Abstract
This study aimed to determine in the canine corpus luteum throughout the dioestrus (1) the influence of insulin on glucose uptake; (2) the regulation of genes potentially involved; and (3) the influence of hypoxia on glucose transporter expression and steroidogenesis, after treatment with cobalt chloride (CoCl2). Glucose uptake by luteal cells increased 2.7 folds (P < 0.05) in response to insulin; a phenomenon related to increased expression of glucose transporter (GLUT) 4 and phosphorylation of protein kinase B (AKT). The gene expression of insulin receptor and SLC2A4 (codifier of GLUT4) genes after insulin stimulation increased on day 20 post ovulation (p.o.) and declined on day 40 p.o. (P < 0.05). Regarding potentially involved molecular mechanisms, the nuclear factor kappa B gene RELA was upregulated on days 30/40 p.o., when SLC2A4 mRNA was low, and the interleukin 6 (IL6) gene was upregulated in the first half of dioestrus, when SLC2A4 mRNA was high. CoCl2 in luteal cell cultures increased the hypoxia-inducible factor HIF1A/HIF1A and the SLC2A4/GLUT4 expression, and decreased progesterone (P4) production and hydroxyl-delta-5-steroid dehydrogenase 3 beta (HSD3B) mRNA expression (P < 0.05). This study shows that the canine luteal cells are responsive to insulin, which stimulates glucose uptake in AKT/GLUT4-mediated pathway; that may be related to local activity of RELA and IL6. Besides, the study reveals that luteal cells under hypoxia activate HIF1A-modulating luteal function and insulin-stimulated glucose uptake. These data indicate that insulin regulates luteal cells’ glucose disposal, participating in the maintenance and functionality of the corpus luteum.
Introduction
The control of the function of canine corpus luteum (CL) is performed by complex mechanisms involving luteotrophic hormones, as well as autocrine and paracrine factors (Okkens et al. 1990, Onclin et al. 2000, Hoffmann et al. 2004, Kowalewski et al. 2011, Papa & Hoffmann 2011, Kowalewski 2014).
According to Sonnack (M Sonnack, unpublished communications) the canine CL lasts 60 days after ovulation (p.o., day 0 is the day of ovulation), formation (day 0–15), maintenance or stage of secretory activity (day 15–45) and regression (day 45–60). During the CL development, a proper nutritional supply for luteal cells must be ensured, which is accomplished by glucose, the major energy substrate for ovarian activity, as demonstrated in granulosa cells of women with polycystic ovary syndrome (Kim et al. 2014) and endometrium (França et al. 2015), follicle and CL of cows, (Nishimoto et al. 2006). In CL of bovines, the expression of solute carriers family 2 members (SLC2A) 1, 3 and 4, which codify the glucose transporters GLUT1, GLUT3 and GLUT4, respectively was reported, and the authors inferred that GLUT1 and GLUT3 would act as main carriers (Nishimoto et al. 2006). In this study, the authors proposed just a supportive role for GLUT4, in spite of (1) the ~3-fold increase observed in SLC2A4 expression through the CL development, (2) the fact that GLUT4 display the highest capacity of transport glucose (Uldry & Thorens 2004) and (3) the fact that it can be rapidly regulated by insulin (Uldry & Thorens 2004). In CL of dogs, our group has demonstrated a differential expression of glucose transporter 1 (SLC2A1/GLUT1), which accompanied hypoxia during the cycle (Papa et al. 2014), pointing out that the CL responds to physiological variations of oxygen tension during dioestrus (Nishimura & Okuda 2015). However, nothing is known about the SLC2A4/GLUT4 expression in CL of dogs.
The longer exposure to hypoxia was reported to increase transcription of the gene Slc2a1 in several cell types (Zhang et al. 1999). Later on, it was described that hypoxia also increases Slc2a3 and Slc2a4 expression in brain (Royer et al. 2000). Finally, it was described that Slc2a4 increases in response to muscle contraction, which reduces the intracellular oxygen tension, and that involves increased binding of the hypoxia-inducible factor 1A (Hif1a) into the Slc2a4 promoter region (Lima et al. 2009). Additionally, some studies further suggest that the Hif1a can also regulate the plasma membrane translocation of GLUT4 (Sakagami et al. 2014), known as the insulin-sensitive glucose transporter, primarily described in insulin-sensitive tissues, as adipose tissue and skeletal and cardiac muscles (James et al. 1988, Slot et al. 1991, Marette et al. 1992, Klip et al. 1996), reinforcing the effect of hypoxia upon regulation of GLUT4.
Although the CL requires high glucose uptake, works so far have explored neither the effect of insulin as inducer of glucose uptake in this organ nor whether physiological hypoxic conditions interfere with that effect when CL is forming, secreting or regressing. The transcriptional regulation of the Slc2a4 gene involves the control of several transcriptional factors including the enhancer Hif1a and the repressor RELA, former transcription factor p65, codified by the gene Rela (Furuya et al. 2013). Besides, interleukin 6 (IL6) has also been suggested as a repressor of Slc2a4 gene (Franckhauser et al. 2008, Poletto et al. 2015).
Thus, this study was designed to investigate the involvement of insulin on glucose uptake by luteal cells, as well as potentially involved positive and negative regulators of this effect. Given the importance of CL hypoxia-induced adaptation and its inductive role of glucose uptake, the effects of CoCl2, a mimicker of hypoxic environment and inducer of HIF1A (Metzen et al. 2003, Grasselli et al. 2005) on the expression of GLUT1/SLC2A1 and GLUT4/SLC2A4, as well as on luteal cells progesterone (P4) production were also investigated.
Materials and methods
Ethics statement
Written consent was obtained from all owners of the dogs and the procedures were performed according to ethical principles approved by the Ethics Committee for the Use of Animals at the School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil (Protocol numbers: 1432/2008 and 2080/2010).
Dogs and sample collection
Corpora lutea (CLs) were collected from 34 healthy cross-bred female dogs aged between 2 and 8 years via ovariohysterectomy (OSH) on specific days of dioestrus. After the onset of pro-oestrous bleeding, blood samples were collected on alternate days to determine P4 concentrations. When plasma P4 levels reached 5 ng/mL, it was considered the day of ovulation (Concannon et al. 1989). For cell culture experiments, CLs were collected as follows: on days 20 (highest P4 plasma concentrations) and 40 (highest oestradiol (E2) plasma concentrations) post ovulation (p.o.) for (insulin treatment (n = 4 per day); on day 30 p.o. (full secretory activity) for glucose uptake assay (n = 3) and over dioestrus (n = 21) for the cobalt chloride treatment.
Anaesthetic and surgical procedures were performed as described previously (Papa et al. 2014). Before anaesthesia, blood samples (5 mL) were collected for metabolic and hormonal analyses. For immunohistochemistry, one CL per dog was fixed in 4% buffered formalin for 24 h and embedded in paraffin. For real-time PCR and Western blotting, one CL per dog per technique was immediately frozen in liquid nitrogen and stored at −80°C, and for cell culture, all CLs from each dog (n = 4 dogs per group) were kept in ice-cold phosphate-buffered saline (PBS) containing 1% penicillin–streptomycin–amphotericin B solution (A5955; Sigma-Aldrich).
Luteal cell culture
CL samples were immediately washed with fresh PBS containing 1% antibiotic-anti-mycotic solution (A5955, Sigma-Aldrich), and cut into small pieces. These pieces were transferred to 1 mL Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Sigma-Aldrich), 1% l-glutamine (Sigma-Aldrich), 20 mM HEPES (Sigma-Aldrich), 1% antibiotic-anti-mycotic solution and 1 mg/mL collagenase type 1 (bib130; Sigma-Aldrich). The incubation lasted for 1 h under stirring (60 rotations/min) at 37°C. The suspension was centrifuged at 200 g for 10 min, re-suspended in DMEM and filtered through a cell strainer (70 µm; BD Falcon; BD Biosciences, Durham, NC, USA). The filtrate was centrifuged at 200 g for 10 min, re-suspended in an erythrocyte lysis buffer (0.16 M NH4Cl, 0.01 M Tris-HCl pH 7.2–7.4) diluted in DMEM (v/v) for 10 min, centrifuged at 200 g for 10 min, and re-suspended in DMEM. For glucose uptake study, luteal cells were seeded in 6-well plates, whereas for insulin and cobalt chloride (CoCl2) treatments, cells were seeded in 24-well plates. Furthermore, for GLUT1 and GLUT4 immunocytochemistry, luteal cells were seeded in 24-well plates containing coverslips and fixed with 95% methanol. Subsequently, cells were kept in an incubator at 37°C and 5% CO2. After ten days of culture (90% confluence), the cells were subjected to serum-free medium for 24 h (except for glucose uptake) and treated following different protocols as described below.
Determination of 2-deoxyglucose uptake
Glucose uptake analysis was performed as described previously (Campello et al. 2012), using 2-deoxy-[1,2-3H]-d-glucose 0.2 mCi·mL−1; Perkin Elmer) for 5 min, after a 20-min stimulus with 100 nM insulin. Incorporated radioactivity was measured, results were calculated and expressed as both cpm/µg protein and cpm/106 cells.
Effect of insulin on luteal INSR and SLC2A4 expression
Luteal cells from CLs obtained on days 20 and 40 p.o. were maintained in DMEM without serum for 24 h. These cells were then treated with 100 nM insulin (I6634; Sigma-Aldrich). After 24 h, the culture medium was removed, TRIzol was added to the cells and they were collected and processed for real-time PCR analysis.
Gene expression assessed by real-time PCR
Total RNA extraction was performed using the TRIzol reagent, following manufacturer’s instructions (Life Technologies). RNA concentration was measured using the NanoDrop 2000 (Thermo Fisher Scientific), whereas the purity and integrity of the samples were determined using 2% agarose gel.
Real-time PCR (qPCR) was carried out as described previously (Kowalewski et al. 2006). Briefly, the total RNA was subjected to reverse transcription using the SuperScript III kit, according to manufacturer’s instructions (Life Technologies). PCR analyses were performed with an automated fluorometer (ABIPrism 7500, Life Technologies), using 96-well optical plates. Each sample (25 ng total RNA) was analyzed at least in duplicates. Primers (Table 1) were ordered from Applied Biosystems (Life Technologies). Relative quantification was performed by normalizing the target genes signals with cyclophilin A signal (as reference gene), as determined by the NormFinder software (Andersen et al. 2004). The amplification efficiency was analyzed by the LinRegPCR programme (Ramakers et al. 2003), followed by Pfaffl’s method (Pfaffl 2001).
List of primers used for real time (TaqMan) PCR.
| Primer | Primer sequence | Accession number | Amplicon length |
|---|---|---|---|
| HIF1A – for | 5′-GCTGCTGGAGACACAATCATATCTT-3′ | XM860420 | 92 |
| HIF1A – rev | 5′ ACATCATTATACAACGGAACTTCCTCAAG-3′ | ||
| HIF1A – probe | 5′-CAATGACACAGAAACTG-3′ | ||
| HSD3B – for | 5′-GGGTACTCAGCTCCTGTTGGAA-3′ | NM_001010954.1 | 102 |
| HSD3B – rev | 5′-GCCACCTCTATGGTGCTGGTAT-3′ | ||
| HSD3B – probe | 5′-TGCCCAGGCTAGTGTGCCGATCTT-3′ | ||
| IL6 – for | 5′-AAAGAGCAAGGTAAAGAATCAGGATG-3′ | XM_843327-1 | 148 |
| IL6 – rev | 5′-GCAGGATGAGGTGAATTGAAGTGTATT-3′ | ||
| IL6 – probe | 5′-GTGATGTCTGCTCGGTAAG-3′ | ||
| INSR – for | 5′-GCAGGARGAGGTGAATTGTTGTG-3′ | AIBJWTL_R | 108 |
| INSR – rev | 5′-CCGAGACCTCAGTTTCCCCA-3′ | ||
| INSR – probe | 5′-AAAACGAGGCCCGAGGATTT-3′ | ||
| RELA – for | 5′-GCAGAAAGAGGACATTGAAGTGTATT-3′ | NW_876266-1 | 118 |
| RELA – rev | 5′-TCGGTGTACATCAGCTTGAGAAA-3′ | ||
| RELA – probe | 5′-CCAGGCTGGGAGGCCCGA-3′ | ||
| SLC2A1 – for | 5′-CAGCCAGAGTCCCCTGTATCTA-3′ | XM_539554 | 99 |
| SLC2A1 – rev | 5′-GTCCCCACCTTCAGGTACTG-3′ | ||
| SLC2A1 – probe | 5′-CACCCCAGACTTCACC-3′ | ||
| SLC2A4 – for | 5′-GCCTGCCAGAAAGAGTCTGAAG-3′ | NM_001159327 | 91 |
| SLC2A4 – rev | 5′-GCTTCCGCTTCTCCTCCTT-3′ | ||
| SLC2A4 – probe | 5′-CAGTGCCCCAGATACAT-3′ | ||
| PPIA | Pre-designed assay from Life Technologies Prod. No. Cf03986523-gH |
Immunohistochemistry for INSR, GLUT4, IL6 and p65 and immunocytochemistry for GLUT4
Immunohistochemistry and immunocytochemistry were performed based on procedures described previously (Mariani et al. 2006). Sections (2 µm thick) from two CL samples per dog, or cells grown over coverslips, analyzed. Primary antibodies are presented in Table 2. Negative controls were established by using the corresponding isotype control (normal rabbit IgG; Santa Cruz Biotechnology) or by replacing the primary antibody with buffer in subsequent reactions. Mouse skeletal muscle was used as a positive control for INSR and GLUT4, and human carcinoma tissue was used as a positive control for p65 and IL6.
List of antibodies used for immunohistochemistry and Western blotting.
| Antibodies | Isotype | Epitope | Dilution IHC | Dilution WB | Supplier (order n°) |
|---|---|---|---|---|---|
| GLUT1 | Goat polyclonal IgG | C-terminus human | – | 0.4 | Santa Cruz (C-20; sc-1605) |
| GLUT4 | Rabbit polyclonal IgG | C-terminus human | 0.5 | 1.4 | Millipore (07-1404) |
| HIF1A | Rabbit polyclonal IgG | Human HIF1A amino acids (432–528) | – | 0.4 | Novus Biologicals (NB100-134) |
| IL6 | Mouse monoclonal IgG2a | Recombinant full length human protein | 0.3 | 0.4 | Abcam (ab 9324) |
| INSR | Rabbit polyclonal IgG | N-terminus human | 0.1 | 0.5 | Abcam (ab 78424) |
| P65 | Mouse monoclonal IgG3 | Human p65 | 0.3 | 0.4 | Millipore (MAB 3026) |
| ACTB | Mouse monoclonal | N-terminal | – | 0.4 | Sigma-Aldrich (Clone AC-15; A1978) |
Protein expression assessed by Western blotting
For total protein extraction, isolated cells and tissue samples were homogenized in NET-2 lysis buffer (50 mM Tris-HCl (pH 7.4), 300 mM NaCl and 0.05% NP-40) containing 1 µL/mL protease inhibitor cocktail (Sigma-Aldrich), and centrifuged at 10,000 g for 10 min at 4°C. The supernatant was used as a total cellular extract. Nuclear protein extraction was prepared based on procedures described previously (Andrews & Faller 1991). The total protein concentration in the samples was determined by the Bradford method (Bradford 1976).
Whole cellular and nuclear extracts (25 or 30 μg) were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% (HIF1A) and 12% (GLUT1, GLUT4, INSR, IL6 and p65) gels and electrophoretically transferred onto a nitrocellulose membrane (Fátima et al. 2013). Immunodetection was performed using the primary antibodies listed in Table 1, and peroxidase-labelled anti-rabbit, anti-mouse and anti-goat antibodies at 1:7500 dilution as secondary antibodies (Amersham Biosciences, GE Healthcare Life Science). The blots were visualized using an Enhanced Chemiluminescence (ECL) Kit (Amersham Biosciences, GE Healthcare Life Science) and images were captured by ChemiDoc MP Image system (Bio-Rad Laboratories). Beta-actin (ACTB) was used as loading control.
Cobalt chloride (CoCl2) treatment
Luteal cells collected on days 10, 30 and 60 p.o. were treated for 24 h with 500 µM cobalt chloride (CoCl2; Sigma-Aldrich). The concentration was chosen based on a preliminary dose–response curve. After the treatment, the medium was collected and stored at −20°C until progesterone determination by validated chemiluminescence immunoassay (Papa et al. 2014). Finally, the cells were processed for RNA or protein extraction as described above.
Statistical analysis
All the experiments were performed at least 3 times with a minimum of three replicates. Data were tested according to their homogeneity and normality of variances, transformed to square to normalize the distributions and presented as mean ± s.e.m. Statistical differences between control and treated groups were calculated using unpaired t-test with Welch’s correction. In each analysis, critical significance level was P < 0.05. All calculations were performed using the GraphPad Prism 5 programme (GraphPad Software).
Results
Glucose uptake by luteal cells under insulin stimulation
To investigate the role of insulin in the canine CL, we tested the capacity of luteal cells from 30-day p.o. CLs to uptake glucose in response to insulin. Glucose uptake was 2.7-fold increased after insulin stimulus (Fig. 1A), expressed by both micrograms of protein (P < 0.01) and number of cells (P < 0.05). Additionally, we verified that despite unchanged total AKT content, insulin increased (P < 0.001) the AKT phosphorylation (Fig. 1A and B). GLUT4 and GLUT1 staining revealed that both were present in luteal cells; however, only GLUT4 showed a qualitative signal increase in the presence of insulin (Fig. 1C and D).
Glucose uptake, AKT, GLUT4 and GLUT1 analyses in canine luteal cells from 30-day p.o. corpora lutea, stimulated or not (basal) with insulin for 20 min. (A) Glucose uptake. (B) Protein expression of phosphorylated AKT (phospho-AKT) and total AKT. Immunocytochemistry for GLUT4 (C and D) and GLUT1 (E and F), before (C and E) and after (D and F) the addition of insulin. Positive signals were observed, as indicated by the brown colour in the luteal cells. Data are mean ± s.e.m. of 4 wells for glucose uptake and 3 wells for AKT per group. In each well, the cells are from CLs from different animals. Different letters represent significant difference (P < 0.001). Bars = 50 µm. Inserts = negative controls.
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
Effect of insulin on INSR and SLC2A4 gene expression in luteal cells
After determining the ability of luteal cells to alter the insulin-stimulated glucose uptake, we investigated the effect of 24-h insulin treatment upon the insulin receptor (INSR) and the SLC2A4 gene expression in luteal cells from 20-day and 40-day p.o. corpora lutea. The results show that in luteal cells from day 20 p.o. insulin increased the expression of INSR and SLC2A4 (Fig. 2A), whereas in cells from day 40 p.o., the insulin effect was completely opposed (Fig. 2B).
Gene expression of the insulin receptor (INSR) and the solute carrier 2A4 (SLC2A4) in canine luteal cells from 20-day (A) and 40-day (B) p.o. and treated with 100 nM insulin for 24 h. Relative expression (RGE) of target genes was normalized by cyclophilin A. Data are mean ± s.e.m. of cells from 4 CLs from different animals in each day. Asterisks indicate significant differences (P < 0.05).
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
INSR, SLC2A4, IL6 and RELA mRNA expression in the CL throughout dioestrus
The mRNA expression of INSR (Fig. 3A) and SLC2A4 (Fig. 3B) in luteal tissue was the lowest on day 40 p.o. (P < 0.05), a moment in which the RELA (Fig. 3D) expression was the highest. Besides, the highest expression of INSR and SLC2A4 was observed on day 20 p.o., when RELA expression was low. In general, the results show that INSR and SLC2A4 expression were parallely regulated throughout the dioestrus, and in opposition to that observed in the RELA expression. Finally, the highest expression of IL6 (Fig. 3C) was concomitant with the highest levels of INSR and SLC2A4 (20 p.o.); however, IL6 expression in other moments of the dioestrus seems to not correlate with the INSR or SLC2A4 expression.
INSR (A), SLC2A4 (B), IL6 (C) and RELA (D) mRNA expression in canine corpora lutea (CLs) from 10 to 60 days after the ovulation, determined by real-time (TaqMan) PCR. Relative expression (RGE) of target genes was normalized by cyclophilin A. Data are mean ± s.e.m. of 4 CLs from different animals. Bars with different letters represent significant differences (P < 0.05).
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
INSR, GLUT4, IL6 and p65 protein expression in the CL throughout dioestrus
The INSR, GLUT4, IL6 and p65 proteins were detected by immunohistochemistry in luteal cells at all studied stages (Fig. 4). To verify whether the mRNA regulations observed in the canine CLs were accompanied by changes in the respective proteins, Western blotting analysis of the proteins was performed. INSR expression (Fig. 5A) was higher on day 10 p.o., which was also observed by immunohistochemistry. GLUT4 expression (Fig. 5B) showed a peak on day 30 p.o., which was also observed by immunohistochemistry. However, these regulations were not in parallel with the mRNA regulation, indicating the occurrence of post-transcriptional modulations.
Immunohistochemical detection of INSR, GLUT4, IL6 and p65 in canine corpora lutea (CLs) from 10 to 60 days after ovulation. Positive INSR, GLUT4, IL6 and p65 signals were observed at all stages of CLs. Inserts = negative controls. Bars = 50 µm.
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
Expression of INSR (A), GLUT4 (B), IL6 (C), total p65 (D) and nuclear p65 (E) proteins in canine corpora lutea (CLs) from 10 to 60 days after ovulation. In each panel, representative Western blotting images are shown. Results were to ACTB expression. Data are expressed as the mean ± s.e.m. of 4 CLs from different animals. Different letters indicate a significant difference (P < 0.05).
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
We also studied the proteins of the above-mentioned genes that are known to regulate GLUT4 expression either positively (IL6) or negatively (p65). Quantification of IL6 showed the highest expression at day 30 p.o., decreasing thereafter (Fig. 5C; P < 0.05); similar to that observed in the immunohistochemistry. We quantified p65 in a total cellular extract and nuclear samples of luteal cells (Fig. 5D and E), and observed that in both the p65 expression was highest on day 30 p.o. This high level of expression was followed by a decline on day 50 p.o. After day 50 p.o., nuclear p65 was undetectable by Western blotting.
Cobalt chloride stimulates HIF1A/HIF1A, SLC2A1/GLUT1 and SLC2A4/GLUT4 expression and inhibits progesterone production in canine luteal cells
It is well determined that CL physiologically undergoes hypoxia; thus, we investigated the effect of CoCl2 on luteal cells obtained on days 10, 30 and 60 p.o. Since no effect of the day (10, 30, 60 p.o.) was observed after statistical analysis over P4 production and hydroxyl-delta-5-steroid dehydrogenase 3 beta (HSD3B) mRNA expression, we considered just the effect of treatment, that is, control cells and CoCl2-treated cells. As expected, CoCl2 increased the expression of HIF1A mRNA and HIF1A protein, and that was accompanied by similar increase in the mRNA and protein expression of SLC2A1/GLUT1 and SLC2A4/GLUT4 (Fig. 6).
Expression of HIF1A, SLC2A1 and SLC2A4 mRNAs (A) and HIF1A, GLUT1 and GLUT4 proteins (B) in canine luteal cells 24-h treated with 500 µM CoCl2. Gene expression was analyzed by qPCR and proteins by Western blotting in a whole cellular homogenate. Relative expression (RGE) of target genes was normalized by cyclophilin A. Representative immunoblots are shown. B-actin (ACTB) was used as loading control. Data are mean ± s.e.m. of 12 plates from 4 different animals from days 10, 30 and 60 p.o. Different letters represent significant difference (P < 0.001).
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
Additionally, 24-h CoCl2 treatment of the luteal cells (Fig. 7) reduced both the P4 secreted to the medium, and the HSD3B mRNA expression, which codifies the key enzyme for P4 synthesis.
Progesterone secretion (A) to the medium (ng·µg−1 de protein) and (B) HSD3B mRNA expression (B) by canine luteal cells 24-h treated with 500 µM CoCl2. Data are mean ± s.e.m. of 12 plates from 4 different animals from days 10, 30 and 60 p.o. Asterisk represents significant difference (P < 0.05).
Citation: Journal of Endocrinology 231, 3; 10.1530/JOE-16-0173
Discussion
To the best of authors’ knowledge, this is the first study to present data that luteal cells are able to uptake glucose under insulin stimulus in an AKT-mediated way, and followed by GLUT4 protein increase. In addition, expression of genes related to insulin biological effectiveness as INSR, SLC2A4, IL6 and RELA has been characterized. Besides, the influence of mimicking low O2 concentrations on the expression of HIF1A/HIF1A, SLC2A1/GLUT1 and SLC2A4/GLUT4 mRNAs and proteins, as well as on the HSD3B mRNA expression and P4 production throughout dioestrus was also analyzed.
GLUT4 expression, the limiting step of insulin-stimulated glucose uptake, was highest in the middle of the CL maintenance period (day 30 p.o.); thus, that moment was chosen to confirm the hypothesis that canine luteal cells are sensitive to insulin. In fact, insulin induced a 2.7-fold increase in the glucose uptake within 20 minutes, similar to that described in classical insulin-sensitive tissues such as adipose tissue and skeletal muscle (James et al. 1988, Klip & Pâquet 1990, Slot et al. 1991, Marette et al. 1992). Besides, in response to insulin, phospho-AKT was 4-fold increased, indicating a classic activation of insulin signalling, as described previously for insulin-induced glucose uptake by oocytes and cumulus cells (Purcell et al. 2012).
As P4 and E2 plasma levels variate throughout dioestrus, and E2 has been reported to be able to modulate SLC2A4/GLUT4 expression, we tested the effect of insulin on INSR and SLC2A4 genes’ expression in luteal cells obtained from CLs harvested at the limits of the maintenance/secretory phase; that is at days 20 and 40 p.o., when the highest levels of P4 and E2, respectively, were observed. Interestingly, insulin increased the SLC2A4 and INSR mRNAs on day 20, but decreased them on day 40 p.o., suggesting that the previous hormonal ambient of the CLs modulated the insulin effect.
SLC2A4 mRNA and GLUT4 protein have been shown to be regulated by E2, and that is differently modulated by the oestrogen receptors 1 (ESR1) and 2 (ESR2), as demonstrated in adipose and muscular tissues (Barros et al. 2009). Using knockout mice for ESR1 and ESR2, Barros and collaborators reported that ESR1 increases, whereas ESR2 represses the expression of GLUT4 (Barros et al. 2009). Using selective ERS1 and ESR2 agonists in 3T3L1 adipocytes, the enhancer effect of ESR1 and the repressor effect of ESR2 on SLC2A4/GLUT4 expression were confirmed (Campello et al. 2012). Therefore, the effect of E2 upon SLC2A4/GLUT4 expression may depend on the balance between ESR1 and ESR2. The expression of ESR1 and ESR2 in canine CL over dioestrus has been already described (Hoffmann et al. 2004, Papa & Hoffmann 2011). During the first half of dioestrus (formation and maintenance/secretory phase, ESR1 is more abundant decreasing in the second half (end of maintenance and regression phases). On the other hand, ESR2 remains unchanged over dioestrus. Associating these findings to this study, we can infer that insulin is able to increase expression of SCL2A4 on day 20 p.o. probably due to the predominant action of ESR1, despite low E2 levels (Papa et al. 2014). Conversely, on day 40 p.o. (end of the maintenance/secretory phase), high E2 levels and predominant action of ESR2 contribute to the repressive effect of insulin on SLC2A4 expression. Therefore, these data we reported for luteal cells regarding insulin effect on SLC2A4 gene seem to be related to E2/ESR1/ESR2 balance, similarly to those described in the adipose and muscle tissues.
Regarding the INSR gene regulation and gonadal hormones, E2 has already been proposed as enhancer of INSR mRNA expression in hypothalamus (Clegg et al. 2006), however, nothing is known about either its hormonal regulation in the female reproductive organs or the ESR1/2 participation.
Comparing the effects of insulin in cultured luteal cells, similar regulations were observed directly in the CLs harvested throughout the dioestrus. SLC2A4 and INSR mRNAs were highest at day 20, and lowest at day 40, suggesting that endogenous insulin is playing the same role that was observed in vitro in luteal cells, and in accordance with the ESR1/ESR2 balance.
Regulation of SLC2A4 expression is dependent on the binding activity of nuclear factor kappa B (NFKB), mainly the isoforms NFKB1 and RELA, to the promoter region of SLC2A4 in muscle and adipose tissues. Besides, NFKB-binding activity also participates on the ESR1- and ESR2-mediated effects of E2 in adipocytes (Campello et al. 2012). Not surprisingly, when evaluating RELA expression in canine CL, we found an upregulation of this gene and respective protein on day 40 p.o., thus suggesting the same regulation of SLC2A4 mRNA that has been described for NFKB in adipose and skeletal muscle tissues or cells (Silva et al. 2005, Campello et al. 2012, Furuya et al. 2013).
Although some studies have demonstrated a negative effect of IL6 on SLC2A4/GLUT4 expression in adipocytes (Lagathu et al. 2003, Poletto et al. 2015), IL6-induced enhancer effect on SLC2A4/GLUT4 expression was reported in skeletal muscle cells (Al-Khalili et al. 2006), indicating that IL6 regulations may vary accordingly to the cell type. Our data support the hypothesis that in luteal cells, IL6 plays an enhancer effect on SLC2A4/GLUT4 during formation/maintenance phases of dioestrus, as the upregulation of IL6 mRNA and protein were highest in the days 20 and 30 p.o., respectively. Besides, we must consider that the variations in IL6 production observed in CLs may be participating on the regulation of ESR1/ESR2 expression, as it has already been reported in a cell line A2780 from human ovarian carcinoma (Wang et al. 2014).
Hypoxia can be considered a physiological state for luteal cells (Nishimura & Okuda 2015) and highly influences the cellular homeostasis. Cells increase HIF1A (hypoxia-inducible factor 1A) expression as the oxygen tension decreases (Tarhonskaya et al. 2015), and the canine CL response to hypoxia and HIF1A expression has been correlated to increased SLC2A1/GLUT1 expression (Papa et al. 2014). The influence of HIF1A in SLC2A4/GLUT4 expression has also been described in skeletal muscle cells (Silva et al. 2005, Lima et al. 2009), in which reduced oxygen tension activates HIF1A and enhances SLC2A4 transcription. This work showed that SLC2A4 expression parallels that of SLC2A1 in canine CL (Papa et al. 2014) over dioestrus, and cells subjected to CoCl2 increased expression of HIF1A, SLC2A1 and SLC2A4 mRNAs and respective proteins. In fact, HIF1A is able to bind to an E-box domain present in the promoter region of SLC2A4 gene, and once activated by reduction in intracellular oxygen tension, as in response to contraction of skeletal muscle, its binding activity increases enhancing SLC2A4 transcription (Lima et al. 2009). Thus, strong regulations observed in GLUT1 and GLUT4 expression, as well as in glucose uptake in CL, seem to be highly related to cellular oxygen disposal througout the dioestrus.
Altogether, our results point towards the ability of luteal cells to uptake glucose in response to insulin stimulus, which includes insulin in the list of luteotrophic factors. Additionally, the data revealed that INSR and GLUT4 expression in luteal cells varies according to the phase of CL lifespan and that can be related to IL6 and RELA/p65 expression. Besides, the study demonstrates that CoCl2 enhances SLC2A4 and HIF1A mRNA expression in luteal cells, indicating that hypoxia plays an important role in the regulation of SLC2A4/GLUT4 expression during the development of CL.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by FAPESP (grant numbers 2008/54835-8, 2010/07373-9 and 2011/17768-3) and CAPES (025/2011).
Author contribution statement
All authors participated in the experimental design, data analysis and interpretation, and revised the manuscript for intellectual content. L M M C S performed canine luteal cell culture, CoCl2 experiments and wrote part of the manuscript. R S S performed canine luteal cell culture, insulin stimulation experiments and wrote part of the manuscript. R M L and T S D V performed ovariohysterectomies and C L sample collection. A B A-V performed glucose uptake analysis in cultured canine luteal cells. U F M critically read the manuscript and participated in very fruitful discussions. P C P conceived the studies, performed IL6 and RELA/p65 analyses and wrote the manuscript.
Acknowledgements
The authors thank Dr Ivana Carvalho from CEPRA NGO and the Zoonosis Control Center of Guarulhos for providing access to the dogs and keeping them in nice atmosphere. We thank all dogs’ guardians, who agreed to dispose ovarian tissue for this study.
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