KR20010053331A - Method for Detecting of a Compound that Mimics, Potentiates or Inhibits the Physiological Effect of Leptin - Google Patents

Abstract

(a) evaluating the effect of a compound on a leptin activated signal transducer and a transcriptional activator (STAT) DNA response element linked to a reporter gene in the case of a compound mimicking the physiological effect of leptin, or

(b) for compounds that enhance or inhibit the physiological effect of leptin, evaluate the effect of the compound on the response provided by leptin on the leptin activated STAT DNA response element linked to the reporter gene,

The response element and reporter are expressed in the white colon epithelial cell line (CACO-2), or

The response element and reporter can be used to determine whether the cell line (treated cell line) can mediate or enhance the stimulation by leptin in the leptin activated STAT DNA response element and that a polypeptide containing the appropriate STAT protein is also expressed in the transfected CACO-2 cells A method for detecting a compound that mimics, enhances, or inhibits the physiological effect of leptin.

Description

TECHNICAL FIELD The present invention relates to a method for detecting a compound that mimics, enhances, or inhibits the physiological effect of leptin,

The present invention relates to a novel method, more particularly to a method for detecting a compound that mimics, enhances or inhibits the physiological effects of leptin. These methods can also be used to measure the functional activity of leptin and leptin mimics, enhancers, and inhibitors.

Obesity is the result of an imbalance in energy consumption and consumption. This increasingly prevalent symptom is typically associated with a number of pathologies, including non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease, hypertension and insulin resistance, characterized by inappropriately elevated plasma levels of insulin, glucose, triglyceride and fat protein Related symptoms. In this symptom, the homeostatic mechanism that regulates the storage and utilization of energy from the constituents of the diet is disturbed, resulting in excessive accumulation of adipose tissue. As a result, it acts on the hypothalamus to suppress food intake and increase energy consumption, and the synthesis and secretion of leptin, a recently discovered factor, is increased to the same extent (1). The importance of leptin in regulating energy balance is stressed by severe early-onset obesity, insulin resistance, and insulin resistance, which are manifested by obesity (ob / ob) mice, a gene strain deficient in functional leptin (1). Administration of recombinant hormones to these mice (with lesser doses in their lean babies) results in significant weight loss and reduced plasma insulin and glucose levels (2, 3). Diabetic (db / db) mice, which are phenotypically similar strains, are deficient in leptin receptors and appear to be insensitive to leptin in many in vivo and in vitro experiments (4-8). Although seemingly rare on the surface, examples of leptin deficiency in humans show that this hormone plays a similar role to humans in rodents (9). The additional role of leptin is hematopoietic and reproductive function (10).

Protein molecules containing four alpha-helical cores forming a bundle of up-top-bottom-bottom morphology consist of a set of cytokines and growth factors. This set of proteins results in homo- and hetero-oligomerization of membrane receptors known to activate the kinase cascade that causes gene transcription. This set of receptors activated by oligomerization is divided into two major classes: receptors such as epithelial growth factor receptors (11) with integral tyrosine kinase activity in the intracellular domain, and Receptors such as IL4 and erythropoietic factor receptors that lack this activity and mediate their response through related protein tyrosine kinases (12). Both receptor subtypes are activated by cytokines, but the 4-helical bundle proteins activate only non-integral tyrosine kinase subtypes. Non-integrative protein tyrosine kinase receptors generally act, at least in part, through a pathway involving Janus kinase (JAK) and its associated signal transducer and transcription activator (STAT) protein. The activated STAT protein binds to the DNA response element and regulates gene transcription. The oligonucleotide sequence containing the DNA regulatory element of the generic sequence TT (N) nAA has been identified as a STAT response element (13). This element binds STAT proteins in response to signal molecules such as cytokines.

The leptin receptor (OB-R) (14) is associated with class I cytokine receptors, including gp 130, a common signal transduction component for cytokines associated with IL-6, known to activate potential cytosolic STAT proteins (15). There are multiple OB-R isoforms, at least two of which, OB-Ra and OB-Rb, are generated by alternate splicing (4,5,7). Short isoforms, OB-Ra, OB-Rc and OB-Rd contain amino acid cytosolic C-termini of 34, 32 and 40, respectively. There is one soluble isoform, OB-Re, which lacks the epidermal region. The OB-Rb isoform contains a full length cytosolic region of 302 amino acids, including the binding motif required for activation of the Janus kinase / signal transducer and the transcription activator (JAK / STAT) signaling pathway. Point mutations in the diabetes (db) gene of mice (G → T) lead to new splice donor sites and immature translation termination (4, 5).

End-truncated OB-R (db) lacks the sequence motif required for interaction with JAK and STAT, and evidence from the db / db mouse model suggests that OB-Rb is involved in JAK / STAT activation and food intake and cell proliferation Suggesting that it is the only receptor capable of mediating the leptin mediated effect.

International patent application publication number WO 96/34885 describes that leptin is characterized by a four-stranded, three-stranded structure. As expected by the present inventors, it now has been found that leptin is one (QB-R _b) to interact with the membrane bound receptor that activates a JAK-STAT kinase cascade communication of which (17). This interaction forms the basis of an analytical system that detects compounds that mimic, enhance or inhibit the physiological effects of leptin. Such assays are useful for screening compounds for the treatment of diseases controlled by weight, energy balance, hematopoiesis, fertility and leptin. This assay is particularly useful for screening for compounds for the treatment of obesity, anorexia, cachexia and diseases associated with diabetes.

International patent application, publication number WO 96/38586, refers to a new detection method using JAK / STAT technology. Co-pending International Patent Application No. PCT GB97 / 02988 relates to a similar technique.

The present inventors have now identified cell lines that are particularly advantageous in identifying leptin mimetics, enhancers, and antagonists comprising compounds that interact with the leptin receptor and its signal transduction pathway in conjunction with JAK-STAT technology. The present inventors have now found the expression of OB-Rb in CAPC-2 cells, an epithelial in the small intestine of the human model, and a separate part of the gastrointestinal tract of the mouse involved in nutrient uptake and lipid uptake. Leptin administration rapidly induced nuclear STAT5 DNA binding activity in the + / + and ob / ob sites (jejunum), but not in OB-Rb deficient db / db mice. Leptin administration results in a 2-fold decrease in the level of apo fat protein AIV (APO-AIV) transcription after 90 minutes of fat administration. Thus, leptin can mediate the region of primary lipid regulation through the negative feedback signal through the OB-Rb and STAT5 mechanisms. The deficiency of leptin or its direct resistance to leptin action at this site may contribute to elevated triglyceride and lipoprotein levels observed in obesity and its related syndromes.

Therefore,

(a) evaluating the effect of a compound on a leptin activated signal transducer and a transcriptional activator (STAT) DNA response element linked to a reporter gene in the case of a compound mimicking the physiological effect of leptin, or

(b) for compounds that enhance or inhibit the physiological effect of leptin, evaluate the effect of the compound on the response provided by leptin on the leptin activated STAT DNA response element linked to the reporter gene,

The response element and reporter are expressed in the white colon epithelial cell line (CACO-2), or

The response element and reporter can be used to determine whether the cell line (treated cell line) can mediate or enhance the stimulation by leptin in the leptin activated STAT DNA response element and that a polypeptide containing the appropriate STAT protein is also expressed in the transfected CACO-2 cells The present invention provides a method for detecting a compound that mimics, enhances or inhibits physiological effects of leptin.

Suitable polypeptides capable of mediating stimulation by leptin in the leptin activated STAT DNA response element are functional isoforms of the ob-gene receptor identified, for example, in the literature (Tartaglia et al., Cell, 1995, 83, 1263) . A suitable polypeptide capable of enhancing stimulation by leptin in the activated STAT DNA response element is JAK kinase, particularly JAK-2, or STAT, particularly STAT 3 or 5.

Suitably, the response element is associated with a promoter gene, preferably a minimal promoter.

Suitable reaction elements are nucleotides of the formula TT (N) _n AA wherein N is any nucleotide and n is 4, 5 or 6.

The preferred response element is selectively activated by an intracellular event that is mediated by leptin, which interacts with its receptor. Such an selective response element can be determined by examining the relative activation of the constitutive region of the response element-reporter gene when leptin versus other cytokines were transfected into the ob-responsive cell line.

The preferred response element is a nucleotide of the formula TT (N) _n AA _where N is any nucleotide and n is 4 or 5.

Another suitable response element is TTCCCGGAA.

Another suitable response element is the part of the promoter of the gene regulated by leptin required for STAT interaction. This gene will depend on the particular therapeutic use of the compound to be selected by analysis.

A suitable reporter gene is firefly luciferase or chloramphenicol acetyltransferase enzyme.

Suitable promoters are minimal promoters such as the herpes simplex virus thymidine kinase or the SV40 promoter.

A brief description of the drawing is as follows.

Brief Description of the Drawings Figure 1 is a diagram showing the expression of leptin receptor (OB-R) mRNA isoforms in tissues from the gastrointestinal tract and human CACO-2 cells.

A. RT-PCR detection of multiple OB-R isoforms in separate sections of ducts of lean (+ / +) and obesity (ob / ob) mice as described in the experimental procedure; OB-Rd (2), OB-Rc (3), OB-Rb (4), OB-Ra (5) and finally a 100 bp DNA ladder.

B. In the left panel, leptin specific primers were used to exclude contamination from fat and transcription. As a positive control, a common sequence from the extracellular domain of OB-R was amplified from the same sample. The right panel shows RT-PCR detection of the functional leptin receptor OB-Rb in human CACO-2 cell line.

Figure 2 is a Western blot analysis of OB-R receptors in the gastrointestinal tract and CACO-2 cells. A. OB-Ra detected in different compartments of the tubes of the + / + and ob / ob mice using antibodies against the carboxy terminal of the short isotype. B. OB-Ra detected in human CACO-2 cells using an antibody to the amino terminus common to all OB-R isoforms. The arrow indicates the approximate size of the 120 kDa band.

Figure 3 shows the relative expression levels of total OB-R transcripts in different compartments of the gastrointestinal tract. The top panel shows a representative gel of PCR amplification of the common OB-R extracellular domain in the various compartments of the gastrointestinal tract of the + / + mouse using different amounts of cDNA as previously described (18). The amount of cDNA in each sample was associated with the beta -actin level and adjusted prior to dilution. The lower panel shows the quantification of OB-R mRNA normalized based on beta -actin mRNA from the optimal linear portion of the PCR in + / + and ob / ob mice.

4 is a graph showing the expression of leptin reactive STAT protein in the gastrointestinal tract of a mouse and human CACO-2 cells.

A. Western blotting analysis of STAT1, 3 and 5 at 25 [mu] g of factory protein solubilis from + / + and ob / ob mice.

B. Western blotting analysis of STAT1, 3 and 5 in CACO-2 cells. Positive controls are human fibroblast, A431 and human endothelial lysates for STAT1, 3 and 5, respectively. STAT2 and 6 were also detected (data not shown).

5 is a graph showing the gel mobility shift analysis of in vivo binding of STAT DNA in response to leptin. The binding activity of the STAT5 DNA to the beta casein promoter STAT5 sympathetic element was induced. Nuclear extracts are prepared and incubated with ³² P end labeled probes and separated on 4% native PAGE gels. Induction was observed in nuclear extracts of + / + and ob / ob plants after 30 min in response to leptin (5 mg / kg). Preliminary incubation with anti-STAT5 antibody revealed a major complex composed of STAT5a. No induction was observed in plant nuclear extracts from db / db mice or control + / + and ob / ob mice.

6 is a graph showing gel migration mobility analysis of STAT DNA binding in CACO-2 cells responsive to leptin. Induction of STAT3 and STAT5 DNA binding activity in response to leptin (20-200 nM, 15 min) is shown. The dosing response relationship is observed after leptin treatment in CACO-2 nuclear extracts at STAT3 activation when assessed by the m67-SIE sympathetic component of the STAT1 / 3 selective high affinity from the c-fos promoter. This complex was identified by preliminary incubation with anti-STAT3 and anti-STAT1 antibodies. STAT5 DNA binding to the beta casein probe was also observed after leptin treatment in the nuclear extract of CACO-2 cells. The major protein complexes were identified by preliminary incubation with anti-STAT5b specific antibodies.

FIG. 7 is a graph showing leptin induction of immediate-type gene transcription in the mouse's plant and human CACO-2 cells. FIG.

A. Northen blotting of total RNA from ob / ob plants from animals treated with A. tris control and intravenous injection of leptin (5 mg / kg). Induction of c-fos mRNA levels 5-fold after 30 minutes of leptin administration is evident.

B. Quantitative PCR for total RNA from CACO-2 cell monolayers treated with leptin (200 nM) for 30 min as described in (18) was performed. Controls and serial dilutions of cDNA from leptin treated cells were used to obtain the optimal linear portion of PCR.

Figure 8 is a graph showing that leptin administration decreases APO-AIV mRNA levels following high fat administration. Leptin (5 mg / kg) treatment resulted in a significant 2-fold reduction of APO-AIV mRNA levels in the plant of ob / ob mice. The mice were starved for 5 hours and received leptin intravenously 15 minutes prior to the administration of fat loading (0.75 ml vegetable oil). After 90 minutes the mice were sacrificed and the plant was removed for RNA extraction. Expression of APO-AIV mRNA was normalized to 28S rRNA and the results were expressed as mean ± SEM (n = 3 and * p <0.05).

The methodology in which the CaCo-2 cell line is identified as a leptin reactive cell line is described below. General methodology for the identification of leptin reactive cell lines is well known and includes, for example, the following.

1. Microphysiometer: This method detects small pH changes resulting from biochemical changes in cells. Ob-protein-responsive cells that are stimulated may undergo biochemical changes that cause small changes in extracellular acidity, which can be detected by a silicon microphysiometer. The microphysiometer biosensor methodology has been reviewed in the literature (McConnell, Science, 1992, 257, 1906).

2. Electrophoretic mobility shift assay (EMSA): nuclear extracts from cells after ob-protein treatment are mixed with radiolabeled oligonucleotides containing indiscriminate or specific STAT response element DNA sequences. Extracts from cells that respond to the ob-protein can cause gel migration to the STAT response element of the oligonucleotide.

(Recombinant DNA, 2nd Edition, Watson et al., 1992, Lamb et al., Blood, 1994, 83, 2063)

3. Protein Sphincter Acid Reaction Assay: Coupling through receptor tyrosine phosphorylation of the intracellular protein of receptor activation and final response can be analyzed using antibodies recognizing the sphingosine-reacted tyrosine. More specifically, since leptin receptors can stimulate the tyrosine dibasic acid response of the JAK / STAT pathway, this method provides a method for detecting leptin responsive cell lines. Specific JAK / STAT antibodies can be used to detect leptin activation in leptin reactive cell lines with antibodies to the tyrosine-sulphonic acid response. Stimulation as well as inhibition of the protein sphingase response can occur. In particular, inhibition by the ob-protein of insulin receptor and insulin-stimulating subunit of acid response of insulin receptor substrate-1 is shown for insulin receptor expression in rat-1 fibroblast (Kroder et al., 1996, Exp. Clin. Endocrinol. Diabetes, 104, suppl 2, p66).

4. Substitution bonds: Nonspecific binding versus specific binding of leptin after incubation of the cell line with radiolabeled leptin, eg, [ ¹²⁵ I] -leptin, can be studied by the addition of unlabeled leptin. High specific vs. non-specific binding ratios suggest that the cell line may contain leptin receptors.

5. Detection by selective antibody use of proteins for functional, preferably functional, long forms of ob-receptors.

6. Detection by Norton, RT-PCR or " Slot Blotting " assays of mRNA for functional, preferably functional, long forms of ob-receptors.

7. Detection of increased c-fos mRNA after leptin treatment. c-fos mRNA can be detected by Norton, RT-PCR or "slot blotting" assays.

The response elements, reporters and preferably the promoter are suitably incorporated into a vector capable of transfecting an ob-responsive cell line.

A suitable vector is a commercially available vector such as the pGL2-basic luciferase vector (Promega).

A suitable constitution of the vector is the STAT DNA response element upstream of the promoter and reporter gene. A more suitable configuration of the vector is the STAT DNA response element, which is a multiple tandem repeat (2-10) above the thymidine kinase promoter and luciferase reporter gene.

The vector is constructed containing reporter genes such as, for example, firefly luciferase or chloramphenicol acetyl transferase enzyme conjugated to a minimal promoter such as, for example, herpes simplex virus thymidine kinase or SV40 promoter. The DNA fragment for the STAT response element is inserted into the vector using the appropriate restriction enzyme site at the top of the minimal promoter.

Reaction elements, reporters and promoters can be incorporated into the vector using conventional expression techniques, if necessary, for example, a DNA fragment for the reaction element can be inserted into the vector using the appropriate restriction enzyme site upstream of the minimal promoter have.

STAT response element-Luciferase enzyme reporter system can be constructed as described in the literature (Lamb et al., Blood, 1994, 8, 2063 and Seidel et al., Proc. Nat. Acad. Sci. USA, 1995, 92, 3041).

Leptin-reactive cells can be transfected with the STAT response element-minimal promoter-luciferase reporter construct using standard methods such as, for example, the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, these cells may be co-transfected with a control plasmid expressing? -Galactosidase activity. After the transfection cycle (12-24 h), these cells are treated with varying concentrations of compound, then harvested and lysed. The lysates are assayed for activity of luciferase and, if appropriate, for beta -galactosidase activity. Enhancing or antagonist activity can be assayed by pre- or simultaneous (simultaneous) addition of an appropriate concentration of leptin to the compound under evaluation and measurement of the enhancement or reduction of the luciferase response relative to leptin alone. There are standard methods for the analysis of luciferase enzyme activity in various commercial kits as well as in literature (Ow et al., Science, 1986, 234, 856 and de Wet et al., Mol. Cell Biol., 1987, 7, 725).

A stable cell line can be generated by transfecting a leptin reactive cell line with a reporter construct and a selectable marker. Selectivity markers are routinely used for stable cell line generation as described in the literature (Recombinant DNA, 2nd edition, J. D. Watson et al., 1992, page 216). This stably transformed cell line can be used to generate high throughput assays for compounds that mimic, enhance or block the physiological effects of leptin.

The invention also extends to compounds that mimic, enhance or inhibit the physiological effects of leptin when identified by the methods disclosed herein.

The invention also extends to a kit of parts adapted for use in the methods disclosed herein.

The present invention also extends to detecting compounds that mimic, enhance or inhibit the physiological effects of leptin by measuring other responses to leptin from such cells using native or treated CACO-2 cells.

The present invention also extends to measuring the functional activity of leptin mimetics, enhancers or inhibitor formulations using native or treated CACO-2 cells.

As used herein, a compound that mimics the physiological effect of leptin is a compound that stimulates the leptin receptor in the absence of leptin to provide substantially the same physiological effect as the ob-protein or can activate the response of the subsequent step (post-receptor) &Lt; / RTI &gt;

As used herein, the compound that enhances the physiological effect of leptin refers to a compound that enhances the efficacy and / or maximum physiological effect of leptin.

As used herein, a compound that inhibits the physiological effect of leptin refers to a compound that reduces or substantially blocks the physiological effect of leptin.

The cDNA encoding the functional form of the polypeptide can be transfected under the control of a constitutive promoter (e.g., a viral promoter) or a regulatable promoter to optimize the expression of the polypeptide for identification of an agonist or antagonist, as needed. In addition, the response elements and reporters are expressed in cell lines in which the constitutive or regulatable promoter is modified by homologous recombination at the site above the chromosomally encoded gene for the leptin receptor. Such methods have been reviewed by Waldman (Critical Reviews in Oncology / Hematology, 1992, 12, 49) and specific examples are given in the literature (Riele et al., Proceedings of the National Academy of Sciences, 1992, 89, 5128).

The following examples are provided to illustrate but not to limit the scope of the invention in any way.

General courses:

CaCo-2 cells were transfected with a reporter plasmid containing the STAT response element in a plurality of tandem copies using standard methods such as the calcium phosphate method, for example, above the minimal promoter, such as the herpes simplex thymidine kinase and the luciferase gene reporter construct (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, the cells may be co-transfected with a control plasmid expressing? -Galactosidase activity. After the transfection cycle (12-24 h), these cells are treated with varying concentrations of compound, then harvested and lysed. The lysates are assayed for the activity of luciferase and, if appropriate, for the activity of beta -galactosidase. Antagonist activity can be assayed by pre- or simultaneous addition of leptin to the compound at an appropriate concentration under the evaluation and measurement of a decrease in luciferase response compared to leptin alone. For example, there are standard methods for the analysis of luciferase enzyme activity in various commercial kits as well as in literature (Ow et al., Science, 1986, 234, 856 and de Wet et al., 1987, 7, 725).

Experimental compartment summary

Leptin, an adipocyte hormone, mediates increased satiety and increased energy expenditure and activates STAT3 in the hypothalamus. To date, leptin mediated activation of the STAT pathway in vivo has not been established in tissues other than the hypothalamus. We now describe in vivo signaling and expression of leptin receptors in separate parts of the gastrointestinal tract of the mouse involved in lipid regulation. Expression of functional isoforms, OB-Rb, is limited to the plant. Intravenous injection of leptin rapidly induces nuclear STAT5 DNA binding activity in the plant of + / + and obesity (ob / ob) mice, but has no effect in diabetic (db / db) mice deficient in OB-Rb isoform . In addition, the induction of the immediate type gene c-fos was observed in vivo. Activation of STAT3 and STAT5 in human models of small epithelial CACO-2 cells and leptin-mediated induction of multiple immediate-type genes confirm this effect. Moreover, intravenous administration of leptin resulted in a significant 2-fold reduction in the APO-AIV transcription level in the plant after 90 minutes of fat administration. Our experimental results suggest that the plant is a direct target of leptin action and that this activity is dependent on the presence of OB-Rb. Resistance to leptin action at this site, or leptin deficiency, can directly contribute to lipid regulation, thereby contributing to obesity and its associated syndromes.

Abbreviations used herein are: NIDDM, non-insulin dependent diabetes mellitus; OB-R, leptin receptor; RT-PCR, reverse transcription polymerase chain reaction; JAK, Janus kinase; STAT, signal transducer and transcription activator; CACO-2, human colon epithelial cell line; APO-AIV, apo fat protein AIV; EMEM, Earls minimum essential badge.

<Procedure>

Animals and Cells-Six-week-old female wild-type and ob / ob mice were housed in the Aston strain and placed in a 12-hour daylight-12 hour dark cycle with water and standard laboratory food access (Beekay rat and mouse toxicology diet, Bantin and Kingman, Hull, UK). The in vivo efficacy of leptin (5 mg / kg) was analyzed using a 10 mM Tris-dissolved recombinant leptin (Peprotech, Rockyville, USA) administered by a 10 mM Tris-vehicle or a tail vein injection into the control. After the designated time, the mice were sacrificed by neck dislocation, the tissue was incised, rinsed and washed with ice-cold physiological saline. Tissues were harvested freshly for extraction of nuclear proteins or frozen quickly in liquid nitrogen and stored at -80 ° C, as described below. Caucasian colonic epithelial cells, CACO-2 (European animal cell culture, Porton Down, UK) were cultured in RPMI medium supplemented with 1% essential amino acid (NEAA), 2 mM L- glutamine, 10% fetal bovine serum, In a minimum essential medium (EMEM) containing 100 μg / ml streptomycin (all from Gibco / BRL (UK)), the cells were fused at 37 ° C humidified 5% carbon dioxide / Lt; / RTI &gt; Cells were preincubated for 24 hours in serum-free supplemented EMEM containing 0.1% BSA. The cells were then incubated in basal EMEM containing 0.1% BSA with or without a given concentration of leptin or Tris control vehicle.

Expression of leptin receptor mRNA - Total RNA was isolated from tissues and cells using RNaid plus kit (BIO 101, Inc. USA) and immediately treated with DNase I (Gibco / BRL, UK). CDNA containing first strand cDNA beads (Pharmacia, Biotech) and oligo (dT) _12-18 (Invitrogen) was obtained using approximately RNA (4 쨉 g). RNA integrity and loading were studied by detection of ribosomal rRNA strips (28S and 18S) in agarose gels. Potential contamination from fat was studied by using leptin specific primers (Table I) as RT-PCR amplification. RT-PCR detection of OB-R transcript isoforms in the gastrointestinal tract of mice as well as OB-Rb transcription in CACO-2 cells was performed using the oligonucleotides described in Table I. [ All PCR amplifications were performed using AmpliTaq (PerkinElmer) in a Progene thermal cycler (Techne / Cambridge) for 45 sec at 95 ° C, annealing at 57 ° C for 45 sec, annealing at 72 ° C for 45 sec (But for 1 min to extend the long OB-Rb isoform). The PCR products were then cloned directly into the pCR-TRAP cloning system (GeneHunter Corp., USA) and the homology of the PCR products was confirmed by sequencing using a kit (ThermoSequenase terminator cycle sequencing kit (American Life Science, UK)). For the sequential PCR, the cDNA samples from the + / + and ob / ob mice are serially diluted and the PCR is carried out as described above with a predetermined number of cycles (37 for OB-R and 30 for beta -actin) Respectively.

Fresh tissue was homogenized in ice-cold RIPA buffer (1X PBS, 1% IGEPAL, 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (Boehringer Mannheim, Germany) and the protein lysate Protein analysis (Sigma Diagnostics, UK) and standard Western analysis. Approximately 20 μg of protein was mixed with gel loading buffer (4% SDS, 100 mM Tris, pH 6.8, 100 mM β-mercaptoethanol) and resolved on 10% SDS-PAGE. For monolayers of CACO-2 cells, proteins were extracted by adding gel loading buffer to the flask. Immunoblotting was performed by electrophoresis of the protein on PVDF (Millipore, UK) membrane and blocking in 2% BSA, 10 mM Tris pH 7.4, 100 mM sodium chloride, 0.1% Tween 20 and OB- STAT-2, -3, and -6 were detected using STAT-2 (Santa Cruz Biothechnology, Inc., USA) (Transduction Laboratories, USA) antibody. The band was visualized by using the ECL kit according to usage (Amersham, UK).

Nuclear extract-CACO-2 cells were placed on ice immediately after treatment and the medium was removed. Cells were washed with PBS containing Then 5mM NaF, 10mM NaMoO ₄ and 1mM activated NaVO ₃ were quickly washed with ice cold PBS. Cells were cultured in RPMI 1640 medium supplemented with ₁₀ mM HEPES, pH 7.9, 10 mM NaMO ₄ , 1 mM NaVO ₃ , 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 0.125 μM okadaic acid, 1 mM DTT, 2 μg / ml rupeptin, ml &lt; / RTI &gt; AEBSF and 0.2% IGEPAL CA-630 detergent (Sigma, UK). The lysate was incubated on ice for 5 minutes and the nuclei were pelleted by microcentrifugation at 14000 rpm for 20 seconds. The supernatant was discarded and the nuclei gently resuspended in 100 μl of high salt buffer (storage buffer plus 200 mM sodium chloride, 20% glycerol). The nucleoprotein was extracted by rotating the tube at 4 ° C for 30 minutes and the debris was pelleted by microcentrifugation at 14000 rpm for 20 minutes. Nuclear extracts were aliquoted and then quenched in liquid nitrogen prior to analysis of gel mobility shifts (see below). Tissues were dissected, washed in ice-cold physiological saline, chopped and homogenized in a 1:10 w / v shelf-lysis buffer, and the nuclear extract was basically obtained as described for CACO-2 cells.

Gel mobility shift analysis - DNA ligating buffer (Tris (R)) containing 1 μg / ml polydeoxyinosine / cytidine (Pharmacia, Sweden) using 100 ng / Re ³² Pγ-ATP end labeled probe using 5 μl nuclear extract 10 mM, pH 7.5, 150 mM sodium chloride, 1 mM DTT) to block non-specific binding. Binding reactions were incubated at room temperature for 20 minutes and then lysed on 4% original PAGE gel. The transient migration experiments included an additional 15 min incubation with the appropriate antibody. Oligonucleotide probes are composed of the inducible element (m67 SIE) of mutated high affinity human serum, 5'-cat ttc ccg taa atc at -3 'from the c-fos promoter and the β-casein promoter element sequence of rat, -gga ctt ctt gga att aag gga-3 '. The complementary oligonucleotide strand (Research Genetics, USA) was annealed in Tris 10 mM, pH 8.0, 1 mM EDTA, 50 mM sodium chloride to a final concentration of 200 ng / microlitre by heating the mixture to 90 ° C for 10 minutes and then slowly cooling to room temperature. The probe was labeled with 1 [mu] l of 32P [gamma] -ATP (6000 Ci / mmol Amersham, UK) using ³ U / ul T4 polynucleotide kinase (Amersham, USB, UK) for one hour at 37 ° C.

Expression of APO-AIV and immediate genomic mRNA species CACO-2 cells and tissues treated for 30 min with or without leptin were immediately quenched in liquid nitrogen and RNA was extracted as described above. Standard norton blotting hybridization was performed using a digoxigenin labeled cDNA probe to measure levels of c-fos and APO-AIV mRNA in vivo. The coupled probe was detected by an anti-digoxigenin detection system using a polyclonal antibody conjugate to alkaline phosphatase (Boehringer Mannheim, Germany) using a chemiluminescent substrate CDP-Star (Tropix, USA) Finally, the band was quantified by scanning laser densitometer. Expression of the immediate-type genes c-fos, c-jun, junB, and junD in response to leptin was established by quantitative PCR using the primers shown in Table 1.

Experiment result

Multiple OB-R mRNA isoforms were detected using RT-PCR in separate compartments of the gastrointestinal tract (see Figure 1A). Different short isoforms, OB-Ra, OB-Rc, OB-Rd and OB-Re were expressed across the digestive tract. Conversely, long functional OB-Rb mRNA was found to be predominantly expressed in plants (FIG. 1A). Using leptin specific primers, RT-PCR amplification of cDNA samples from the factory of + / + and ob / ob mice showed negative detection, whereas PCR amplification of the common extracellular domain of OB-R of the same sample (Fig. 1B). This suggests that the OB-R mRNA shown in Fig. IA is not the result of contamination from fat tissue or the transfer of antigens. Expression of the functional leptin receptor OB-Rb mRNA was readily detected using RT-PCR in the small intestinal epithelium CACO-2 of the human model (Fig. 1B). Western blotting analysis of 20 μg crude protein lysate using an antibody against the carboxy terminal sequence of the short isoform OB-Ra, which is predominantly isoform in many tissues, resulted in predominant expression in the small intestine of both the + / + and ob / ob mice A band of the expected size of approximately 120 kDa appeared (Figure 2A). Inconsistencies between the results obtained by detecting the expression of OB-R using RT-PCR or Western analysis reflect the differences in detection limits of these two methods. Thus, Western analysis shows the difference in the abundance of OB-R in different compartments of the digestive tract (see FIG. 2A). The antibody to the amino terminus of the human OB-R receptor in general detected a ~ 120 kDa protein in CACO-2 cells, possibly explaining the dominant short OB-R isoform (Figure 2B).

The relative level of expression of the entire OB-R transcript normalized to endogenously expressed beta -actin is then quantified using the primer described in the quantitative PCR assay described above, using primers that recognize sequences common to all isoforms encoding the amino terminal end of the receptor In a separate compartment of the gastrointestinal tract (22). Here, the ratios of OB-R and beta -actin PCR products from the optimal linear portion of PCR conditions generated from the same volume of cDNA were compared (Figure 3A). No difference in OB-R mRNA levels was detected between the + / + and ob / ob mouse tissues, but OB-R transcription occurred 2-3 times more in the small intestine than in the gastric and transverse colon (Fig. 3B). These results are in good agreement with the results observed with the Western analysis of the OB-Ra distribution as shown above (see Figure 2A).

To address the leptin-signaling mechanism of the small intestine expressing OB-Rb, the presence of a STAT protein known to transmit the leptin signal was sought. Western blotting analysis of the + / + and ob / ob plants readily detects STAT5 but STAT1 and STAT3 are weaker (FIG. 4A). Furthermore STAT5 was also well expressed in the colon and duodenum (Fig. 4A). CACO-2 cells also expressed leptin-reactive STAT isoforms (Fig. 4B). Overnight fasted + / + and ob / ob mice were treated with 5mg / kg recombinant mouse leptin by intravenous injection. After 30 minutes of injection, the mice were sacrificed and the plant was removed to prepare the nuclear extract. The present inventors used two selective probes to evaluate STAT activation by EMSA analysis. The STAT sympathetic element with high affinity of the m67-SIE mutant from the c-fos promoter preferentially binds STAT1 and STAT3 but binds with low affinity to STAT5. This probe did not reveal the STAT DNA binding activity in response to in vivo leptin treatment in the gastrointestinal tract of mice (data not shown). The rat beta-casein promoter STAT DNA binding vaginal element was used as an optional probe for in vivo STAT5 activity. Here, treatment with 5 mg / kg leptin resulted in significant activation of DNA binding activity in + / + and ob / ob mice, which was not observed in animal controls treated with Tris alone (Fig. 5A). In contrast, the same amount of leptin injected into db / db mice deficient in OB-Rb isoform did not cause any activation of STAT5 DNA binding in the nuclear extract of the plant (Fig. 5A). The STAT5 DNA binding activity derived from the + / + and ob / ob plants was abolished by preincubation with certain anti-STAT5a antibodies and less abruptly with anti-STATb antibodies (ob / ob plant in Figure 5B See results from. These results suggest that the plant is a direct target of leptin action and that this activity is mediated through the OB-Rb and STAT5 mechanisms.

15 min of leptin exposure resulted in dose-dependent activation of STAT DNA binding to m67-SIE probe in serum-free CACO-2 cells. This activity was abolished by the anti-STAT3 antibody while the anti-STAT1 antibody had no effect (Fig. 6). In addition, leptin-treated CACO-2 cells showed activation of DNA binding activity of STAT5 to the? -Casein probe, and transient migration study identified STAT5b as a major factor of the complex (Fig. 6). Thus, leptin induced nuclear STAT5 DNA-binding activity in both mouse plant and CACO-2 cells, but the isoforms of STAT5 that differ in these two systems appear to form protein-DNA complexes.

Leptin administration also resulted in the elongation of the genotype c-fos immediately after 30 minutes in the plant of ob / ob mice (Fig. 7A). Intravenous injection of 5 mg / kg leptin resulted in a 5-fold increase in c-fos mRNA levels upon detection by Norton blotting analysis. Similarly, leptin treatment for 30 min resulted in a dramatic increase in the expression of the immediate type genes c-fos, c-jun, junB and junD in serum-free CACO-2 cells as measured by quantitative PCR 7B). Since activation of STAT5 is not normally associated with c-fos induction, these results may indicate that in addition to the STAT5 pathway in vivo, other pathways are active.

We then investigated the effect of leptin on APO-AIV mRNA levels on whether leptin could mediate transcription of genes involved in lipid regulation, one of the plant's major functions. A 5 mg / kg leptin injection in ob / ob mice fed high fat diets resulted in a significant 2-fold reduction in APO-AIV mRNA levels; APO-AIV mRNA /? -Actin mRNA mean ± SEM: control vehicle = 2.23 ± 0.16 and leptin treatment = 1.04 ± 0.16 (p <0.05, n = 3) 90 minutes after high fat administration (FIG. These results suggest that leptin can act as an immediate break in fat accumulation by reducing the transport of dietary triglycerides into plasma.

Discussion of experiments

Leptin, an adipocyte hormone, signals to the hypothalamus to inhibit food intake and increase energy consumption (1). Leptin has also been reported to play a role in the surrounding tissue ecosystems (8, 20, 23, 24). Leptin treatment in + / + and ob / ob mice results in a reduction in body weight and fat in excess of the reduction resulting from suppression of food intake (25). This suggests that leptin may affect energy homeostasis, fat accumulation and metabolism, which are partially independent of food intake control. The present inventors now demonstrate the presence of the full-length leptin receptor OB-Rb in a unique portion of the small intestine associated with lipid ingestion. Leptin administration resulted in the activation of the STAT5 signaling pathway in the plant of + / + and ob / ob mice after 30 min, but not in db / db mice deficient in OB-Rb. Leptin also caused a decrease in APO-AIV mRNA after 90 minutes of high fat dosing. Immediate and direct effects of leptin on plant lipid regulation may represent a frontline mechanism for fat accumulation.

Long OB-Rb isoforms are more limited in tissue distribution, whereas short OB-R isoforms are widely expressed in tissues (7). OB-Rb is relatively well expressed in the hypothalamus, particularly in parts involved in the regulation of weight homeostasis (26). However, the present inventors and others have identified the expression of OB-Rb in peripheral tissues such as islets, lymph nodes and hematopoietic stem cells (7, 8, 24). The current view is that only the OB-Rb receptor isoform activates JAK / STAT cascade information transfer and mediates the physiological effects of leptin. This is based on a number of in vivo and in vitro experiments demonstrating that OB-Rb is required for food intake, insulin secretion, or stimulation of cell proliferation (8, 16, 19). In this study, predominantly short receptor isoforms in most tissues are unable to mediate such responses to leptin, and so far no physiological function has been assigned to this isotype. It has been suggested that shorter OB-R isoforms can alter the activity of OB-Rb during homodimerization of the cytosolic domain followed by aggregation (7). However, according to recent data from White et al., Which demonstrated that the signaling ability of OB-Rb is significantly resistant to inhibition by shorter receptor isoforms, this effect of shorter isoforms seems weak (27). In the present study, we demonstrate that functional OB-Rb is predominantly expressed weaker in the ileum than in the two major regions involved in lipid regulation, while several short isoforms are expressed throughout the gastrointestinal tract. Moreover, both Western blotting and quantitative PCR analysis show that OB-R is more abundant in the small intestine than in stomach or colon. In fact, recent studies on the effects of leptin on gastric emptation suggest that leptin does not regulate the delivery rate of juice to the small intestine (27). This is consistent with the lack of OB-Rb expression at this site in recent studies. The results for OB-R expression are presented to the present inventors that the small intestine, in particular the plant, is an important target for leptin action.

In vivo studies have shown that intravenous injection of leptin induces STAT3 DNA binding activity in the hypothalamus of ob / ob mice, but not in the hypothalamus of db / db mice (19). Nuclear extracts from the hypothalamus and various surrounding tissues failed to shift STAT5-specific? -Casein promoter elements. We showed that intravenous injection of leptin induced STAT5 DNA binding activity in the + / + and ob / ob mouse plants, but not in db / db mice. Here, we chose the sub-maximal leptin concentration (5 mg / kg) and the optimal time regimen for 30 minutes based on the in vivo reports on STAT activation (19). The inventors therefore do not exclude the activation of other leptin mechanisms that do not significantly overlap this time regime in factories. The 5 mg / kg dose is somewhat higher than the physiological concentration of leptin, but the activity of recombinant leptin from different sources varies considerably. Thus, the literature report suggests that a dose of leptin between 0.1 mg / kg and 5 mg / kg results in a 50% reduction in food intake. This suggests that recombinant leptin may be significantly less potent than endogenous leptin.

Rapid activation of immediate gene transcription and STAT5 DNA binding is consistent with the direct effect of leptin on the digestive tract. Our in vivo results show that the plant effect of leptin requires the presence of long OB-Rb isoforms. In addition, STAT5 activation and immediate gene induction in response to leptin in CACO-2 cells, the small epithelium of the human model, can be interpreted as confirmation of the direct effect of leptin on enterocytes. It is also a recognized phenomenon of the in vitro system that the associated activation of STAT3 and STAT5 is more widespread in this cell line than in the primary gastrointestinal tract (7). Requirements for the removal of serum cells before and during leptin treatment and the use of high concentrations of leptin for the proven activation of STAT DNA binding in CACO-2 cells have already been observed in other cell lines (29). The significance of STAT5 in mediating the effect of leptin on weight homeostasis is evidenced by the fact that in vitro from obesity (fa / fa) Zucker rats containing a mismatch mutation (Gln → Pro) in the extracellular region of OB- It is further supported by evidence (30). OB-R (fa) mediates leptin-independent (structural) activation of STAT1 and STAT3, whereas activation of STAT5 is completely abolished (31). This animal model, together with leptin deficiency (ob / ob) and OB-Rb deficient (db / db) mice all develop early-onset obesity, and the inventors believe that a deficiency of leptin effect in the small intestine of this animal model, I believe we can make a partial contribution.

The small intestine forms the primary contact between the ingested nutrients and the carefully controlled internal environment of the organism. During obesity symptoms, endogenous intestinal triglyceride production is increased and contributes to elevated plasma triglyceride levels (32). Our results have shown for the first time that leptin may result in rapid activation of STAT5 in the plant associated with a decrease in APO-AIV transcription levels 90 minutes after a meal of fat. This suggests that leptin may play a physiological role in lipid regulation in vivo at this site. The APO-AIV system serves as a conduit for the transport of triglycerides as a particulate in circulation and for delivery to the receptor membrane in various tissues. Abolition of this function can be part of the mechanism leading to elevated levels of obesity and fat protein. Moreover, hyperglycemia and hyperlipoproteinemia are risk factors for cardiovascular disease and atherosclerosis (33). However, in physiological conditions, postprandial elevation of plasma leptin (34) may serve as a buffering system to reduce the level of colloidal triglyceride by reducing APO-AIV. It is also possible that leptin can serve as an inhibitory enhancing mechanism for the APO-AIV system to reduce the level of secreted triglyceride. It is known that leptin can induce enzymes of fatty acid oxidation (35), thus switching the fuel metabolism to β-oxidation of fatty acids (36). Therefore, the present inventors have demonstrated a novel peripheral effect on the plant function of in vivo leptin mediated through the OB-Rb receptor and the STAT5 mechanism. This may represent a negative feedback signal from fat storage to the primary site of lipid regulation, a fat-intestinal loop that contributes to the anti-obesity effect of leptin.

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Claims (11)

(a) evaluating the effect of a compound on a leptin activated signal transducer and a transcriptional activator (STAT) DNA response element linked to a reporter gene in the case of a compound mimicking the physiological effect of leptin, or (b) for compounds that enhance or inhibit the physiological effect of leptin, evaluate the effect of the compound on the response provided by leptin on the leptin activated STAT DNA response element linked to the reporter gene, The response element and reporter are expressed in the white colon epithelial cell line (CACO-2), or The response element and reporter can be used to determine whether the cell line (treated cell line) can mediate or enhance the stimulation by leptin in the leptin activated STAT DNA response element and that a polypeptide containing the appropriate STAT protein is also expressed in the transfected CACO-2 cells Wherein the method comprises the steps of: (a) detecting a compound that mimics, enhances or inhibits the physiological effect of leptin. 2. The method of claim 1, wherein the response element is linked to a promoter gene, preferably a minimal promoter. 3. The method of claim 2, wherein the reaction element is a nucleotide of the formula TT (N) _n AA wherein N is any nucleotide and n is 4, 5 or 6, preferably 5. 3. The method of claim 2, wherein the reaction element is a nucleotide of the formula TTCCCGGAA. The method according to claim 1, wherein the reporter gene is firefly luciferase or chloramphenicol acetyltransferase enzyme. 2. The method of claim 1, wherein the promoter is a herpes simplex virus thymidine kinase or SV40 promoter. 2. The method of claim 1, wherein the ob-responsive cell line is a liver or liver tumor derived cell line. 2. The method of claim 1, wherein the response element, reporter, and promoter are incorporated into a vector capable of transfecting an ob-responsive cell line. 9. The method of claim 8, wherein the vector is a pGL2-basic luciferase vector (Promega). 9. The method according to claim 8 or 9, wherein the vector is constructed so that the STAT DNA response element is located above the promoter and reporter gene. (a) evaluating the effect of a compound on a leptin activated signal transducer and a transcriptional activator (STAT) DNA response element linked to a reporter gene in the case of a compound mimicking the physiological effect of leptin, or (b) for compounds that enhance or inhibit the physiological effect of leptin, evaluate the effect of the compound on the response provided by leptin on the leptin activated STAT DNA response element linked to the reporter gene, The response element and reporter are expressed in the white colon epithelial cell line (CACO-2), or The response element and reporter can be used to determine whether the cell line (treated cell line) can mediate or enhance the stimulation by leptin in the leptin activated STAT DNA response element and that a polypeptide containing the appropriate STAT protein is also expressed in the transfected CACO-2 cells Wherein the kit is adapted for use in a method of detecting a compound mimicking, enhancing or inhibiting the physiological effect of leptin.