Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Abstract

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM. Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM. Metformin is a widely used antidiabetic drug for treating type 2 diabetes mellitus (T2DM) that enhances insulin regulation of glucose, promotes weight loss, and reduces appetite (1Bailey C.J. Turner R.C. Metformin.N. Engl. J. Med. 1996; 334: 574-579Crossref PubMed Google Scholar, 2Kirpichnikov D. McFarlane S.I. Sowers J.R. Metformin: An update.Ann. Intern. Med. 2002; 137: 25-33Crossref PubMed Scopus (867) Google Scholar, 3Matthaei S. Stumvoll M. Kellerer M. Haring H.U. Pathophysiology and pharmacological treatment of insulin resistance.Endocr. Rev. 2000; 21: 585-618Crossref PubMed Scopus (313) Google Scholar). These effects are generally explained by an increase in peripheral insulin sensitivity, and hence a decrease in blood glucose levels rather than a direct promotion of pancreatic insulin release. Overall, although metformin has been used as a drug for more than half a century, its antidiabetic mechanisms, particularly in the pancreas, have not been much documented (4Yang X. Xu Z. Zhang C. Cai Z. Zhang J. Metformin, beyond an insulin sensitizer, targeting heart and pancreatic beta cells.Biochim. Biophys. Acta Mol. Basis Dis. 2016; 1863: 1984-1990Crossref PubMed Scopus (51) Google Scholar). Glycerol is metabolized in the pancreas and shows the potential to stimulate insulin production and secretion (5Skelly R.H. Wicksteed B. Antinozzi P.A. Rhodes C.J. Glycerol-stimulated proinsulin biosynthesis in isolated pancreatic rat islets via adenoviral-induced expression of glycerol kinase is mediated via mitochondrial metabolism.Diabetes. 2001; 50: 1791-1798Crossref PubMed Scopus (15) Google Scholar). Aquaporins comprise a family of 13 members of water channels (AQP0-12) that facilitate the rapid transport of water across cell membranes. In some cases, these pores are also permeated by small solutes, particularly solutes such as glycerol, urea, or nitric oxide, which are known as aquaglyceroporins. AQP7, a member of aquaglyceroporin family, is expressed in pancreatic β-cells, and murine studies have confirmed its participation in insulin secretion, triacylglycerol synthesis, and proliferation of these endocrine cells. Therefore, AQP7 is now considered as a β-cell protein and critical regulator of intraislet glycerol content (6Matsumura K. Chang B.H. Fujimiya M. Chen W. Kulkarni R.N. Eguchi Y. Kimura H. Kojima H. Chan L. Aquaporin 7 is a beta-cell protein and regulator of intraislet glycerol content and glycerol kinase activity, beta-cell mass, and insulin production and secretion.Mol. Cell. Biol. 2007; 27: 6026-6037Crossref PubMed Scopus (70) Google Scholar). However, nothing is known about whether metformin could regulate this member protein in pancreatic islets and its possible mechanism. Several molecular abnormalities are linked to T2DM, in which mutations in important regulatory networks remain substantial (7Galant D. Gaborit B. Desgrouas C. Abdesselam I. Bernard M. Levy N. Merono F. Coirault C. Roll P. Lagarde A. Bonello-Palot N. Bourgeois P. Dutour A. Badens C. A heterozygous ZMPSTE24 mutation associated with severe metabolic syndrome, ectopic fat accumulation, and dilated cardiomyopathy.Cells. 2016; 5: 21Crossref PubMed Scopus (20) Google Scholar, 8de Gonzalo-Calvo D. Kenneweg F. Bang C. Toro R. van der Meer R.W. Rijzewijk L.J. Smit J.W. Lamb H.J. Llorente-Cortes V. Thum T. Circulating long-non coding RNAs as biomarkers of left ventricular diastolic function and remodelling in patients with well-controlled type 2 diabetes.Sci. Rep. 2016; 6: 37354Crossref PubMed Scopus (89) Google Scholar, 9Cristobal I. Madoz-Gurpide J. Rojo F. Garcia-Foncillas J. Comment on Goldsworthy et al. Haploinsufficiency of the insulin receptor in the presence of a splice-site mutation in Ppp2r2a results in a novel digenic mouse model of type 2 diabetes. Diabetes 2016;65:1434-1446.Diabetes. 2016; 65: e22-e23Crossref PubMed Scopus (2) Google Scholar). Pancreatic β-cell injury and insulin resistance appear to be partially triggered by inflammatory (10Lawrence M.C. Borenstein-Auerbach N. McGlynn K. Kunnathodi F. Shahbazov R. Syed I. Kanak M. Takita M. Levy M.F. Naziruddin B. NFAT targets signaling molecules to gene promoters in pancreatic beta-cells.Mol. Endocrinol. 2015; 29: 274-288Crossref PubMed Scopus (16) Google Scholar), oxidative (11Sidarala V. Kowluru A. The regulatory roles of mitogen-activated protein kinase (MAPK) pathways in health and diabetes: Lessons learned from the pancreatic beta-cell.Recent Pat. Endocr. Metab. Immune Drug Discov. 2017; 10: 76-84Crossref PubMed Scopus (33) Google Scholar), and endoplasmic reticulum stress-induced pathways (12Yang G. Yang W. Wu L. Wang R. H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.J. Biol. Chem. 2007; 282: 16567-16576Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), including the mitogen-activated protein kinases (MAPK) signaling cascade. However, whether MAPK modulations affect AQP7 expression in pancreatic tissue of T2DM treated with metformin remains poorly understood. The present study aimed to investigate whether metformin improves and alleviates the symptoms through modulation of AQP7 and the MAPK pathway in pancreas of T2DM. Significant increases were observed for glucose (Glu), triglycerides (TG), and total cholesterol (TC) in the T2DM group compared with the normal control group (Ctrl) group (p < 0.05). After metformin (Met) treatment, serum Glu, TG, and TC were significantly decreased in T2DM+metformin (Met) groups [T2DM+M1 (100 mg/kg/d), T2DM+M2 (300 mg/kg/d) and T2DM+M3 (500 mg/kg/d)] compared with the T2DM + 0.9% NaCl group (T2DM+NS) (p < 0.05 for all; Table S1), supporting the beneficial effects of metformin on the treatment of T2DM. The pancreatic sections stained with hematoxylin and eosin (HE) are shown in Fig. S1. In the control group (Ctrl), pancreatic islet was normal without edema or inflammatory cells infiltration. In contrast, islets in the T2DM group showed disturbed cellular arrangement, necrotic changes in islets (karyolysis and loss of nuclei), and smaller islet cell masses. All these changes were alleviated in the T2DM+Met groups by metformin, especially in T2DM+M2 and T2DM+M3 group. We examined whether metformin could regulate AQP7 expression. To confirm AQP7 expression in the pancreas, we used immunohistochemistry to localize AQP7 and insulin in the pancreas by fluorescence microscopy and detected immunoreactive AQP7 in insulin-producing cells. Immunohistochemical staining of pancreatic tissues from the Ctrl group showed fairly well immunoreactive AQP7 in insulin-producing cells. However, immunostaining sample of T2DM group, T2DM+NS group, or T2DM+M1 group stained faintly for the few immunoreactive insulin-positive cells. On the contrary, immunostaining with AQP7 antibody in the T2DM+M2 group and T2DM+M3 group detected more intense immunostaining in insulin-positive cells than T2DM+NS group To whether metformin effects on AQP7 was pancreatic islets from A reduction of AQP7 levels was in the T2DM group as compared with the Ctrl group (p < 0.05). An increase of AQP7 expression was observed in treatment groups in a for group (p < A and In we a and glucose model in INS-1 cells on studies Z. H. Z. W. M. Wang J. Yang F. Y. Y. Xu T. J. of pancreatic β-cells in response to and Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. X. Z. J. K. improves insulin production and reduces apoptosis in J. 2017; PubMed Scopus Google Scholar, Y. S. Y. and metformin effects on pancreatic β-cell apoptosis via J. Biol. 2015; PubMed Scopus Google Scholar). to the in INS-1 cells in a reduction of AQP7 protein which was increased in the presence of and 2 metformin, (p < and of pancreatic islets or INS-1 cells were that with the total or of of the MAPKs to investigate whether these MAPKs are activated or inhibited in diabetic pancreas to The total of MAPK p38 and JNK was significantly increased in T2DM group compared with the control group, metformin the activation of p38 and JNK in diabetic rats in a However, changes different groups (p < In INS-1 cells with and glucose for JNK p38 and MAPK metformin the activation of JNK and p38 MAPK (p < We also the changes in MAPK signaling molecules treated with and glucose for and The results showed that the levels of JNK and p38 increased treatment with glucose and for and which were The levels of increased treatment with glucose and for and to normal we examined the glycerol of group in INS-1 cells. Glycerol was by the of the glycerol content of INS-1 cells to a glycerol glycerol this metformin could the of on glycerol and increase glycerol content (p < To the effects of on insulin secretion and whether metformin could insulin secretion function in pancreatic β-cells, INS-1 cells were to and glucose with or without metformin for has shown that and were inhibited the the metformin insulin secretion (p < shown in and as of JNK and the decrease of AQP7 protein expression in INS-1 cells of and we observed that pharmacological activation of JNK and p38 by of p38 and 2 also significantly decreased AQP7 protein expression (p < 0.05). To whether AQP7 was to the insulin secretion, we INS-1 cell or AQP7 by The or of AQP7 was the protein we whether or of AQP7 could glycerol content, insulin secretion in INS-1 cells. The showed both glycerol content and insulin secretion were inhibited by AQP7 and Overexpression of AQP7 could increase the content of glycerol, not insulin secretion and In the present we demonstrated that a metformin in T2DM rats the metabolic abnormalities by hyperglycemia and and islet cell mass, that metformin could pancreatic islet was shown that AQP7 expression was decreased, whereas p38 and JNK MAPK were activated in response to hyperglycemia and in vivo and in vitro. of p38 and JNK MAPK pathway could AQP7 expression. metformin upregulated AQP7 expression and inhibited p38 and JNK MAPK that metformin AQP7 expression by MAPK pathway as to some for rat pancreatic islets. Metformin glycerol content and insulin secretion by Overexpression of AQP7 increased glycerol influx into and inhibition of AQP7 could glycerol influx into thereby decreasing insulin secretion. Although studies have on the roles of metformin in glucose and promoting insulin sensitivity, the mechanism of metformin on the pancreas remains studies demonstrated that a decrease in β-cell or β-cell function could to insulin thereby promoting glucose and diabetes (6Matsumura K. Chang B.H. Fujimiya M. Chen W. Kulkarni R.N. Eguchi Y. Kimura H. Kojima H. Chan L. Aquaporin 7 is a beta-cell protein and regulator of intraislet glycerol content and glycerol kinase activity, beta-cell mass, and insulin production and secretion.Mol. Cell. Biol. 2007; 27: 6026-6037Crossref PubMed Scopus (70) Google Scholar). The present study showed that metformin alleviated pancreatic islet and increased β-cell in type 2 diabetic its possible direct protective on the gene of glycerol kinase to cell or isolated rat β-cells showed that proinsulin biosynthesis and insulin secretion glycerol is metabolized (5Skelly R.H. Wicksteed B. Antinozzi P.A. Rhodes C.J. Glycerol-stimulated proinsulin biosynthesis in isolated pancreatic rat islets via adenoviral-induced expression of glycerol kinase is mediated via mitochondrial metabolism.Diabetes. 2001; 50: 1791-1798Crossref PubMed Scopus (15) Google Scholar, Antinozzi P.A. of insulin secretion in islet beta cells. metabolic of glucose and glycerol into mechanisms of Biol. Chem. Full Text Full Text PDF PubMed Scopus (70) Google Scholar). To of the known glycerol channels is as which glycerol as well as including and A. V. J. G. as metabolic in and insulin resistance 10: PubMed Scopus Google Scholar). AQP7 is a aquaglyceroporin in pancreatic islets β-cells (6Matsumura K. Chang B.H. Fujimiya M. Chen W. Kulkarni R.N. Eguchi Y. Kimura H. Kojima H. Chan L. Aquaporin 7 is a beta-cell protein and regulator of intraislet glycerol content and glycerol kinase activity, beta-cell mass, and insulin production and secretion.Mol. Cell. Biol. 2007; 27: 6026-6037Crossref PubMed Scopus (70) Google Scholar). AQP7 is associated with a reduction in islet cell and total β-cell (6Matsumura K. Chang B.H. Fujimiya M. Chen W. Kulkarni R.N. Eguchi Y. Kimura H. Kojima H. Chan L. Aquaporin 7 is a beta-cell protein and regulator of intraislet glycerol content and glycerol kinase activity, beta-cell mass, and insulin production and secretion.Mol. Cell. Biol. 2007; 27: 6026-6037Crossref PubMed Scopus (70) Google Scholar). the AQP7 gene is in and has been linked to T2DM S. F. D. S. V. V. A. F. F. L. B. V. A of the glycerol 7 gene is associated with and metabolic 2007; PubMed Scopus Google Scholar). These studies that glycerol mediated by AQP7 is in glucose in the pancreas S. F. D. S. V. V. A. F. F. L. B. V. A of the glycerol 7 gene is associated with and metabolic 2007; PubMed Scopus Google Scholar). in AQP7 permeated glycerol influx into The present study also demonstrated that of AQP7 or reduction in AQP7 expression could decrease glycerol influx into thereby decreasing insulin secretion. 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Diabetes 2017; PubMed Scopus Google Scholar). metformin AQP7 expression through or some pathway or whether these pathways with the MAPK in pancreas, In we a mechanism by which metformin glucose via pancreatic in type 2 diabetic is associated with of pancreatic AQP7 expression to glycerol influx to insulin secretion by inhibition of of JNK and p38 MAPK pathway in T2DM Our findings that the novel mechanism for metformin treatment of T2DM and the modulation of AQP7 function have important for of T2DM. Our be more for whether the of AQP7 could the of metformin in the of metformin on glycerol and insulin response in tissues in the study was by the of All were on the of the of for the and of rats were from the were for in with the of of were a rats were on a with an of from from and from To rats were with the high-fat diet with a total of from from and from for in a new rat model of type 2 diabetes high-fat diet with PubMed Scopus Google Scholar, K. B. L. P. of high-fat and A model for type 2 diabetes and pharmacological PubMed Scopus Google Scholar, K. L.J. A new rat model of type 2 diabetes: The 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and diabetic rats were by of from in blood glucose levels were by and diabetic model was considered in rats with blood glucose levels than were into the normal control group (Ctrl), diabetes mellitus group diabetes mellitus group NaCl group and and mg/kg/d) (Met) groups [T2DM+M1 (100 mg/kg/d), T2DM+M2 (300 mg/kg/d), and T2DM+M3 (500 mg/kg/d)] were with metformin for T2DM+NS group was treated with an of 0.9% The pancreatic was in a as M. R. R. S. L. as for pancreatic islet in a diabetic rat PubMed Scopus Google Scholar, Zhang Wang X. M. and mechanism of on the of insulin secretion by on isolated rat Med. J. PubMed Scopus Google Scholar). After rats were with to were was into the of the The pancreas with the was and into a with for 13 to The by the was through and with with serum were from rats by the INS-1 cell was from the were in glucose with serum and in a with cells were to for the The glucose was glucose INS-1 cells. the control group cells were in of and presence of and group cells were in the presence of glucose and with or without pathway cells were with the JNK or the p38 MAPK for or with of p38 and 2 C. S. Z. P. X. Y. Yang G. Z. INS-1 rat pancreatic beta cells from apoptosis through of Biophys. 2015; PubMed Scopus Google Scholar, X. C. K. P. H. J. through of the JNK and p38 signaling 2015; PubMed Scopus Google for 2 and glucose treatment. 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