Silvina Beatriz Meroni

The AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-b-d-ribonucleoside, regulates lactate production in rat Sertoli cells

The aim of the present study was to investigate whether the AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in Sertoli cells and whether its activation by 5-aminoimidazole-4-carboxamide-1-b-d-ribonucleoside (AICAR) results in the regulation of cell metabolism to ensure lactate supply for germ cell development. Sertoli cell cultures from 20-day-old rats were used. Western blot analysis for the alpha-subunit of AMPK showed that high levels of AMPK are present in Sertoli cells. Treatment of the cultures with AICAR resulted in a dose- and time-dependent increase of P-AMPK levels indicating activation of the enzyme. A possible effect of AICAR on Sertoli cell lactate production was then analyzed. A dose- and time-dependent increment in lactate secretion was observed. The participation of AMPK activation in different biochemical processes that may be implicated in the regulation of lactate production was also analyzed. AICAR stimulated glucose uptake in a dose- and time-dependent manner. Additionally, AICAR increased the glucose transporter 1 (GLUT1) and decreased the glucose transporter 3 (GLUT3) mRNA levels. As for the role of AMPK in the regulation of the monocarboxylate transporters 1 and 4 (MCT1 and MCT4), it has been observed that AICAR treatment decreased MCT1 and increased MCT4 mRNA levels. In summary, the results presented herein show that AMPK is present in Sertoli cells and that its activation by AICAR increases lactate production as a result, at least in part, of a) an increase in glucose uptake, b) an increase in GLUT1 expression, and c) a decrease in MCT1 and an increase in MCT4 levels. Altogether, these results suggest an important role of AMPK in modulating the nutritional function of Sertoli cells.

Molecular mechanisms involved in Sertoli cell adaptation to glucose deprivation.

Sertoli cells provide the physical support and the necessary environment for germ cell development. Among the products secreted by Sertoli cells, lactate, the preferred energy substrate for spermatocytes and spermatids, is present. Considering the essential role of lactate on germ cell metabolism, it is supposed that Sertoli cells must ensure its production even in adverse conditions, such as those that would result from a decrease in glucose levels in the extracellular milieu. The aim of the present study was to investigate 1) a possible effect of glucose deprivation on glucose uptake and on the expression of glucose transporters in rat Sertoli cells and 2) the participation of different signal transduction pathways in the above-mentioned regulation. Results obtained show that decreasing glucose levels in Sertoli cell culture medium provokes 1) an increase in glucose uptake accompanied by only a slight decrease in lactate production, 2) an increase in GLUT1 and a decrease in GLUT3 expression, and 3) an activation of AMP-activated protein kinase (AMPK)-, phosphatidylinositol 3-kinase (PI3K)/PKB-, and p38 MAPK-dependent pathways. Additionally, by using specific inhibitors of these pathways, a possible participation of AMPK- and p38MAPK-dependent pathways in the regulation of glucose uptake and GLUT1 expression is shown. These results suggest that Sertoli cells adapt to conditions of glucose deprivation to ensure an adequate lactate concentration in the microenvironment where germ cell development occurs.

Signal transduction pathways in FSH regulation of rat Sertoli cell proliferation

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH is the major Sertoli cell mitogen; however, little is known about the signal transduction pathways that regulate the proliferation of Sertoli cells. The hypothesis of this investigation was that FSH regulates proliferation through a PI3K/Akt/mTORC1 pathway, and additionally, AMPK-dependent mechanisms counteract FSH proliferative effects. The present study was performed in 8-day-old rat Sertoli cell cultures. The results presented herein show that FSH, in addition to increasing p-Akt, p-mTOR, and p-p70S6K levels, increases p-PRAS40 levels, probably contributing to improving mTORC1 signaling. Furthermore, the decrease in FSH-stimulated p-Akt, p-mTOR, p-p70S6K, and p-PRAS40 levels in the presence of wortmannin emphasizes the participation of PI3K in FSH signaling. Additionally, the inhibition of FSH-stimulated Sertoli cell proliferation by the effect of wortmannin and rapamycin point to the relevance of the PI3K/Akt/mTORC1 signaling pathway in the mitotic activity of FSH. On the other hand, by activating AMPK, several interesting observations were made. Activation of AMPK produced an increase in Raptor phosphorylation, a decrease in p70S6K phosphorylation, and a decrease in FSH-stimulated Sertoli cell proliferation. The decrease in FSH-stimulated cell proliferation was accompanied by an increased expression of the cyclin-dependent kinase inhibitors (CDKIs) p19INK4d, p21Cip1, and p27Kip1. In summary, it is concluded that FSH regulates Sertoli cell proliferation with the participation of a PI3K/Akt/mTORC1 pathway and that AMPK activation may be involved in the detention of proliferation by, at least in part, a decrease in mTORC1 signaling and an increase in CDKI expression.

Lactate Regulates Rat Male Germ Cell Function through Reactive Oxygen Species

Besides giving structural support, Sertoli cells regulate the fate of germ cells by supplying a variety of factors. These factors include hormones, several pro- and anti-apoptotic agents and also energetic substrates. Lactate is one of the compounds produced by Sertoli cells, which is utilized as an energetic substrate by germ cells, particularly spermatocytes and spermatids. Beyond its function as an energy source, some studies have proposed a role of lactate in the regulation of gene expression not strictly related to the energetic state of the cells. The general hypothesis that motivated this investigation was that lactate affects male germ cell function, far beyond its well-known role as energetic substrate. To evaluate this hypothesis we investigated: 1) if lactate was able to regulate germ cell gene expression and if reactive oxygen species (ROS) participated in this regulation, 2) if different signal transduction pathways were modified by the production of ROS in response to lactate and 3) possible mechanisms that may be involved in lactate stimulation of ROS production. In order to achieve these goals, cultures of germ cells obtained from male 30-day old rats were exposed to 10 or 20 mM lactate. Increases in lactate dehydrogenase (LDH) C and monocarboxylate transporter (MCT)2 expression, in Akt and p38-MAPK phosphorylation levels and in ROS production were observed. These effects were impaired in the presence of a ROS scavenger. Lactate stimulated ROS production was also inhibited by a LDH inhibitor or a NAD(P)H oxidase (NOX) inhibitor. NOX4 expression was identified in male germ cells. The results obtained herein are consistent with a scenario where lactate, taken up by germ cells, becomes oxidized to pyruvate with the resultant increase in NADH, which is a substrate for NOX4. ROS, products of NOX4 activity, may act as second messengers regulating signal transduction pathways and gene expression.

Possible involvement of ceramide in the regulation of rat Leydig cell function

In the present study, a possible role of a ceramide-dependent pathway in the regulation of Leydig cell function was investigated. Intracellular ceramide levels were increased by: (a) adding ceramide analogs; (b) inhibiting ceramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permeable ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S,2R-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Sphingomyelinase from S. aureus origin was utilized. Leydig cells were cultured for 3 or 24 h with or without the different drugs (10 microM) and SMase (0.3 U/ml) in the presence or absence of hCG (10 ng/ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-stimulated testosterone production was observed at 3 and 24 h in all cases. The inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibition in hCG-stimulated testosterone production. TNFalpha and IL1beta, two possible inducers of sphingomyelin hydrolysis, produced similar effects on hCG-stimulated testosterone production. In experiments performed in the presence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultures indicating that this pathway inhibits post-cAMP formation events. To study possible loci for the action of ceramide on the steroidogenic pathway, cells were incubated with C6 and MAPP in the presence of different testosterone precursors. The drugs inhibited testosterone produced from 22(R)-hydroxycholesterol (22R-OHChol), pregnenolone and 17alpha-hydroxyprogesterone (17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events.

Activation of PPAR α and PPAR β/δ regulates Sertoli cell metabolism

The purpose of this study was to evaluate the existence of a possible simultaneous regulation of fatty acid (FA) metabolism and lactate production by PPAR α and PPAR β/δ activation in Sertoli cells (SC). SC cultures obtained from 20-day-old rats were incubated with WY14643 or GW0742-pharmacological activators of PPAR α and PPAR β/δ respectively. The fatty acid transporter CD36, carnitine palmitoyltransferase 1, long- and medium-chain 3-hydroxyacyl-CoA dehydrogenases mRNA levels were analyzed. An increase in the above-mentioned genes in response to activation of both nuclear receptors was observed. Additionally, PPAR β/δ activation increased lactate production as a consequence of increased pyruvate availability by inhibiting the Pyruvate Dehydrogenase Complex. Altogether, these results suggest that in SC, PPAR α activation participates in the regulation of FA metabolism. On the other hand, PPAR β/δ activation regulates FA metabolism and lactate production ensuring simultaneously the energetic metabolism for SC and germ cells.

Adenosine regulates Sertoli cell function by activating AMPK

This work evaluates adenosine effects on Sertoli cell functions, which are different to those resulting from occupancy of purinergic receptors. The effects of adenosine and N(6)-cyclohexyladenosine (CHA) - an A(1) receptor agonist resistant to cellular uptake - on Sertoli cell physiology were compared. Adenosine but not CHA increased lactate production, glucose uptake, GLUT1, LDHA and MCT4 mRNA levels, and stabilized ZO-1 protein at the cell membrane. These differential effects suggested a mechanism of action of adenosine that cannot be solely explained by occupancy of type A(1) purinergic receptors. Activation by adenosine but not by CHA of AMPK was observed. AMPK participation in lactate production and ZO-1 stabilization was confirmed by utilizing specific inhibitors. Altogether, these results suggest that activation of AMPK by adenosine promotes lactate offer to germ cells and cooperates in the maintenance of junctional complex integrity, thus contributing to the preservation of an optimum microenvironment for a successful spermatogenesis.

Possible involvement of microfilaments in the regulation of Sertoli cell aromatase activity

Recent observations indicate that Sertoli cell aromatase activity decreases when cultures are performed at high density. Increasing cell density modifies cell shape in culture from flat cells with visible anchorage sites and abundant intercellular spaces to cells with higher profiles that form a uniform epithelial sheet with no intercellular spaces. Changes in cell architecture are associated with reorganization of the cytoskeleton components. In this report, we have tested whether disruption of microfilaments and microtubules by cytochalasin B and colchicine, respectively, has any effect on the ability of FSH to stimulate aromatase activity. Cytochalasin B, but not colchicine, significantly enhanced aromatase activity in FSH and dbcAMP stimulated cells. The increase in aromatase activity was accompanied by a striking change in cell morphology. Time course studies suggested that microfilament organization is involved in some metabolic event which occurs sometime between 2 and 4 h after the initial steps of FSH action. The reversibility of the biochemical and morphological changes induced by cytochalasin B was demonstrated. The effect of cytochalasin B was observed in high but not in low-density cultures, suggesting that microfilament organization in high-density cultures constrains FSH stimulation of aromatase activity. The last two observations made suggest the existence of a dynamic interplay between microfilament organization and FSH action in Sertoli cells.

Immunodetection of Cell Adhesion Molecules in Rat Sertoli Cell Cultures

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell‐matrix and cell‐to‐cell interactions in Sertoli cell cultures was examined.

Participation of HIFs in the regulation of Sertoli cell lactate production.

Hypoxia Inducible Factors (HIFs) are master regulators of glycolytic metabolism. HIFs consist of a constitutive HIFbeta (HIFβ) subunit and a HIFalpha (HIFα) subunit, whose half-life depends on prolyl-hydroxylases activity. Inhibition of prolyl-hydroxylases by hypoxia or transition metals, or augmentation of HIFα subunit levels by hormonal stimuli lead to a higher HIF transcriptional activity. On the other hand, it is well known that lactate produced by Sertoli cells is delivered to and used by germ cells as an energy substrate. The aim of this work was to investigate whether HIFs participate in the regulation of lactate production in rat Sertoli cells and whether they are involved in the FSH mechanism of action. In order to reach a higher HIF transcriptional activity, Sertoli cells were treated with CoCl2. We observed that a higher HIF transcriptional activity leads to an augmentation of: lactate production, glucose uptake and LDH activity. Besides, an increase in Glut1, Pkm2 and Ldha mRNA levels was observed. These findings suggested that HIFs may participate in the modulation of Sertoli cell nutritional function. As FSH regulates lactate production, we evaluated whether HIFs were involved in FSH action. Sertoli cells were stimulated with FSH in the absence or presence of LW6, a drug which promotes HIFα subunit degradation. On the one hand, we observed that FSH increases HIF1α protein, Hif1α and Hif2α mRNA levels and, on the other hand, that LW6 inhibits FSH-stimulated lactate production, glucose uptake, Glut1, Pkm2 and Ldha expression. It is proposed that HIFs are key components of the intricate pathways utilized by FSH to regulate the provision of lactate for germ cells. Considering that FSH is the master endocrine regulator of Sertoli cells, it is not surprising that this hormone may employ several regulatory mechanisms to fulfill the nourishing functions of this cell type.