Abstract
Hypogonadism in men is associated with an increased incidence of type 2 diabetes. Supplementation with testosterone has been shown to protect pancreatic β-cell against apoptosis due to toxic substances including streptozotocin and high glucose. One of the pathological mechanisms of glucose-induced pancreatic β-cell apoptosis is the induction of the local rennin–angiotensin–aldosterone system (RAAS). The role of testosterone in regulation of the pancreatic RAAS is still unknown. This study aims to investigate the protective action of testosterone against glucotoxicity-induced pancreatic β-cell apoptosis via alteration of the pancreatic RAAS pathway. Rat insulinoma cell line (INS-1) cells or isolated male mouse islets were cultured in basal and high-glucose media in the presence or absence of testosterone, losartan, and angiotensin II (Ang II), then cell apoptosis, cleaved caspase 3 expression, oxidative stress, and expression of angiotensin II type 1 receptor (AGTR1) and p47phox mRNA and protein were measured. Testosterone and losartan showed similar effects in reducing pancreatic β-cell apoptosis. Testosterone significantly reduced expression of AGTR1 protein in INS-1 cells cultured in high-glucose medium or high-glucose medium with Ang II. Testosterone decreased the expression of AGTR1 and p47phox mRNA and protein in comparison with levels in cells cultured in high-glucose medium alone. Furthermore, testosterone attenuated superoxide production when co-cultured with high-glucose medium. In contrast, when cultured in basal glucose, supplementation of testosterone did not have any effect on cell apoptosis, oxidative stress, and expression of AGT1R and p47phox. In addition, high-glucose medium did not increase cleaved caspase 3 in AGTR1 knockdown experiments. Thus, our results indicated that testosterone prevents pancreatic β-cell apoptosis due to glucotoxicity through reduction of the expression of ATGR1 and its signaling pathway.
Introduction
Diabetes mellitus is a chronic metabolic disease that affects the quality of life of millions of people worldwide. Type 2 diabetes (T2D) is characterized by increased levels of glucose due to impairment of insulin action and/or insulin secretion (Lin & Sun 2010). The progressive failure of pancreatic β-cells to secrete adequate amounts of insulin to compensate for insulin resistance leads to hyperglycemia (Weir et al. 2001). Prolonged or chronic hyperglycemia contributes to detrimental effects in various tissues, such as kidneys, eyes, peripheral nerves, and microvasculature, that lead to diabetic complications (Forbes & Cooper 2013).
Chronic hyperglycemia generates glucotoxicity which causes pancreatic β-cell dysfunction and death, leading to reductions in pancreatic β-cell mass (Federici et al. 2001). There are several pathways causing glucotoxicity of pancreatic β-cells, including increased production of reactive oxygen species (ROS; Evans et al. 2002), protein glycation (Tajiri et al. 1997), and glucose auto-oxidation (Wolff & Dean 1987). Although the accumulation of oxidative stress has been proposed to be a major mechanism of glucotoxicity, antioxidant substances have been shown to confer only partial protection against pancreatic β-cell death due to glucotoxicity (Lonn et al. 2005). Therefore, it has been proposed that there are still unidentified pathways involved in glucotoxicity. One of the novel pathological pathways of glucotoxicity is the local rennin–angiotensin–aldosterone system (RAAS). Local RAAS has been reported in several organs including liver and pancreas (Leung & Carlsson 2001). In the pancreas, the local RAAS is divided into the acinar RAAS and islet RAAS. The acinar RAAS regulates exocrine function and the islet RAAS has a role in glucose homeostasis by regulating glucose-induced insulin secretion (Leung 2007). The expression of RAAS components, such as angiotensinogen (AGT), angiotensin-converting enzyme, angiotensin II type 1 receptor (AGTR1) and AGTR2, in human and mouse islets has been demonstrated (Lam & Leung 2002, Lau et al. 2004). RAAS blockers decreased blood glucose levels in diabetes patients (Scheen 2004). Also, the expression of Agtr1 mRNA is upregulated due to hyperglycemia in obesity-induced T2D mice. In these mice, local pancreas angiotensin II (Ang II) can inhibit insulin secretion by decreasing production of (pro)insulin via AGTR1 (Chu et al. 2006). In addition, AGTR1 is expressed in rat insulinoma cell line (INS-1) cells and Ang II decreased glucose-stimulated insulin secretion (Leung & Leung 2008). Also, knockout of Nox2, a NADPH oxidase components, in mice conferred a protective effect against streptozotocin (STZ)-induced diabetes (Xiang et al. 2010). These results indicate that pancreatic RAAS plays an important role in high-glucose-induced pancreatic β-cell death.
The incidence of T2D has been found to increase in males with hypogonadism (Rhoden et al. 2005, Grossmann et al. 2009). Results from previous studies have indicated that testosterone replacement therapy ameliorated insulin resistance and improved glycemic control in hypogonadal men with T2D (Kapoor et al. 2006). Also, testosterone treatment has been shown to improve pancreatic β-cell function by decreasing proinflammatory cytokines such as interleukin-6 (IL6), IL1β, and tumor necrosis factor-α in hypogonadal men with T2D (Kapoor et al. 2007). In addition, it has been shown that the insulin concentration, insulin receptor mRNA, and glucose oxidation were decreased in orchidectomized rats, and these conditions were reversed by testosterone replacement (Muthusamy et al. 2007). In an in vitro study, testosterone has been shown to induce uptake of calcium and to stimulate insulin secretion in isolated pancreatic islets (Grillo et al. 2005). Moreover, testosterone replacement conferred protection against pancreatic β-cell apoptosis in STZ-diabetic male rats (Palomar-Morales et al. 2010). However, it remains unclear whether or not testosterone protects against pancreatic β-cell death via an effect on local RAAS.
Materials and methods
Animals
This work was approved by the Siriraj Animal Care and Use Committee (SI-AUCC). Male imprinting control region (ICR) outbred 8–12-week-old mice were purchased from the National Laboratory Animal Center, Mahidol University, Bangkok, Thailand.
Mouse pancreatic islet isolation
Pancreatic islets were isolated by collagenase digestion according to a modification of the method of Lacy & Kostianovsky (1967) and Gotoh et al. (1985). Briefly, pancreases were infused with collagenase-P and digested at 37 °C. The islets were separated by using histopaque gradient and manually picked under a stereomicroscope.
Cell culture
Isolated islets and INS-1 cells were grown in RPMI-1640 supplemented with 10% heat-activated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. The culture medium was changed every 2 days. The cells were normally cultured in medium containing 11.1 mM glucose, which was used as the control condition, and they were cultured in approximately four times the concentration (40 mM) of glucose to create the condition of glucotoxicity.
Analysis of cell apoptosis using Annexin V–FITC/propidium iodide staining
INS-1 cells were cultured in 11.1 mM or 40 mM glucose in the presence or absence of 1 μM Ang II, with or without 0.05 μg/ml testosterone or 1 μM losartan or a combination of both. After incubation for 72 h, cells were collected and stained with Annexin V–FITC and propidium iodide (PI) (Becton Dickinson, Franklin Lakes, NJ, USA). The apoptotic cells were then analyzed using a FACSort flow cytometer (Becton Dickinson).
Analysis of cell apoptosis by measurement of cleaved caspase 3 expression
INS-1 cells were cultured in 11.1 mM or 40 mM glucose in the presence or absence 0.05 μg/ml testosterone or 1 μM losartan. After incubation for 72 h, cells were lysed in radioimmunorecipitation assay (RIPA) buffer and western blotting analysis of cleaved caspase 3 expression was performed.
RNA isolation and real-time RT-PCR
Total RNA of isolated male mouse pancreatic islets and INS-1 cells were extracted using a High Pure RNA isolation kit (Roche Diagnostic Corporation) according to the manufacturer's instructions. The RNA concentration was measured using a Nano Drop-1000 spectrophotometer. RT was carried out on 1 μg of RNA using the SuperScript III RT kit with random hexamer primer (Invitrogen), according to the manufacturer's instructions. Quantitative real-time PCR was then performed using SYBR Green reaction mix and an Mx3005P instrument (Stratagene Agilent, Cedar Creek, TX, USA). The specific primer sequences for rat AGTR1 were forward: 5′-CAGTGTGCGCGTTTCATTATG-3′ and reverse: 5′-TGGTAAGGCCCAGCCCTAT-3′; those for rat p47phox were forward: 5′-GGAGCTTATGAATGACCTCGAT-3′ and reverse: 5′-CTACGCAGGTGAACCGTATGTA-3′; those for mouse AGTR1 were forward: 5′-CCATTGTCCACCCGATGAAG-3′ and reverse: 5′-TGCAGGTGACTTTGGCCAC-3′; those for mouse p47phox were forward: 5′-AGCCTGAGACATACCTGGTG-3′ and reverse: 5′-AGACTTCTGCAGATACATGG-3′ (Leung & Leung 2008); those for mouse β-actin were forward: 5′-ATGAAGTGTGACGTTGACATCGTC-3′ and reverse: 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′. The comparative threshold cycles (Ct) of tested mRNA and β-actin were measured and their difference (ΔCt) was calculated. The relative expression was then calculated by 2−ΔΔCt method, compared with the control sample.
Knock down Agtr1
Transfection of siRNA directed against Agtr1 mRNA (Dharmacon, Lafayette, CO, USA) was performed using Lipofectamine 2000 (Invitrogen) as detailed by the manufacturer. INS-1 cells were seeded into six-well plates for 24 h before transfection. The double-stranded siRNA were transfected. After 6 h, the medium was changed to a complete culture medium. As a control, cells were treated with siRNA-control (Dharmacon) under identical conditions. At 24 h after siRNA transfection, cells were treated with 11.1 mM or 40 mM glucose for 72 h. Then, the cells were harvested for further examination by western blotting analysis.
Western blotting analysis
The cells were lysed in a RIPA buffer (Pierce, Waltham, MA, USA) and total proteins were quantified using a micro bicinchoninic acid protein assay kit. Total proteins were separated by SDS–PAGE and transferred to polyvinylidene fluoride membrane (Bio-Rad). After that, the membrane was blocked with 5% skimmed milk. The membrane was incubated with one of the following primary antibodies: rabbit polyclonal anti-AGTR1 (Santa Cruz Biotechnology), goat polyclonal anti-p47phox (Santa Cruz Biotechnology), rabbit monoclonal anti-cleaved caspase 3 (Cell Signaling, Boston, MA, USA), or mouse monoclonal anti-β-actin (Santa Cruz Biotechnology), overnight at 4 °C. Then, the membrane was washed and incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology). The protein bands were detected by ECL (Pierce). The band intensities of proteins were analyzed using Image J densitometry software version 1.43.
Measurement of intracellular superoxide generation
Superoxide production was detected using the nitroblue tetrazolium (NBT) assay. Briefly, INS-1 cells were cultured in 11.1 mM or 40 mM glucose with or without 0.05 μg/ml testosterone for 72 h. Next, cells were incubated with NBT for 90 min. The cells were then lysed in potassium hydroxide and were dissolved in DMSO. The amount of superoxide production was determined from optical density at a wavelength of 630 nm measured using a PowerWave microplate scanning spectrophotometer (BIO-TEK, Winooski, VT, USA).
Statistical analyses
All data were generated from triplicated experiments and expressed as mean±s.e.m. The differences between the groups of results were determined by one-way ANOVA, followed by Tukey's post hoc test. A P value of less than 0.05 was considered to be statistically significant.
Results
Testosterone decreased apoptosis of pancreatic β-cells cultured in high-glucose (40 mM) medium
To examine whether testosterone protected against high-glucose-induced pancreatic β-cell apoptosis, apoptotic cells were measured by Annexin V–FITC/PI staining. As shown in Fig. 1A, high-glucose (40 mM) medium significantly increased the percentage of apoptosis in INS-1 cells compared with those cultured in basal-glucose (11.1 mM) medium. The presence of testosterone in INS-1 cells cultured in high-glucose medium significantly reduced the percentage of cell apoptosis. However, testosterone had no effect on the percentage of apoptosis in INS-1 cells cultured in basal-glucose medium.
Effect of testosterone, losartan, and Ang II on apoptosis of pancreatic β-cells cultured in basal- and high-glucose media. INS-1 cells were cultured in basal-glucose (11.1 mM) or high-glucose (40 mM) medium in the presence or absence of testosterone (0.05 μg/ml) for 72 h. Apoptotic INS-1 cells were detected using Annexin V–FITC/PI staining. The bar graph shows the average percentage undergoing early apoptosis (A). INS-1 cells were cultured in basal or high-glucose medium in the presence or absence of Ang II, with or without testosterone or losartan or a combination of both for 72 h. This bar graph shows the percentage of early apoptosis (B). INS-1 cells were cultured in basal- or high-glucose media in the presence or absence of testosterone or losartan for 72 h. These bar graphs show caspase 3 activity (C and D). All data are expressed as mean±s.e.m. of three to four independent experiments. +++P<0.001 compared with the basal glucose-treated group. †††P<0.001, *P<0.05, **P<0.01 compared with the high-glucose-treated group. ***P<0.001 compared with the high glucose- and Ang II-treated group.
Citation: Journal of Endocrinology 224, 3; 10.1530/JOE-14-0397
Testosterone and angiotensin II receptor blocker decreased apoptosis of pancreatic β-cells induced by high-glucose medium and Ang II
Previous studies have reported that Ang II production was increased in mice with T2D, which might be produced by pancreatic islet RAAS (Lau et al. 2004, Leung 2007). To examine whether Ang II or AGTR1 is important for pancreatic β-cell apoptosis, INS-1 were cultured in basal-glucose or high-glucose medium with or without Ang II and in the presence or absence of testosterone, losartan, or both. As shown in Fig. 1B, the percentage of apoptotic cells was significantly increased in INS-1 cells cultured in high-glucose medium or high-glucose medium with Ang II, when compared with those cultured in basal glucose medium. Importantly, testosterone or losartan in INS-1 cells cultured in high-glucose medium with Ang II significantly reduced the percentage of apoptotic cells. However, testosterone and losartan did not have synergic effect on apoptosis of INS-1 cells cultured in high-glucose medium with Ang II. Surprisingly, Ang II did not trigger a significant increase in the percentage of cell apoptosis in basal-glucose medium.
We further confirmed our cell apoptosis results using the levels of cleaved caspase 3. INS-1 cells were cultured in basal- and high-glucose media with or without testosterone or in the presence or absence of losartan. Then, cleaved caspase 3 levels were determined by western blot analysis. Cleaved caspase 3 was significantly increased in INS-1 cells cultured in high-glucose medium, while treatment with testosterone or losartan of INS-1 cells cultured in high-glucose medium significantly reduced the levels of cleaved caspase 3 compared with those of cells cultured with basal-glucose with or without testosterone or losartan.
Testosterone decreased AGTR1 expression in pancreatic β-cells activated by high-glucose medium and Ang II
To further examine effect of Ang II on expression of AGTR1 protein in basal-glucose medium, INS-1 cells were cultured in basal-glucose medium with or without Ang II and expression of AGTR1 protein was determined by western blotting analysis. Ang II did not affect the expression of AGTR1 protein in INS-1 cells cultured in basal-glucose medium (Fig. 2A). In contrast, the presence of Ang II in INS-1 cells cultured with high-glucose medium significantly increased the expression of AGTR1 compared with that in cells cultured in high-glucose medium alone (Fig. 2B). To verify whether testosterone decreased the expression of AGTR1 in INS-1 cells induced by high-glucose medium or high-glucose medium with Ang II, AGTR1 protein was assessed by western blotting analysis. Testosterone markedly reduced the expression of AGTR1 protein in INS-1 cells co-cultured in high-glucose medium or high-glucose medium with Ang II.
Effect of basal- and high-glucose media, Ang II, and testosterone on expression of AGTR1 protein in INS-1 cells. INS-1 cells were cultured with basal-glucose medium with or without Ang II for 72 h. The image shows a representative western blot of AGTR1 and β-actin proteins. The bar graph below shows the fold changes in AGTR1 protein normalized to β-actin protein (A). INS-1 cells were cultured in high-glucose medium in the presence or absence of Ang II, with or without testosterone for 72 h. The image shows a western blot of AGTR1 and β-actin proteins. The bar graph below shows fold changes in AGTR1 protein relative to the control group, and normalized to β-actin protein (B). All data are expressed as mean±s.e.m. of three independent experiments. +P<0.05,++P<0.01 compared with the high-glucose-treated group. ***P<0.001 compared with the high-glucose- and Ang II-treated group. NS, not significant.
Citation: Journal of Endocrinology 224, 3; 10.1530/JOE-14-0397
Testosterone attenuated the expression of AGTR1 mRNA and protein in pancreatic β-cells cultured in high-glucose medium
To investigate whether high-glucose-induced pancreatic β-cells apoptosis was associated with upregulation of the AGTR1 pathway and whether testosterone could reduce AGTR1 expression, we assessed the expression of AGTR1 mRNA and protein by real-time RT-PCR and western blotting analysis respectively. As shown in Fig. 3A and B, AGTR1 mRNA and protein expression were markedly increased in INS-1 cells cultured in high-glucose medium compared with those cultured in basal-glucose medium. Testosterone significantly reduced the expression of AGTR1 mRNA and protein in INS-1 cells cultured in high-glucose medium. In contrast, testosterone did not show any effect on expression of AGTR1 mRNA and protein in INS-1 cells cultured with basal-glucose medium. Similarly, expression of AGTR1 mRNA and protein was upregulated in isolated male mouse pancreatic islets cultured in high-glucose medium, whereas addition of testosterone to high-glucose medium downregulated AGTR1 mRNA and protein expression (Fig. 3C and D).
Effect of testosterone on the expression of Agtr1 mRNA and protein in INS-1 cells (A and B) and isolated male mouse pancreatic islets (C and D) cultured in high-glucose medium. Fold changes in Agtr1 mRNA normalized to β-actin mRNA at 48 h (A and C). The images show representative western blots of AGTR1 and β-actin from INS-1 cells. The bar graphs below show AGTR1 protein levels normalized to β-actin protein (B and D). The data are presented as mean±s.e.m. of four independent experiments. +P<0.05,++P<0.01 compared with the basal-glucose-treated group. *P<0.05, **P<0.01 compared with the high-glucose-treated group.
Citation: Journal of Endocrinology 224, 3; 10.1530/JOE-14-0397
Testosterone decreased p47phox mRNA and protein expression in pancreatic β-cells cultured in high-glucose medium
AGTR1 activation was shown to induce oxidative stress via activation of NADPH oxidase. P47phox is a subunit of activated NADPH oxidase. To examine whether testosterone decreased NADPH oxidase activation, p47phox mRNA and protein were measured by real-time RT-PCR and western blot analysis respectively. As shown in Fig. 4A and B, expression of p47phox mRNA and protein was markedly increased in INS-1 cells cultured in high-glucose medium compared with those cultured in basal-glucose medium. However, testosterone reduced the expression of p47phox mRNA and protein in INS-1 cells cultured in high-glucose medium but did not affect the expressions of p47phox mRNA and protein in basal-glucose medium. Moreover, the expression of p47phox mRNA and protein was significantly increased in isolated male mouse pancreatic islets cultured in high-glucose medium when compared with those cultured in basal-glucose medium. As expected, testosterone in high-glucose medium significantly decreased the expression of p47phox mRNA and protein (Fig. 4C and D).
Effect of testosterone on the expression of p47phox mRNA and protein in INS-1 cells (A and B) and isolated male mouse pancreatic islet cells (C and D) cultured in high-glucose medium. Fold change in p47phox mRNA normalized to β-actin mRNA at 48 h (A and C). The images show representative western blots of AGTR1 and β-actin from INS-1 cells. The bar graphs below show p47phox protein levels normalized to β-actin protein (B and D). The data is presented as mean±s.e.m. of four independent experiments. ++P<0.01,+++P<0.001 compared with the basal-glucose-treated group. *P<0.05, **P<0.01 compared with the high-glucose-treated group.
Citation: Journal of Endocrinology 224, 3; 10.1530/JOE-14-0397
Testosterone reduced superoxide production in pancreatic β-cells cultured in high-glucose medium
To further examine whether the reduction in high-glucose-induced pancreatic β-cell apoptosis due to testosterone was mediated by decreased NADPH oxidase activity that reduced production of ROS, superoxide anion in INS-1 cells was measured by NBT assay. The superoxide anion production in INS-1 cells cultured with high-glucose medium was significantly increased compared with those cultured with basal-glucose medium, whereas testosterone decreased superoxide production in INS-1 cells cultured in high-glucose medium. No significant changes were found in INS-1 cells cultured in basal-glucose medium in the presence or absence of testosterone (Fig. 5).
Superoxide production in INS-1 cells cultured in high-glucose medium. Superoxide production was measured using an NBT assay. The amount of superoxide was determined from the optical density (OD). The bar graph shows production of superoxide in INS-1 cells. The data are presented as mean±s.e.m. of three independent experiments. ++P<0.01 compared with the basal-glucose-treated group (11.1 mM); *P<0.05 compared with the high-glucose-treated group (40 mM).
Citation: Journal of Endocrinology 224, 3; 10.1530/JOE-14-0397
AGTR1 knockdown rescued pancreatic β-cells apoptosis from high-glucose medium
To confirm the role of testosterone in reducing pancreatic β-cells apoptosis and AGTR1 expression, AGTR1 silencing were performed in INS-1 cells cultured with basal- or high-glucose media (Fig. 6). After AGTR1 knockdown, cellular apoptosis was determined by detection of cleaved caspase 3 using western blotting analysis. Expression of AGTR1 protein was not observed in INS-1 cells cultured in basal- and high-glucose media with si-AGTR1. Cleaved caspase 3 was not detected in INS-1 cells cultured in high-glucose medium with AGTR1 knockdown, which was similar to the results for cells cultured in basal-glucose medium with mock treatment, si-control, and si-AGTR1. Whereas INS-1 cells cultured in high-glucose medium with mock treatment and si-control showed markedly increased cleaved caspase 3 and AGTR1 protein levels when compared with those cultured in basal-glucose medium. These results indicated that AGTR1 silencing protected pancreatic β-cells against glucotoxicity.
Effect of AGTR1 knockdown on levels of cleaved caspase 3. INS-1 cells cultured in basal- and high-glucose media with mock treatment, si-control, and si-AGTR1. Cleaved caspase 3 and AGTR1 were detected by western blot analysis. The image shows a representative western blot of cleaved caspase 3, AGTR1, and β-actin from INS-1 cells. The bar graph below shows cleaved caspase 3 protein levels normalized to β-actin protein. The results are presented as mean±s.e.m. of four independent experiments. ***P<0.001 compared with the high-glucose-treated group.
Citation: Journal of Endocrinology 224, 3; 10.1530/JOE-14-0397
Discussion
There have been increasing evidence indicative of a role for the pancreatic RAAS in the pathophysiology of T2D (van der Zijl et al. 2012). It has been shown in previous studies that Ang II production was increased in a rodent model of T2D, and that Ang II modulated AGTR1 expression in pancreatic β-cells (Singh et al. 2008, Chu & Leung 2009, Hirata et al. 2009). Addition of Ang II to β-cells cultured in basal-glucose medium has been shown to reduce glucose-stimulated insulin secretion (Chu et al. 2006). Ang II binds to AGTR1, which is a major Ang II receptor in pancreatic β-cells. AGTR1 was also upregulated in animal models of T2D (Nakayama et al. 2005, Chu et al. 2006) and AGTR1 blocker improved β-cell mass and function in T2D (Chu et al. 2006, Hasegawa et al. 2009). Therefore, pancreatic RAAS may be another pathological mechanism of glucotoxicity-induced pancreatic β-cell apoptosis.
In this study, we have shown that testosterone protected pancreatic β-cells against apoptosis when these cells were cultured in high-glucose medium. Also, it has been revealed in this study that the level of pancreatic β-cell apoptosis was approximately the same for INS-1 cells cultured either in high-glucose medium or in high-glucose medium with addition of Ang II, which indicates that a similar pathway might be exploited for either condition. Either testosterone or losartan decreased pancreatic β-cell apoptosis, which was induced by high-glucose medium alone or high-glucose medium with Ang II to approximately equal levels. Interestingly, treatment with both testosterone and losartan reduced pancreatic β-cell apoptosis similarly to testosterone or losartan alone, indicating that testosterone and losartan had no synergistic action in reducing β-cell apoptosis. This result was confirmed by detection of cleaved caspase 3 by western blot analysis. In correspondence with the previous result, INS-1 cells cultured in high-glucose media with testosterone or losartan displayed significantly decreased levels of the active form of caspase 3 when compared with those cultured in basal-glucose media. The results of Annexin V/PI staining also indicated that there was no difference in the level of pancreatic β-cell apoptosis for the INS-1 cells cultured in basal-glucose medium with or without testosterone, losartan, or both. Hence, it is possible that testosterone and losartan may exploit the same pathway to protect against glucotoxicity. Another interesting finding was that Ang II did not induce β-cell apoptosis in cells cultured in basal-glucose medium. This result indicates that expression of AGTR1 may be an important factor in Ang II-increased β-cell apoptosis. To verify this observation, expression of AGTR1 in the INS-1 cells cultured in basal-glucose medium with or without Ang II was determined. AGTR1 was found to be expressed at similar levels in the INS-1 cells cultured in basal-glucose medium or in basal-glucose medium with Ang II. This result indicates that addition of Ang II to basal-glucose medium does not increase pancreatic β-cell apoptosis as it does not influence the AGTR1 signaling pathway. While the addition of Ang II to high-glucose medium increased AGTR1 expression and testosterone decreased AGTR1 expression under the same extent in both conditions that was induced by high-glucose medium and high-glucose medium with the addition of Ang II. Again, expression of AGTR1 correlated with β-cell apoptosis, which supports the hypothesis that both testosterone and losartan attenuate the AGTR1 signaling pathway. Altogether, our results indicate that expression of AGTR correlates with pancreatic β-cell apoptosis while Ang II has an effect on pancreatic β-cell apoptosis when AGTR1 is upregulated. Similarly, in separate experiments, expression of AGTR1 mRNA and protein were increased in both INS-1 cells and isolated male mouse islets when they were cultured under high-glucose conditions. Interestingly, testosterone ameliorated the effect of high-glucose-medium-induced AGTR1 expression in pancreatic β-cells but had no effect on AGTR1 expression in cells-cultured in basal-glucose medium. These results indicate that testosterone reduces both AGTR1 mRNA and protein levels in high-glucose medium.
The mechanistic effect of AGTR1 activation on pancreatic β-cell death is the induction of NADPH oxidase and ROS generation (Shao et al. 2006, Leung 2010). NADPH oxidase subunits in pancreatic β-cells are composed of p22phox, gp91phox, and p47phox (Oliveira et al. 2003). The results of this study also indicated that high-glucose medium increased expression of p47phox mRNA and its protein in both a pancreatic β-cell line and isolated male mouse islets and also increased ROS production in the β-cell line. Supplementation of the high-glucose medium with testosterone reduced the expression of p47phox mRNA and protein and production of ROS. These results are consistent with the previous finding that for human pancreatic islets prolonged culture in high-glucose increased expression of p47phox and p22phox (Lastra & Manrique 2007). Also, culturing INS1-E cells in high-glucose medium has been shown to induce expression of p47phox mRNA (Leung & Leung 2008). Furthermore, inhibition of AGTR1 decreased Ang II-induced ROS production in pancreatic β-cells (Hirata et al. 2009). Taken together, the results from this study indicate that chronic high levels of glucose induce AGTR1 and NADPH oxidase activation, which produces ROS in pancreatic β-cells, and that testosterone protects against high-glucose-induced β-cell apoptosis through the inhibition of AGTR1-mediated NADPH-oxidase-induced ROS production both in vitro and ex vivo.
The results from our previous study indicated that testosterone conferred protection against high-glucose-induced pancreatic β-cell apoptosis via decreased endoplasmic reticulum (ER) stress (Hanchang et al. 2013). The results of this study indicated that testosterone protected against high-glucose-induced pancreatic β-cell apoptosis via suppression of AGTR1 and its signaling pathway. Thus, there may be a connection between AGTR1 activation and ER stress. This notion was supported by results from a recent study which indicated that high levels of glucose up-regulated the expression of AGTR1 and AGT mRNA in human islets, leading to the activation of islet RAAS (Madec et al. 2013). AGTR1 was activated through phospholipase C and caused the depletion of ER calcium. The reduction in ER calcium levels induced ER stress, which then induced β-cell dysfunction and apoptosis. Losartan showed a protective effect against glucotoxicity via inhibition of expression of AGTR1 and reduction of ER stress (Madec et al. 2013). All of these lines of evidence indicated that testosterone and losartan inhibit similar aspects of high-glucose activated AGTR1. To verify this notion, induction of AGTR1 silencing was performed. High-glucose medium induced cleaved caspase 3. In contrast, cleaved caspase 3 was diminished by siRNA against AGTR1 but not si-control in INS-1 cultured in high-glucose medium. Thus, testosterone and losartan protect pancreatic β-cells against apoptosis due to glucotoxicity through reduction of expression of AGTR1 and ER stress.
Conclusion
Our results indicated that testosterone protects against glucotoxicity-induced β-cell apoptosis by means of a decrease in the action of the AGTR1 signaling pathway both in vitro and ex vivo. The molecular mechanism by which testosterone inhibits the activation of AGTR1 is still unknown. Identification of the molecular mechanism by which testosterone reduces AGTR1 signaling merits further investigation.
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 Siriraj Research Development Grant (to S K) and Siriraj Chalearmprakiat Fund (to S K, T L, and P Y), Mahidol University Grant (to S K), Siriraj Graduate Thesis Scholarship (to W H), TRF Mid-Carreer Research Grant (to T L), and TRF Senior Research Scholar Grant (to P Y).
Acknowledgements
The authors thank to Malika Churintaraphan and Samarn Onreabroi for their technical assistances.
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