To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2. To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24 h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2. Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse β-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.
Living animals cannot live without oxygen, which is required to efficiently produce ATP in mitochondria in cells. Recent studies have clarified that low oxygen concentrations affect the expression of genes in cells and tissues that allow organisms to adapt to life-threatening hypoxic environments. The family of hypoxia-inducible factors (HIFs), which are involved in the hypoxia-signaling pathways and have been intensively studied at the molecular and cell biological levels, are key players in the cellular responses to hypoxic states (<5% O2). For example, HIF-1 regulates the expression of a wide variety of genes, including VEGF, MMP2, cytochrome c, and the glucose transporters that are involved in angiogenesis, tumor invasion and metastasis, apoptosis, and cell metabolism [1–3].
Although the O2 concentration in air is 21%, the local concentration of O2 in tissues is reduced, the oxygen concentrations in the lung parenchyma and circulation range from 3.6 to 12.8% . Several studies in stem cells have indicated that low oxygen concentrations may facilitate cell proliferation compared with room-air oxygen concentrations [5,6]. Considering the decrease in the oxygen concentration in peripheral cells or tissues of living animals, the optimal concentration of oxygen for functionally differentiated cells or tissues is possibly much lower than we expected.
In differentiated neuro-endocrine cells, peptide hormones and biologically active peptides are synthesized as large precursors at the endoplasmic reticulum. Then, the precursor proteins sequentially undergo a wide variety of post-translational modifications, such as removal of the signal peptide, formation of disulfide linkages, and glycosylation, through the secretory pathway to the trans-Golgi network (TGN). In the TGN, prohormones are sorted to secretory granules (SGs), where they are processed to mature active hormones through proteolytic cleavage at mono- or dibasic amino acids followed by the removal of C-terminal basic amino acids . Mature peptide hormones are then exocytosed from cells in a regulated manner upon appropriate stimulation from the outside of the cells. These biological processes in secretory pathways have been investigated in vitro using cultured cell lines derived from endocrine tissues and tumors. However, these cells are mostly cultured in 21% O2 with 5% CO2. Whether in vitro studies with endocrine cell lines cultured in 21% O2 accurately reflect the functional states of differentiated endocrine cells in living animals remains unclear.
In the present study, we attempted to examine whether the ambient 21% O2 is optimal for endocrine function in animals. We analyzed the expression, processing, and secretion of peptide hormones both in endocrine cell lines and tissues excised from mice cultured in a wide range of oxygen concentrations. Based on our results, we discuss the physiological and ideal oxygen concentrations to accurately assess the potential of endocrine cells or tissues in living animals.
Materials and methods
The mouse corticotrope-derived AtT-20 cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The mouse pancreatic β-cell-derived MIN6 cell line was cultured in DMEM with 15% FBS and 50 µM 2-mercaptoethanol. For hypoxic conditions, an oxygen concentration-changeable multigas incubator APM-30D (ASTEC, Fukuoka, Japan) was used. Cells were cultured under five different oxygen concentrations (1, 5, 10, 15, and 21%) for 24 h at 37°C.
Rabbit polyclonal anti-SgIII (SgIII-C#6; diluted 1:1000) was prepared and characterized as described in our previous studies . The following antibodies were purchased: anti-adrenocorticotropic hormone (ACTH) (Ab-1; mouse monoclonal, Thermo Scientific, Waltham, MA; diluted 1:1000), anti-C-peptide (4020-01; rabbit polyclonal, Linco, St. Charles, MO; diluted 1:1000), anti-CgA 94-130 (Y291; rabbit polyclonal, Yanaihara Institute, Inc., Fujinomiya, Japan; diluted 1:1000), anti-PC1/3 (AB10553; rabbit polyclonal, Merck Millipore Corporation, Darmstadt, Germany; diluted 1:1000), anti-PC2 (AB15610; rabbit polyclonal, Merck Millipore Corporation; diluted 1:500), anti-CPE (610759; mouse monoclonal, BD Transduction Laboratories; diluted 1:1000), anti-furin (sc-20801; rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; diluted 1:1000), anti-HIF1-α (NB100-479; rabbit polyclonal, Novus Biological, Littleton, CO; diluted 1:1000), and anti-β-actin (A5316; mouse monoclonal, Sigma–Aldrich, St. Louis, MO; diluted 1:5000). Secondary antibodies, including anti-mouse and anti-rabbit IgG, labeled with horseradish peroxidase (HRP) were purchased from Jackson ImmunoResearch (West Grove, PA; diluted 1:5000).
ACTH secretion assays using AtT-20 cells
ACTH secretion from AtT-20 cells was measured as described previously [9,10]. Briefly, AtT-20 cells were cultured on a six-well plate until 80% confluency under 21% O2 for 48 h after passage. Then, the cells were cultured under different oxygen concentrations (10 or 21%) for 24 h. After culturing under different oxygen concentrations, AtT-20 cells were further cultured in a low KCl Krebs–Ringer bicarbonate glucose solution [15 mM HEPES (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 24 mM Na2HCO3, 2.8 mM glucose, and 0.1% bovine serum albumin] for 30 min in 10 or 21% O2 to minimize the effects of the culture media on secretion. Then, the medium was changed to a fresh high KCl Krebs–Ringer bicarbonate glucose solution [15 mM HEPES (pH 7.4), 65 mM NaCl, 60 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 24 mM Na2HCO3, 2.8 mM glucose, and 0.1% bovine serum albumin], and cells were cultured for an additional 30 min in 10 or 21% O2 to measure the amount of ACTH released from the cells released by regulated secretion (stimulated secretion). Alternatively, the medium was changed to a fresh low KCl Krebs–Ringer bicarbonate glucose solution instead of the high KCl solution to measure the amount of ACTH released from cells without stimulation (basic secretion). Following the secretion assay, the number of cells in each sample was assessed using a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA). After cell counting, ACTH was extracted from the cells in an acid-ethanol solution (70% ethanol and 0.18 M HCl) to determine the ACTH contents in cells. Immunoreactive (IR) ACTH was measured using an ACTH ELISA assay kit (ACTH-EIA Kit; Phoenix Pharmaceuticals, Inc., Burlingame, CA), and the amount of ACTH was standardized based on the number of cells. Of note, the ACTH ELISA assay kit (ACTH-EIA) is highly specific for mature ACTH and does not react with proopiomelanocortin (POMC; manufacturer's information; Phoenix Pharmaceuticals, Inc.).
Insulin secretion assays using MIN6 cells
Insulin secretion from MIN6 cells was measured as described previously [11,12]. Briefly, MIN6 cells were cultured in a six-well plastic plate until 80% confluency for 48 h. Then, cells were cultured under different oxygen concentrations (10 or 21%) for 24 h in low-glucose (LG) DMEM (1.0 g/l d-glucose). After culturing MIN6 cells under different oxygen concentrations, the cells were further cultured in a LG Krebs–Ringer bicarbonate solution [LG: 15 mM HEPES (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 24 mM Na2HCO3, 2.8 mM glucose, and 0.1% bovine serum albumin] under the same oxygen concentrations (10 or 21%) for 30 min to minimize the effects of the culture media on secretion. Then, the medium was changed to a high-glucose (HG) Krebs–Ringer bicarbonate solution [HG: 15 mM HEPES (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 24 mM Na2HCO3, 25 mM glucose, and 0.1% bovine serum albumin] for 30 min in 10 or 21% O2 to determine the amount of insulin released from the cells by regulated secretion (stimulated secretion). To measure the amount of insulin released from the cells without stimulation (basic secretion), LG was used instead of HG. Following incubation, the number of cells in each sample was assessed to standardize insulin secretion and content. The IR-insulin levels secreted into media or that remained in cells were measured using a mouse Insulin ELISA KIT (S-type) (AKRIN-011S; Shibayagi Co., Ltd., Gunma, Japan). Of note, the insulin ELISA assay kit (S-type) is highly specific for mature insulin and does not react with proinsulin (manufacturer's information; Shibayagi Co., Ltd.).
To assess the effects of low oxygen concentrations on mouse endocrine cells, pituitary glands and pancreas were obtained from C57BL/6J male mice (Charles River Laboratories Japan, Inc., Yokohama, Japan) for experiments. We conducted our animal experiments in accordance with the guidelines for the Care and Use of Laboratory Animals of the Medical Research Council of Akita Prefectural University (approval number 15-03 and 17-03). ACTH secretion from mouse pituitary was measured as described previously . Briefly, whole pituitary glands and pancreata were aseptically removed from male mice (n = 6 per experimental group) anesthetized with sodium pentobarbitone (5 mg/mouse). The pituitaries were cultured in minimum essential medium (M4655; Sigma–Aldrich, St. Louis, MO) with 5% FBS and 10% normal horse serum for 24 h and then incubated in different oxygen concentrations (10 or 21% O2) for 24 h. ACTH secretion assays and IR-ACTH measurements in medium and pituitaries were performed as described above for AtT-20 cells.
Insulin secretion from mouse islets isolated from the pancreas was measured as described recently . Briefly, isolated islets were cultured in a 35-mm plastic plate for 24 h after passage in 21% O2 and then cultured under different oxygen concentrations (10 or 21%) for 24 h in RPMI 1640 (2.0 g/l d-glucose: 11835-030; Thermo Scientific, Waltham, MA). Before performing the secretion assay, the islets were cultured under different oxygen concentrations (10 or 21%) for 30 min in media prepared by adding 1.0 g/l d-glucose to DMEM no glucose media (11966-025; Thermo Scientific). To evaluate stimulated insulin secretion, IR-insulin was measured in medium and islets as described above for MIN6 cells.
Quantitative real-time PCR
Total RNA was extracted using Sepazol RNA I Super G (Nacalai Tesque, Inc., Kyoto, Japan). Briefly, 1 µg of RNA was reverse-transcribed into cDNA by a SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA), and then, quantitative PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) in the CFX manager (Bio-Rad, Hercules, CA). The following primers were used to amplify β-actin: forward 5′-GGCTGTATTCCCCTCCATCG-3′ and reverse 5′-CCAGTTGGTAACAATGCCATGT-3′. The following primers were used to amplify 18S ribosomal RNA: forward 5′-CTCAACACGGGAAACCTCAC-3′ and reverse 5′-CGCTCCACCAACTAAGAACG-3′. The following primers were used to amplify PC1/3: forward 5′-AGACAGCATTTACACCATCTCTATCAG-3′ and reverse 5′-AGAACACTTCTCTGCATACCAAGGT-3′. The following primers were used to amplify PC2: forward 5′-CCCAGAGACGACGACTCCAA-3′ and reverse 5′-CCAGGTGTGGGTGGTCATG-3′. The following primers were used to amplify CPE: forward 5′-TTCGAGTACCACCGCTATCCA-3′ and reverse 5′-CCCACTGTGTAGATTCTGCTGATG-3′. All of the primers used in this study were designed according to a previous report [14–16]. The relative expression levels were calculated using β-actin and 18S ribosomal RNA as references. The quantification of gene expression was performed by using the ΔΔCT calculation with CT as the threshold cycle. Samples were analyzed in triplicate after each quantitative real-time PCR with β-actin and 18S ribosomal RNA for normalization, and similar experiments were independently repeated five times (n = 5). The averages of triplicate samples for each experiment were first calculated, and then, the mean of these values from five independent experiments was calculated and statistically analyzed.
Protein expression analysis
Cells/tissues cultured under different oxygen concentrations for 24 h were solubilized in 20 mM HEPES (pH 7.4) containing 0.1 M NaCl, 1 mM EDTA, 1% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride and a protease inhibitor cocktail (Nakalai Tesque, Inc., Kyoto, Japan) in a glass Teflon-homogenizer. The cell/tissue lysates were centrifuged at 11000×g for 10 min at 4°C to achieve protein sample solutions. After the protein concentrations in the aliquots of soluble extracts were quantified by the Bradford protein assay using Protein Assay CBB Solution (Nacalai Tesque, Inc.), the extracts were treated in a sample buffer solution containing 1.7% (w/v) sodium dodecyl sulfate and 1.55% (w/v) dithiothreitol at 100°C for 5 min. The samples (20 µg protein/lane) were then loaded onto 7% (for the detection of HIF1-α), 10% (for the detection of PC1/3, PC2, CPE, CgA, SgIII, furin, and β-actin), and 14% (for the detection of POMC, ACTH, and proinsulin) SDS-polyacrylamide gels (73 × 80 × 1.5 mm) and were electrophoresed using a Mini-Protean II (Bio-Rad, Hercules, CA). After electrophoresis, the proteins separated on the gels were transferred onto a methanol-activated polyvinylidene difluoride membranes (with a pore size of 0.45 µm, Millipore) for 3 h using a TE22 Mighty Small Transfer system (GE Healthcare, Chicago, IL) at 250 mA (current constant) against transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol. The blots were probed with primary antibodies against ACTH (for the detection of ACTH and POMC), C-peptide (for the detection of proinsulin), PC1/3, PC2, CPE, CgA, SgIII, furin, HIF1-α, and β-actin, followed by incubation with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. The protein bands on the membranes were detected by chemiluminescence using an ECL-Plus Western Blotting Detection System (GE Healthcare). The quantities of proteins were estimated via densitometry of the immunoblot bands of the appropriate molecular mass using LAS4000 (General Electric Company, CT, U.S.A.) software.
Trypan blue exclusion test of cell viability
AtT-20 and MIN6 cells were cultured for 48 h until 80% confluency before viability assays were performed. For cell viability assays, cells were cultured in freshly changed media in 1, 5, 10, 15, or 21% O2 for up to 48 h, and then the media with dissociated cells were collected in a new tube. After washing the culture dishes with PBS, the adherent cells were enzymatically detached from the dishes. The dissociated and detached cells were stained with 0.4% Trypan Blue (Bio-Rad, Hercules, CA) to obtain initial seeding density for normalization. Cells cultured for 48 h were dissociated as described above and subject to survival assays using the TC20 Automated Cell Counter (Bio-Rad). All experiments were performed in triplicate.
Statistical significance was evaluated using Student's t-tests, and P < 0.05 was considered significant. Statistical significance is indicated in each figure legend as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.
Culture in 10% O2 increased the secretion and intracellular storage of ACTH and insulin in AtT-20 and MIN6 cells, respectively
To evaluate the effects of oxygen concentrations less than that of atmospheric oxygen on the production and secretion of peptide hormones in neuro-endocrine cells, we first compared the levels of ACTH secreted from AtT-20 cells cultured for 24 h in 10 or 21% O2 (Figure 1A, left). The basal secretion of ACTH from AtT-20 cells cultured for 30 min with 5 mM KCl in 10% O2 was slightly increased compared with that from AtT-20 cells cultured in 21% O2, and the stimulated secretion of ACTH from cells cultured with 60 mM KCl in 10% O2 was significantly increased (∼3-fold) compared with that in 21% O2. The storage of ACTH in the cells also markedly increased upon culture in 10% O2 (Figure 1A, right). The effects of culturing AtT-20 cells cultured under various oxygen concentrations (21, 15, 10, 7.5, 5, and 1%) on ACTH secretion and storage were further examined in detail. KCl-induced ACTH secretion peaked when the cells were cultured in 10% O2, whereas the basal secretion of ACTH was not significantly altered by culturing cells under different oxygen concentrations (Supplementary Figure S1A). ACTH storage in cells also peaked when they were cultured in 10% O2 based on the immunoblotting results (Supplementary Figure S1B).
Effects of culture in 10% O2 on hormone secretion and intracellular levels in neuro-endocrine cell lines.
To examine whether the increase in intracellular storage and stimulated the release of the mature hormone in endocrine cells cultured in 10% O2 concentration is a common feature in neuro-endocrine cells, insulin secretion and storage were similarly analyzed by using MIN6 β-cells cultured in 10% O2 (Figure 1B). The stimulated secretion of insulin in the HG (25 mM) medium was increased in MIN6 cells cultured in 10% O2 compared with cells cultured in 21% O2, whereas the basal secretion of insulin in the LG (2.8 mM) medium was unaffected by the oxygen concentration (Figure 1B, left). The insulin content in cells cultured in 10% O2 also significantly increased by ∼1.8-fold compared with that in cells cultured in 21% O2 (Figure 1B, right). These findings suggest that oxygen concentrations less than that in the atmosphere commonly increase the stimulated release and intracellular storage of mature hormones in endocrine cells.
To evaluate the effects of lower oxygen conditions on the growth and survival rates of neuro-endocrine cells, the numbers of living and dead AtT-20 and MIN6 cells 24 and 48 h after being cultured under different oxygen concentrations (21, 10, 5, and 1% O2) were assessed (Supplementary Figure S2). No significant differences in the number of the living AtT-20 (Supplementary Figure S2A) and MIN6 (Supplementary Figure S2B) cells were noted among the experimental groups at 24 and 48 h (Supplementary Figure S2, left panels). The survival rates of MIN6 cells were significantly reduced both at 24 and 48 h when cells were cultured in 5 and 1% O2 (Supplementary Figure S2B, right panel). However, the survival rates of both AtT-20 and MIN6 cell lines cultured in 10% O2 for 24 and 48 h were similar to those cultured in 21% O2 (Supplementary Figure S2, right panels). Based on these growth and survival rates, we concluded that 10% O2 was an acceptable concentration for estimating the effect of reduced oxygen conditions on the secretion and storage of peptide hormones in neuro-endocrine cells.
Culture in lower oxygen conditions increased the processing enzymes for prohormones in neuro-endocrine cells
Given that culturing AtT-20 and MIN6 cells in 10% O2 did not affect their proliferation and survival rates of AtT-20 and MIN6 cells, we next examined the expression levels of prohormones and intra-granular proteins in AtT-20 cells cultured under 20, 15, or 10% O2 (Figure 2) and MIN6 cells cultured under 20 or 10% O2 (Figure 3).
Expression and processing of intra-granular proteins in AtT-20 cells cultured at lower oxygen concentrations.
Expression and processing of intra-granular proteins in MIN6 cells cultured in 10% O2.
No significant differences in the amount of POMC, the precursor form of ACTH, were observed in AtT-20 cells cultured under 20, 15, and 10% O2, whereas the HIF-1α expression levels slightly increased in response to hypoxic culture conditions (Figure 2A,B). In contrast with POMC, the levels of the precursor and processed forms of secretogranin III (SgIII) and chromogranin A (CgA) significantly changed depending on the oxygen concentration (Figure 2A). As the oxygen concentration in the culture decreased from 21 to 10%, the amount of the precursor forms of SgIII (fragment a; ∼70 kDa) and CgA (fragment a′; ∼97 kDa) significantly decreased, whereas the levels of the processed forms of SgIII (fragment b; ∼35 kDa) and CgA (fragment b′; ∼20 kDa) significantly increased. These findings suggested that culture under lower oxygen concentrations promoted the processing of granins in endocrine cells.
Given that prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) are involved in the proteolytic processing of granins and prohormones, including POMC, in the pituitary gland [17,18], we next examined the changes in the expression levels of these processing enzymes in AtT-20 cells cultured under lower oxygen concentrations. As the oxygen concentration in the culture decreased from 21 to 10%, the levels of the mature forms of PC1/3 (66 kDa) and CPE (∼55 kDa) increased by ∼2.5-fold (at 10% O2) compared the control level in 21% O2 (Figure 2A,B), suggesting that the active forms of PC1/3 and CPE are up-regulated by lower oxygen culture conditions and promote the proteolytic processing of granins (SgIII and CgA) and prohormones, including POMC, in the SGs of AtT-20 cells. The up-regulation of the PC1/3 and CPE expression levels in AtT-20 cells in 10% O2 plateaued at 24 h (Supplementary Figure S3A,B), and 10% oxygen most effectively up-regulated both PC1/3 and CPE at 24 h (Supplementary Figure S3C,D).
In addition to the production of ACTH in AtT-20 cells, the expression levels of prohormone, granins, and processing enzymes were similarly examined in MIN6 cells producing insulin (Figure 3). As noted in AtT-20 cells, no significant difference in the amount of proinsulin was observed between cells cultured in 21 and 10% O2, whereas the levels of the precursor and processed forms of SgIII and CgA were significantly altered depending on the oxygen concentration. As the oxygen concentration in the culture decreased from 21 to 10%, the levels of the processed forms of SgIII (fragment b) and CgA (fragment b′) significantly increased. The levels of both PC1/3 and CPE significantly increased as the oxygen concentration in the culture decreased from 21 to 10% (Figure 3A,B). As previously established, proinsulin in pancreatic islet β-cells is cleaved by PC1/3 (at the B-chain/C-peptide junction) and PC2 (at the C-peptide/A-chain junction) followed by the removal of the COOH-terminal basic residues by CPE to produce mature insulin and C-peptide [18–20]. The level of the mature form of PC2 (∼70 kDa) in MIN6 cells cultured in 10% O2 were also significantly increased compared with that in MIN6 cells cultured in 20% O2 (Figure 3A,B), suggesting that lower oxygen culture conditions commonly up-regulate processing enzymes in endocrine cells, facilitating the proteolytic processing of granins and prohormones.
Processing enzyme expression was commonly up-regulated at the transcription level in neuro-endocrine cells in response to lower oxygen concentrations
Although mature forms of PC1/3 and PC2, which are responsible for the activation of prohormones in neuro-endocrine cells, were increased in response to lower oxygen concentrations, it should be clarified whether these processing enzymes are transcriptionally up-regulated or activated post-translationally by another member of the subtilisin/kexin-like proprotein convertase family, namely, furin. In both AtT-20 and MIN6 cells, the furin expression levels were not altered by the oxygen concentration used in culture (Figure 4), whereas the expression levels of the mature 66 kDa PC1/3 [in AtT-20 (Figure 4A,B) and MIN6 (Figure 4C,D) cells] and PC2 [in MIN6 (Figure 4C,D) cells] in 10% O2 increased by 2- to 3-fold compared with those cultured in 21% O2. Based on these findings, furin is not involved in the activation of PC1/3 and PC2 processing enzymes in response to being cultured under lower oxygen concentrations.
Expression and processing of PC1/3, PC2, and furin in AtT-20 or MIN6 cells cultured in 10% O2.
On the other hand, quantitative real-time PCR analyses clearly demonstrated that culturing AtT-20 cells in 10% O2 increased the PC1/3 and CPE mRNA levels normalized to β-actin by 1.4- and 1.3-fold, respectively, compared with the control levels in 21% O2 (Figure 5A). The PC1/3, PC2, and CPE levels normalized to β-actin also increased in MIN6 cells cultured in 10% O2 (by 1.5-, 2.2-, and 1.2-fold compared with the control levels in 21% O2, respectively; Figure 5B). These observations were consistent with the immunoblotting results (Figures 2 and 3) and suggest that the up-regulation of processing enzymes at the transcription level occurs commonly in neuro-endocrine cells in response to lower oxygen concentrations in culture.
Quantitative RT-PCR analysis of processing enzyme transcripts in AtT-20 and MIN6 cells.
Lower oxygen conditions also induced a response in mouse endocrine tissues
To examine whether the up-regulation of processing enzymes observed in cell lines cultured under lower oxygen conditions also occurs in vivo, we analyzed the secretion of peptide hormones and the expression of processing enzymes in mouse pituitary glands and pancreatic islets cultured under lower oxygen conditions.
Pituitary tissues excised from mice were cultured in 21 or 10% O2 for 24 h after dissection. No significant difference in basal ACTH secretion from pituitary tissues cultured for 30 min with 5 mM KCl was noted between the 21 and 10% O2 culture conditions. However, the stimulation of ACTH secretion from tissues cultured with 60 mM KCl in 10% O2 was significantly increased compared with that in 21% O2 (Figure 6A, left). The ACTH levels in pituitary tissues also increased approximately by 1.6-fold compared with the control level in 21% O2 (Figure 6A, right). No significant difference in the POMC expression levels was noted between pituitary tissue cultured in 21 and 10% O2; however, the PC1/3 and CPE expression levels significantly increased in tissues cultured in 10% O2 compared with those cultured in 21% O2 (Figure 6B,C). These findings indicated that the proteolytic processing of POMC to ACTH in the SGs of mouse pituitary corticotropes was accelerated by lower oxygen conditions.
Effect of 10% O2 on ACTH secretion and intracellular levels and the expression of processing enzymes in mouse pituitaries.
Similarly, pancreatic islets excised from mice were also cultured in 21 or 10% O2 for 24 h, and the basal and stimulated secretion of insulin from the islets were evaluated (Figure 7). After the islets were cultured in LG medium for 30 min in 21 or 10% O2, the islets were further incubated in LG and HG media under the same oxygen conditions for 30 min to measure the basal and stimulated secretion, respectively. Basal insulin secretion did not differ between 21 and 10% O2 culture conditions, whereas HG-induced insulin secretion in 10% O2 was significantly increased by 2.0-fold compared with that in 21% O2 (Figure 7A, left). The insulin content in the islets also increased by up to 1.6-fold after culture in 10% O2 compared with that in 21% O2 (Figure 7A, right). No significant difference in proinsulin expression levels was noted between the 21 and 10% O2 conditions, whereas the PC1/3, PC2, and CPE expression levels significantly increased with culture in 10% O2 compared with that in 21% O2 (Figure 7B,C). Thus, the proteolytic processing of proinsulin to mature active insulin in the SGs of the mouse pancreatic islets was also accelerated by lower oxygen conditions.
Effect of 10% O2 on insulin secretion and intracellular levels and the expression of processing enzymes in mouse pancreatic islets.
The up-regulation of processing enzymes in endocrine cells cultured in 10% O2 occurs independently of HIF-1α
We next examined whether HIF-1α was involved in the transcriptional activation of processing enzymes in AtT-20 cells cultured in 10% oxygen. HIF-1α is continuously degraded via the ubiquitin-proteasome pathway in 21% O2. In contrast, hypoxic conditions stabilize HIF-1α, resulting in the translocation of stabilized HIF-1α into the nucleus and the subsequent formation of a heterodimer with HIF-1β that functions as HIF-1 . Transition metals, such as CoCl2, artificially stabilize HIF-1α even in 21% O2 by blocking degradation . Thus, we analyzed the changes in the amount of processing enzymes in AtT-20 cells cultured in the presence of CoCl2 to evaluate the contribution of HIF-1α to the up-regulation of processing enzymes in cells cultured under lower oxygen conditions. Similar to those noted in AtT-20 cells, no significant differences in the PC1/3 and CPE levels were noted between cells cultured either with or without CoCl2; however, the HIF-1α levels significantly changed depending on the CoCl2 concentration (Figure 8). The CoCl2 culture concentration also did not affect SgIII processing (Figure 8). These data suggest that the up-regulation of processing enzymes under low oxygen culture conditions occurs independently of HIF-1α.
Up-regulation of processing enzymes in response to lower oxygen concentrations occurs independently of the HIF-1α expression levels.
In neuro-endocrine cells, prohormones, which are precursors of active hormones, are sorted at the TGN to the SGs, where the prohormones are processed into mature active hormones through the proteolytic cleavage by the PC1/3 and/or PC2 followed by the removal of C-terminal basic amino acids by CPE . Several transcription factors have been implicated in the regulation of PC1/3, PC2, and CPE. The nescient helix-loop-helix (Nhlh)-2 transcription factor was identified as the first transcription factor regulating PC1/3 and PC2 in both hypothalamic neurons , and the STAT3/Nhlh-2 heterodimer is required for the full transcription response of PC1/3 upon leptin stimulation in the hypothalamus . In pancreatic β-cells, PAX6 regulates PC1/3 and PC2 expression . Furthermore, EGR-1 and Sirt1 possibly enhance PC2 expression [26,27], and FoxO1 and Sirt1 regulate CPE expression [28,29]. However, to date, very few reports have demonstrated that lower oxygen concentrations in cell culture affect the expression levels of the transcription factors and processing enzymes that are involved in the production/secretion of the mature active hormone. The previous report demonstrated that the bioactivity of peptidyl-glycine α-amidating monooxygenase (PAM), an enzyme that catalyzes the conversion of the C-terminal glycine of peptide hormones into their bioactive amidated forms, highly depends on the change in oxygen concentration . The enzymatic activity of PAM was strongly reduced under hypoxic conditions (<4% O2), although the expression level of PAM was not changed depending on the oxygen conditions . Another report further suggested a putative relationship between the expression levels of PAM and hypoxia-dependent changes in gene regulation .
The present study first demonstrated that culture under 10% O2 significantly increased the production and stimulated the release of mature active hormones in neuro-endocrine cells, including both established cell lines (AtT-20 and MIN6) and primary cultures of mouse endocrine tissues (pituitary glands and pancreatic islets). Culture in 10% O2 up-regulated the prohormone-processing enzymes PC1/3, PC2, and CPE, thus increasing the efficiency of the proteolytic conversion of prohormones to their active form in the SGs, while culture in extremely low oxygen conditions, such as hypoxic conditions (<5% O2), disrupted the regulated secretion in endocrine cells  (Supplementary Figure S1).
In general, the cellular responses to hypoxia (<5% O2) are mainly controlled by the HIF family [1–3]. HIF-1, a representative member of the HIF protein family, consists of constitutively expressed HIF-1β and oxygen-regulated HIF-1α subunits. The stability of HIF-1α is regulated by post-translational modifications, such as hydroxylation, ubiquitination, acetylation, and phosphorylation, which are crucial for its bioactivity . Under normoxic conditions, the oxygen-dependent degradation domain of HIF-1α triggers its association with pVHL, leading to HIF-1α degradation via the ubiquitin-proteasome pathway. In contrast, the HIF-1α subunit becomes stable under hypoxic conditions forms the HIF-1 complex by binding to the HIF-1β subunit. The HIF-1 complex functions as a master transcription factor by binding to the promoter regions of many genes that are up-regulated in response to hypoxic conditions [34,35]. Thus, HIFs heavily influence gene expression profiles and phenotypic changes under hypoxic conditions. However, the up-regulation of processing enzymes in 10% oxygen demonstrated in the present study appeared to occur independently of HIFs; the PC1/3 and CPE expression levels in normoxic cultures were not affected by CoCl2, which can promote the HIF-1 signaling by preventing HIF-1α degradation [22,36]. These findings strongly suggest that the up-regulated levels of the prohormone-processing enzymes induced under 10% O2 culture conditions are not a pathological response to hypoxia that occurs downstream of HIF-1 signaling but rather a physiological state that possibly reflects the microenvironment in a living body.
Although the oxygen concentration in the air we breathe is ∼21%, the concentration of oxygen in the body is significantly reduced during gas exchange in the lung and transportation via circulation to the peripheral organs/tissues where oxygen is consumed in the intracellular mitochondria to produce ATP. The physiological oxygen levels in the blood range between 10% and 13% , and those in normal peripheral tissues are estimated to be 3.6–12.8% [4,38–42]. These oxygen concentrations in the body of living animals are considerably <21%, which has been conventionally used for cell culture in laboratories. Thus, conventional conditions for cell culture under ambient 21% atmospheric O2 are hyperoxic compared with the putative physiological condition of differentiated tissues/cells in the body. The findings in the present study indicate that 10% O2 is the optimal microenvironment for endocrine tissues/cells to efficiently generate and secrete active peptide hormones.
As demonstrated in the present study, the protein levels of processing enzymes normalized to β-actin in 10% O2 culture increased by 2.8-fold compared with that in 21% O2 culture, whereas mRNA levels normalized to β-actin increased by up to ∼1.5-fold in 10% O2 compared with those in 21% O2. This discrepancy in the ratio of the increase between the protein contents and transcription levels suggests that protein stability contributes to an increase in the activities of processing enzymes under the 10% O2 culture conditions. Similar to many other secreted proteins, processing enzymes, including PC1/3, PC2, and CPE, are modified via glycosylation events that may impose enhanced stability [43,44]. The previous study reported that hypoxia promotes the glycosylation of proteins  and favors the involvement of the post-translational stabilization of processing enzymes at relatively lower O2 concentrations. In addition to the up-regulation of processing enzymes at both the transcriptional and translational levels, the enzymatic activity of these enzymes might be post-translationally enhanced in response to the 10% O2 culture condition. The precise mechanisms by which 10% oxygen conditions up-regulates the expression of processing enzymes and whether 10% oxygen up-regulates the enzymatic activities of processing enzymes should be further investigated in detail in the future.
In summary, we first demonstrated in the present study that the production of active hormones in neuro-endocrine cells/tissues is accelerated under the 10% O2 culture conditions by the up-regulation of prohormone-processing enzymes. However, this up-regulation in 10% O2 culture is not a pathological response to hypoxia but rather a physiological state given that this phenomenon occurred independently of the HIF-1 signaling induced by CoCl2. Judging from the present findings, the conventional culture conditions of ambient 21% O2 that is commonly used in laboratories is possibly hyperoxic and does not facilitate the maximal potential of endocrine cells/tissues. For a more appropriate assessment of the functional states of endocrine cells in differentiated living tissues, experiments performed in 10% O2 culture conditions are preferable to those performed under ambient 21% O2 conditions. If the oxygen concentrations in living animal tissues could be precisely measured and determined in real time with the aid of various methods, including invasive and non-invasive methods, such as oxygen needle electrodes, immunohistochemical staining (e.g. nitroimidazole and its derivatives), positron emission tomography, and optical imaging , we could further determine the physiological state of neuro-endocrine cells/tissues under various experimental conditions in a more appropriate microenvironment. The optimal/physiological concentration of O2 in the microenvironment of functionally differentiated cells/tissues in living animals should be further clarified.
M.H. and T.W. conceived and designed the experiments and wrote the manuscript. E.S. performed most of the experiments, analyzed the data, and prepared the figures. E.S. and Y.S. performed the mouse studies, including tissue culture. E.S., Y.M., A.H., and D.K. performed the biochemical experiments. H.G. and S.T. contributed to the experimental design and data analysis.
This work was supported by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (JP25440052 and JP18K05443 to M.H.); the Joint Research Program of the Institute for Molecular and Cellular Regulation, Gunma University (#18011 to M.H. and S.T.); and Akita Prefectural University President's Research Project Fund to M.H.
The Authors declare that there are no competing interests associated with the manuscript.