of a number of key metabolic enzymes through phosphorylation. It protects cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. Alternatively spliced transcript variants encoding distinct isoforms have been observed. [provided by RefSeq, Jul 2008]
Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation. Puri P et al. (2016) Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation.//////////////////
Activation of 5' Adenosine Monophosphate-Activated Protein Kinase Blocks Cumulus Cell Expansion Through Inhibition of Protein Synthesis During In Vitro Maturation in Swine. Santiquet N 2014 et al.
The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study was undertaken to analyze AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in cumulus-oocyte complexes (COC). Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis through the hexosamine biosynthetic pathwaywhich ruled out possible depletion of substrates. Increased gap junction communication and phosphodiesterase activity in COC are both dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor, cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian physiology and to the development of new oocyte culture media.
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A Requirement for Fatty Acid Oxidation in the Hormone-Induced Meiotic Maturation of Mouse Oocytes. Valsangkar D 2013 et al.
We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with 3H-palmitic acid as substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with AICAR and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into mitochondria. The carnitine palmitoyltransferase 1 (CPT1B) activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor (EGF), and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated hCG-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO.
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AMPK Activation by Dihydrotestosterone Reduces FSH-Stimulated Cell Proliferation in Rat Granulosa Cells by Inhibiting ERK Signaling Pathway. Kayampilly PP et al. We have previously reported that 5a-dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. The present study investigated the role of AMP activated protein kinase (AMPK) in DHT-mediated inhibition of granulosa cell proliferation. Granulosa cells harvested from 3-d estradiol primed immature rats were exposed to different concentrations of DHT (0, 45, and 90 ng/ml) for 24 h. Western blot analysis of immunoprecipitated AMPK showed a dose-dependent activation (P < 0.05) as evidenced by the increased phosphorylation at thr 172. In addition, time-courses studies (0, 6, 12, and 24 h) using DHT (90 ng/ml) showed a time-dependent increase in AMPK activation with maximum effect at 24 h. FSH inhibited AMPK phosphorylation and promoted granulosa cell proliferation, but pretreatment with DHT (90 ng/ml) for 24 h prior to FSH treatment reduced this effect. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-?-ribofuranoside abolished FSH-mediated ERK phosphorylation, indicating that AMPK is a negative upstream regulator of ERK. Furthermore, inhibition of AMPK activation by compound C reversed the DHT-mediated reduction in positive cell cycle regulator, cyclin D2, and 5-bromo-2'-deoxyuridine incorporation. These results suggest that elevated levels of DHT activate AMPK, which in turn inhibits ERK phosphorylation. Thus, inhibition of ERK phosphorylation by activated AMPK in response to DHT might contribute to decreased granulosa cell mitogenesis and ovulatory dysfunction seen in hyperandrogenic states.
AMPK regulates progesterone secretion in rat granulosa cells Tosca L, et al .
The AMP-activated protein kinase (AMPK) is a major regulator of energy metabolism involved in fatty acid and cholesterol synthesis. In the ovary, cholesterol plays a key role in steroid production. We report the presence of AMPK in rat ovaries and we have investigated its role in granulosa cells. We show using RT-PCR and western-blot that the mRNAs for the alpha1/2 and beta1/2 subunits and the proteins are found in the ovaries. Immunohistochemistry localized the alpha1 AMPK subunit in granulosa cells, corpus luteum, oocyte, and less abundantly in theca cells. Treatment with AICAR (5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside, 1 mM), an activator of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKalpha1 on Thr 172 in primary granulosa cells. Simultaneously, phosphorylation of Acetyl-CoA Carboxylase at Ser-79 was also increased. AICAR treatment for 48 h halved progesterone secretion, 3beta-HSD protein and mRNA levels and phosphorylation of both basal MAPK ERK1/2 and p38 and in response to IGF-1 and/or FSH in granulosa cells. AICAR treatment (1 mM) had no detectable effect on basal and FSH- and/or IGF-1-induced estradiol production and on granulosa cell proliferation or viability. Adenovirus-mediated expression of dominant negative AMPK totally abolished the effects of AICAR on progesterone secretion, 3beta-HSD protein production, and MAPK ERK1/2 and p38 phosphorylation. Moreover, we showed using specific inhibitors of ERK1/2 and p38 MAPK that the MAPK ERK1/2 and not p38 is involved in progesterone secretion and 3beta-HSD expression, strongly suggesting that the activation of AMPK in response to AICAR reduces progesterone production through the MAPK ERK1/2 signaling pathway in rat granulosa cells.
Gene whose expression is detected by cDNA array hybridization: transporters, signal transduction Rozenn Dalbi?Tran and Pascal Mermilloda
Activation of Adenosine Monophosphate-Activated Protein Kinase Is an Additional Mechanism That Participates in Mediating Inhibitory Actions of Prostaglandin F2 Alpha in Mature, but Not Developing, Bovine Corpora Lutea. Bowdridge EC et al. (2015) Elevated cytosolic calcium and protein kinase C are well-established mediators of luteolytic actions of prostaglandin F 2 alpha (PGF2α). The objectives of this study were to determine: (1) if calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) participates in mediating PGF2α actions in developing (d-4) and mature (d-10) bovine CL, (2) distal targets of CAMKK2, (3) developmental expression of adenosine monophosphate activated protein kinase (AMPK), and (4) effects of AMPK activation on progesterone (P4) production. Expression of AMPK increased as the CL matured. Activation of the prostaglandin receptor (FP) induced rapid phosphorylation of AMPK, which was blocked by a CAMKK2 inhibitor. Changes in basal P4 secretion in vitro were determined in response to AMPK activation via metformin (met) or 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) in d-4 and d-10 CL. Production of P4 in d-10 CL decreased with met or AICAR compared to control, similar to activation by PGF2α. Therefore, potential distal targets of AMPK in d-10 CL were examined during induced functional regression via exogenous PGF2α. Serum and luteal P4 decreased at 2 and 4 h after administration of PGF2α. Protein expression of LDLR decreased at 2 and 4 h, while ACAT1 and STAR increased 4 h after PGF2α. During induced regression, alterations of cholesterol transport proteins, contributed to decreased luteal and serum P4. Therefore, developmental differences in signal transduction associated with FP, specifically CAMKK2 and AMPK, partially contribute; to differences in the ability of PGF2α to induce regression in mature, but not developing, bovine CL. Multiple cholesterol transport proteins, including LDLR, were altered by PGF2α and could be potential AMPK targets.//////////////////
Follicle Stimulating Hormone Inhibits AMPK Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture Through an Akt Dependent Pathway. Kayampilly PP et al. FSH, acting through multiple signaling pathways regulates the proliferation and growth of granulosa cells which are critical for ovulation. The present study investigated whether AMP Kinase, which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum free, phenol red free DMEM-F12 and were treated with FSH (50 ng/mL) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (AICAR, 0.5mM) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20microM) and FSH reduced p27kip expression significantly compared to control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 while reducing thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by AICAR treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression whereas FSH increased the expression by two fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway.
AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro. Chen J et al. We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo.
Metformin-induced Stimulation of AMP-Activated Protein Kinase (PRKA) Impairs Progesterone Secretion in Rat Granulosa Cells. Tosca L et al. Metformin is an anti-diabetic drug commonly used to treat cycle disorders and anovulation in women with polycystic ovary syndrome. However, the effects and molecular mechanism of metformin in the ovary are not entirely understood. We investigated the effects of this drug on steroidogenesis and proliferation in rat granulosa cells. Metformin (10 mM) treatment for 48 h reduced progesterone and oestradiol production in both basal conditions and under FSH stimulation. It also decreased the levels of the HSD3B, CYP11A1, STAR and CYP19A1 proteins in response to FSH (10-8M) and of HSD3B in the basal state only. Metformin treatment (10 mM, 24 h) also reduced cell proliferation and the levels of CCND2 and CCNE proteins, without affecting cell viability, both in the basal state and in response to FSH. Furthermore, metformin treatment for 1 h simultaneously increased the Thr172 phosphorylation of PRKAA (Adenosine 5' Monophosphate- activated protein kinase alpha) and the Ser79 phosphorylation of ACACA (acetyl-Coenzyme A carboxylase). The adenovirus-mediated production of dominant negative PRKAA totally abolished the effects of metformin on progesterone secretion, HSD3B and STAR protein production and MAPK3/1 phosphorylation. Conversely, total inhibition of PRKAA Thr172 phosphorylation with the dominant negative PRKAA adenovirus did not restore the decrease in oestradiol production and cell proliferation induced by metformin. Our results therefore strongly suggest that metformin reduces progesterone production via a PRKAA- dependent mechanism, whereas PRKAA activation is not essential for the decrease in oestradiol production and cell growth induced by Metformin in rat granulosa cells.
Ovarian localization
Oocyte, Granulosa, Luteal cells
Comment
AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles. Tosca L et al. AMP-activated protein kinase (AMPK) is a fuel sensor in glucose, lipid, and cholesterol metabolism. Using RT-PCR and Western blot, AMPK subunits mRNAs (alpha1/2, beta1/2, and gamma1/2) and proteins (alpha1/2 and beta1/2) can be found in the hen preovulatory follicles and precisely in both granulosa and theca cells. These preovulatory follicles are organized in a hierarchy according to their size (F5/6 to F1). The smallest number (F1) corresponds to the largest size and the latest mature stage. Phosphorylation of AMPKalpha on Thr172 and of acetyl-CoA carboxylase on Ser79 are higher in F4 and F3 than in F1 granulosa cells. However, they are not affected in F4-F1 theca cells. Treatment with 1 mM 5-amino-imidazole-4-carboxyamide-1-beta-d-ribofuranoside (AICAR), an activator of AMPK, dose dependently increased phosphorylation of AMPKalpha on Thr172 in primary F3/4 and F1 granulosa cells. In the absence of FSH, AICAR treatment increased progesterone, P450 side chain cleavage and steroidogenic acute regulatory (StAR) production in both F3/4 and F1 granulosa cells. However, in the presence of FSH, AICAR treatment for 36 h increased progesterone secretion, StAR protein levels and reduced extracellular signal-regulated kinase (ERK)1/2 phosphorylation in F3/4 granulosa cells. Opposite data were observed in F1 granulosa cells. Adenovirus-mediated expression of dominant-negative AMPK totally restored the effects of AICAR on FSH-induced progesterone secretion, StAR protein production, and ERK1/2 phosphorylation in F3/4 and F1 granulosa cells. Using a specific inhibitor of ERK1/2 (U0126), we also showed that this kinase is a negative regulator of the FSH-induced progesterone secretion in F3/4 and F1 granulosa cells, suggesting that AICAR-mediated AMPK activation modifies FSH-induced progesterone secretion differently through the ERK1/2 signaling pathway in hen F3/4 and F1 granulosa cells.
AMPK regulation of mouse oocyte meiotic resumption in vitro. Chen J et al. We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200-212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2'-deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro.
Follicle stages
Secondary, Antral, Preovulatory, Corpus luteum
Comment
Effects of Metformin on Bovine Granulosa Cells Steroidogenesis: Possible Involvement of Adenosine 5' Monophosphate-Activated Protein Kinase (AMPK). Tosca L et al. In mammals, insulin-like growth factors (IGFs) are important for the proliferation and steroidogenesis of ovarian cells. Metformin is an insulin sensitizer molecule used for the treatment of the infertility of women with polycystic ovary syndrome. It is, however, unclear whether metformin acts on ovarian cells. Adenosine 5' monophosphate-activated protein kinase (AMPK) is involved in metformin action in various cell types. We investigated the effects of metformin on bovine granulosa cell steroidogenesis in response to IGF1 and FSH, and studied AMPK in bovine ovaries. In granulosa cells from small follicles, metformin (10 mM) reduced production of both progesterone and estradiol and decreased the abundance of HSD3B, CYP11A1, and STAR proteins in presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). In cows, the different subunits of AMPK are expressed in various ovarian cells including granulosa and theca cells, corpus luteum and oocytes. In bovine granulosa cells from small follicles, metformin, like AICAR (1 mM) a pharmaceutical activator of AMPK, increased phosphorylation of both Thr172 of AMPK alpha and Ser 79 of ACACA (Acetyl-CoA Carboxylase). Both metformin and AICAR treatment reduced progesterone and estradiol secretion in presence or absence of FSH and IGF1. Metformin decreased phosphorylation levels of MAPK3/MAPK1 and MAPK14 in a dose- and time-dependent manner. The adenovirus-mediated production of dominant negative AMPK abolished the effects of metformin on secretion of progesterone and estradiol and on MAPK3/MAPK1 phosphorylation but not on MAPK14 phosphorylation. Thus, in bovine granulosa cells, metformin decreases steroidogenesis and MAPK3/MAPK1 phosphorylation through AMPK activation.
Phenotypes
Mutations
1 mutations
Species: mouse
Mutation name: type: null mutation fertility: subfertile Comment: Specific Deletion of AMP-Activated Protein Kinase (α1AMPK) in Murine Oocytes Alters Junctional Protein Expression and Mitochondrial Physiology. Bertoldo MJ et al. (2015) Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.//////////////////