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Neutralizing Interleukin-1β (IL-1β) Induces β-Cell Survival by Maintaining PDX1 Protein Nuclear Localization*

Open AccessPublished:May 13, 2011DOI:https://doi.org/10.1074/jbc.M110.210526
      The transcription factor PDX1 plays a critical role during β-cell development and in glucose-induced insulin gene transcription in adult β-cells. Acute glucose exposure leads to translocalization of PDX1 to the nucleoplasm, whereas under conditions of oxidative stress, PDX1 shuttles from the nucleus to the cytosol. Here we show that cytosolic PDX1 expression correlated with β-cell failure in diabetes. In isolated islets from patients with type 2 diabetes and from diabetic mice, we found opposite regulation of insulin and PDX1 mRNA; insulin was decreased in diabetes, but PDX1 was increased. This suggests that elevated PDX1 mRNA levels may be insufficient to regulate insulin. In diabetic islets, PDX1 protein was localized in the cytosol, whereas in non-diabetic controls, PDX1 was in the nucleus. In contrast, overexpression of either IL-1 receptor antagonist or shuttling-deficient PDX1 restored β-cell survival and function and PDX1 nuclear localization. Our results show that nuclear localization of PDX1 is essential for a functional β-cell and provides a novel mechanism of the protective effect of IL-1 receptor antagonist on β-cell survival and function.

      Introduction

      New therapies for diabetes that lead to protection of the insulin-producing β-cell are urgently needed. Only when the β-cell compensates for the higher insulin demand during insulin resistance can normoglycemia be maintained. Recent studies suggest that the low grade inflammation in type 2 diabetes mellitus (T2DM)
      The abbreviations used are: T2DM, type 2 diabetes mellitus; HFD, high fat diet; ND, normal diet; NES, nuclear export signal; IL-1Ra, IL-1 receptor antagonist; PARP, poly(ADP-ribose) polymerase; OE, overexpressing; JNKi, JNK inhibitor.
      contributes to β-cell failure (
      • Donath M.Y.
      • Schumann D.M.
      • Faulenbach M.
      • Ellingsgaard H.
      • Perren A.
      • Ehses J.A.
      ). Especially, interleukin-1β (IL-1β), whose secretion has been postulated from intraislet macrophages (
      • Ehses J.A.
      • Böni-Schnetzler M.
      • Faulenbach M.
      • Donath M.Y.
      ) and from β-cells themselves when exposed to double-stranded RNA (
      • Heitmeier M.R.
      • Arnush M.
      • Scarim A.L.
      • Corbett J.A.
      ), to elevated glucose concentrations (
      • Maedler K.
      • Sergeev P.
      • Ris F.
      • Oberholzer J.
      • Joller-Jemelka H.I.
      • Spinas G.A.
      • Kaiser N.
      • Halban P.A.
      • Donath M.Y.
      ,
      • Böni-Schnetzler M.
      • Thorne J.
      • Parnaud G.
      • Marselli L.
      • Ehses J.A.
      • Kerr-Conte J.
      • Pattou F.
      • Halban P.A.
      • Weir G.C.
      • Donath M.Y.
      ,
      • Zhou R.
      • Tardivel A.
      • Thorens B.
      • Choi I.
      • Tschopp J.
      ), or to free fatty acids (
      • Böni-Schnetzler M.
      • Boller S.
      • Debray S.
      • Bouzakri K.
      • Meier D.T.
      • Prazak R.
      • Kerr-Conte J.
      • Pattou F.
      • Ehses J.A.
      • Schuit F.C.
      • Donath M.Y.
      ), initiates β-cell destruction.
      The receptor for IL-1β, IL-1R1, is highly expressed in the β-cell, and more than 10-fold higher expression of IL-1R1 mRNA was observed in isolated islets than in total pancreas, which is attributed to the expression in β-cells (
      • Böni-Schnetzler M.
      • Boller S.
      • Debray S.
      • Bouzakri K.
      • Meier D.T.
      • Prazak R.
      • Kerr-Conte J.
      • Pattou F.
      • Ehses J.A.
      • Schuit F.C.
      • Donath M.Y.
      ). This may explain the high sensitivity of the β-cell to IL-1β. A recent study shows that glucose-induced IL-1β secretion involves caspase 1 activation mediated by the NALP3 inflammasome. The inflammasome is activated by bacterial toxins or endogenous stress signals (e.g. ATP and β-amyloid (
      • Donath M.Y.
      • Böni-Schnetzler M.
      ,
      • Masters S.L.
      • Dunne A.
      • Subramanian S.L.
      • Hull R.L.
      • Tannahill G.M.
      • Sharp F.A.
      • Becker C.
      • Franchi L.
      • Yoshihara E.
      • Chen Z.
      • Mullooly N.
      • Mielke L.A.
      • Harris J.
      • Coll R.C.
      • Mills K.H.
      • Mok K.H.
      • Newsholme P.
      • Nuñez G.
      • Yodoi J.
      • Kahn S.E.
      • Lavelle E.C.
      • O'Neill L.A.
      ,
      • Mandrup-Poulsen T.
      )) through the formation of reactive oxygen species (
      • Zhou R.
      • Tardivel A.
      • Thorens B.
      • Choi I.
      • Tschopp J.
      ,
      • Schroder K.
      • Zhou R.
      • Tschopp J.
      ). Glucose-induced IL-1β secretion by the β-cell is prevented in NALP3−/− mice, indicating that IL-1β is generated through glucose-induced reactive oxygen species production and oxidative stress (
      • Zhou R.
      • Tardivel A.
      • Thorens B.
      • Choi I.
      • Tschopp J.
      ). The thioredoxin-interacting protein, which has been linked to insulin resistance (
      • Parikh H.
      • Carlsson E.
      • Chutkow W.A.
      • Johansson L.E.
      • Storgaard H.
      • Poulsen P.
      • Saxena R.
      • Ladd C.
      • Schulze P.C.
      • Mazzini M.J.
      • Jensen C.B.
      • Krook A.
      • Björnholm M.
      • Tornqvist H.
      • Zierath J.R.
      • Ridderstråle M.
      • Altshuler D.
      • Lee R.T.
      • Vaag A.
      • Groop L.C.
      • Mootha V.K.
      ), functions as an activator of NALP3. In line with these data, another recent study shows that thioredoxin-interacting protein is highly increased by elevated glucose in β-cells and that thioredoxin-interacting protein-deficient islets are protected against glucose toxicity (
      • Chen J.
      • Fontes G.
      • Saxena G.
      • Poitout V.
      • Shalev A.
      ). Although glucose-induced IL-1β production in the β-cell was not observed in all studies (
      • Cnop M.
      • Welsh N.
      • Jonas J.C.
      • Jörns A.
      • Lenzen S.
      • Eizirik D.L.
      ), IL-1β expression in islets from patients with T2DM has been confirmed (
      • Böni-Schnetzler M.
      • Thorne J.
      • Parnaud G.
      • Marselli L.
      • Ehses J.A.
      • Kerr-Conte J.
      • Pattou F.
      • Halban P.A.
      • Weir G.C.
      • Donath M.Y.
      ,
      • Igoillo-Esteve M.
      • Marselli L.
      • Cunha D.A.
      • Ladrière L.
      • Ortis F.
      • Grieco F.A.
      • Dotta F.
      • Weir G.C.
      • Marchetti P.
      • Eizirik D.L.
      • Cnop M.
      ), supporting the concept of blocking IL-1β signals as a target for diabetes treatment. Indeed, daily injection of IL-1Ra in mice fed a high fat diet (HFD) improves glycemia, glucose-stimulated insulin secretion, and survival (
      • Sauter N.S.
      • Schulthess F.T.
      • Galasso R.
      • Castellani L.W.
      • Maedler K.
      ); reduces hyperglycemia; and reverses the islet inflammatory phenotype in the GK rat (
      • Ehses J.A.
      • Lacraz G.
      • Giroix M.H.
      • Schmidlin F.
      • Coulaud J.
      • Kassis N.
      • Irminger J.C.
      • Kergoat M.
      • Portha B.
      • Homo-Delarche F.
      • Donath M.Y.
      ). Treatment with an IL-1β antibody also improves glycemic control in diet-induced obesity in mice (
      • Osborn O.
      • Brownell S.E.
      • Sanchez-Alavez M.
      • Salomon D.
      • Gram H.
      • Bartfai T.
      ,
      • Owyang A.M.
      • Maedler K.
      • Gross L.
      • Yin J.
      • Esposito L.
      • Shu L.
      • Jadhav J.
      • Domsgen E.
      • Bergemann J.
      • Lee S.
      • Kantak S.
      ). Results from a clinical study in patients with T2DM showed that IL-1Ra improves glycemic control and β-cell function (
      • Larsen C.M.
      • Faulenbach M.
      • Vaag A.
      • Vølund A.
      • Ehses J.A.
      • Seifert B.
      • Mandrup-Poulsen T.
      • Donath M.Y.
      ,
      • Donath M.Y.
      • Böni-Schnetzler M.
      • Ellingsgaard H.
      • Halban P.A.
      • Ehses J.A.
      ,
      • Maedler K.
      • Dharmadhikari G.
      • Schumann D.M.
      • Storling J.
      ). Blocking IL-1β signals reduces expression of inflammatory marker in fat tissue and in islets (
      • Sauter N.S.
      • Schulthess F.T.
      • Galasso R.
      • Castellani L.W.
      • Maedler K.
      ). None of the studies have studied the mechanisms of the protective effect of IL-1Ra at the level of β-cell gene regulation. IL-1β affects expression of the transcription factor PDX1 (pancreatic duodenal homeobox-1, previously called IPF1, IDX1, STF1, or IUF1) (
      • Eizirik D.L.
      • Mandrup-Poulsen T.
      ), a key factor in pancreas development and function. Reduced PDX1 expression levels negatively regulates insulin expression and secretion and predispose islets to apoptosis (
      • Ahlgren U.
      • Jonsson J.
      • Jonsson L.
      • Simu K.
      • Edlund H.
      ,
      • Brissova M.
      • Shiota M.
      • Nicholson W.E.
      • Gannon M.
      • Knobel S.M.
      • Piston D.W.
      • Wright C.V.
      • Powers A.C.
      ,
      • Johnson J.D.
      • Ahmed N.T.
      • Luciani D.S.
      • Han Z.
      • Tran H.
      • Fujita J.
      • Misler S.
      • Edlund H.
      • Polonsky K.S.
      ). Its homozygous mutations result in pancreas agenesis associated with neonatal diabetes (
      • McKinnon C.M.
      • Docherty K.
      ,
      • Stoffers D.A.
      • Zinkin N.T.
      • Stanojevic V.
      • Clarke W.L.
      • Habener J.F.
      ). PDX1 directly binds to the promoter and controls expression of important β-cell genes, which are vital for β-cell function, such as insulin, Glut2, and glucokinase (
      • McKinnon C.M.
      • Docherty K.
      ). PDX1 deficiency contributes to impaired proliferation and enhanced apoptosis via transcriptional mechanisms in models of type 2 diabetes, such as Psammomys obesus and the leptin receptor-deficient (Leprdb/db) db/db mice (
      • Leibowitz G.
      • Ferber S.
      • Apelqvist A.
      • Edlund H.
      • Gross D.J.
      • Cerasi E.
      • Melloul D.
      • Kaiser N.
      ). Overexpression of PDX1 restores β-cell mass and function, thereby preventing the onset of diabetes in IRS2 knock-out mice, showing the critical role of PDX1 in β-cell survival (
      • Kushner J.A.
      • Ye J.
      • Schubert M.
      • Burks D.J.
      • Dow M.A.
      • Flint C.L.
      • Dutta S.
      • Wright C.V.
      • Montminy M.R.
      • White M.F.
      ).
      Complicated signaling networks control PDX1 regulation, and nucleo-cytoplasmic shuttling plays a major role in the regulation of PDX1 function (
      • Kawamori D.
      • Kaneto H.
      • Nakatani Y.
      • Matsuoka T.A.
      • Matsuhisa M.
      • Hori M.
      • Yamasaki Y.
      ,
      • Meng Z.
      • Lv J.
      • Luo Y.
      • Lin Y.
      • Zhu Y.
      • Nie J.
      • Yang T.
      • Sun Y.
      • Han X.
      ). Well characterized PDX1 nuclear import signal and nuclear export signal (NES) suggest that PDX1 might be regulated at the level of cellular localization (
      • Kawamori D.
      • Kaneto H.
      • Nakatani Y.
      • Matsuoka T.A.
      • Matsuhisa M.
      • Hori M.
      • Yamasaki Y.
      ,
      • Moede T.
      • Leibiger B.
      • Pour H.G.
      • Berggren P.
      • Leibiger I.B.
      ). Post-translational modification of proteins is the most abundant form of cellular regulation, affecting many cellular signal pathways, including metabolism, growth, differentiation, and apoptosis. In response to acute elevation of glucose and survival factors, such as insulin, PDX1 is phosphorylated and translocates to the nucleus (
      • Elrick L.J.
      • Docherty K.
      ,
      • Macfarlane W.M.
      • Shepherd R.M.
      • Cosgrove K.E.
      • James R.F.
      • Dunne M.J.
      • Docherty K.
      ). By contrast, stimuli associated with diabetes, such as oxidative stress (
      • Kawamori D.
      • Kaneto H.
      • Nakatani Y.
      • Matsuoka T.A.
      • Matsuhisa M.
      • Hori M.
      • Yamasaki Y.
      ) and free fatty acids (
      • Hagman D.K.
      • Hays L.B.
      • Parazzoli S.D.
      • Poitout V.
      ), cause nuclear exclusion of PDX1 (
      • Hagman D.K.
      • Hays L.B.
      • Parazzoli S.D.
      • Poitout V.
      ). This suggests that cytoplasmic accumulation may represent a mechanism to reduce the nuclear action of PDX1 under pathologic conditions rather than to promote a specific cytoplasmic function.
      We have shown previously that a diet enriched with fat and sucrose (“Surwit”; HFD) induces impaired glucose tolerance after 4 weeks of feeding, impaired fasting glucose after 8 weeks, and hyperglycemia after 12 weeks in C57Bl/6J mice (
      • Sauter N.S.
      • Schulthess F.T.
      • Galasso R.
      • Castellani L.W.
      • Maedler K.
      ,
      • Owyang A.M.
      • Maedler K.
      • Gross L.
      • Yin J.
      • Esposito L.
      • Shu L.
      • Jadhav J.
      • Domsgen E.
      • Bergemann J.
      • Lee S.
      • Kantak S.
      ,
      • Glas R.
      • Sauter N.S.
      • Schulthess F.T.
      • Shu L.
      • Oberholzer J.
      • Maedler K.
      ). These changes in glycemia were accompanied by fluctuations in β-cell mass. Despite the reduction in β-cell proliferation and the increase in β-cell apoptosis, islets showed a compensatory increase in β-cell mass up to 12 weeks of diet. After 16 weeks, apoptosis was increased, and β-cell mass was reduced in the HFD-treated mice. IL-1β antagonism by anti-IL-1β antibody treatment, IL-1Ra injections, or overexpression restored normoglycemia and β-cell function and survival (
      • Sauter N.S.
      • Schulthess F.T.
      • Galasso R.
      • Castellani L.W.
      • Maedler K.
      ,
      • Owyang A.M.
      • Maedler K.
      • Gross L.
      • Yin J.
      • Esposito L.
      • Shu L.
      • Jadhav J.
      • Domsgen E.
      • Bergemann J.
      • Lee S.
      • Kantak S.
      ,
      • Glas R.
      • Sauter N.S.
      • Schulthess F.T.
      • Shu L.
      • Oberholzer J.
      • Maedler K.
      ), but also changes in the inflammatory state of the fat tissue were involved in the protective effects of IL-1β antagonism. Therefore, we asked how IL-1β signals regulate gene transcription in the β-cell. We tested the effect of IL-1Ra on regulating glucose homeostasis in another animal model, the obese diabetic leptin receptor-deficient Leprdb/db mice (db/db). Commonly, we detected that IL-1Ra was able to maintain the cellular localization of PDX1. A diabetic milieu in vitro as well as T2DM in vivo induced a switch of PDX1 from the nucleus to the cytosol, which was accompanied by a loss in β-cell mass and function.
      Whether PDX1 is altered in its localization in β-cells during the progression to diabetes and whether these changes may affect β-cell function in different levels was previously unknown. Because PDX1 regulates insulin and specific β-cell genes, altered localization may contribute to β-cell death and loss of function. In the short term, this stressful response may be tolerated, but under chronic situations in T2DM, prolonged stress conditions ultimately affect β-cell survival.

      DISCUSSION

      Human mutations of PDX1 as well as changes in its transactivation are strongly associated with diabetes (
      • Liu A.
      • Oliver-Krasinski J.
      • Stoffers D.A.
      ,
      • Stoffers D.A.
      • Stanojevic V.
      • Habener J.F.
      ). In the present study, we show that localization of PDX1 correlates with T2DM, β-cell function, and survival. PDX1 shuttling is one of the mechanisms that may explain the dual role of glucose on β-cell function and survival. Although acute elevated glucose induces β-cell proliferation and insulin secretion, chronically elevated glucose concentrations impair β-cell function, induce apoptosis (
      • Maedler K.
      • Spinas G.A.
      • Lehmann R.
      • Sergeev P.
      • Weber M.
      • Fontana A.
      • Kaiser N.
      • Donath M.Y.
      ), and thus may accelerate diabetes. Upon acute exposure of β-cells to glucose, PDX1 translocates to the nucleus, leading to insulin gene transcription (
      • Elrick L.J.
      • Docherty K.
      ). In contrast, oxidative stress induces PDX1 shuttling to the cytosol (
      • Kawamori D.
      • Kajimoto Y.
      • Kaneto H.
      • Umayahara Y.
      • Fujitani Y.
      • Miyatsuka T.
      • Watada H.
      • Leibiger I.B.
      • Yamasaki Y.
      • Hori M.
      ) and impairs insulin secretion (
      • Robertson R.P.
      ). We show here that in diabetic islets or under conditions of chronic hyperglycemia or IL-1β exposure for 3 days in vitro, conditions of impaired β-cell survival and function shifted PDX1 expression to the cytoplasm. These data show the impact of PDX1 localization in regulating β-cell turnover and function.
      Whenever it was shifted to the cytosol, PDX1 showed a weaker staining and signal intensity, which suggests the possibility of PDX1 degradation after translocalization. This is also supported by the observation that overexpression of PDX1-NES, which remains in the nucleus, prevents PDX1 shuttling together with degradation at conditions of chronically elevated glucose. Although transfection could not be achieved in all islet cells, nuclear PDX1 overexpression prevented the deleterious effects of glucose and IL-1β on β-cell survival and function. Previously, we have observed such quantitative decrease in PDX1 induced by elevated glucose concentrations in human and rat islets. At this time, whole islet lysates were used for the analysis, and we did not investigate PDX1 localization. PDX1 decrease was dose-dependent on glucose concentrations (5.5–33.3 mm) (
      • Maedler K.
      • Schumann D.M.
      • Schulthess F.
      • Oberholzer J.
      • Bosco D.
      • Berney T.
      • Donath M.Y.
      ). In the same previous study, we also show an age-dependent PDX1 decrease in human and rat islets, which was confirmed in pancreatic biopsy samples (
      • Reers C.
      • Erbel S.
      • Esposito I.
      • Schmied B.
      • Büchler M.W.
      • Nawroth P.P.
      • Ritzel R.A.
      ). Such PDX1 decrease correlated with the increased susceptibility to glucose-induced apoptosis and with a decline in β-cell proliferation at an older age. Neither Reers et al. (
      • Reers C.
      • Erbel S.
      • Esposito I.
      • Schmied B.
      • Büchler M.W.
      • Nawroth P.P.
      • Ritzel R.A.
      ) nor we were able to show nuclear PDX1 localization. It was suggested that the human pancreas embedding method prevented detection of nuclear PDX1. Here we improved tissue permeabilization and clearly detected nuclear PDX1 even in sections from human autopsy and biopsy, with strong PDX1 signals in the nucleus in non-diabetic control pancreases and a shift of PDX1 to the cytosol in diabetic conditions, a signal that appeared much weaker in sections from patients with T2DM. Together, the data support PDX1 shuttling in response to chronic glucose to the cytosol and its subsequent degradation as one deleterious factor contributing to β-cell failure.
      Strategies to block these deleterious effects on the β-cell are needed for a successful diabetes therapy. Blocking IL-1β signals has been suggested as a novel treatment for diabetes. The anti-inflammatory cytokine IL-1Ra prevents glucose-induced apoptosis by blocking proapoptotic IL-1β signaling in vitro (
      • Maedler K.
      • Sergeev P.
      • Ris F.
      • Oberholzer J.
      • Joller-Jemelka H.I.
      • Spinas G.A.
      • Kaiser N.
      • Halban P.A.
      • Donath M.Y.
      ) and improves glycemia and β-cell function and survival in vivo (
      • Sauter N.S.
      • Schulthess F.T.
      • Galasso R.
      • Castellani L.W.
      • Maedler K.
      ,
      • Ehses J.A.
      • Lacraz G.
      • Giroix M.H.
      • Schmidlin F.
      • Coulaud J.
      • Kassis N.
      • Irminger J.C.
      • Kergoat M.
      • Portha B.
      • Homo-Delarche F.
      • Donath M.Y.
      ,
      • Larsen C.M.
      • Faulenbach M.
      • Vaag A.
      • Vølund A.
      • Ehses J.A.
      • Seifert B.
      • Mandrup-Poulsen T.
      • Donath M.Y.
      ).
      In this study, we provide further mechanisms of the protective effect of IL-1Ra directly on the β-cell transcriptional regulation in C57BL/6J mice fed a high fat/high sucrose diet (Surwit) and in db/db mice, serving as two animal models of T2DM and in human islets exposed to a diabetic milieu. 12 weeks of high fat feeding induced impaired glucose tolerance, which was inhibited in the IL-1Ra-OE mice. Unexpectedly, insulin and PDX1 mRNA levels were oppositely regulated in the HFD-treated mice; insulin was reduced, and PDX1 was significantly increased in islets after 12 weeks of high fat/high sucrose diet. IL-1Ra prevented such changes. Many previous studies have observed no changes or increases in insulin mRNA in response to a high fat diet without increase in sucrose (“Western diet”) in rat or mouse models (e.g. see Refs.
      • Briaud I.
      • Kelpe C.L.
      • Johnson L.M.
      • Tran P.O.
      • Poitout V.
      and
      • Hansotia T.
      • Maida A.
      • Flock G.
      • Yamada Y.
      • Tsukiyama K.
      • Seino Y.
      • Drucker D.J.
      ). The addition of sucrose to the diet (Surwit) causes β-cell failure together with reduced insulin mRNA in the β-cell (
      • Mulder H.
      • Mårtensson H.
      • Sundler F.
      • Ahrén B.
      ).
      Although our results are in contrast to previous data showing PDX1 down-regulation in response to hyperglycemia in β-cells in culture as well as in type 2 diabetic animal models (e.g. in ZDF rats (
      • Robertson R.P.
      • Harmon J.
      • Tran P.O.
      • Tanaka Y.
      • Takahashi H.
      ), in P. obesus (
      • Leibowitz G.
      • Ferber S.
      • Apelqvist A.
      • Edlund H.
      • Gross D.J.
      • Cerasi E.
      • Melloul D.
      • Kaiser N.
      ), and in partially pancreatectomized rats (
      • Zangen D.H.
      • Bonner-Weir S.
      • Lee C.H.
      • Latimer J.B.
      • Miller C.P.
      • Habener J.F.
      • Weir G.C.
      )), studies in isolated human islets from patients with T2DM show a similar opposite regulation, with reduced insulin mRNA and increased PDX1 mRNA levels compared with islets isolated from non-diabetic controls (
      • Del Guerra S.
      • Lupi R.
      • Marselli L.
      • Masini M.
      • Bugliani M.
      • Sbrana S.
      • Torri S.
      • Pollera M.
      • Boggi U.
      • Mosca F.
      • Del Prato S.
      • Marchetti P.
      ).
      PDX1 expression seems to be important for the β-cell response to a higher insulin demand (e.g. in insulin resistance and β-cell compensation may occur through increased PDX1 mRNA). But suppression of PDX1 expression in MIN6 cells did not lead to a decrease of insulin or glucokinase mRNA (
      • Kajimoto Y.
      • Watada H.
      • Matsuoka T.
      • Kaneto H.
      • Fujitani Y.
      • Miyazaki J.
      • Yamasaki Y.
      ). To activate insulin transcription, PDX1 translocates into the nucleus, where it binds to the insulin gene (
      • Macfarlane W.M.
      • McKinnon C.M.
      • Felton-Edkins Z.A.
      • Cragg H.
      • James R.F.
      • Docherty K.
      ,
      • Rafiq I.
      • Kennedy H.J.
      • Rutter G.A.
      ). Therefore, post-translational changes, which define PDX1 localization, rather than mRNA expression levels may play a more important role under diabetic conditions. For instance, oxidative stress induces PDX1 shuttling from the nucleus to the cytosol and thus causes a severe reduction of PDX1 activity (
      • Kawamori D.
      • Kajimoto Y.
      • Kaneto H.
      • Umayahara Y.
      • Fujitani Y.
      • Miyatsuka T.
      • Watada H.
      • Leibiger I.B.
      • Yamasaki Y.
      • Hori M.
      ). Also, 24-h exposure of rat islets to palmitic acid at elevated glucose concentrations causes PDX1 localization to the cytosol together with a decrease in MafA expression and inhibition of insulin expression (
      • Hagman D.K.
      • Hays L.B.
      • Parazzoli S.D.
      • Poitout V.
      ). In line with these previous observations, we detected PDX1 predominantly expressed in the cytosol in HFD-treated and in hyperglycemic db/db mice. In contrast, in IL-1Ra-treated as well as the normal diet groups, PDX1 was localized in the nucleus. Because IL-1Ra protected from the prodiabetic effect of the diet, we propose that this is a result of maintaining PDX1 functionally in the nucleus.
      Homozygous Leprdb/db mice (db/db) on the C57BLKS/J background with this depletion in the leptin receptor become obese, hyperglycemic, and hyperinsulinemic within the first month of age. In these mice, IL-1β-mediated innate immunity is augmented, which results from a diabetes-associated loss of IL-1β counterregulation (
      • O'Connor J.C.
      • Satpathy A.
      • Hartman M.E.
      • Horvath E.M.
      • Kelley K.W.
      • Dantzer R.
      • Johnson R.W.
      • Freund G.G.
      ). To test the hypothesis that IL-1Ra would prevent diabetes progression in this model of T2DM, we injected db/db mice daily with IL-1Ra or with vehicle from 4 weeks of age on. db/db mice that received IL-1Ra showed improved glucose tolerance during intraperitoneal glucose tolerance test experiments after 2 weeks of treatment as compared with their vehicle-treated littermates (p < 0.05 at time points 30 and 60 min; data not shown). From 3 weeks of treatment on, we did not observe any differences in glucose levels between the two groups anymore. Considering the short half-life of IL-1Ra (6–8 h) and the 10–100-fold excess that is needed to block IL-1β-mediated effects (
      • Eizirik D.L.
      • Tracey D.E.
      • Bendtzen K.
      • Sandler S.
      ), we tested whether constitutive endogenous overexpression of IL-1Ra would improve the outcome of elevated IL-1Ra levels in db/db mice. We could confirm in the db/db mouse model that IL-1Ra overexpression was protective against the development of diabetes and β-cell failure, although the effects of IL-1Ra in db/db mice were quite modest. However, considering the db/db mouse as a model of severe diabetes with rapid development of hyperglycemia, any significant rescue in the model and a combined effect on glucose tolerance, insulin secretion, β-cell mass, and apoptosis confirms a protective effect on the β-cell. This was paralleled by IL-1Ra-induced PDX1 stabilization in the nucleus in the db/db mice. Already at 6 weeks of age, PDX1 was predominantly expressed in the cytosol in the db/db mice, whereas at the age of 10 weeks, PDX1 was strongly decreased. IL-1Ra restored nuclear PDX1 in 6-week-old mice, and also in 10-week-old mice, nuclear PDX1 expression could be detected. Previous cytochemistry analyses of db/db mouse pancreases show a nuclear loss of MafA rather than of PDX1 (
      • Harmon J.S.
      • Bogdani M.
      • Parazzoli S.D.
      • Mak S.S.
      • Oseid E.A.
      • Berghmans M.
      • Leboeuf R.C.
      • Robertson R.P.
      ). To exclude differences in the background, we also analyzed PDX1 in male BKSdb/db mice and could confirm the switch of PDX1 localization to the nucleus already after 6 weeks of age.
      Activation of JNK is a hallmark in glucose and IL-1β effect on the β-cell and is involved in PDX1 regulation (
      • Størling J.
      • Zaitsev S.V.
      • Kapelioukh I.L.
      • Karlsen A.E.
      • Billestrup N.
      • Berggren P.O.
      • Mandrup-Poulsen T.
      ,
      • Kaneto H.
      • Matsuoka T.A.
      • Nakatani Y.
      • Kawamori D.
      • Matsuhisa M.
      • Yamasaki Y.
      ). We used such well known results to confirm our study design and could identify JNK as cellular component also involved in glucose- and IL-1β-regulated PDX1 localization. In wild type islets, JNK was phosphorylated after short term (30 min (data not shown) and 1 day) as well as long term (3 days) incubations with elevated glucose. Therefore, it seems that acute JNK activation does not necessarily cause impairment in β-cell function and survival, whereas chronic activation correlates with glucotoxicity. IL-1Ra protected islets from prolonged JNK activation. JNK activity has previously been linked to PDX1 shuttling under conditions of oxidative stress (
      • Kawamori D.
      • Kajimoto Y.
      • Kaneto H.
      • Umayahara Y.
      • Fujitani Y.
      • Miyatsuka T.
      • Watada H.
      • Leibiger I.B.
      • Yamasaki Y.
      • Hori M.
      ) and in prostaglandin E2-induced β-cell dysfunction (
      • Meng Z.
      • Lv J.
      • Luo Y.
      • Lin Y.
      • Zhu Y.
      • Nie J.
      • Yang T.
      • Sun Y.
      • Han X.
      ), together with Foxo1 as a key player (
      • Kawamori D.
      • Kaneto H.
      • Nakatani Y.
      • Matsuoka T.A.
      • Matsuhisa M.
      • Hori M.
      • Yamasaki Y.
      ). Foxo1 cellular localization determines PDX1 localization, and both are reversely expressed. Foxo1 itself is regulated by JNK and AKT activity; JNK induces Foxo1 nuclear import, which leads to PDX1 export, whereas AKT-mediated Foxo1 phosphorylation results in Foxo1 cytoplasmic and PDX1 nuclear localization. Our findings suggest the JNK-PDX1 pathway as a critical signaling network that transduces short term as well as long term glucose stimulation and therefore might partly mediate the dual effect of glucose on β-cell function and survival.
      The fact that IL-1Ra potentially prevented hyperglycemia and improved β-cell function favors the critical role of IL-1β signaling in the β-cell not only in a type 1 but also in a type 2 diabetic environment. Our data provide new insights into mechanisms of the protective effect of IL-1Ra on β-cell function and turnover, establish the important role of nuclear PDX1 localization, and support IL-1Ra as a potential therapy for diabetes.

      Acknowledgments

      We thank Dr. Ingo Leibiger for the PDX1 plasmids, Dr. Chris Wright for the PDX1 antibody, Jennifer Bergemann and Anke Meyer for excellent assistance, Madhura Panse for the analysis of PDX1 in isolated islets, Silvia Del Guerra for the islet RNA preparation, and the National Disease Research Interchange for providing human pancreatic sections. Human islets were provided through the Islet Cell Resource Consortium (administered by the Administrative and Bioinformatics Coordination Center (ABCC) and supported by the National Center for Research Resources (NCRR); NIDDK, National Institutes of Health; and the Juvenile Diabetes Research Foundation) and through the European Consortium for Islet Transplantation Islets for Research Distribution Program.

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