Lactate-induced effects on bovine granulosa cells are mediated via PKA signaling

l-lactate acts as a signaling molecule in bovine granulosa cells (GCs). The initiated alterations depend on the transport of l-lactate into the cells via monocarboxylate transporters. In the present study, we further elucidated the intracellular actions of l-lactate and tested whether the PKA signaling pathway is involved. Therefore, we treated cultured bovine GCs with l-lactate and PKA inhibitors H-89 and KT5720, and with an activator of PKA, 6-Bnz-cAMP. l-lactate treatment resulted in decreased estradiol production and downregulation of CYP19A1, FSHR, and LHCGR as well as in the upregulation of the markers of early luteinization PTX3, RGS2, and VNN2. These specific l-lactate effects were almost completely abolished by pre-treatment of the GCs with both inhibitors of PKA signaling. In addition, also the l-lactate-induced upregulation of LDHA and of the monocarboxylate transporters SLC16A1 and SLC16A7 was abolished after PKA inhibition. An activation of the PKA with 6-Bnz-cAMP revealed similar effects on the gene expression like l-lactate alone. In summary, the presented data demonstrate that l-lactate-induced effects on GCs are mediated via PKA signaling thus supporting the role of l-lactate as signaling molecule during the folliculo-luteal transition. Supplementary information The online version contains supplementary material available at 10.1007/s00441-021-03569-7.


Introduction
The folliculo-luteal transition of the ovarian follicle is triggered by LH (luteinizing hormone) and leads to profound morphological and physiological changes (Adams et al. 2008;Bao and Garverick 1998). This reorganization is initiated by a precise regulation of gene expression, in particular of genes involved in steroidogenesis (Christenson et al. 2013;Lussier et al. 2017). The key enzyme of estradiol synthesis, aromatase, encoded by CYP19A1, is massively downregulated along with the gonadotropin receptors FSHR and LHCGR in the granulosa cell (GC) layer. Other genes are known to be remarkably upregulated, among them RGS2 (regulator of G protein signaling 2), VNN2 (vanin 2), and PTX3 (pentraxin 3). Therefore, these genes can be considered as marker genes for an LH-induced transformation of bovine GCs. As GCs are the major source of estrogen production and proved to be primarily targeted by LH (Christenson et al. 2013), we established a bovine GC culture model to study effects on steroidogenesis Vanselow 2013, 2018b). Previously, we could show that l-lactate is able to induce a specific differentiation process in cultured bovine GCs that is dependent upon l-lactate transport into the cells via specific monocarboxylate transporters (Baufeld and Vanselow 2018a). Studies in other cell types also support the role of l-lactate as signaling molecule affecting the expression of genes (Constant et al. 2000;Yang et al. 2014). In an mRNA microarray approach, we could confirm that l-lactate led to genome-wide alterations that might initiate differentiation of GCs and gathered first hints of "cAMP-mediated signaling" in these cells (Baufeld et al. 2019). Consequently, here, we investigated whether the PKA signaling cascade might be involved. This pathway is known as a main component involved in the regulation of the LH-and FSH (follicle-stimulating hormone)-induced changes in the follicle (Escamilla-Hernandez et al. 2008;Richards and Pangas 2010). Therefore, we analyzed the effects of PKA inhibitors and of an activator of PKA on l-lactate-treated GCs using steroid hormone production and transcriptional changes of marker genes as readout.
All reagents were purchased from Merck Millipore (Berlin, Germany) if not stated otherwise. Granulosa cells were cultured in 24-well collagen R coated plates (0.02%; Serva, Heidelberg, Germany) for 8 days at 37 °C and 5% CO 2 with media exchange of two thirds every second day. The long duration of cell culture is necessary for the GCs to recover from isolation and plating stress and to resume E2 production and thus develop an estradiol-active status in vitro Yenuganti and Vanselow 2017).

Statistics
Each experiment was performed in triplicates from different GC preparations. For statistical analysis, SigmaPlot 11.0 Statistical Analysis System (Jandel Scientific, San Rafael, CA, USA) was used. Untransformed data were used for statistical analysis of qPCR and hormone measurement. The student's t test or Mann-Whitney test depending on fulfillment of t test criteria was applied. Multiple comparisons were performed using one-way ANOVA with an appropriate post hoc test. The level of significance was set at P < 0.05. The data are shown as log2-transformed values relative to the respective control.

Results
The treatment with l-lactate confirmed the previously described specific regulation of marker transcripts for luteinization, e.g., downregulation of CYP19A1, FSHR, and LHGCR , and upregulation of PTX3, RGS2, and VNN2 (Fig. 1, grey bars). This is in accordance with the observation or Mann-Whitney test was used comparing treatment to control (*) and ANOVA was used for multiple comparison between different treatments (letters), P < 0.05 of lower estradiol concentrations after l-lactate treatment. When the GCs were pre-treated with H-89, an established PKA inhibitor, E2 concentrations nearly reached control levels thus abolishing the l-lactate effect (Fig. 1a, black bars). In addition, effects on transcript abundances of CYP19A1 and of the other marker genes for luteinization were largely abolished even though in particular the upregulated transcripts (PTX3, RGS2, and VNN2) did not fully reach control levels (Fig. 1b, black bars). Treatment with another PKA inhibitor, KT5720, could confirm the partial or complete abolishment of the l-lactate effects on the concentration of estradiol or gene expression of marker genes ( Fig. 1c and d). Treatment of GCs with the inhibitors H-89 and KT5720 alone did not result in significant changes compared to the vehicle control.
We also investigated effects of l-lactate and of PKA inhibitors on genes involved in lactate metabolism. Therefore, expression of LDHA, coding for the lactate dehydrogenase and of two known transporters for l-lactate, SLC16A1 and SLC16A7, was analyzed. As observed previously, l-lactate enhanced expression of LDHA and SLC16A1 (Fig. 2, grey bars). When the GCs were pretreated with H-89, the l-lactate effects were significantly reduced (Fig. 2a, black bars). Inhibition of PKA with KT5720 again resulted in a reduction or near abolishment of the l-lactate effect.
The activation of PKA with the cAMP analogon 6-Bnz-cAMP revealed a massive downregulation of CYP19A1, FSHR, and LHCGR compared to the untreated control (Fig. 3a). Other marker transcripts were highly upregulated, especially RGS2 showed a nearly 50-fold increase after PKA activation. The genes of the lactate metabolism, LDHA and SLC16A1, were upregulated probably due to the activation of PKA in culture (Fig. 3b).

Discussion
PKA signaling is a major pathway mediating the FSH-and LH-dependent changes during folliculogenesis (Escamilla-Hernandez et al. 2008;Morris and Richards 1995;Murphy 2000). Involvement of PKA and PKC signaling in mediating l-lactate effects was also shown in skeletal muscle cells (Narumi et al. 2010). Therefore, in our present study, we aimed to elucidate if this functionally important pathway is also involved in the observed l-lactate effects in GCs. Application of the PKA inhibitors H-89 and KT5720 reduced or nearly abolished the l-lactate-induced effects on cultured bovine GCs. The direct activation of the PKA signaling pathways via the cAMP analogon 6-Bnz-cAMP in the cell culture model Fig. 2 Effects of l-lactate and PKA inhibition on genes related to lactate metabolism. Cultured GCs were treated with 30 mM l-lactate (grey bars) and additionally pre-treated with a 15 µM H-89 (black bars) and b 200 nM KT5720 compared to the control (NaCl, dashed line). In addition, effects of both inhibitors alone (light grey bars) were checked. Means and SEM are shown of n = 3 replicates. Student's t-test or Mann-Whitney test was used comparing treatment to control (*), and ANOVA was performed for multiple comparison between different treatments (letters), P < 0.05 revealed a similar regulation as observed with l-lactate. This indicates that l-lactate effects are mediated via PKA signaling. Typically, the activation of PKA is known to be the result of G-protein coupled receptor activation. In neuronal cells and adipocytes, it was shown that l-lactate indeed acts on such a receptor, HCAR1 (Liu et al. 2009;Mosienko et al. 2015). HCAR1 is the only so far known G-protein coupled receptor that can bind l-lactate. The studies on HCAR1 in adipocytes reveal a decrease of cAMP concentration and inhibition of lipolysis (Cai et al. 2008;Liu et al. 2009). However, analysis of HCAR1 transcripts in the follicle leads to the suggestion that this receptor is not expressed in GCs but only in theca cells of the bovine follicle. Accordingly, the results further support our previous hypothesis that l-lactate uptake into cells is mandatory to facilitate a change in GCs' gene expression profile. In our previous study, we observed an abolishment of the l-lactate effects by inhibiting the transporters for lactate (Baufeld and Vanselow 2018a). Moreover, the inhibition of PKA abolished the upregulation of l-lactate-related genes, especially of the transcripts encoding the monocarboxylate transporters, SLC16A1 and SLC16A7 (MCT1 and MCT2). Additionally, the presented data provide evidence that l-lactate induces effects comparable to that of a PKA activation. This further supports our hypothesis that the l-lactate effect is mediated via intracellular PKA signaling. Main transcription factors activated by PKA are CREB (cAMP response elementbinding protein), CREM (cAMP-responsive modulator), and ATF1 (Sassone-Corsi 2012). The observed upregulation of transcripts due to l-lactate or PKA activator treatment is in line with the fact that CRE binding sites are present in the promotor region of the genes LDHA, RGS2, and VNN2 (Larabee et al. 2018;Perschbacher et al. 2020;Short et al. 1994). Within the CYP19A1 promoter, a CRE-like sequence (CLS) was found relevant to mediate the cAMP response (Hinshelwood et al. 1997;Michael et al. 1997). In the wake of the LH surge, the inducible cAMP early repressor (ICER) is expressed supporting the downregulation of CYP19A1 (Morales et al. 2003). Together with a subsequent and more detailed investigation of involved transcription factors, it would be also interesting to analyze the protein alterations of the lactate transporters to strengthen our hypothesis further.

Conclusions
Taken together, these data extend our previous knowledge on the l-lactate-mediated differentiation of bovine GCs in culture. Besides the already demonstrated involvement of active transport of l-lactate into GCs, an interaction via PKA becomes evident (Fig. 4). In GCs, a possible way of l-lactate action might be the following: l-lactate enters the cells via specific transporters (MCTs). Subsequently, PKA is activated thus enhancing the action of the preovulatory LH surge.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Fig. 4 Suggested signaling model of l-lactate action in bovine GCs. As a first essential step, l-lactate crosses the cell membrane via specific transporters (MCTs, see Baufeld and Vanselow 2018a, b). Specific effects on gene expression are then mediated by the PKA pathway thus supporting the LH initiated folliculo-luteal transition