KR20050016494A - Sgk and nedd used as diagnostic and therapeutic targets - Google Patents

Abstract

The present invention provides the use of a substance for the diagnostic detection of Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2, and for the treatment of diseases associated with disturbed glucose transport and for fattening animals. It relates to the use of active ingredients which affect glucose transport. The invention also relates to pharmaceutical compositions and diagnostic kits containing an active amount of at least one active ingredient that affects glucose transport. The invention also relates to a method of making a transgenic animal.

Description

SG and NEDD used as diagnostic and therapeutic targets {SGK AND NEDD USED AS DIAGNOSTIC AND THERAPEUTIC TARGETS}

1 : Control of Na ⁺ -coupled glucose transporter Sglt1 by Nedd4-2 and Sgk1.

Top: original measured data; Lower part: Mathematical statistics ± SEM (n = 6-15). Xenopus laevis oocytes were injected with sglt1, nedd4-2 and / or sgk1 cRNA. While Nedd4-2 downregulated the current induced by 20 mM glucose in oocytes expressing Sglt1, Sgk1 stimulated the current to reverse the effect of Nedd4-2.

* Indicates a significant difference compared to the current measured in oocytes expressing Sglt1 only.

# Shows significant differences compared with corresponding values in oocytes expressing Sglt1 and Nedd4-2.

2 : Control of Na ⁺ -coupled glucose transporter Sglt1 by Nedd4-2, always active ^S422D Sgk1 and inactive ^K127N Sgk1.

Top: original measured curve; Bottom: Mathematical Statistics ± SEM (n = 8-71). Xenopus laby oocytes were injected with sglt1, nedd4-2 and / or ^S422D sgk1 or ^K127N sgk1 cRNA. Nedd4-2 significantly down-regulated the current induced by 20 mM glucose in oocytes expressing Sglt1, whereas ^S422D Sgk1 rather than ^K127N Sgk1 stimulated the current to reverse the effect of Nedd4-2.

* Indicates a significant difference compared to the current measured in oocytes expressing only Sglt1 itself.

# Shows significant differences compared with corresponding values in oocytes expressing Sglt1 and Nedd4-2.

3 : Control of Na ⁺ -coupled glucose transporter Sglt1 by Nedd4-2, ^{T308D, S473D} PKB and Sgk3.

Top: original measured curve; Lower part: Mathematical Statistics ± SEM. Xenopus laby oocytes were injected with sglt1, nedd4-2, ^{T308D, S473D} PKB and / or sgk3 cRNA. Nedd4-2 significantly downregulated the current induced by 20 mM glucose in oocytes expressing Sglt1. ^{T308D, S473D} PKB and Sgk3 stimulated the current to reverse the effect of Nedd4-2.

* Indicates a significant difference compared to the current measured in oocytes expressing only Sglt1 itself.

# Shows significant differences compared with corresponding values in oocytes expressing Sglt1 and Nedd4-2.

4 : Control of Na ⁺ -coupled glucose transporter Sglt1 by Nedd4-2 and Sgk1. Mathematical Statistics ± SEM (n = 18). Xenopus oocytes were injected with sglt1, nedd4-2, and / or ^S422D Sgk1 (SD) cRNA. The current induced by the addition of 5 mM glucose reduced Nedd4-2 co-expression, while the current increased significantly by co-expression of the always active kinase ^S422D sgk1.

5 : Control of Na ⁺ -coupled glucose transporter Sglt1 by Nedd4-2, Sgk3 and PKB. Mathematical Statistics ± SEM (experimental procedure is the same as in FIG. 4).

Way

1. Expression in Xenopus laby oocytes and 2-electrode voltage clamp

Wild type Sgk1 [Waldegger S, Barth P, Raber G, Lang F: Cloning and characterization of a putative human serine / threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 1997; 94; 4440-4445], always active Sgk1 ( ^S422D Sgk1) and inactive Sgk1 ( ^K127N Sgk1) [Kobayashi T, Cohen P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phophatidylinositide 3-kinase is mediated by 3- phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 1999; 339: 319-28], wild-type Sgk3 and PKB [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; 334: 189-97], always active ^{T308D, S473D} PKB [Alessi DR, Cohen P. Mechanism of activation and function of protein kinase B. Burr. Opin Genet Dev. 1998; 8: 55-62], wild type Nedd4-2 [Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, Staub O Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na (+) channel cell surface expression. EMBO J. 2001; 20: 7052-7059] and wild type Sglt1 [Hediger MA, Coady MJ, Ikeda TS, Wright EM. Expression cloning and cDNA sequencing of the Na + / glucose co-transporter. Nature. 1987; 330: 379-381] was synthesized in vitro [Wagner CA, Friedrich B, Setiawan I, Lang F, Broeer S: The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 2000; 10: 1-12]. Ovarian incision, and collection and processing of oocytes of Xenopus rabbi have already been described in detail [Wagner CA, Frierdrich B, Setiawan I, Lang F, Broeer S: The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 2000; 10: 1-12]. Oocytes were injected with human sglt1 5 ng, human sgk1, ^K127N sgk1, ^S422D sgk1, sgk3, PKB or ^T308D, 7.5 ng of ^S473D PKB, and / or xenopus nedd4-2 5 ng. Control oocytes were injected with water. Three days after each cRNA injection, electrophysiological experiments were performed at room temperature. Current induced by extracellular administration of 20 mM or 5 mM glucose was measured using a two-electrode voltage clamp [Wagner CA, Friedrich B, Setiawan I, Lang F, Broeer S: The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 2000; 10: 1-12], taken on a glucose transport scale. Data was filtered at 10 Hz and analyzed using MacLab Digital with Analog Converter and corresponding software (AD Instruments, Castle Hill, Australia). Control bath solution (ND96) included 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ and 5 mM HEPES, pH 7.4. All materials were used at the concentrations indicated. The final solution was titrated to pH or pH 7.4 expressed in HCl or NaOH. The flow rate of the perfusion solution was 20 ml / min and complete exchange of the solution was performed within 10 s.

For the calculations, data are presented as mathematical mean ± SEM. n is the number of oocytes examined. All experiments were performed with at least three different groups of oocytes. Qualitatively similar data were obtained in all replicates. The results were evaluated for significance using the Student's t test. Only results with P <0.05 were used as statistically significant.

2. The study of twins

126 pairs of identical and 70 pairs of fraternal twins were recruited for blood pressure control and cardiovascular phenotyping studies. The parents of fraternal twins were also included. All participants were German Caucasians from other parts of Germany. Parents of all twins and fraternal twins were bled for conjugation determination and other molecular genetic studies. Each participant underwent medical and physical examination. None of the participants had a family history of chronic medical disease. Single nucleotide polymorphisms (SNPs) were identified at exon 8 (C → T), and the second SNP was identified at 551 base pair distances at the donor site (extron 6) of exon 7 (T → C) [Busjahn A, Aydin A, Uhlmann R et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 2002; 40: 256-260). The two individual SNPs, intron 6 (T → C) and exon 8 (C → T), were analyzed.

The factual statistics on the two SNPs showed a recessive mode of action. Therefore, based on the comparison of the two groups, the association analysis was performed on mutant homozygous versus heterozygous and non-carriers. Independence of the two SNPs was assessed using the chi ² test. Correlation between SNPs and phenotypes was assessed by unidirectional ANOVA, introducing both polymorphisms simultaneously. The analysis involved both random pairs of fraternal twins and randomly selected sides of identical twin pairs. The assessment is more reliable than the t assessment and allows for both polymorphisms at the same time, including their interactions. It was also possible to reduce the number of irradiations in this manner. Since the two pairs of fraternal twins are not independent of each other, familial effects as well as age and gender are included in ANOVA as covariates. Significance was set to 0.05. In the confirmation group, both SNPs were used simultaneously to assess the association effect with unidirectional ANOVA.

result

20 mM glucose administration resulted in an inward current (I _glc ) of 48.6 ± 11.5 nA (n = 18) in Sglt1 mRNA-injected Xenopus oocytes but not in water injected oocytes. In contrast, glucose treatment produced a 1.3 ± 0.7 nA (n = 6) current in oocytes injected with water. In (co-expressed) Xenopus oocytes injected with Sglt1 mRNA and Nedd4-2 mRNA, I _glc significantly decreased to 49.2 ± 6.8% (n = 15). As a result, Sglt1 was downregulated by ubiquitin ligase Nedd4-2 (FIG. 1).

Coexpression of wild type Sgk1 upregulated glucose-induced currents by 81.3 ± 19.0% (n = 15) and reversed the effect of Nedd4-2. In oocytes expressing Sgk1 and Nedd4-2 with Sglt1, the glucose-induced current was 34.8 ± 11.8% (n = 14) higher than that observed for oocytes expressing Sglt1 itself (FIG. 1).

Always active ^S422D Sgk1 showed a similar effect to wild type Sgk1 (FIG. 2). Co-expression of ^S422D Sgk1 increased glucose-induced current by 72.4 ± 9.1% (n = 57). In this series of experiments, coexpression of Nedd4-2 lowered the current by 35.3 ± 4.4% (n = 46). The effect was reversed by additional coexpression of ^S422D Sgk1. In oocytes co-expressing Nedd4-2 and ^S422D Sgk1, the current was 59.2 ± 19.8% (n = 16) higher than in oocytes expressing their own Sglt1 (FIG. 2). In contrast to wild-type or always active Sgk1, inactive mutant ^K127N Sgk1 did not significantly change substrate-induced currents (-2.0 ± 5.3%, n = 14) and did not reverse the effect of Nedd4-2. In oocytes expressing ^K127N Sgk1 and Nedd4-2 with ^Sglt1 , glucose-induced current was 54.9 ± 9.7% (n = 8) lower than that observed for oocytes expressing Sglt1 itself (FIG. 2).

The effect of ^Sgk1 was mimicked with ^{T308D, S473D} PKB (FIG. 3). In this series of experiments, coexpression of Nedd4-2 reduced the current by 26.5 ± 5.5% (n = 42). ^{Coexpression of the} always active ^{T308D, S473D} PKB significantly increased glucose-induced current 117.4 ± 15.8% (n = 31) in Sglt1 expressing oocytes and reversed the effect of Nedd4-2. ^{In xenopus} oocytes co-expressing ^{T308D, S473D} PKB and Nedd4-2 with Sglt1, glucose-induced current was 106.5 ± 18.2% (n = 27) higher than that in Xenopus oocytes expressing their own Sglt1 ( 3).

^In a manner comparable to that in ^{T308D, S473D} PKB and Sgk1, Sgk3 stimulated glucose-induced currents and reversed the effects of Nedd4-2. Glucose-induced currents were 123.6 ± 15.0% (n = 22) higher in glucose expressing Sglt1 and Sgk3 than in glucose-induced oocytes expressing Sglt1 itself, Sglt1, Nedd4-2 and In oocytes expressing Sgk3, they were 112.4 ± 19.4% higher (n = 22).

4 shows that coexpression of Sglt1 and ^S422D Sgk1 (SD) increases I _glc from 77 ± 23% to 65.4 ± 10.6 nA (n = 18). In oocytes expressing Sgk1 and Nedd4-2 with Sglt1, the glucose-induced current was 61 ± 21% greater than the corresponding value in oocytes injected with Sglt1 only, compared to that in oocytes injected with Sglt1 and Nedd4-2 mRNA A 126 ± 23% large 60.5 ± 9.9 nA (n = 18) was reached. In this experiment, current was induced with 5 mM glucose.

In a further series of experiments, the isoforms of Sgk, ie Sgk2 and Sgk3, as well as protein kinase B (PKB) were assessed in addition to the always active ^S422D Sgk1 (SD). Glucose-induced current is the coexpression of ^{S422D Sgk1 55 ± 12% (n} = 44), co-expression of Sgk3 117 ± 16% (n = 16), and co-expression of 101 ± 18% (n = 24 of PKB ), Whereas Sgk2 did not have a statistically significant effect. Coexpression of Nedd4-2 lowered glucose transport by 23 ± 4% (n = 79), whereas ^S422D Sgk1 (+ 48 ± 11%, n = 48), Sgk3 (+114 ± 26%, n = 16) And stimulation by further coexpression of PKB (+ 107 ± 20%, n = 24) was not prevented. Again, Sgk2 did not have a significant effect.

To investigate the functional relevance of Sgk1 in Sglt1 and weight control, the weight index of twins carrying the Sgk1 gene polymorphism was related. The average body weight of twins with polymorphic E8CC / CT; I6CC was 26.7 ± 1.4 kg / m ² (n = 13). The value was significantly higher (P <0.008) compared to the corresponding mean value for the whole twin (23.3 ± 0.2 kg / m ² , n = 263).

Overall, experiments showed that Sgk1, Sgk3 and PKB had a strong stimulatory effect on Sglt1. Increasing Sglt1 activity accelerated glucose intestinal uptake in a faster way to increase glucose concentration in plasma. This increases insulin release, stimulating the accumulation of lipids in adipose tissue. In contrast, inhibitors of Sglt1 counteracted hypertrophy.

Studies of twins showed that the same polymorphism associated with elevated blood pressure was also associated with higher weight indices.

The present invention relates to Sgk (serum and glucocorticoid dependent kinases), in particular Sgk1 and / or Sgk3, and / or protein kinase B (PKB) and / or Nedd (genetically downregulated genes expressed in neural progenitor cells, neural precursors). cell-expressed developmentally down-regulated genes, in particular the use of substances for the diagnostic detection of Nedd4-2. The present invention also relates to the use of active compounds that affect glucose transport, in particular for the therapeutic treatment of diseases associated with disturbed glucose transport and for gaining weight in animals during fattening. The invention also relates to a diagnostic kit.

Na ⁺ -coupled transporter Sglt1 (sodium glucose transporter) in the epithelium of epithelial cells is involved in intestinal and renal transport of glucose. The disturbance of glucose transport can cause various diseases such as obesity and diabetes.

To date, little is known about the regulation of Sglt1. Recently, a novel mechanism for regulating renal epithelial Na ⁺ channel ENaC has been discovered: the channel is ubiquitinated by the ubiquitin ligase Nedd4-2, ready to internalize and degrade [Debonneville C, Flores SY, Kamynina E]. , Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, Staub O. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na (+) channel cell surface expression. EMBO J. 2001; 20: 7052-7059. Nedd4-2 is phosphorylated and inactivated by serum- and glucocorticoid-induced kinase 1 (Sgk1). As a result, Sgk1 is a potent stimulant of renal epithelial Na ⁺ channels [De la Rosa et al., 1999, Boehmer et al., 2000, Chen et al., 1999, Naray-Fejes-Toth et al., 1999, Lang et al., 2000, Chigaev et al., 2000 , Wagner et al., 2001].

Recently, studies on twins have shown that certain single nucleotide polymorphisms (SNPs) in the sgk1 gene (E8CC / CT; I6CC) are associated with elevated blood pressure [Busjahn A, Aydin A, Uhlmann R. et al., Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 2002; 40: 256-260.

In general, kinases are proteins that deliver phosphate groups to individual substrates. Serum- and glucocorticoid-dependent kinases (Sgk) were originally cloned in rat breast carcinoma cells [Webster MK, Goya L, Firestone GL, Y. Biol. Chem. 268 (16): 11482-11485, 1993; Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL, Mol. Cell. Biol. 13 (4): 2031-2040, 1993].

Sgk1 was originally cloned into glucocorticoid sensitive genes [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 1993; 13: 2031-2040. Several studies have shown that Sgk1 is affected by multiple stimuli [Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE. 2001 Nov 13; 2001 (108): RE17], for example, has been shown to be particularly affected by mineral corticoids [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 Natl Acad Sci USA 1999; 96: 2514-2519; Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G: Sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na ⁺ channels. J Biol Chem 199; 274: 16973-16978; Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, Hemmings BA: Serum and glucocorticoid-inducible kinase (Sgk) is a target of the PI3-kinase-stimulated signaling pathway. EMBO J 1999; 18: 3024-3033; 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; 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]. Sgk1 is stimulated by insulin-like growth factor IGF1, insulin, and oxidative stress by signal transduction, and by phosphinositol-3-kinase (PI3-kinase) and phosphoinositol-dependent kinase (Pdk1) [Kobayashi T, Cohen P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phophatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1) and pdk2. Biochem J 1999; 339: 319-328; Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, Hemmings BA: Serum and glucocorticoid-inducible kinase (Sgk) is a target of PI3-kinase stimulated signaling pathway. EMBO J 1999; 18: 3024-3033; Kobayashi T, Deak M, Morrice N, Cohen P. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kianse. Biochem. J. 1999; 344: 189-197. Activation of Sgk1 by Pdk1 involves phosphorylation of serine at position 422. The mutation of serine to aspartate ( ^S422D Sgk1) produces an kinase that is always active [Kobayashi T, Cohen P: Activation of serum- and glucocorticoid-regulated protein kianse by agonists that activate phophatidylinositide 3-kinase is mediated by 3- phophoinositide-dependent protein kinase-1 (Pdk1) and pdk2. Biochem J 1999; 339: 319-328.

Since then, two isoforms of Sgk1, Sgk2 and Sgk3, have been cloned [Kobayashi T, Deak M, Morrice N, and Cohen P. 1999. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kianse. Biochem J. 344: 189-197]. All three Sgk isotypes and protein kinase B (PKB) are activated by PI3 kinase and Pdk1 [Kobayashi, T., and Cohen, P. 1999. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phophatidylinositide 3-kinase is mediated by 3-phophoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 339: 319-328.

It is an object of the present invention to provide novel diagnostic and therapeutic applications for modulating glucose uptake. It is also an object of the present invention to provide an application that increases the weight of an animal by regulating glucose absorption.

Surprisingly, two electrode voltage clamp experiments have shown that Nedd4-2 also inactivates renal and intestinal Na ⁺ glucose transporter Sglt, the effect being inhibited by Sgk1 and / or Sgk3 and / or PKB. Since accelerated glucose uptake promotes the development of obesity, it is concluded that, for example, Nedd4-2, Sgk1, Sgk3 and PKB play a causal role in the development of obesity. By detecting Nedd4-2 and / or Sgk1 and / or Sgk3 and / or PKB, for example, the cause of obesity can be identified and treated or prevented by appropriate treatment and prevention methods. Obesity induced due to accelerated intestinal glucose uptake, and also hyperglycemia, also promotes the development of diabetes. Finally, simultaneous dysregulation of renal Na ⁺ channels will result in hypertension. The development of obesity, hypertension and diabetes is a major feature called metabolic syndrome.

In contrast, inhibition of Sgk1 and / or Sgk3 and / or PKB in turn results in inhibition of the renal and intestinal Na ⁺ glucose transporter Sglt.

Accordingly, the object of the present invention is achieved on the subject of the independent claims 1, 10, 13, 23, 28, 30, 31, 32, 34 and 46. Preferred embodiments are specified in the dependent claims. All terms of the claims are incorporated herein by reference.

The present invention claims the use of one or more substances for detecting the expression and / or function of active and / or inactive Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. This makes it possible to diagnose vaginal involvement, in particular associated with disturbed glucose transport. The substance is preferably at least one substance from the antibody and / or nucleotide group. For example, the substance may be an antibody produced against Sgk1, Sgk3, PKB and / or Nedd4-2, and may be used for detection methods known to those skilled in the art, such as ELISA (enzyme-linked immunosorbent assay). In the immunoassay, the specific antibody (or homologous test antigen, if antibody determinant) generated against the antigen to be determined (eg Sgk1, Sgk3 and / or PKB) is bound to a support material (eg cellulose or polystyrene) and incubated with the sample. Thereafter an immune complex is formed. In a subsequent step, the immune complex is supplied with a labeled antibody. By adding a chromogenic substrate to the reaction mixture, the immune complex-binding enzyme / substrate complex can be visualized or confirmed by photometric determination of the immune complex-binding labeling compound by comparing the antigen concentration in the sample with a standard of known enzymatic activity. have. As already mentioned, nucleotides, in particular suitable for providing quantitative detection of Sgk1 for diagnostic detection, for example by molecular genetic methods in which specific DNA fragments are selectively amplified, for example using polymerase chain reactions. Oligonucleotides can be used.

Preference is given to using antibodies generated against one or more phosphorylated and / or nonphosphorylated kinase consensus sequences in the Nedd protein. In this regard, “common sequence” is understood to mean an amino acid sequence that forms the substrate site of a kinase that is a phosphorylation site (s). In this regard, the Sgk1 consensus sequence in the Nedd protein is particularly preferred.

In Nedd proteins, inactivation mutations (such as ^S338D Nedd4-2 or ^S444D Nedd4-2) can also be detected, particularly in the kinase consensus sequence. In addition, activating mutations such as ^S422D Sgk1 and / or ^{T308D, S473D} PKB are detected in the DNA of the patient. In further uses, the mutation is detected in the RNA of the patient. Finally, the mutation is detected in the patient's Sgk, in particular Sgk1 and / or Sgk3, PKB and / or Nedd proteins, in particular Nedd4-2 protein. As probes for such detection, preference is given to using suitable antibodies and / or suitable nucleotides, in particular oligonucleotides.

Diseases that are associated with disturbed glucose transport and to be diagnosed are metabolic syndrome or obesity in particular.

The invention also includes methods for diagnosing tendency to hypertrophy or obesity. The diagnostic method is characterized in that one or more polymorphisms are detected in sgk, in particular sgk1 and / or sgk3, the PKB gene, nedd, in particular nedd4-2, and / or sglt, in particular sglt1. In this regard, it is particularly preferable to detect the E8CC / CT; I6CC polymorphism in sgk1. The polymorphism is directly related to the body weight index as a particularly suitable marker for emphasizing tendency to hypertrophy. The abbreviation represents the SNP at exon 8 (C → T) and the second SNP (T → C) located 551 base pairs away from the donor site (Intron 6) of exon 7. In order to detect the polymorphism, it is preferable to collect the appropriate experimental animal or patient and determine the sequence at the site by appropriate sequencing using the genetic material contained therein or by using other methods familiar to those skilled in the art. In addition to blood, all other biological samples from which genetic material can be isolated are also suitable in principle.

The invention also claims the use of one or more active compounds to influence glucose transport, in particular intestinal and / or renal glucose transport. The glucose transporter Sglt, in particular Sglt1, is preferably at least partly involved in the glucose transport. According to the invention, glucose transport can be carried out by affecting the expression and / or activity of Sglt, in particular Sglt1. The active compound preferably affects one or more Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or affects one or more Nedd, in particular Nedd4-2. The active compound is preferably for Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. In another preferred embodiment of the invention, the active compound is Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular for activators, inhibitors, modulators and / or biological precursors of Nedd4-2 will be.

In a preferred embodiment of the invention, the active compound is a polynucleotide. The polynucleotides can include, for example, antisense sequences that reduce or inhibit the expression of one or more of the proteins. In another preferred embodiment, the polynucleotide encodes a peptide, preferably a polypeptide, which peptide expresses and / or expresses Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. Or affect function. In addition, the active compound may itself be a peptide or polypeptide which preferably affects the expression and / or function of said protein. The active compound may be a "small molecule compound", preferably a "small molecule compound" having a molecular weight <1000.

Each effect must be treated in a different manner, depending on whether it is intended to treat diseases associated with disturbed glucose transport or to gain weight in animals associated with fattening. In order to prevent or treat diseases associated with disturbed glucose transport, the active compound must inhibit and / or stimulate one or more Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or stimulate one or more Nedd, in particular Nedd4-2 . Since Sgk and PKB are kinases, kinase inhibitors known to those skilled in the art, such as staurosporin and / or kelerythrin, or one or more analogs thereof are particularly suitable. Since Nedd is a ligase, ligase activators are suitable for stimulating them. The active compound is preferably used for the manufacture of a drug or pharmaceutical composition. The disease to be treated is preferably metabolic syndrome, in particular obesity.

On the other hand, in contrast to the prophylaxis or treatment of the above-described diseases, whose purpose is to lower glucose transport, for example, when an increase in glucose transport aimed at gaining weight in animals associated with fattening should be achieved, The active compound preferably stimulates one or more Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or inhibits one or more Nedd, in particular Nedd4-2. Stimulation of Sgk1, for example, leads to inhibition of Nedd4-2, which in turn delays the degradation of the glucose transporter Sglt1. This, in turn, increases glucose transport. In a preferred embodiment of the invention, the active compound is at least one Sgk activator and / or PKB activator, in particular a growth factor, preferably IFG1 and / or insulin.

In another preferred embodiment of the invention, the active compound is one or more stimulants of sgk1 and / or sgk3 and / or PKB gene transcription, preferably one or more glucocorticoids, mineral corticoids, gonadotropins and / or cytokines, Especially TGFβ.

The invention also relates to a diagnostic kit. The kits detect the expression and / or function of active and / or inactive Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2, for diagnosing diseases associated with disturbed glucose transport. One or more materials are included. The disease is preferably metabolic syndrome, in particular obesity. Kits may include, in particular, antibodies and / or oligonucleotides for detection of corresponding proteins and / or nucleic acids. For example, the antibodies and / or oligonucleotides can be used to analyze the amount and / or activity of different proteins or enzymes. It is also possible to detect the mutation in the gene. See the rest of the description for additional features of the kit.

In addition to the above, the present invention includes antibodies generated against one or more phosphorylation kinase consensus sequences of the Nedd protein. The kinase consensus sequence is a sequence that is phosphorylated by the corresponding kinase, in particular Sgk1. The antibody preferably recognizes the kinase consensus sequence in the Nedd4-2 protein. The antibody can be used to analyze whether Nedd4-2 was phosphorylated by Sgk1 and thus inactivated. Therefore, the active state of Nedd4-2 can be investigated. The present invention also encompasses antibodies generated against the corresponding non-phosphorylated kinase consensus sequences in the Nedd protein. Particular preference is given to combining two antibodies according to the invention in one test setup, which can give very useful results on the active state of Nedd.

The invention also includes antibodies generated against one or more mutated kinase consensus sequences in the Nedd protein. Again preferably said consensus sequence is thus a Sgk1 consensus sequence mutated. The kinase consensus sequence is preferably located in the Nedd4-2 protein. Particularly preferred mutations in this regard are ^S338D Nedd4-2 and / or ^S444D Nedd4-2. The effect of this mutation is that Nedd can no longer be phosphorylated by the corresponding kinase, in particular Sgk1. The antibody can be used as a useful tool for investigating the mutation.

Antibodies according to the invention are prepared using methods familiar to those skilled in the art. In particular, polyclonal or monoclonal antibodies can be prepared, and monoclonal antibodies are generally preferred because of their higher specificity.

The antibodies described above can be used particularly advantageously in diagnostic kits according to the invention. In addition, the antibodies described above can be used very advantageously in the use according to the invention for detecting the expression and / or function of Sgk, PKB and / or Nedd. In this regard, the antibodies can be used according to conventional immunological methods. In particular, the antibodies can be used to perform the ELISA already mentioned.

The present invention additionally encompasses compositions, preferably pharmaceutical compositions, containing one or more active compounds which affect glucose transport, in particular intestinal and / or renal glucose transport, and where appropriate pharmaceutically acceptable excipients. Particularly preferably, the active compound affects one or more Sgk and / or PKB and / or one or more Nedd. In another preferred embodiment, the active compound affects activators, inhibitors, modulators and / or biological precursors of Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2.

The active compound is advantageously a polynucleotide. The polynucleotide may comprise or form an antisense sequence that reduces or inhibits expression of the gene of interest. In addition, as is known to those skilled in the art, a dominant negative approach can be used to select a polynucleotide in such a way as to inhibit the expression of the gene or genes or to limit the function of the gene product. The polynucleotide may also encode a peptide, preferably a polypeptide, which affects the expression and function of Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. . Corresponding molecular biological procedures required for this approach are available to those skilled in the art. In another preferred embodiment, the active compound is the described peptide itself. The active compound is preferably a "small molecule compound", preferably a "small molecule compound" having a molecular weight <1000.

In particular for the treatment of diseases associated with disturbed glucose transport, the active compounds inhibit one or more Sgk and / or PKB and / or stimulate one or more Nedd. To treat such a disease, the active compound is particularly preferably at least one kinase inhibitor, preferably staurosporin and / or kelerytrin or one analog thereof, and / or at least one ligase activator.

In order to increase glucose transport, particularly in connection with animal fattening, the active compounds preferably stimulate one or more Sgk and / or PKB and / or inhibit one or more Nedd. To increase glucose transport, the active compound is advantageously an Sgk1 activator, in particular a growth factor, preferably IGF1, and / or insulin. In another preferred embodiment of the invention, the active compound is a transcriptional stimulator of Sgk1 and / or Sgk3 and / or PKB, preferably at least one glucocorticoid, mineral corticoid, gonadotropin and / or cytokine, in particular TGFβ .

The different possibilities described may be combined with each other.

The present invention also includes a method of producing a transgenic animal that exhibits an increase in lipid accumulation in adipose tissue. Humans are excluded from this aspect of the invention. The animals are very important for food production, especially since they grow very quickly. The use of such animals makes fattening much faster and more efficient. The production method of the animal is characterized in that the expression and / or function of Sglt, in particular Sglt1 in the animal is increased. This accelerates intestinal absorption of glucose and increases glucose concentration in plasma more rapidly. This results in higher secreted insulin levels, which in turn stimulates lipid accumulation in adipose tissue.

In a particularly preferred embodiment of this aspect of the invention, sglt, in particular sglt1, is overexpressed in an animal for this purpose. This is achieved, for example, by the introduction of suitable gene constructs, in particular vectors, with appropriately strong promoters functionally located upstream of the appropriate sglt sequence. It is also desirable to clone animals that exhibit moderately strong sglt, particularly sglt1 expression. Methodological procedures for carrying out this are available to those skilled in the art.

In another preferred embodiment, the expression and / or function of Sgk, in particular Sgk1 and / or Sgk3 and / or PKB, is increased. The end result, this also increases the activity or protein amount of Sglt, in particular Sglt1, which means that glucose transport is increased. To accomplish this, the gene of interest can be overexpressed by conventional molecular biological methods. On the other hand, gene constructs expressing appropriate always active mutations may also be introduced or inserted into an individual. In this connection, the mutations ^S422D sgk1 and / or ^{T308D, S473D} PKB are particularly preferred. Since the activity of this mutation is independent of other activating enzymes, especially kinases, the mutation is always active. They inhibit the degradation of Sglt, in particular Sglt1 caused by the ubiquitin ligase Nedd, in particular Nedd4-2, thus increasing glucose transport.

In another preferred embodiment, the expression and / or function of the ubiquitin ligase Nedd, in particular Nedd4-2, is reduced. It also has the effect of increasing glucose transport as a result of the degradation of Sglt, in particular Sglt1, resulting in reduced amounts. Likewise appropriate reductions in Nedd expression and / or function can be achieved using conventional molecular biological methods such as antisense or dominant-negative approaches. Stably introducing suitable mutations of nedd, in particular nedd4-2, or switching off negative genes for Nedd is particularly preferred in order to reduce or inhibit the enzyme expression over a long period of time in this manner. Appropriate procedures are known to those skilled in the art. In this regard, it is particularly preferred to insert one or more inactivating mutations into Nedd, in particular Nedd4-2. In this regard, the mutations ^S338D nedd4-2 and / or ^S444D nedd4-2 can be used very advantageously. Likewise, the present invention includes animals which can be prepared by the method according to the present invention.

The above and other features of the invention are apparent from the following description of the preferred embodiments, in conjunction with the claims and drawings. In this regard, the individual features in each case may be implemented in themselves or in combination with some of them.

Claims (52)

For diagnosing diseases associated with disturbed glucose transport, for detecting the expression and / or function of active and / or inactive Sgk, in particular Sgk1 and / or Sgk3, and / or PKB, and / or Nedd, in particular Nedd4-2 Use of one or more substances. 2. Use according to claim 1, wherein the substance is at least one substance from antibodies and nucleotides. Use according to claim 1 or 2, characterized in that it uses antibodies produced against phosphorylated and / or nonphosphorylated sequences at Sgk, in particular Sgk1 and / or Sgk3, PKB and / or Nedd, in particular Nedd4-2. 4. Use according to claim 3, characterized by using antibodies generated against one or more phosphorylated and / or nonphosphorylated kinase consensus sequences, in particular Sgk1 consensus sequences, in Nedd proteins, in particular Nedd4-2 proteins. The method according to any one of the preceding claims, wherein one or more mutations, in particular inactivated mutations, are detected in the DNA, RNA and / or Nedd proteins of nedd, in particular nedd4-2, from a biological sample, in particular a sample of the patient. , Mutation is preferably present in the nedd segment encoding the Sgk1 consensus sequence in the Nedd protein. 6. Use according to claim 5, wherein the mutations are ^S338D Nedd4-2 and / or ^S444D Nedd4-2. The DNA of a gene according to any one of claims 1 to 6, wherein the one or more mutations, in particular the activating mutations, are sgk, in particular sgk1 and / or sgk3, and / or PKB from a biological sample, in particular a patient's sample, Use as detected in RNA and / or Sgk protein and / or PKB protein. 8. Use according to claim 7, wherein the mutation is ^S422D Sgk1 and / or ^{T308D, S473D} PKB. 9. Use according to any one of the preceding claims, characterized in that the disease is metabolic syndrome, in particular obesity. A method for diagnosing obesity tendency, wherein at least one polymorphism is detected in sgk, in particular sgk1 and / or sgk3, genes for PKB, nedd, in particular nedd4-2, and / or sglt, in particular sglt1. The method of claim 10, wherein the polymorphism is a single nucleotide polymorphism (SNP). 12. The method of claim 10 or 11, wherein the polymorphism is E8CC / CT in Sgk1; I6CC. Use of one or more compounds to affect glucose transport, in particular intestinal and / or renal glucose transport. Use according to claim 13, characterized in that the active compound affects one or more Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or affects one or more Nedd, in particular Nedd4-2. Use according to claim 13 or 14, characterized in that the active compound is produced for Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. The active compound according to claim 13, wherein the active compound is Sgk, in particular Sgk1 and / or Sgk3, and / or activator, inhibitor, modulator and / or biological of PKB and / or Nedd, in particular Nedd4-2. And generated for the precursor. Use according to any of claims 13 to 16, characterized in that the active compound is preferably a polynucleotide encoding a peptide, in particular a polypeptide. 18. Use according to any of the claims 13 to 17, characterized in that the active compound is a peptide, preferably a polypeptide. Use according to claim 17 or 18, characterized in that the peptide affects the expression and / or function of Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. 20. Use according to any of the claims 13 to 19, characterized in that the active compound is a "small molecule compound", preferably a "small molecule compound" with a molecular weight (MW) <1000. 21. The method according to any one of claims 13 to 20, wherein the active compound inhibits one or more Sgk, in particular Sgk1 and / or Sgk3, and / or PKB, in particular to prevent or treat diseases associated with disturbed glucose transport. / Inhibiting or stimulating one or more Nedd, in particular Nedd4-2. 22. The method according to any one of claims 13 to 21, wherein the active compound is one or more kinase inhibitors, preferably staurosporin and / or kelerytrin, or one analog thereof, and / or one or more ligase activators. Use, characterized in that. Influencing, in particular inhibiting and / or inhibiting or influencing one or more Nedds, in particular stimulating one or more Sgk, and / or PKB, for the manufacture of a drug or pharmaceutical composition for the treatment of diseases associated with disturbed glucose transport Use of at least one active compound for. 24. Use according to any of claims 21 to 23, characterized in that the disease is metabolic syndrome, in particular obesity. 21. The method according to any one of claims 13 to 20, wherein the active compound stimulates one or more Sgk, in particular Sgk1 and / or Sgk3, and / or PKB to increase glucose transport, in particular to increase animal body weight. / Stimulating or inhibiting one or more Nedd, in particular Nedd4-2. Use according to claim 25, characterized in that the active compound is at least one Sgk activator and / or PKB activator, in particular a growth factor, preferably IGF1, and / or insulin. The method according to claim 25 or 26, wherein the active compound is at least one stimulant of sgk1 and / or sgk3 and / or PKB gene transcription, preferably at least one glucocorticoid, mineral corticoid, gonadotropin and / or cytokine, in particular TGFβ. One for detecting the expression and / or function of active and / or inactive Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2, for diagnosing diseases associated with disturbed glucose transport Diagnostic kit containing the above substances. 29. The diagnostic kit of claim 28, wherein the disease is metabolic syndrome, in particular obesity. An antibody, characterized in that it is generated for at least one phosphorylation kinase consensus sequence, in particular the Sgk1 consensus sequence, in a Nedd protein, in particular a Nedd4-2 protein. An antibody, characterized in that it is generated for at least one non-phosphorylated kinase consensus sequence, in particular the Sgk1 consensus sequence, in a Nedd protein, in particular a Nedd4-2 protein. An antibody, characterized in that it is generated for at least one mutated kinase consensus sequence, in particular the Sgk1 consensus sequence, in a Nedd protein, in particular a Nedd4-2 protein. 33. The antibody of claim 32, wherein the Nedd protein having the mutated kinase consensus sequence is ^S338D Nedd4-2 and / or ^S444D Nedd4-2. Compositions, in particular pharmaceutical compositions, containing an effective amount of at least one active compound which affects glucose transport, in particular intestinal and / or renal glucose transport, and where appropriate pharmaceutically acceptable excipients. 35. The composition of claim 34, wherein the active compound affects one or more Sgk and / or PKB and / or one or more Nedd. The active compound according to claim 34 or 35, wherein the active compound affects the activators, inhibitors, modulators and / or biological precursors of Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. Composition, characterized in that. 37. The composition according to any one of claims 34 to 36, wherein the active compound is preferably a polynucleotide encoding a peptide, in particular a polypeptide. 38. The composition according to any one of claims 34 to 37, wherein the active compound is a peptide, preferably a polypeptide. 39. The composition according to claim 37 or 38, wherein the peptide affects the expression and / or function of Sgk, in particular Sgk1 and / or Sgk3, and / or PKB and / or Nedd, in particular Nedd4-2. 40. The composition according to any one of claims 34 to 39, wherein the active compound is a "small molecule compound", preferably a "small molecule compound" having a molecular weight (MW) <1000. 41. The composition of any one of claims 34-40, wherein the active compound inhibits one or more Sgk and / or PKB and / or stimulates one or more Nedd. 42. The method according to any one of claims 34 to 41, wherein the active compound is one or more kinase inhibitors, preferably staurosporin and / or keletritin, or one analog thereof, and / or one or more ligase activators A composition, characterized in that. 43. The composition of any one of claims 34 to 42, wherein the active compound stimulates one or more Sgk and / or PKB and / or inhibits one or more Nedd. The composition according to claim 43, wherein the active compound is at least one Sgk activator and / or PKB activator, in particular a growth factor, preferably IGF1, and / or insulin. The method according to claim 43 or 44, wherein the active compound is at least one stimulant of sgk1 and / or sgk3 and / or PKB gene transcription, preferably at least one glucocorticoid, mineral corticoid, gonadotropin and / or cytokine, in particular TGFβ composition. A method for producing transgenic animals other than humans, which exhibits increased lipid accumulation in adipose tissue, characterized in that the expression and / or function of Sglt, in particular Sglt1, is increased. 47. The method of claim 46, wherein Sglt, in particular Sglt1, is overexpressed. 48. The method according to claim 46 or 47, wherein the expression and / or function of one or more Sgk, in particular Sgk1 and / or Sgk3 and / or PKB is increased. 49. The method according to claim 48, wherein one or more sgk, in particular sgk1 and / or sgk3 and / or one or more PKB genes are overexpressed. The method according to claim 48 or 49, characterized in that one or more activating mutations of genes of sgk, in particular sgk1 and / or sgk3, and / or PKB, in particular ^S422D sgk1 and / or ^{T308D, S473D} PKB are used. . 51. The method according to any one of claims 46 to 50, wherein the expression and / or function of one or more Nedd, in particular Nedd4-2, is reduced. The method according to claim 51, wherein at least one inactivating mutation of nedd, in particular nedd4-2, in particular ^S338D nedd4-2 and / or ^S444D nedd4-2 is used.