Altered Insulin Signaling in Retinal Tissue in Diabetic States

Both type 1 and type 2 diabetes can lead to altered retinal microvascular function and diabetic retinopathy. Insulin signaling may also play a role in this process, and mice lacking insulin receptors in endothelial cells are protected from retinal neovascularization. To define the role of diabetes in retinal function, we compared insulin signaling in the retinal vasculature of mouse models of type 1 (streptozotocin) and type 2 diabetes (ob/ob). In streptozotocin mice, in both retina and liver, insulin receptor (IR) and insulin receptor substrate (IRS)-2 protein and tyrosine phosphorylation were increased by insulin, while IRS-1 protein and its phosphorylation were maintained. By contrast, in ob/ob mice, there was marked down-regulation of IR, IRS-1, and IRS-2 protein and phosphorylation in liver; these were maintained or increased in retina. In both mice, Phosphatidylinositol 3,4,5-trisphosphate generation by acute insulin stimulation was enhanced in retinal endothelial cells. On the other hand, protein levels and phosphorylation of PDK1 and Akt were decreased in retina of both mice. Interestingly, phosphorylation of p38 mitogen-activated protein kinase and ERK1 were responsive to insulin in retina of both mice but were unresponsive in liver. HIF-1α and vascular endothelial growth factor were increased and endothelial nitric-oxide synthase was decreased in retina. These observations indicate that, in both insulin-resistant and insulin-deficient diabetic states, there are alterations in insulin signaling, such as impaired PDK/Akt responses and enhanced mitogen-activated protein kinases responses that could contribute to the retinopathy. Furthermore, insulin signaling in retinal endothelial cells is differentially altered in diabetes and is also differentially regulated from insulin signaling in classical target tissues such as liver. Both type 1 and type 2 diabetes can lead to altered retinal microvascular function and diabetic retinopathy. Insulin signaling may also play a role in this process, and mice lacking insulin receptors in endothelial cells are protected from retinal neovascularization. To define the role of diabetes in retinal function, we compared insulin signaling in the retinal vasculature of mouse models of type 1 (streptozotocin) and type 2 diabetes (ob/ob). In streptozotocin mice, in both retina and liver, insulin receptor (IR) and insulin receptor substrate (IRS)-2 protein and tyrosine phosphorylation were increased by insulin, while IRS-1 protein and its phosphorylation were maintained. By contrast, in ob/ob mice, there was marked down-regulation of IR, IRS-1, and IRS-2 protein and phosphorylation in liver; these were maintained or increased in retina. In both mice, Phosphatidylinositol 3,4,5-trisphosphate generation by acute insulin stimulation was enhanced in retinal endothelial cells. On the other hand, protein levels and phosphorylation of PDK1 and Akt were decreased in retina of both mice. Interestingly, phosphorylation of p38 mitogen-activated protein kinase and ERK1 were responsive to insulin in retina of both mice but were unresponsive in liver. HIF-1α and vascular endothelial growth factor were increased and endothelial nitric-oxide synthase was decreased in retina. These observations indicate that, in both insulin-resistant and insulin-deficient diabetic states, there are alterations in insulin signaling, such as impaired PDK/Akt responses and enhanced mitogen-activated protein kinases responses that could contribute to the retinopathy. Furthermore, insulin signaling in retinal endothelial cells is differentially altered in diabetes and is also differentially regulated from insulin signaling in classical target tissues such as liver. One of the major long term complications of both type 1 (insulin-deficient) and type 2 (insulin-resistant) diabetes is proliferative retinopathy, which is characterized by increased neovascularization and neuronal degeneration in the retina. Clinical studies have indicated that hyperglycemia and poor metabolic control are important factors in the development of diabetic retinopathy (1Adler A.I. Stevens R.J. Neil A. Stratton I.M. Boulton A.J. Holman R.R. Diabetes Care. 2002; 25: 894-899Crossref PubMed Scopus (307) Google Scholar, 2Koya D. King G.L. Diabetes. 1998; 47: 859-866Crossref PubMed Scopus (1161) Google Scholar). Multiple mechanisms have been implicated, including relative hypoxia in retina, resulting in the induction of vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; STZ, streptozotocin; HIF, hypoxia-inducible factor; ERK, extracellular signal-regulated protein kinase; PDK, 3-phosphoinositide-dependent kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; eNOS, endothelial nitric-oxide synthase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; IR, insulin receptor; IRS, insulin receptor substrate; PI, phosphatidylinositol. and other vascular mediators that stimulate proliferation of retinal endothelial cells in preretinal area (3Lu M. Amano S. Miyamoto K. Garland R. Keough K. Qin W. Adamis A.P. Invest. Ophthalmol. Vis. Sci. 1999; 40: 3281-3286PubMed Google Scholar, 4Poulaki V. Qin W. Joussen A.M. Hurlbut P. Wiegand S.J. Rudge J. Yancopoulos G.D. Adamis A.P. J. Clin. Invest. 2002; 109: 805-815Crossref PubMed Scopus (240) Google Scholar, 5Kuboki K. Jiang Z.Y. Takahara N. Ha S.W. Igarashi M. Yamauchi T. Feener E.P. Herbert T.P. Rhodes C.J. King G.L. Circulation. 2000; 101: 676-681Crossref PubMed Scopus (531) Google Scholar, 6Cardillo C. Nambi S.S. Kilcoyne C.M. Choucair W.K. Katz A. Quon M.J. Panza J.A. Circulation. 1999; 100: 820-825Crossref PubMed Scopus (272) Google Scholar, 7Kondo T. Vicent D. Suzuma K. Yanagisawa M. King G.L. Holzenberger M. Kahn C.R. J. Clin. Invest. 2003; 111: 1835-1842Crossref PubMed Scopus (203) Google Scholar). Although the risk of diabetic retinopathy over the long term is correlated with the degree of metabolic control (8Henricsson M. Nilsson A. Janzon L. Groop L. Diabetes Med. 1997; 14: 123-131Crossref PubMed Scopus (91) Google Scholar), several clinical studies (9Lauritzen T. Frost-Larsen K. Larsen H.W. Deckert T. Diabetes. 1985; 34: 74-79Crossref PubMed Google Scholar, 10Dahl-Jorgensen K. Brinchmann-Hansen O. Hanssen K.F. Sandvik L. Aagenaes O. Br. Med. J. (Clin. Res. Ed.). 1985; 290: 811-815Crossref PubMed Scopus (299) Google Scholar, 11Roysarkar T.K. Gupta A. Dash R.J. Dogra M.R. Am. J. Ophthalmol. 1993; 115: 569-574Abstract Full Text PDF PubMed Scopus (46) Google Scholar) have demonstrated that insulin may also play a role and that intensive insulin therapy may cause a transient worsening of retinopathy in some individuals, even when compared with treatment with oral hypoglycemic agents. This is supported by our previous observation that mice with a vascular endothelial cell specific knock-out of the insulin receptor (VENIRKO) are protected from retinal neovascularization (7Kondo T. Vicent D. Suzuma K. Yanagisawa M. King G.L. Holzenberger M. Kahn C.R. J. Clin. Invest. 2003; 111: 1835-1842Crossref PubMed Scopus (203) Google Scholar). Furthermore, in general, rates of development of retinopathy are somewhat lower in patients with insulin-resistant type 2 diabetes than in those with insulin-deficient type 1 diabetes (8Henricsson M. Nilsson A. Janzon L. Groop L. Diabetes Med. 1997; 14: 123-131Crossref PubMed Scopus (91) Google Scholar, 12Chaturvedi N. Sjoelie A.K. Porta M. Aldington S.J. Fuller J.H. Songini M. Kohner E.M. Diabetes Care. 2001; 24: 284-289Crossref PubMed Scopus (169) Google Scholar). Insulin action at a molecular level is created by a complex signaling network using alternative or complementary pathways and multiple molecular isoforms of key signaling molecules (13Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3935) Google Scholar). Insulin and insulin-like growth factor 1 receptors almost ubiquitously expressed are present in endothelial cells and other cells of the retina (14Reiter C.E. Gardner T.W. Prog. Retin. Eye Res. 2003; 22: 545-562Crossref PubMed Scopus (89) Google Scholar). How insulin signaling in these tissues might be altered in diabetes, as compared with classical target tissues, i.e. liver, muscle, and fat, is unknown. To define the potential role of these pathways in development of diabetic retinopathy, we have assessed retinal insulin signaling in type 1 (streptozotocin (STZ)-induced: insulin-deficient hyperglycemic model) and type 2 (ob/ob: hyperglycemic insulin-resistant model) diabetic model mice. We find that circulating insulin can stimulate the insulin signaling cascade in retinal tissue primarily in endothelial cells and that these pathways are differentially altered in insulin-resistant and insulin-deficient diabetes, as compared with peripheral tissues. We also find that hyperglycemia in both types of diabetes is associated with increased expression of hypoxia-inducible factor (HIF)-1α and VEGF. Thus, the combination of hyperglycemia and altered insulin activation of the MAP kinase pathway in retinal endothelial cells and VEGF induction may contribute to altered retinal vascular function in diabetes and development of diabetic retinopathy. Animals—8-10-week-old male obese hyperglycemic mice (C57Bl/6J ob/ob) and their lean matched controls (ob/+) were purchased from Jackson Laboratory (Bar Harbor, ME). For the generation of the STZ mouse model, 8-week-old C57Bl/6J mice were given STZ (Sigma), 180 mg/kg as a single intraperitoneal injection, and studied 7 days later after blood sugars were elevated more than 250 mg/dl. All mice were fed standard rodent chow and water ad libitum. Food was withdrawn 16 h before the experiments. Mice were anesthetized with 100 mg/kg of sodium pentobarbital injected intraperitoneally. Following loss of pedal and corneal reflexes, 5 units of regular human insulin or its diluent was injected into the inferior vena cava. The retina and liver were excised 5 min after insulin or its diluent was injected and frozen in liquid nitrogen. Immunoprecipitation and Western Blotting—Frozen tissues were homogenized in T-PER Tissue Protein Extraction Reagent (Pierce) with Halt Protease Inhibitor Mixture (Pierce), and cytoplasmic or nuclear fractions were isolated. Equal amounts of the protein supernatant were subjected to immunoprecipitation for 2 h using the indicated antibodies, followed by adding of protein A-Sepharose for another 1 h. The samples were processed for SDS-PAGE electrophoresis and Western blotting as described previously (15Folli F. Saad M.J. Backer J.M. Kahn C.R. J. Clin. Invest. 1993; 92: 1787-1794Crossref PubMed Scopus (218) Google Scholar). Retinal protein was isolated in pools from three mice. At least three independent experiments were performed for each condition. Antibodies—Rabbit polyclonal anti-insulin receptor antibody, rabbit polyclonal anti-IRS-1 antibody, rabbit polyclonal anti-IRS-2 antibody, rabbit polyclonal anti-HIF-1α antibody, and mouse monoclonal anti-VEGF antibody were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Rabbit polyclonal anti-phospho-3-phosphoinositide-dependent kinase 1 (PDK1) (Ser-241) antibody, rabbit polyclonal anti-phospho-Akt (Ser-473) antibody, rabbit polyclonal anti-phospho-p38 (Thr-180/Tyr-182) antibody, and mouse monoclonal anti-phospho extracellular signal-regulated protein kinase 1 (ERK1) (Thr-202/Tyr-204) were purchased from Cell Signaling Technology, Inc., Beverly, MA. Mouse monoclonal anti-endothelial nitric-oxide synthase (eNOS) antibody was purchased from BD Transduction Laboratories, Franklin Lakes, NJ. Mouse monoclonal anti-phosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology, Lake Placid, NY. Immunohistochemistry for Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) and Vascular Marker—PIP3 and a vascular marker, Fluorescein Griffonia (Bandeiraea) Simplicifolia Lectin I (Isolectin B4), were assessed by immunohistochemical analysis of the retinas. After a PBS equilibration, 6-μm retinal frozen sections were incubated with 3% normal goat serum in 2.5% Triton X-100/PBS for 2 h at room temperature. Incubations with a primary antibody to PIP3 (Echelon, Salt Lake City, UT) were performed with 1/50 dilution in blocking solution (3% normal goat serum in 2.5% Triton for at After with were incubated with a antibody to for Inc., with dilution and Lectin Laboratories, Inc., with dilution in blocking solution for 2 h at room temperature. The sections were using a and with a of ob/ob and STZ diabetic mice were and The of STZ mice was levels were and and insulin levels were ob/ob mice were and The of ob/ob mice was as compared with controls of levels were and insulin levels were Protein and of IR, IRS-1, and the level of insulin receptor IRS-1 and of retina and liver of STZ, and their controls were subjected to immunoprecipitation and Western blotting with or stimulation 5 min after of insulin into the inferior vena cava. expression in liver of ob/ob mice was decreased by by Western and insulin injection, tyrosine phosphorylation of was by By contrast, expression and tyrosine phosphorylation were altered in retina of the mice In STZ diabetic mice, expression was increased by and in both retina and liver, and tyrosine phosphorylation of in retina and liver of these mice was also increased by Thus, insulin receptors in retina in type 1 diabetes but to be protected from the down-regulation and in type 2 diabetes as in peripheral tissues. of IRS-1 protein was decreased by in ob/ob liver and was increased by in STZ liver By contrast, IRS-1 expression in retina of ob/ob and STZ mice was altered and phosphorylation of IRS-1 the in protein expression and was decreased in ob/ob liver increased in STZ liver but was in retina of both ob/ob and STZ mice and In with previous IRS-2 protein and phosphorylation were decreased by and in liver of ob/ob mice. By contrast, IRS-2 protein was increased by in ob/ob retina and tyrosine phosphorylation of IRS-2 was almost in ob/ob retina In STZ diabetic mice, IRS-2 protein expression was increased by and in retina and liver, phosphorylation of IRS-2 was also increased by in both tissues in this model of insulin-deficient diabetes protein expression and its tyrosine phosphorylation in retina and liver of ob/ob and STZ mice as compared with their and liver from these mice were isolated and subjected to with or immunoprecipitation with followed by with antibody indicate at are the of at least three independent experiments using for each and are expressed as relative to control which were at protein expression and its tyrosine phosphorylation in retina and liver of ob/ob and STZ mice as compared with their and liver from these mice were isolated and subjected to with or immunoprecipitation with followed by with antibody indicate at are the of at least three independent experiments using for each and are expressed as relative to control which were at of PIP3 in the cell type in retina to insulin we assessed as by of PIP3, using and compared this with the of the vascular marker, the PIP3 was and this to retinal endothelial cells as by Following 5 min of insulin the PIP3 level increased in retinal endothelial cells of and this was enhanced in ob/ob and STZ retinal endothelial cells stimulation of PIP3 was in retinal at this Thus, in to the in insulin signaling in liver of ob/ob mice, PIP3 generation in to insulin is or enhanced in retina of ob/ob and STZ diabetic mice, and this primarily in retinal retinal PIP3 induction by insulin 6-μm retinal frozen sections were and incubated with a primary antibody of PIP3 in 3% normal goat serum in 2.5% Triton X-100/PBS at the were incubated with a antibody of and Lectin for 2 h. The were using a and protein expression and phosphorylation of PDK1 and Akt in retina and liver of ob/ob and STZ mice as compared with their of retina and liver from these mice were isolated and subjected to with or and or indicate at are the of at least three independent experiments using for each and are expressed as relative to control which were at Protein and of PDK1 and kinase is of the key of of Akt is regulated by PDK1 In to the down-regulation of signaling, PDK1 phosphorylation was decreased by and phosphorylation of Akt was decreased by in liver of ob/ob mice and by in liver of STZ diabetic mice and PIP3 levels were both PDK1 and Akt phosphorylation in retina were in both ob/ob mice by and and in STZ diabetic mice by and and These observations can be by a of PDK1 and Akt protein levels by in the of ob/ob and STZ diabetic mice and Thus, that the retinal tissue is more to the diabetic in of PDK1 and Akt protein expression or than classical tissues such as liver. of p38 and the of signaling of were impaired in retina of ob/ob and STZ diabetic mice, which activation C.J. S. R.J. C.M. Diabetes. 2003; PubMed Scopus Google Scholar) of both p38 and were increased by in ob/ob retina of both these kinase was also enhanced in liver of these mice In STZ diabetes, phosphorylation of p38 was even more increased in retina, and this was by a in liver of ERK1 was also increased by in both retina and liver Interestingly, p38 and ERK1 phosphorylation were in retinal tissues of both ob/ob and STZ mice, the diabetic in liver, levels were but there was insulin and Protein of VEGF, and hyperglycemia and diabetic can a relative we the levels of VEGF, and protein expression in retina of ob/ob and STZ mice. HIF-1α expression was increased by in both ob/ob and STZ retina. VEGF expression in retina was also increased by in both ob/ob and STZ mice. By contrast, expression was decreased by in ob/ob and decreased by in STZ retina of these at the protein level in acute to and the metabolic alterations associated with diabetes are factors in the development of diabetic retinopathy (1Adler A.I. Stevens R.J. Neil A. Stratton I.M. Boulton A.J. Holman R.R. Diabetes Care. 2002; 25: 894-899Crossref PubMed Scopus (307) Google Scholar, 2Koya D. King G.L. Diabetes. 1998; 47: 859-866Crossref PubMed Scopus (1161) Google Scholar). The relative of insulin and insulin present in both type 1 and type 2 diabetic patients may also contribute to the risk of proliferative diabetic retinopathy N. Sjoelie A.K. Porta M. Aldington S.J. Fuller J.H. Songini M. Kohner E.M. Diabetes Care. 2001; 24: 284-289Crossref PubMed Scopus (169) Google Scholar, M. A. M. J. 2000; 14: Scopus Google Scholar), the is Although the risk of in diabetic retinopathy over the long term is to the degree of control (8Henricsson M. Nilsson A. Janzon L. Groop L. Diabetes Med. 1997; 14: 123-131Crossref PubMed Scopus (91) Google Scholar), several clinical studies have demonstrated that intensive insulin therapy may cause a transient worsening of retinopathy (9Lauritzen T. Frost-Larsen K. Larsen H.W. Deckert T. Diabetes. 1985; 34: 74-79Crossref PubMed Google Scholar, 10Dahl-Jorgensen K. Brinchmann-Hansen O. Hanssen K.F. Sandvik L. Aagenaes O. Br. Med. J. (Clin. Res. Ed.). 1985; 290: 811-815Crossref PubMed Scopus (299) Google Scholar, 11Roysarkar T.K. Gupta A. Dash R.J. Dogra M.R. Am. J. Ophthalmol. 1993; 115: 569-574Abstract Full Text PDF PubMed Scopus (46) Google Scholar). We have that mice lacking or insulin-like growth factor 1 receptors in vascular endothelial cells are protected from development of retinal neovascularization a relative (7Kondo T. Vicent D. Suzuma K. Yanagisawa M. King G.L. Holzenberger M. Kahn C.R. J. Clin. Invest. 2003; 111: 1835-1842Crossref PubMed Scopus (203) Google Scholar). vascular endothelial cells to be important insulin target in of in D. J. T. K. S.J. S. Yanagisawa M. King G.L. Kahn C.R. J. Clin. Invest. 2003; 111: PubMed Scopus Google Scholar), these cells insulin receptors and are to such as hypoxia and hyperglycemia A. R. R. Invest. Ophthalmol. Vis. Sci. Google Scholar). in the of insulin signaling have been as important of insulin-resistant insulin decreased receptor kinase decreased IRS-1 and and decreased have been demonstrated in ob/ob mice and other models of type 2 diabetes (15Folli F. Saad M.J. Backer J.M. Kahn C.R. J. Clin. Invest. 1993; 92: 1787-1794Crossref PubMed Scopus (218) Google Scholar, Kahn C.R. J. Full Text PDF PubMed Google Scholar, C.R. J. J. Full Text PDF PubMed Google Scholar, M.J. M. Kahn C.R. J. Clin. Invest. PubMed Scopus Google Scholar, M.J. F. Kahn J.A. Kahn C.R. J. Clin. Invest. 1993; 92: PubMed Scopus Google Scholar, D. S. Kahn C.R. J. Clin. 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Interestingly, protein levels of PDK1 and Akt in the retinal tissue of ob/ob and STZ mice were These observations indicate that retinal tissue is more to the diabetic in of PDK1 and Akt protein expression PDK1 activation to PIP3 in the of the is that PDK1 is and in diabetic endothelial cells F. 2001; PubMed Scopus Google Scholar). is also that the multiple such as with tissue may contribute to the of insulin signaling in tissue F. 2001; PubMed Scopus Google Scholar). The kinase pathway is a of growth factor signaling and is also in the of The p38 is by insulin, the MAP kinase kinase M. A. M. M. T. J. Clin. Invest. 2000; PubMed Scopus Google Scholar, M. T. M. M. K. M. T. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). ERK1 is a major signaling in kinase of p38 and ERK1 is increased insulin stimulation in retina of ob/ob or STZ mice, these are enhanced in ob/ob and STZ liver. 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The induction of HIF-1α in retina of ob/ob and STZ mice that this tissue a relative hypoxia The that this is the of hyperglycemia is indicated by the that HIF-1α expression in the of these mice The induction of HIF-1α with the in VEGF, and VEGF can stimulate retinal neovascularization. On the other hand, expression in retina of ob/ob and STZ mice is This might the decreased Akt Akt in endothelial cells T. S. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and could cause vascular such as decreased retinal blood our demonstrated that insulin signaling in retina of type 1 and type 2 diabetic mice are differentially regulated from signaling in peripheral tissues and may cell The induction of vascular such as VEGF, also the retinal neovascularization. Thus, these observations in insulin in vascular tissues other tissues and the combination of hyperglycemia and altered insulin activation of the MAP kinase pathway in retinal endothelial cells and VEGF induction may contribute to altered retinal vascular function in diabetes and development of diabetic retinopathy. We for and for in the of the