Abstract
It has been shown that angiotensin(1–7) (Ang(1–7)) produces several effects related to glucose homeostasis. In this study, we aimed to investigate the effects of genetic deletion of Ang(1–7), the GPCR Mas, on the glucagon-producing cells. C57BL6/N Mas−/− mice presented a significant and marked increase in pancreatic α-cells (number of cells: 146 ± 21 vs 67 ± 8 in WT; P < 0.001) and the percentage per islet (17.9 ± 0.91 vs 12.3 ± 0.9% in WT; P < 0.0001) with subsequent reduction of β-cells percentage (82.1 ± 0.91 vs 87.7 ± 0.9% in WT; P < 0.0001). Accordingly, glucagon plasma levels were increased (516.7 ± 36.35 vs 390.8 ± 56.45 pg/mL in WT; P < 0.05) and insulin plasma levels were decreased in C57BL6/N Mas−/− mice (0.25 ± 0.01 vs 0.31 ± 56.45 pg/mL in WT; P = 0.02). In order to eliminate the possibility of a background-related phenotype, we determined the number of glucagon-producing cells in FVB/N Mas−/− mice. In keeping with the observations in C57BL6/N Mas−/− mice, the number and percentage of pancreatic α-cells were also significantly increased in these mice (number of α-cells: 260 ± 22 vs 156 ± 12 in WT, P < 0.001; percentage per islet: 16 ± 0.8 vs 10 ± 0.5% in WT, P < 0.0001). These results suggest that Mas has a previously unexpected role on the pancreatic glucagon production.
Introduction
Besides its role in the regulation of blood pressure and the cardiovascular system, the renin–angiotensin system (RAS) plays an important role in the control of glucose and lipid metabolism (1). Moreover, it is a determinant in the pathophysiology of diabetes and metabolic syndrome (2).
In the past few years, our understanding of the role of the heptapeptide angiotensin(1–7) has been extended to include other organs and systems well beyond the heart, vessels and the kidney (3). A relevant part of this new understanding is derived from the phenotypic characterization of Mas-deficient mice in a variety of models, tissues and conditions, including the cardiovascular system (4), liver (5), testis (6), memory process (7), erectile dysfunction (8) and inflammation (9, 10).
Angiotensin(1–7) has been described to improve the glucose homeostasis by influencing different mechanisms including improvement of insulin signalling in skeletal muscle (11), endothelium (12), adipose tissue and liver (13). Moreover, Mas deletion has been reported to have deleterious effects on glucose and lipid metabolism (14). Although it is now clear, in contrast to ACE/Ang II/AT1R, the ACE2/Ang(1–7)/Mas axis has a beneficial action on glucose metabolism (15, 16, 17, 18), the mechanism of the effect of Ang(1–7)/Mas action regarding insulin-related mechanisms is not fully understood.
In this study we aimed to further address this issue by determining the effect of Mas deletion on the α- and β-cell population in the pancreas. In order to rule out background dependency the study was performed in two mouse lines: C57BL6/N and FVB/N Mas-deficient (Mas-KO) mice.
Research design and methods
Animals
12-week-old male Mas-KO (both FVB/N and BL6/N, n = 10) and wild-type (WT, n = 8) mice, weighting approximately 25 g, were obtained from the Biological Sciences Institute of Federal University of Minas Gerais (ICB-UFMG, Brazil). Mice were kept under standard and controlled environment conditions of temperature (20–25°C), light and darkness cycles (12 h each, 06:00–18:00) with standard chow and water ad libitum. The experimental protocol was approved by the ethic committee in animal experimentation of Federal University of Minas Gerais (147/2008).
Pancreas dissection and immunohistochemistry
After overnight fasting, mice were killed by i.p. administration of a ketamine (130 mg/kg) and xylazine (0.3 mg/kg) solution. The pancreas was dissected and the splenic part was fixed in 10% formalin for 48 h, dehydrated in ethanol and embedded in paraffin. Tissue sections were cut 5 µm thick, with 150 µm interval between the cuts to obtain different pancreatic islets, and mounted on glass slides previously prepared to prevent detachment of cuts during the immunostaining. The slices were deparaffinized in xylene, rehydrated in alcohol baths of decreasing concentration and washed in deionized water. Subsequently, the slices were processed for retrieving epitopes using a microwave oven and citric acid solution, pH 6.0 for 15 min. Next, the sections were treated with 3% hydrogen peroxide (H2O2) for 30 min to inhibit endogenous peroxidase. The tissues were incubated with anti-glucagon primary antibody (1:200; Abcam) at 4°C overnight. The next day, the sections were incubated with the secondary antibody biotinylated (1:200; Vector Laboratories) for 1 h, and the slices were incubated with reagents of the ABC detection system, as specified by the manufacturer. Peroxidase activity was visualized by exposing the sections to DAB solution ((3-3)-diaminobenzidine tetrahydrochloride (Sigma)). Thereafter, the sections were counterstained with Mayer haematoxylin dehydrated in ethanol and mounted. In the negative control, the primary antibody was replaced by non-immune serum.
Glucagon levels and insulin
Enzyme-linked immunosorbent assay kit was used to measure serum insulin (Linco Research) and plasma glucagon (ALPCO immunoassays) levels.
Image analysis
Positive immunostaining for pancreatic α-cell was examined using a light microscope (Zeiss). Ten islets per mouse pancreas were randomly chosen and all the cells present in the islet were counted. After that, each marked α-cell was also counted. The estimated β-cell content was obtained by subtracting the total cells from the α-cells.
Statistical analysis
Data were analysed using non-paired Student t-test in the GraphPad Prism (GraphPad Software). Data are expressed as mean ± s.e.m. and statistical significance was accepted at the level of P < 0.05.
Results
As shown in Fig. 1A, Mas deficiency in the FVB/N background resulted in an increase in the total number and percentage of the pancreatic α-cell when compared to WT mice (number of α-cells: 260 ± 22 vs 156 ± 12 in WT, P < 0.001; percentage per islet: 16 ± 0.8 vs 10 ± 0.5% in WT, P < 0.0001). Likewise, the number and percentage of α-cells were significantly higher in the C57BL6/N in comparison to the WT mice (Fig. 1B). The number of α-cells in C57BL6/N mice averaged is 146 ± 21 vs 67 ± 8 in WT (P < 0.001) and the percentage per islet is 17.9 ± 0.91 vs 12.3 ± 0.9% in WT (P < 0.0001). Regarding the estimated β-cell number, no significant differences was evidenced in FVB/N and C57BL6/N mice; however, the percentage of β-cells was reduced in both backgrounds (C57BL6/N: 82.1 ± 0.91 vs 87.7 ± 0.9% in WT P < 0.0001; FVB/N: 83.53 ± 1.2 vs 88.6 ± 0.9% in WT, P < 0.001, Fig. 2). No significant differences were observed in the total amount of cells in pancreatic islets, although a trend for increase in Mas-KO was observed (Fig. 3). These results indicate that the increment in the total cell number, independently of the strain background, is linked to increase in the α-pancreatic cells. In keeping with the morphological data, glucagon plasma level was increased in C57BL6/N background: 516.7 ± 36.35 vs 390.8 ± 56.45 pg/mL vs WT 0.25 ± 0.01 vs 0.31 ± 56.45 pg/mL (P < 0.05) while insulin 0.25 ± 0.01 vs 0.31 ± 56.45 pg/mL (P = 0.02) in WT was reduced (Fig. 4).
(A) Total number and percentage of pancreatic α-cells in FVB/N Mas-KO in comparison with WT mice; (B) total number and percentage of α-cells in C57BL6 Mas-KO mice vs WT. (A and B) Values are presented as mean ± s.e.m. Images represent the immunohistochemical staining of α-cell in mouse pancreatic islet of FVB/N and C57BL6 Mas-KO mice. (A) Negative control: replacement of primary antibody with buffer; (B) immunostaining for pancreatic α-cells in WT; (C) Mas-KO mice, respectively. Magnification: 40×.
Citation: Endocrine Connections 6, 1; 10.1530/EC-16-0098
Estimated β-cell number in FVB/N and C57BL6. Values are means ± s.e.m.
Citation: Endocrine Connections 6, 1; 10.1530/EC-16-0098
Total amount of cells in pancreatic islets in Mas-KO mice from FVB/N and C57BL6 background. Values are means ± s.e.m.
Citation: Endocrine Connections 6, 1; 10.1530/EC-16-0098
Glucagon and insulin plasma levels in C57BL6 Mas-KO mice. Values are means ± s.e.m.
Citation: Endocrine Connections 6, 1; 10.1530/EC-16-0098
Discussion
In this study, we report for the first time that genetic ablation of Mas produced a marked alteration in the total number and percentage of glucagon-producing cell in the pancreas. This was associated to a significant increase of fasting plasma glucagon levels. As consequence of the increase of α-cells, the percentage of β-cells was reduced in the pancreas since the absolute number did not differ from that of WT mice. In the past few years, the role of Ang(1–7)/Mas axis in the attenuation of metabolic diseases has been investigated in different models of diabetes and also in different experimental contexts (19, 20). Wang and colleagues shed light on the pivotal role of ACE2/Ang(1–7)/Mas axis in organogenesis during embryonic development of pancreatic islets. In this study, the authors also demonstrated a major role of Mas receptor in the pancreatic development and insulin secretion in early postnatal life reiterating the potential implications of the Ang(1–7)/Mas axis for the development of therapies to handle pathological metabolic states (21).
In keeping with our current observation, we have previously described that Mas-KO mice presented significant changes in blood glucose homeostasis (14). In this previous study, however, we did not address the possibility of changes in glucagon as one of the mechanisms of such alterations. Although, functional studies involving glucagon-related mechanisms are obviously needed to fully understand its role in the metabolic changes observed in Mas-KO mice, our current data indicate that an imbalance of α- and β-cells is involved in the functional phenotypic changes of Mas-KO mice.
In previous studies, significant differences were reported for the phenotypic variation in the two backgrounds where Mas has been deleted: FVB/N and C57BL6/N. For instance, increase in blood pressure was observed in FVB/N but not in C57BL6/N mice (22), and the metabolic syndrome-like state observed in FVB/N mice was absent in the BL6/N background (14). Here, we show that the increase in glucagon-producing cells is a common feature of both backgrounds, clearly indicating that this phenotypic alteration is mostly linked to Mas deletion. A further implication of this finding is that Mas appears to play a key role in the glucagon-producing pancreatic cells.
Diabetes mellitus is characterized by insulin insufficiency due to either decreased insulin release or end organ insulin resistance. Among the major existing categories of diabetes, type I (T1DM) usually occurs in children and young adults and is characterized by pancreatic β-cell failure or autoimmune destruction of the pancreatic β-cells (18). Indeed, it has been shown that glucagon antagonism, glucagon neutralizing antibodies and antisense oligonucleotides lower plasma glucose level in many rodent models of diabetes (23, 24, 25, 26). Similarly, the reversal of diabetes induced by leptin therapy in rodents has been attributed to a reduction in plasma glucagon (27, 28, 29). In keeping with studies in rodents, suppression of glucagon in patients with diabetes improves glucose tolerance (30, 31). These observations support a role for glucagon, and probably Mas, in diabetes hyperglycaemia.
Conclusion
The main finding of this report is the marked increase in the number and per cent of pancreatic α-cells in Mas-deficient mice. This was associated to a significant increase in glucagon plasma levels. These findings suggest a crucial role for Mas in the pancreatic morphology with subsequent impact in glucose homeostasis. Although further studies are necessary to fully understand the role of Ang(1–7)/Mas in the endocrine pancreas, our results provide a clear basis for understanding the role of Mas in glucagon production.
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
Funding agencies: CAPES, Fundação de Amparo à Pesquisa do estado de Minas Gerais, (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ).
Author contribution statement
J F B coordinated the study. She participated in all experimental procedures, data collections and analysis and has drafted this manuscript. D R D participated in the data collection, analysis and has drafted this manuscript. N A and M B participated in the all experimental procedures, data interpretation and reviewed and approved this manuscript. R A S is the main investigator of the study. He participated in the coordination of all tasks; designed and led the study; raised funding for the study; participated in data collection and analysis procedures; and has drafted, reviewed and approved the manuscript.
Acknowledgements
This work was supported by CAPES, Programa Nacional de Pós Doutorado (PNPD-CAPES), Fundação de Amparo à Pesquisa do estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ).
References
- 1↑
Favre GA, Esnault VL & Van Obberghen E. Modulation of glucose metabolism by the renin-angiotensin-aldosterone system. American Journal of Physiology: Endocrinology and Metabolism 2015 308 E435–E449. (doi:10.1152/ajpendo.00391.2014)
- 2↑
Cabandugama PK, Gardner MJ & Sowers JR. The renin angiotensin aldosterone system in obesity and hypertension: roles in the cardiorenal metabolic syndrome. Medical Clinics of North America 2017 101 129–137. (doi:10.1016/j.mcna.2016.08.009)
- 3↑
Passos-Silva DG, Verano-Braga T & Santos RA. Angiotensin-(1-7): beyond the cardio-renal actions. Clinical Science 2013 124 443–456. (doi:10.1042/CS20120461)
- 4↑
Santos RA. Angiotensin-(1-7). Hypertension 2014 63 1138–1147. (doi:10.1161/HYPERTENSIONAHA.113.01274)
- 5↑
Santos RA, Ferreira AJ & Simões E Silva AC. Recent advances in the angiotensin-converting enzyme 2-angiotensin(1-7)-Mas axis. Experimental Physiology 2008 93 519–527. (doi:10.1113/expphysiol.2008.042002)
- 6↑
Xu P, Santos RA, Bader M & Alenina N. Alterations in gene expression in the testis of angiotensin-(1-7)-receptor Mas-deficient mice. Regulatory Peptides 2007 138 51–55. (doi:10.1016/j.regpep.2006.11.017)
- 7↑
Lazaroni TL, Raslan AC, Fontes WR, de Oliveira ML, Bader M, Alenina N, Moraes MF, Dos Santos RA & Pereira GS. Angiotensin-(1-7)/Mas axis integrity is required for the expression of object recognition memory. Neurobiology of Learning and Memory 2012 97 113–123. (doi:10.1016/j.nlm.2011.10.003)
- 8↑
da Costa Gonçalves AC, Leite R, Fraga-Silva RA, Pinheiro SV, Reis AB, Reis FM, Touyz RM, Webb RC, Alenina N & Bader M et al. Evidence that the vasodilator angiotensin-(1-7)-Mas axis plays an important role in erectile function. American Journal of Physiology: Heart and Circulatory Physiology 2007 293 H2588–H2596. (doi:10.1152/ajpheart.00173.2007)
- 9↑
da Silveira KD, Coelho FM, Vieira AT, Sachs D, Barroso LC, Costa VV, Bretas TL, Bader M, de Sousa LP & da Silva TA et al. Anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, MAS, in experimental models of arthritis. Journal of Immunology 2010 185 5569–5576. (doi:10.4049/jimmunol.1000314)
- 10↑
Souza LL, Duchene J, Todiras M, Azevedo LC, Costa-Neto CM, Alenina N, Santos RA & Bader M. Receptor MAS protects mice against hypothermia and mortality induced by endotoxemia. Shock 2014 41 331–336. (doi:10.1097/SHK.0000000000000115)
- 11↑
Muñoz MC, Giani JF, Burghi V, Mayer MA, Carranza A, Taira CA & Dominici FP. The Mas receptor mediates modulation of insulin signaling by angiotensin-(1-7). Regulatory Peptides 2012 177 1–11. (doi:10.1016/j.regpep.2012.04.001)
- 12↑
Verano-Braga T, Schwämmle V, Sylvester M, Passos-Silva DG, Peluso AA, Etelvino GM, Santos RA & Roepstorff P. Time-resolved quantitative phosphoproteomics: new insights into Angiotensin-(1-7) signaling networks in human endothelial cells. Journal of Proteome Research 2012 11 3370–3381. (doi:10.1021/pr3001755)
- 13↑
Giani FC, Mayer MA, Muñoz MC, Silberman EA, Höcht C, Taira CA, Gironacci MM, Turyn D & Dominici FP. Chronic infusion of angiotensin-(1-7) improves insulin resistance and hypertension induced by a high-fructose diet in rats. American Journal of Physiology: Endocrinology and Metabolism 2009 296 E262–E271. (doi:10.1152/ajpendo.90678.2008)
- 14↑
Santos SH, Fernandes LR, Mario EG, Ferreira AV, Pôrto LC, Alvarez-Leite JI, Botion LM, Bader M, Alenina N & Santos RA. Mas deficiency in FVB/N mice produces marked changes in lipid and glycemic metabolism. Diabetes 2008 57 340–347. (doi:10.2337/db07-0953)
- 15↑
Shoemaker R, Yiannikouris F, Thatcher S & Cassis L. ACE2 deficiency reduces β-cell mass and impairs β-cell proliferation in obese C57BL/6 mice. American Journal of Physiology: Endocrinology and Metabolism 2015 309 E621–E631. (doi:10.1152/ajpendo.00054.2015)
- 16↑
He J, Yang Z, Yang H, Wang L, Wu H, Fan Y, Wang W, Fan X & Li X. Regulation of insulin sensitivity, insulin production, and pancreatic β cell survival by angiotensin-(1-7) in a rat model of streptozotocin-induced diabetes mellitus. Peptides 2015 64 49–54. (doi:10.1016/j.peptides.2014.12.012)
- 17↑
Sahr A, Wolke C, Maczewsky J, Krippeit-Drews P, Tetzner A, Drews G, Venz S, Gürtler S, van den Brandt J & Berg S et al. The Angiotensin-(1-7)/Mas axis improves pancreatic β-cell function in vitro and in vivo. Endocrinology 2016 157 4677–4690. (doi:10.1210/en.2016-1247)
- 18↑
Dominici FP, Burghi V, Muñoz MC & Giani JF. Modulation of the action of insulin by angiotensin-(1-7). Clinical Science 2014 126 613–630. (doi:10.1042/CS20130333)
- 19↑
Bindom SM & Lazartigues E. The sweeter side of ACE2: physiological evidence for a role in diabetes. Molecular and Cellular Endocrinology 2009 302 193–202. (doi:10.1016/j.mce.2008.09.020)
- 20↑
Santos SH, Giani JF, Burghi V, Miquet JG, Qadri F, Braga JF, Todiras M, Kotnik K, Alenina N & Dominici FP et al. Oral administration of angiotensin-(1-7) ameliorates type 2 diabetes in rats. Journal of Molecular Medicine 2014 92 255–265. (doi:10.1007/s00109-013-1087-0)
- 21↑
Wang L, Liang J & Leung PS. The ACE2/Ang-(1-7)/Mas axis regulates the development of pancreatic endocrine cells in mouse embryos. PLoS ONE 2015 10 e0128216. (doi:10.1371/journal.pone.0128216)
- 22↑
Xu P, Costa-Goncalves AC, Todiras M, Rabelo LA, Sampaio WO, Moura MM, Santos SS, Luft FC, Bader M & Gross V et al. Endothelial dysfunction and elevated blood pressure in MAS gene-deleted mice. Hypertension 2008 51 574–580. (doi:10.1161/HYPERTENSIONAHA.107.102764)
- 23↑
Ali S & Drucker DJ. Benefits and limitations of reducing glucagon action for the treatment of type 2 diabetes. American Journal of Physiology: Endocrinology and Metabolism 2009 296 E415–E421. (doi:10.1152/ajpendo.90887.2008)
- 24↑
Edgerton DS & Cherrington AD. Glucagon as a critical factor in the pathology of diabetes. Diabetes 2011 60 377–380. (doi:10.2337/db10-1594)
- 25↑
Lee Y, Wang M-Y, Du XQ, Charron MJ & Unger RH. Glucagon receptor knockout prevents insulin-deficient type 1 diabetes in mice. Diabetes 2011 60 391–397. (doi:10.2337/db10-0426)
- 26↑
Conarello SL, Jiang G, Mu J, Li Z, Woods J, Zycband E, Ronan J, Liu F, Roy RS & Zhu L et al. Glucagon receptor knockout mice are resistant to diet-induced obesity and streptozotocin-mediated beta cell loss and hyperglycaemia. Diabetologia 2007 50 142–150. (doi:10.1007/s00125-006-0481-3)
- 27↑
Wang MY, Chen L, Clark GO, Lee Y, Stevens R, Ilkayeva O, Wenner B, Bain J, Charron M & Newgard C et al. Leptin therapy in insulin deficient type I diabetes. PNAS 2010 107 4813–4819. (doi:10.1073/pnas.0909422107)
- 28↑
Yu X, Park BH, Wang MY, Wang ZV & Unger RH. Making insulin-deficient type 1 diabetic rodents thrive without insulin. PNAS 2008 105 14070–14075. (doi:10.1073/pnas.0806993105)
- 29↑
Stevenson RW, Williams PE & Cherrington AD. Role of glucagon suppression on gluconeogenesis during insulin treatment of the conscious diabetic dog. Diabetologia 1987 30 782–790. (doi:10.1007/bf00275744)
- 30↑
Dinneen S, Alzaid A, Turk D & Rizza R. Failure of glucagon suppression contributes to postprandial hyperglycaemia in IDDM. Diabetologia 1995 38 337–343. (doi:10.1007/BF00400639)
- 31↑
Shah P, Vella A, Basu A, Basu R, Schwenk WF & Rizza RA. Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus. Journal of Clinical Endocrinology and Metabolism 2000 85 4053–4059. (doi:10.1210/jc.85.11.4053)
