MXPA06010018A - Methods for altering insulin secretion - Google Patents

Abstract

Modulation of the activity of glucocorticoid inducible kinase SGK1 in pancreatic islet cells restores insulin release. Also disclosed are methods and compounds useful for the treatment of glucocorticoid induced diabetes mellitus type-2.

Description

METHODS TO ALTER THE INSULIN SECRETION Field of the Invention A method for altering insulin secretion comprising contacting a pancreatic islet cell expressing SGK1 with a substance that modulates SGK1 is described, and wherein the inhibition of SGK1 includes the reversal of the depolarizing effect of the glucose, causing the activation of calcium channels regulated by voltage and the release of insulin. BACKGROUND OF THE INVENTION Treatment with glucocorticoids induces type 2 diabetes mellitus which is easily reversible after stopping the drug (Hoogwerf and Dnes 1999; Schac et al., 2002). In addition to peripheral insulin resistance and increased hepatic glucose production by stimulating gluconeogenesis (McMahon et al., 1988), glucocorticoids interfere with the secretion of insulin from pancreatic cells (Lambillotte et al., 1997; Pierluissi et al. al., 1986). Despite extensive studies, the molecular mechanism is still a matter of debate. The antiprogestin mifepristone (RU486), a nuclear glucocorticoid receptor antagonist, completely stopped the inhibition of insulin secretion induced by dexamethasone (Lambillotte et al., 1997), pointing to the implication of a REF: 174711 gene expression dependent on glucocorticoids. Among the glucocorticoid-sensitive genes is the serum-inducible kinase and glucocorticoids SGK1 (Webster et al., 1993b, Webser et al., 1993a, US6326181). SGK1 is influenced by a number of stimuli (Lang et al., 2001) such as, for example, mineral corticosteroids (Chen et al., 1999, Naray-Fejes-Toth et al., 1999, Shigaev et al., 2000, Brennan et al., 2000, cowling et al., 2000). The SGK1 has been shown to be regulated through insulin-like growth factor IGF1, insulin and oxidant stress passing through a cascade of signals including phosphoinositol-3-cinsa (PI3 kinase) and phosphoinositide-dependent kinase PDKI (Kobayashi and Cohen) 1999, Park et al., 1999, Kobayashi et al., 1999). Activation of SGK1 through PDK1 includes phosphorylation of serine 422. It has further been shown that a mutation from being 422 to aspartate (S 22DSGK1) results in a continuously activated kinase (Kobayashi et al., 1999). Several assay systems are available to measure the activity of glucocorticoid-inducible SGK1 kinase. In the scintillation proximity assay (Sorg et al., J. of Biomolecular Screening, 2002, 7, 11-19) and the vaporization plate assay, the radioactive phosphorylation of a protein or peptide is measured as an? ATP substrate. . In the presence of an inhibitor compound, a radioactive signal is not detected or a reduced radioactive signal is detected. In addition, fluorescence resonance energy transfer technologies result in homogeneous time (HTR-FRET) and fluorescence polarization (FP) are useful for assay methods (Sills et al., J. of Biomolecular Screening, 2002, 191 - 214). Other non-radioactive ELISA test methods use phospho-antibodies (AB) specific. Phospho-AB binds only to the phosphorylated substrate. This binding is detectable with a second anti-lamb antibody conjugated to peroxidase by chemiluminescence (Ross et al., 2002, biochem J., immediate publication, document BJ20020786). Previous studies demonstrated that SGK1 is a potent, renal epithelial Na + channel stimulator (De la Rosa et al., 1999, Boehmer et al., 2000, Chen et al., 1999, Naray-Fejes-Toth et al., 1999 , Lang et al., 2000, Shigaev et al., 2000, Wagner et al., 2001). Another discovery related to SGK1 was that the polymorphism of a single nucleotide (SNP) in exon 8 with combinations of nucleotides (CC / CT) and additional polymorphism in intron 6 (CC) are associated with increased blood pressure (Busjahn et al. ., 2002) and from this it was concluded that SGKl may be important for the regulation of blood pressure and hypertension. Because the increased activity of SGK1 correlates with renal epithelial Na + channel activity leading to hypertension through increased renal sodium reabsorption (Lifton 1996; Staessen et al. , 2003; Warnock 2001), it was concluded that depending on the combination of allelic variants of SGKl, an increase in renal Na + resorption may occur, which in turn will increase blood pressure (Busjahn et al., 2002). Up to now, the insulin secreting cells of the pancreatic islets have not been shown to express significant amounts of SGK1 (Klingel et al., 2000) and it is generally believed that the cells of the untreated islets do not express SGK1 or only express it to a lesser degree. . Treatment with glucocorticoids at high doses for an extended period of time predisposes to the development of diabetes mellitus at least in part through the deterioration of insulin secretion. The underlying mechanism has remained elusive and the objectives that could allow a therapeutic intervention are currently unknown. The present application defines this new mechanism and molecular objective, and at the same time shows how to identify new compounds that interfere with the pathological mechanism mentioned above, with the purpose of overcoming diabetes mellitus. BRIEF DESCRIPTION OF THE INVENTION The present application unexpectedly demonstrates that the cells of the pancreatic islets show a pronounced increase in the levels of transcription of SGK1 and the expression in cells of the insulin secreting islands, which have been pretreated by glucocorticoids. Excess glucocorticoids predispose the development of diabetes mellitus at least in part through the deterioration of insulin secretion, and the "method of the present invention is to modulate the activity of SGK1 in cells of pancreatic islets thus reducing diabetes mellitus .. type 2 induced glucocorticoid in a subject requiring such treatment The invention shows, among other aspects, methods for the identification of therapeutically active compounds that are useful for restoring insulin secretion by contacting a cell of the pancreatic islets that express SGKl with a substance that modulates SGKl Thus, the depolarizing effect of glucose is reversed resulting in the activation of voltage regulated calcium channels and a subsequent insulin release.The modulation of SGKl is especially useful when applied to a clinically relevant phenotype or genotype that is defined by a Single-nucleotide limorfism of the SGK1 gene. Therefore, the analysis of a polymorphic SNP variant of SGK1 in samples derived from an individual that requires treatment could be another application. Furthermore, the invention provides a method for determining the progression, regression or onset of a disease by measuring SGK1 expression. Samples taken from the sick individuals may also allow the analysis of selected SNK variants of SGK1 and their correlation with the predisposition to a disease or other conditions induced for example by prolonged treatment with glucocorticoids. Another aspect is related to methods of analysis or screening to identify new candidates for drugs that modulate diseases related to SGKl. Especially useful modulators are compounds that interfere with the function of SGK1 thus resulting in an over-regulation of insulin secretion. SGK1 inhibitors are especially useful for treating subjects suffering from symptoms of type 2 diabetes mellitus. SGKl modulators are also useful for treating subjects with stress-induced hyperglycemia or subjects having hypoglycaemia. The method of drug analysis carried out in accordance with this invention has led to the discovery of therapeutic compounds directed to SGK1. Two classes of different compounds have been identified, one belonging to the class of acylhydrazone derivatives and another belonging to pyridopyrimidine derivatives. Selected SGK1 inhibitor compounds in pharmaceutical compositions comprising a carrier, pharmaceutically effective excipient or diluent, are useful for the treatment of type 2 diabetes mellitus induced by glucocorticoids. It is crucial for this invention that the methods of analysis used to identify new drugs with the desired therapeutic profile are not restricted to the compounds described in this application. Moreover, it is clear to the expert that it could be useful to apply a one-step approach or a two-step approach to screen or analyze SGK1 modulator compounds. The first stage of this analysis includes the identification of compounds that interfere with the activity of SGKl kinase. Several assay formats are available and a preferred assay uses the measurement of SGK1-catalyzed radioactive phosphorylation of a protein or peptide as a substrate together with? ATP. In the presence of a SGK1 inhibitor compound no radioactive signal is detected or a reduced radioactive signal is detected. In a second step of the analysis, SGK1 inhibitor compounds are tested to verify their potential to re-establish insulin secretion in cells of pancreatic islets treated with glucocorticoids such as for example INS-1 cells. After the inhibition of SGKl, the release of insulin is measured. However, measuring other reading activities could be useful as well. The underlying mechanism of glucocorticoid-induced diabetes has remained elusive to date. In this invention it is demonstrated that glucocorticoids such as dexamethasone over-regulate the transcription and expression of SGK1 kinase inducible by serum and glucocorticoids in insulin-secreting cells, an effect that can be reversed by mifepristone (RU486), a nuclear glucocorticoid receptor antagonist. . When co-expressed in Xenopus oocytes, SGKl increases the activity of the voltage regulated K + channel (Kvl.5). In INS-1 cells, dexamethasone stimulates the transcription of Kvl. 5, increases the outward current of repolarization and reduces the release of insulin induced by glucose. These last two effects are reversed by the channel blockers of K + 4-AP and TEA. Dexamethasone virtually stops glucose-induced insulin release from islets isolated from wild-type mice, a significantly attenuated effect in isolated islets of mice with suppressed SGK1 gene. In conclusion, glucocorticoids stimulate the transcription of SGK1, which in turn over-regulates the activity of voltage regulated K + channels. The subsequent hyperpolarization counteracts the depolarizing effect of glucose and prevents the activation of voltage-regulated Ca2 + channels, Ca2 + entry and insulin release. The present invention relates to the role of SGK1 and the activity of the SGK1-dependent channel in the regulation of insulin secretion.

According to real-time PCR, the transcription level of SGK1 is low in untreated INS-1 cells (figure 1A), a finding that parallels the low levels of transcription reported previously for human pancreatic islets (Klingel et al., 2000). However, incubation of INS-1 cells with 100 nM dexamethasone during 2 to 23 hours increased the levels of mRNA transcription, an effect that was completely abrogated by the glucocorticoid receptor antagonist RU486 (Fig. IA). Within 23 hours dexamethasone increased the levels of transcription of cellular SGKl increased in mouse islets after treatment with dexamethasone (Fig. 1A). A similarly strong stimulation of SGK1 transcription by glucocorticoids was observed in other cell types (Itani et al., 2002; Rozansky et al., 2002). As it is apparent from the Western blotting analysis, the SGK1 protein was not detectable in untreated cells but appeared within 2 hours and increased more within the next 23 hours of exposure to dexamethasone (100 nM) (Fig. IB). The increase in the abundance of SGKl protein was completely reversed by RU486. Thus, dexamethasone stimulates the expression of SGK1 in insulin-secreting cells. As shown in FIG. ID, the co-expression of the SGK1 and Kv channels in Xenopus oocytes, over-regulates approximately 2-fold the activity of heterologously expressed Kvl.5 channels (Fig. ID). These channels have previously been shown to be expressed in 1NS-1 cells (Su et al., 2001) as well as in rodent and human ß cells (Philipson et al., 1994, Roe et al., 1996). In INS-1 cells, the channels are inhibited by the K + 4-AP channel blocker (Su et al., 2001). As illustrated in Figures 2A and 2B, the dexamethasone treatment was in fact followed by an increase in outward current regulated by 4-AP responsive voltage. In untreated cells, the K + 4-AP channel blocker inhibited only 10% (0.1 mM) and 28% (1 mM) of the outward current. After a 4 hour treatment with 100 nM dexamethasone, the 4-AP sensitive current was increased to 28% (0.1 mM of 4-AP) and 40% (1 mM of 4-AP). These data demonstrate that the activity of the Kvl .5 channel is increased by dexamethasone in insulin secreting cells. It has been found that glucocorticoids increase the expression of Kvl .5 channels in heart (Takimoto and Levitan 1994), in skeletal and pituitary muscle, but not in hypothalamus and lung (Levitan et al., 1996). Moreover, dexamethasone was necessary for T3 to increase the levels of Kvl.5 and mRNA in rat left ventricle of adrenalectomized animals made hypothyroid (Nishiyama et al., 1997). Real-time PCR reveals that treatment with dexamethasone (100 nM) within 4 hours increases the abundance of Kvl .5 mRNA in INS cells by a factor of approximately 1-0. Thus, dexamethasone stimulates the expression of SGK1, which in turn increases the activity of the Kv channel. Additional experiments were carried out to elucidate the impact of the Kv and SGKl channels on the release of insulin by dexamethasone. As illustrated in Figure 3, pretreatment of INS-1 cells with dexamethasone (100 nM) inhibited glucose-induced insulin secretion by 62%. This inhibition was reversed by blockers of the Kv, TEA and 4-AP channels, demonstrating the inhibition of insulin secretion mediated by dexamethasone. depends on the activity of the Kv channel. To estimate the contribution of SGK1 to the inhibitory effect of dexamethasone on insulin secretion, the effects of dexamethasone on insulin secretion in mice with suppressed gene SGK1 [sgk1] were compared with those of their partners in wild type bait. { sgkl + / +). Without dexamethasone treatment, insulin secretion after glucose exposure (16.7 nM), activation of adenylate cyclase (5 μm forskolin) or stimulation of protein kinase C (100 nM PMA) was not significantly different in isolated islands of sgkl ~ ~ and sgkl + / + mice (Figure 4 A and B, black bars). Treatment with dexamethasone, however, reduced the stimulatory effect of glucose, forskolin or PMA on insulin secretion significantly more in the islets of sgkl + / + mice than in the islets of the sgkl ~ / ~ mice. These data indicate that SGK1 participates in the sub-regulation of insulin secretion by dexamethasone. In conclusion, the present experiments describe a new mechanism in the regulation of insulin secretion. The glucocorticoid dexamethasone increases the transcription and expression of SGK1 in insulin-secreting cells. The kinase over-regulates voltage regulated K + channels including Kvl .5. The over-expression of the Kv channels hyperpolarizes the plasma membrane of ß cells thus preventing the activation of the voltage regulated Ca2 + channels. Consequently, the kinase contributes to the inhibition of insulin release during an excess of glucocorticoids. BRIEF DESCRIPTION OF THE FIGURES Figures IA-IB: Dexamethasone induces the expression of SGK1 in insulin secreting INS-1 cells. INS-1 cells were treated with 100 nM dexamethasone or vehicle (DMSO) in culture for the indicated periods of time. Dexamethasone significantly reduced SGK1 expression at 2 hours. RU486 at 1 μmol / 1 completely inhibited the effect of dexamethasone. Figure IA cellular RNA was transcribed into cDNA using M-MuLV reverse transcriptase (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). SGK1 mRNA was quantified by real-time PCR in a light cycle system (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). The primers used were: SGKl above: 5'-TTT tt CCA AC CTT GC-3 '; down: 5 '-AAT GAA CAÁ AGG TTG GGG GG-3. The mean ± SEM of the indicated number of experiments is shown. Figure IB Whole cell lysates were subjected to 1% SDS-PAGE and plotted on a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). The plots were incubated with antibodies against SGK1 (New England Biolabs, Beverly, MA, E.U.A). The bound antibody was visualized using a second antibody coupled to horseradish peroxidase. Figure 1C Real-time PCR for Kv 1.5 was carried out using a light cycle system (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). The same RNA preparations were analyzed as for the experiments described in FIG. 1A. The means ± SEM of three independent experiments are shown. Figure ID The coexpression of the SGK1 and Kv channel in Xenopus oocytes increases the K + currents. MRNA for SGKl (μg / ml) and human Kv 1.5 (μg / ml) were injected into the oocytes and whole cell streams were measured using the 2-volt clamp method two days after the injection. The representative traces and the mean ± SEM are shown. Figures 2A-2B: Dexamethasone increases the activity of the kv channel in INS-1 cells. The cells were treated before the experiment with 100 nM dexamethasone for 4 hours. Figure 2A The entire cell stream was induced by voltage pulses of 200 ms incrementing stepwise from 10 mv from -70 mV to +50 mV. Figure 2B Sensitivity to 4-ap (0.1 and 1 mm) and tea (1 and 10 mm) was tested on cells before (black columns) and after (white columns) of dexamethasone treatment. Voltage pulses of 200 ms duration from -70 to 50 mv were applied. The means ± sem are shown for the indicated number of experiments. * indicates significance (p <0.05) to the current in control, untreated cells at the same concentration of inhibitor. INS-1 cells were cultured as described before (Abel et al., 1996; Asfari et al., 1992). The external patch clamp solution contained (in mmoles / l): 140 NaCl, 5.6 KCl, 1.2 MgCl2 / 2.6 CaCl2, 0.5 glucose and 10 HEPES, pH 7.4. The internal solution contained (in mmoles / l): 30 KCL, 95 KAgi conato, 1 of MgCl2, 1.2 of NaH2P04, 4.8 of NaH2P04, 5 of Na2ATP, 1 of Na3GTP, 5 mmoles / l of EGTA, pH 7.2. An Epc9 Patch Clamp Amplifier amplifier (Heka Electronic, Lambrecht, Germany) was used for current measurements. Figure 3: Inhibition of the Kv channel reverses the inhibition of insulin secretion induced by dexamethasone in INS-1 cells. Before the experiments INS-1 cells were treated in culture with dexamethasone, 100 nM, for 4 hours. Cells were washed twice and preincubated in pH regulated saline with HEPES containing: (in mmol / l): 140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 0.5 glucose, 10 HEPES and 5 g / 1 of bovine serum albumin, pH 7.4 at 37 ° C. t Subsequently the cells were incubated for 30 minutes at 37 ° C in fresh solution containing the test substances at the appropriate concentrations. Insulin was measured by radioimmunoassay using rat insulin antiserum (Lineo, Biot-rend Chemikalien GmbH, Cologne, Germany), l125-insulin (CIS Diagnostik GmbH, Dreieich, Germany) and rat insulin (Novo Nordisk, Mainz, Germany) as a parameter or by means of a device for insulin Elisa (Mercodia, Uppsala, Sweden). Figures 4A and 4B: Dexamethasone did not affect the secretion of the islets of mice with suppressed gene SGKl The isolated islets were grown overnight in RPMI 1640 containing 11 mmoles / 1 glucose. Dexamethasone (100 ng / ml) or DMSO (control) was added 5 hours before the experiment. After cultivation the islets were preincubated for one hour at 37 ° C in pH buffer incubation containing (in mmoles / l): 140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 2.8 glucose, 10 of HEPES, PH 7.4 and 5 g / 1 of bovine serum albumin (fraction V, Sigma, Deisenhofen). Subsequently, batches of 5 islets / 0.5 ml were incubated for 30 minutes at 37 ° C in the presence of test substances as indicated for each experiment. Insulin was measured using a Elisa kit (Mercodia, Uppsala, Sweden). Detailed Description of the Invention Example 1 Generation of Sg l - / - mice A conditional direction vector was generated from a 7-kb fragment spanning the entire transcribed region on 12 exons (Wulff et al., 2002). The neomycin resistance cassette was flanked by two loxP sites and inserted into intron 11. Exons 4-11, which code for the sgkl kinase domain, were "flocked" by inserting a third loxP site into intron 3 A clone with a recombination between the first and third loxP sites (type I recombination) was injected into C57BL / 6 blasts. Male chimeras were crossed with females C57B / 6 and 129 / SvJ. Mice deficient in heterozygous sgk-1 were cross-re-crossed with wild-type 129 / SvJ mice for two generations and then cross-linked to generate bait partners sgkl - / - and sgkl + / + homozygous. EXAMPLE 2 Cell culture and measurement of insulin secretion INS-1 cells (kindly provided by CB Wollheim, University of Geneva, Switzerland) derived from a rat insulinoma were grown in RPMI 1640 pH regulated with HEPES supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 1 mmol / l of HEPES, 1 mmol / l of sodium pyruvate, 10 μmol / l of β-mercaptoethanol (Sigma, Munich, Germany) and antibiotics as described in any side (Abel et al., 1996; Asfari et al., 1992). The cells were plated at a cell density of 2.0-2.5-105 cells / ml in 24-well culture plates and cultured for 2 days before the experiment. The cells were washed twice with saline of pH regulated with HEPES containing (in mmoles / 1): 140 of NaCl, 5.6 of KCl, 1.2 of MgCl2, 2.6 of CaCl2, 0.5 of glucose, 10 of HEPES and 5 g / 1 of bovine serum albumin, pH 7.4, and preincubated for 30 minutes at 37 ° C. Subsequently the medium was discarded and a fresh medium containing the test substances was added at the appropriate concentrations. The cells were incubated for 30 minutes at 37 ° C. The incubations were stopped on ice, the medium was removed and frozen at -20 ° C until the insulin released in the supernatant was measured by radioimmunoassay using rat insulin antiserum (Lineo, Biotrend Chemikalien GmbH, Cologne, Germany), l125-insulin (CIS Diagnostik GmbH, Dreieich, Germany) and rat insulin (Novo Nordisk, Mainz, Germany) as a parameter or a device for insulin Elisa (Mercodia, Uppsala, Sweden). Insulin content was measured after extraction with acid ethanol (1.5 (v / v)% HCl / 75% ethanol) overnight at 4 ° C. For the isolation of islets from SGKl KO mice and wild-type bait partners, 3 ml of collagenase solution containing 1 mg / ml collagenase (Serva, Heidelgerg, Germany) was injected into the pancreas in situ through the cododectric duct. The entire gland was removed and digested for 10 minutes at 37 ° C. The islands were subsequently isolated from the tissue, exocrine when collected in fresh medium under a dissecting microscope. The islets were grown overnight in RPMI 1640 containing 11 mmoles / 1 glucose and dexamethasone (100 ng / ml) or DMSO (control) . After cultivation, the islets were pre-incubated for 1 hour at 37 ° C in an incubation pH buffer containing (in mmoles / l): 140 of NaCl, 5.6 of KCl, 1.2 of MgCl2, 2.6 of CaCl2, 2.8 of glucose, 10 of HEPES, pH 7.4 and 5 g / 1 of bovine serum albumin (fraction V, Sigma, Deisenhofen). Subsequently, batches of 5 islets / 0.5 ml were incubated for 30 minutes at 37 ° C in the presence of test substances as indicated for each experiment. Insulin was measured using a Elisa kit (Mercodia, Uppsala, Sweden). Example 3 Measurement of membrane currents INS-1 cells were cultured for 2-4 days on glass slides coated with poly-L-ornithine (10 mg / l Sigma, Munich, Germany) at the appropriate cell densities (1.2 x 106 cells / ml). The slides were mounted in a bath chamber on the support of an inverted microscope (IM, Zeiss, Jena, Germany). Cells were maintained at room temperature or at 34 ° C as indicated for each experiment and superinfused with a solution containing (in mmoles / l): 140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 0.5 glucose and 10 HEP? S, PH 7.4. Patch fastener pipettes (Clark-Medical, Reading, Great Britain) with a resistance of 4-6 MO were pulled using a universal DMZ squeegee (Zeitz, Augsburg, Germany). They were filled with an internal solution containing (in mmoles / l): 30 KCL, 95 K + -gluconate, 1 MgCl, 1.2 NaH2P04, 4.8 NaH2P04, 5 Na2ATP, 1 Na3GTP, 5 mmol / L EGTA, pH 7.2. An Epc9 patch clamp amplifier (Heka Electronics, Lambrecht, Germany) was used for current measurements. Only the stable current measurements, that is, when the currents reached at least 90% control current after the removal of the respective inhibitory drug, were used for the analysis. Example 4 Real-time PCR INS-1 cells were cultured in 70 cm2 flasks, the medium was removed and 600 μl of lysis pH buffer (Mini kit, Qiagen, Hilden, Germany) was added. The lysed cells were scraped and the lysate was collected in an Eppendorf tube. Cell RNA was isolated using the Qiagen Mini kit and 2 μg of RNA transcribed into cDNA using M-MuLV reverse transcriptase (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). An aliquot of cDNA, which corresponded to the amount of RNA as indicated in each experiment, was used for quantification of the mRNA by real-time PCR using a light cycle system (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany ) with specific primers for the rat Kv 1.5 channel, direction: 5 '-ATC TTC AAG CTC TCC CGC CAC TCC AAG GG-3'; antisense: 5 '-GGG TTA TGG AAA GAG GAG TTA-3 '. The used rat SGKl primers - were: sense: 5 '-TTT TTT TTC CCA ACC CTT GC-3'; antisense: 5'AAT GAA CAÁ AGG TTG GGG GG-3. The isolated mouse islets were cultured and treated with dexamethasone as indicated. Subsequently, the islets were collected and lysed in lysis pH regulator (Mini kit, Qiagen, Hilden, Germany) and by repeatedly sucking the islets into an insulin syringe. Example 5 Western blotting analysis INS-1 cells were grown in 70 cm2 flasks without control) or with 100 ng / ml dexamethasone for the indicated period of time. Subsequently, the culture medium was removed and the cells were lysed in a solution containing: 300 mM NaCl, 20 mM Tris HCl, pH 7.4, 1% (v / v) Triton X-100, 1% deoxycholate sodium, 0.1% SDS, 2.5 mM EDTA, 10 μg / ml pepstatin A, 10 μg / ml aprotinin and 0.1 mM PMSF. Total cellular protein, 50 μg, quantified by staining with Coomassie Blue G (Bradford stain test, Biorad Laboratories GmbH, Munich Germany) was subjected to SDS-PAGE (1%), and plotted on a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). The plots were incubated with antibodies against SGKl (New England Biolabs, Beverly, MA, E.U.A). The bound antibody was visualized using a second antibody coupled to horseradish peroxidase. EXAMPLE 6 SGK1 Modulator Compounds 6.1 Compounds of the general formula I and pharmaceutically useful derivatives, salts, solutions and stereoisomers thereof, including mixtures wherein R1, R5 is either H, OH, OA, OAc or methyl, R2 'R3, R4, R6, R7, R8, R9, R10 is either H, OH, OA, OAc, OCF3, Hal, N02, CF3, A, CN, OS02CH3, S02CH3, NH2 or COOH, R11 is H or CH3, A is alkyl with 1, 2, 3 or 4 carbon atoms, X is CH2 CH2CH2, OCH2 or -CH (OH) -, Hal is F, Cl, Br or I Compounds according to formula I selected from the following group of compounds: acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-hydroxy-phenyl) -acid acid- [1 - (4-hydroxy-2-methoxy-phenyl) -ethylidene] -hydrazide (3-hydroxy-phenyl) -acetic acid (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-ethoxy-phenyl) - acid, acid- (3-fluoro-4-hydroxy-benzylidene) -hydrazide phenylacetate, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (4-hydroxy-phenyl) -acid, acid- (4-hydroxy) -2-methoxy-benzylidene) -hydrazide (3,4-dichlorophenyl) -acid, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide m-tolyl-acid, acid- (4-hydroxy-2) -methoxy-benzylidene) -hydrazide o-tolyl-acid, acid- (4-h idroxy-2-methoxy-benzylidene) -hydrazide (2-chloro-phenyl) -acetic, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-chloro-phenyl) -acid, acid- (4- hydroxy-2-methoxy-benzylidene) -hydrazide (4-fluoro-phenyl) -acetic acid (4-hydroxy-2-methoxy-benzylidene) -hydrazide (2-chloro-4-fluoro-phenyl) -acid, acid - (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-fluoro-phenyl) -acetic acid (4-hydroxy-benzylidene) -hydrazide (3-methoxy-phenyl) -acid, acid- (4-) hydroxy-2, 6-dimethyl-benzylidene) -hydrazide (3-methoxy-phenyl) -acid, acid- (3-fluoro-4-hydroxy-benzylidene) -hydrazide (3-methoxy-phenyl) -acid, acid- [ 1- (4-hydroxy-2-methoxy-phenyl) -ethylidene] -hydrazide (3-methoxy-phenyl) -acetic acid (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-methylsulfonyloxy-phenyl) -acid, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3,5-dihydroxy-phenyl) -acid, acid- (3-fluoro-4-hydroxy-benzylidene) -hydrazide (3-fluoro- phenyl) -acid, acid- (4-acetoxy-2-methoxy-benzylidene) -hydrazide (3- ethoxy-phenyl) -acid, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-trifluoromethyl-phenyl) -acetic, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide 3- ( 3-methoxy-pheny1) -propionic acid- (2, -dihydroxy-benzylidene) -hydrazide (3-methoxy-phenyl) -acetic, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide "(3- methoxy-phenoxy) -acid, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3-nitrophenyl) -acid, acid- (5-chlor-2-hydroxy-benzylidene) -hydrazide (3- methoxy-phenyl) -acetic, acid- (2-hydroxy-5-nitro-benzylidene) -hydrazide (3-methoxy-phenyl) -acetic, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide 2-hydroxy -2-phenyl-acid, acid- (2-ethoxy-4-hydroxy-benzylidene) -hydrazide (3-methoxy-phenyl) -acetic, acid- (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3- bromo-phenyl) -acid, [1- (4-hydroxy-phenyl) -ethylidene] -hydrazide (3-methoxy-phenyl) -acetic acid (4-hydroxy-2-methoxy-benzylidene) -hydrazide (3 , 5-difluoro-phenyl) -acid, acid- (4-hydroxy-2-methyl-benzyl) lden) -hydrazide (3-hydroxy-phenyl) -acid, acid- (2-ethoxy-4-hydroxy-benzylidene) -hydrazide (3-hydroxy-phenyl) -acid, acid- (2-methoxy-4-hydroxy) 6-methyl-benzylidene) -hydrazide (3-hydroxy-phenyl) -acetic acid (2-methoxy-4-hydroxy-benzylidene) -hydrazide (2-fluoro-phenyl) -acid. 6.2 Compounds of the general formula II and pharmaceutically useful derivatives, salts, solutions and stereoisomers thereof, including mixtures. wherein R1, R2, R3, R4, R5 is either H, A, OH, OA, alkenyl, alkynyl, N02, NH2, NHA, NA2, Hal, CN, COOH, COOA, -OHet, -O-alkylene- Het, 0-alkylene-NR8R9 or CONR8R9, two groups selected from R1, R2, R3, R4, R5 or also -0-CH2-CH2-, -0-CH2-0- or -0-CH2-CH2-0- , R5, R7 is either H, A, Hal, OH, OA or CN, R8, R9 is either H or, Het is a saturated or unsaturated heterocycle with 1 to 4 N, O and / or S atoms, substituted by one or several groups Hal, A, OA, COOA, CN or carboniloxygen (= 0), A is • alkyl having 1 to 10 carbon atoms, wherein 1-7 hydrogen atoms. can be replaced by fluorine and / or chlorine, X, X '-is either NH or is absent Hal is F, Cl, Br or I Compounds according to formula II selected from the following group of compounds: 1- [4- (4-amino-5-oxo- 5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2-fluoro-5-trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo -5-pyrido [2, 3-d] pyrimidin-8-yl) -phenyl] -3- (4-chloro-5-trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo -5 f -pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2,4-difluoro-phenyl) -urea, 1- [4- (4-amino-5-oxo-5i β-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2,6-difluoro-phenyl) -urea, 1- [4- (4-amino-5-oxo-5H- pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (3-fluoro-5-trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo-5iT- pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (4-fluoro-5-trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo-5-JT- pyrido [2, 3-d] pyrimidin-8-yl) -phenyl] -3- (4-methyl-5-trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo-5í- pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2,3,4,5,6-pentafluoro-phenyl) - urea, 1- [4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2, -dibrom-6-fluoro-phenyl) -urea, 1- [4- (4-amino-5-oxo-5-fluoro-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2-fluoro-6-trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2-fluoro-5-methyl-phenyl) -urea, 1- [4- (4-amino-5-oxo-5-f-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2,3,4-trifiuor- phenyl) -urea, 1- [4- (4-amino-5-oxo-5-yl-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (4-brom-2, 6- difluoro-phenyl) -urea, 1- [4- (4-amino-5-oxo-5-y-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- (2-fluoro-3-) trifluoromethyl-phenyl) -urea, 1- [4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [2- (1-ter -butyloxycarbonyl-piperidin-4-yl) -phenyl] urea, N- [4- (4-amino-5-oxo-5-pyrido [2,3-d] pyrimidin-8 ~ yi) -phenyl] -2, -dichloro-benzamide, N- [4- (4-amino-5-oxo-5ff-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -4-chloro-5-trifluoromethyl-benzamide, N - [4- (4-amino-5-oxo-5-pyrido [2, 3 -d] pyrimidin-8-y1) -phenyl] -2-fluoro-5-trifluoromethyl-benzamide, 1- [4- (4-amino-5-oxo-5-pyrido [2,3-d] pyrimidin-8] ~ il) -phenyl] -3- [3-chloro-5-trifluoromethyl-2- (piperidin-4-yloxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5H-pyrido] [2,3-d] pyrimidin-8-yl) -phenyl] -3- [(2-fluoro-5- (2-dimethylamino-ethoxy) -phenyl] -urea, 1- [4- (4-amino- 5-oxo-5-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [5-fluoro-2- (piperidin-4-yloxy) -phenyl] -urea, 1- [4 - (4-amino-5-oxo-5i-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [4-chloro-5-trifluoromethyl-2- (piperidin-4-yloxy) - phenyl] -urea, 1- [4- (4-amino-5-oxo-5-T-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [2- (piperidin-4- iloxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5-yl-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [2-fluoro-5] - (2-diethylamino-ethoxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [2-fluoro-5- [2- (piperidin-1-yl) -ethoxy] -phenyl] -urea, 1- [4- (4-amino-5-oxo-5? -pyrido [2, 3-d] ] pyrimidin-8-yl) -phenyl] -3- [4-fl or-2- (2-dimethylamino-ethoxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [4-Fluoro-2- (2-diethylamino-ethoxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8) -yl) -phenyl] -3- [3-chloro-4- [2- (morpholin-4-yl) -ethoxy] -phenyl] -urea, 1- [4- (4-amino-5-oxo-5H - pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [4-fluoro-2- [2- (morpholin-4-yl) -ethoxy] -phenyl] -urea, 1- [ 4- (4-amino-5-oxo-5H-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [3-chloro-4- (2-dimethylamino-ethoxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [3-chloro-4- (2-diethylamino -ethoxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5iT-pyrido [2,3-d] pyrimidin-8-yl) -phenyl] -3- [4-cioro- 2- (2-dimethylamino-ethoxy) -phenyl] -urea, 1- [4- (4-amino-5-oxo-5i? -pyrido [2,3-d] pyrimidin-8-yl) -phenyl] - 3- [2-Chloro-5- (2-diethylamino-ethoxy) -phenyl] -urea, and pharmaceutically useful derivatives, solvates, salts, tautomers and stereoisomers thereof, including its mixtures Example 8 Nucleotide polymorphism of SGKl The nucleotide sequence defining intron 6 of facultative hypertensive patients is ... aattacattgCgcaacccag ..., while the nucleotide sequence representing a healthy population is ... aattacattgTgcaacccag .... Both sequences are available through the registration number Gl 2463200 Position 2071. The sequences of exon 8 of facultative hypertensive patients are either homozygous ... tactgaCtcggact ... or ... tactgaTttcggact ... or heterozygous ... tactgaCttcggact ... and ... tactgaTttcggact. The sequences are available through registration number NM__005627.2, Position 777. A homozygous individual with a combination of TT nucleotides is protected even if simultaneously a single nucleotide CC polymorphism occurs in intron 6. Example 9 Statistics are presented the data as mean ± SEM. ANOVA for several groups and student t tests were used for statistical analyzes. The p <values; 0.05 were accepted to indicate the statistical significance. References Bohrner, C, Wagner, CA, Beck, S., Moschen, I., Melzig, J., Werner, A., Lin, J.-T., Lang, F., Wehner, F. The Shrinkage-activated Na + - Conductance of Rat Hepatocytes and its Possible Correlation to rENaC. Cell Phys Biochem. 2000: 10: 187- 194. Brenan FE, Fuller PJ. Rapid upregulation of serum and glucocorticoid-regulated kinase (sgk) gene expression by corticosteroids in vivo. Mol Cell Endocrinol. 2000; 30; 166: 129-36. Busjahn A, Aydin A, Uhlmann R, Feng Y, Luft FC, Lang F. Serum- and glucocorticoid-regulated kinase (SGKl) gene and blood p ressure Hypertension 40 (3) : 256-260, 2002. Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D: Epithelial sodium channel regulated by aldosterone-induced protein sgk Proc Nati Acad Sci USA 1999: 96: 2514- 2519. Cowling RT, Birnboim HC, Expression of serum-and glucocorticoid-regulated kinase (sgk) mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes J Leukoc Biol. 2000; 67 : 240-248. De la Rosa DA, Zhang P, Naray-Fejes-Toth A, Fejes-Toth G, Canessa CM: The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. Biol Chem 1999; 274: 37834-37839, Hoogwerf B, Dáñese RD: Drug selection and the management of corticosteroid-related diabetes mellitus Rheum Dis Clin North Am 1999: 25: 48 9-505. Klingel K, Wárntges S, Bock J, Wagner CA, Sauter M, Waldegger S, Kandolf R, Lang F. Expression of the cell volume regulated kinase h-sgk in pancreatic tissue. Am J Physiol (Gastroint, Liver-Physiol.) 2000; 279: G998- G1002. Kobayashi • T, Cohen P: Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activates phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDKl) and PDK2. Biochem J 1999: 339: 319-328. Kobayashi T, Deak M, Morrice N, Cohen P. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem. J. 1999: 344: 189-197. Lambillotte C, Gilon P, Henquin JC: Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets. J Clin Invest 1997: 99: 414-423. Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE. 2001 Nov 13; 2001 (108): RE17. Lang F, Klingel K, Wagner CA, Stegen C, Warntges S, Friedrich B, Lanzendorfer M, Melzig J, Moschen I, Steuer S, Waldegger S, Sauter M, Paulmichi M, Gerke V, RislerT, Gamba G, Capasso G, Kandolf R, Hebert SC, Massry SG, Broer S; Deranged transcriptional regulation of cell volume - sensitive kinase hSGK in diabetic nephropathy. Proc Nati Acad Sci USA 2000: 97: 8157-8162. Lifton RP. Molecular genetics of human blood pressure 5. variation. Science 1996: 272: 676-680. McMahon M, Gerich J, Rizza R: Effects of glucocorticoids on carbohydrate metabolism. Diabetes Metab Rev 1988: 4: 17-30. Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G: sgk is an aldosterone-induced kinase in the 0 renal collecting duct. Effects on epithelial Na + channels. J Biol Chem 1999: 274: 16973-16978. Park J, Leong ML, Buse P, MaiyarAC, Firestone GL, Hemmings BA: Serum and glucocorticoid-inducible kinase (SGK) is a target of the Pl 3- kinase-stimulated signaling pathway. EMBO J 1999: 18: 3024-3033. Pierluissi J, Navas FO, Ashcroft SJ: Effect of adrenal steroids on insulin relay from cultured rat islets of Langerhans. Diabetology 1986: 29: 119-121. Schacke H, Docke WD, Asadullah K: Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther 2002: 96: 23-43. Shigaev A, Asher C, Latter H, Garty H, Reuveny E: Regulation of sgk by aldosterone and its effects on the epithelial Na (+) channel. Am J Physiol 2000; 278: F613-F619. Staessen JA, Wang J, Bianchi G, BirkenhagerWH. Essential hypertension. Lancet. 2003: 361: 1629-1641. Wagner CA, Ott M, Klingel K, Beck S, Melzig J, Friedrich B, Wild NK, Broer S, Moschen I, Albers A, Waldegger S, Tumler B, Egan?, Geibel JP, Kandolf R, Lang F. Effects of serine / threonine kinase SGKl on the epithelial Na + channel (EnaC) and CFTR. Cell Physiol Biol 2001: 11: 209-218. Warnock DG. Liddle syndrome: genetics and mechanisms of Na + channel defects. Am J Med Sci. 2001: 322: 302-307. Webster MK, Goya L, Firestone GL: Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine / threonine protein kinase. J Biol Chem 1993a; 268: 11482-11485. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL: Characterization of sgk, a novel member of the serine / threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 1993b; 13: 2031 -2040. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. It gives a method for altering insulin secretion, characterized in that it comprises contacting a cell of the pancreatic islets expressing SGK1 with a substance that modulates SGK1. .
2. 2. The method according to claim 1, characterized in that the expressed SGK1 comprises a selected SNP variant.
3. 3. The method according to claim 1-2, characterized in that the SGK1 modulator is an inhibitor.
4. 4. The method according to claim 1-2, characterized in that the modulator is an activator of SGK1.
5. 5. The method according to claim 1, characterized in that the inhibition of SGK1 comprises the reversal of the depolarizing effect of glucose, the activation of the voltage-regulated Ca channels and the release of insulin.
6. 6. The method according to claim 5, characterized in that the SNP variant of the polymorphic SGK-1 is diagnosed before the inhibition.
7. 7. The method according to claims 1-4, characterized in that it comprises the over-regulation of insulin secretion.
8. 8. The method according to claims 1-4, characterized in that the subject suffers from symptoms of type 2 diabetes mellitus.
9. 9. A method characterized in that it is to reduce type 2 diabetes mellitus induced by glucocorticoids in a subject requiring this treatment by modulating the activity of SGK1 in cells of the pancreatic islets.
10. 10. The method according to claims 1-4, characterized in that the subject treated has hyperglycemia induced by stress.
11. 11. The method according to claims 1-4, characterized in that the subject treated has hypoglycemia.
12. 12. A method for determining the progression, regression or onset of a disease by measuring SGK1 expression, characterized in that it comprises taking a sample from the sick individual.
13. The method according to claim 12, characterized in that the SGKl comprises a selected SNP variant.
14. 14. A pharmaceutical composition characterized in that it comprises an inhibitory agent of SGK1 together with a pharmaceutically effective carrier, excipient or diluent.
15. 15. Use of SGKI inhibitors selected from the listed compounds having the general formula I or II, in the preparation of a medicament for the treatment of disorders caused by impaired insulin secretion.