METHOD FOR DETECTING OF A COMPOUND THAT MIMICS, POTENTIATES OR INHIBITS THE PHYSIOLOGICAL EFFECT OF LEPTIN

Abstract: A method for detection of a compound that mimics, potentiates or inhibits the physiological effect of leptin, which method comprises: (a) for a compound which mimics the physiological effect of the leptin, assessing the effect of the compound upon a leptin activated signal transducer and activator of transcription (STAT) DNA response element coupled to a reporter gene; or (b) for a compound which potentiates or inhibits the physiological effect of leptin, assessing the effect of the compound upon the response provided by leptin upon a leptin activated STAT DNA response element coupled to a reporter gene; wherein, the response element and the reporter are expressed in the human Caucasian colon epithelial cell line (CACO-2) or the response element and the reporter are expressed in CACO-2 cells, which cell line (the engineered cell line) is also transfected with a polypeptide which is capable of mediating or enhancing the stimulation by leptin of a leptin activated STAT DNA response element and contains the appropriate STAT proteins.

Description: METHOD FOR DETECΗNG OF A COMPOUND THAT MIMICS, POTENΗATES OR INHIBITS THE PHYSIOLOGICAL EFFECT OF LEPTIN The invention relates to a novel method and more particularly to a method for the detection of compounds that mimic, potentiate or inhibit the physiological effects of the 5 leptin. These methods can also be used to measure the functional activity of preparations of leptin and leptin mimetics, potentiators or inhibitors. Obesity is the result of an imbalance in energy intake and expenditure. Increasingly prevalent, this condition is associated with a number of pathological conditions including non-insulin dependent diabetes (NIDDM) 1 , cardiovascular disease, 10 hypertension and insulin resistance that are typically characterised by inappropriately elevated plasma levels of insulin, glucose, triglycerides and lipoproteins. Homeostatic mechanisms that co-ordinate the storage and utilisation of energy from the constituents of a meal are disturbed in this condition, leading to exaggerated adipose tissue deposition. As a result there is a commensurate increase in the synthesis and secretion of the adipose 15 tissue hormone leptin, a recently discovered factor that acts on the hypothalamus to inhibit food intake and increase energy expenditure (1). The importance of leptin in the regulation of energy balance is accentuated by the profound early onset obesity, hyperinsulinaemia and insulin resistance exhibited by obese (ob/ob) mice, the genetic strain that lacks a functional leptin (1). Upon administration of the recombinant hormone 20 these mice, and to a lesser extent their lean littermates, exhibit a marked loss in weight and a lowering of plasma insulin and glucose concentrations (2,3). A phenotypically similar strain, the diabetic (db/db) mouse has a defect in the leptin receptor and has been shown to be unresponsive to leptin in a number of in vivo and in vitro experiments (4-8). Although seemingly rare, examples of leptin deficiency in humans demonstrate that the 25 hormone plays a similar role in man as it does in rodents (9). Additional roles for the leptin in haematopoietic and reproductive function have been (10). Protein molecules that contain a core composed of four α-helices forming a bundle of up-up-down-down topology comprise a family of cytokines and growth factors. Proteins of this family cause homo- and hetero-oligomerisation of membrane receptors 30 known to activate kinase cascades resulting in gene transcription. Receptors of the family which are activated by oligomerisation fall into two broad classes; those such as epidermal growth factor receptor, which possess integral tyrosine kinase activity in their intracellular domains (11), and those such as the IL4 and erythropoietin receptors, which lack this activity and mediate their response by way of an associated protein tyrosine 35 kinase (12). Both receptor subtypes are activated by cytokines, but the 4-helix bundle proteins activate only the non-integral tyrosine kinase subtype. The non-integral protein tyrosine kinase receptors generally act, at least in part, through a pathway involving Janus kinase (JAK) and their associated signal transducers and activators of transcription (STAT) proteins. On activation STAT proteins bind to DNA response elements thereby 40 controlling gene transcription. Oligonucleotide sequences comprising DNA regulatory elements of the general sequence TT(N)nAA have been identified (13) as STAT response elements. These elements bind STAT proteins in response to signalling molecules such as cvtokines. The leptin receptor (OB-R) (14) is related to class I cytokine receptors which include gpl30, the common signal transducing component for IL-6 related cytokines that are known to activate latent cytosolic STAT proteins (15). Multiple OB-R isoforms exist, of which at least two, OB-Ra and OB-Rb, are generated by alternative splicing (4,5,7). The short isoforms, OB-Ra, OB-Rc and OB-Rd contain 34, 32 and 40 amino acid cytosolic C-terminals, respectively. One soluble isoform exists, OB-Re, which lacks the transmembrane domain. The OB-Rb isoform contains a full-length cytosolic domain of 302 amino acids which includes binding motifs required for activation of the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) signalling pathway. A point mutation (G- T) in the mouse diabetes (db) gene results in a novel splice donor site and a premature translation termination (4,5). The truncated OB-R(db) lacks the sequence motifs required for the interaction with JAK and STAT and evidence from the db/db mouse model suggests that OB-Rb is the sole receptor that can mediate JAK/STAT activation and leptin-mediated effects on food intake and cell proliferation (4,5,16). International patent application, publication number WO 96/34885 describes that leptin is characterised by a four helix bundle tertiary structure. In accordance with our predictions, it has now been shown that leptin interacts with membrane-bound receptors, one of which(QB-Rb),activates a JAK-STAT kinase cascade (17). This interaction forms the basis for an assay system for the detection of compounds that mimic, potentiate or inhibit the physiological effects of leptin. Such an assay has utility in selecting compounds for the treatment of weight, energy balance, haematopoietic, fertility and other disorders modulated by leptin. The assay is especially useful for selecting compounds for the treatment of those disorders related to obesity, anorexia, cachexia and diabetes. International patent application, publication number WO 96/38586 relates to a novel detection method which uses JAK-STAT technology. Copending International patent application number PCT GB97/02988 also relates to similar technology. We have now identified a cell line that is particularly advantageous for use with the JAK-STAT technology to identify leptin mimetics, potentiators or antagonists including compounds that interact with the leptin receptor and its signal transduction pathway. We have now discovered that OB-Rb expression in discrete regions of the mouse gut associated with nutrient absorption and lipid uptake and in a human model of small intestinal epithelium, CACO-2 cells. Administration of leptin rapidly induced nuclear STAT5 DNA binding activity in +/+ and ob/ob jejunum, but had no effect in the OB-Rb deficient db/db mouse. Leptin administration results in a 2-fold reduction in apolipoprotein AIV (APO-AIV) transcript levels, 90 minutes after fat load. Thus, leptin can mediate, through a OB-Rb and a STAT5 mechanism, a negative-feedback signal to the primary site of lipid handling. Lack of leptin or resistance to direct leptin action in this site may contribute to elevated triglyceride and lipoprotein levels observed in obesity and its related syndromes. .?- Accordingly, the invention provides a method for the detection of a compound that mimics, potentiates or inhibits the physiological effect of leptin, which method comprises: (a) for a compound which mimics the physiological effect of the leptin, assessing the effect of the compound upon a leptin activated signal transducer and activator of transcription (STAT) DNA response element coupled to a reporter gene; or (b) for a compound which potentiates or inhibits the physiological effect of leptin, assessing the effect of the compound upon the response provided by leptin upon a leptin activated STAT DNA response element coupled to a reporter gene; wherein, the response element and the reporter are expressed in the human Caucasian colon epithelial cell line (CACO-2) or the response element and the reporter are expressed in CACO-2 cells, which cell line (the engineered cell line) is also transfected with a polypeptide which is capable of mediating or enhancing the stimulation by leptin of an leptin activated STAT DNA response element and contains the appropriate STAT proteins. A suitable polypeptide which is capable of mediating the stimulation by leptin of a leptin activated STAT DNA response element is a functional isoform of the ob-gene receptor, for example that identified in Tartaglia et al, Cell, 1995, 83, 1263. A suitable polypeptide which is capable of enhancing the stimulation by leptin of an activated STAT DNA response element is a JAK kinase, especially JAK-2, or a STAT, especially STAT 3 or 5. Suitably, the response element is coupled to a promoter gene, preferably a minimal promoter. A suitable response element is a nucleotide of formula TT(N) n AA, where N is any nucleotide and n is 4, 5 or 6. A favoured response element is selectively activated by the intracellular events mediated by leptin interacting with its receptor. Such selective response elements can be determined by examining the relative activation of a range of response element-reporter gene constructs when transfected into an ob-responsive cell line by leptin versus other cytokines. A favoured response element is a nucleotide of formula TT(N) n AA, where N is any nucleotide and n is 4 or 5. A further suitable response element is TTCCCGGAA. A further suitable response element is that region of the promoter of a gene regulated by the leptin that is required for STAT interactions. This gene will depend on the particular therapeutic use of the compounds to be selected by the assay. A suitable reporter gene is firefly luciferase or chloramphenicol acetyltransferase enzyme. A suitable promoter is a minimal promoter such as the herpes simplex virus thymidine kinase or SV40 promoter. A brief description of the figures follows: Figure 1 : Expression of the leptin receptor (OB-R) mRNA isoforms in tissues from the gastrointestinal tract and in human CACO-2 cells. A. RT-PCR detection, as described in experimental procedures, of the multiple OB-R isoforms in discrete regions of the tract in lean (+/+) and obese (ob/ob) mice; OB- Re (1), OB-Rd (2), OB-Rc (3), OB-Rb (4), OB-Ra (5) and finally a lOObp DNA ladder. B. In the left panel leptin specific primers were used to rule out contamination from fat and detection of illegitimate transcription. As a positive control, a common sequence from the extracellular domain of OB-R was amplified from the same samples. The right panel shows RT-PCR detection of the functional leptin receptor OB-Rb in the human CACO-2 cell line. Figure 2: Western blot analysis of the OB-R receptor in the gastrointestinal tract and CACO-2 cells. A. OB-Ra detected in different sections of the tract of both +/+ and ob/ob mice using a antibody raised against the carboxy terminal of the short isoform. B. OB-R detected in the human CACO-2 cells using antibody raised against the amino terminus that is common to all OB-R isoforms. Arrows point at an approximate size of a 120kDa band. Figure 3: Relative expression levels of the total OB-R transcript in different sections of the gastrointestinal tract. Top panel shows representative gels of PCR amplification of the common OB-R extracellular domain in various sections of the gastrointestinal tract of the +/+ mouse, using different amounts of cDNA as described previously (18). The amount of cDNA in each sample was related to β-actin levels and adjusted before dilutions were performed. Lower panel shows the quantitation of OB-R mRNA normalised to the β-actin mRNA, from the optimised linear regions of PCR, in +/+ and ob/ob mice. Figure 4: Expression of leptin responsive STAT proteins in mouse gut and human CACO-2 cells. A. Western blot analysis of STAT1, 3 and 5 in 25 μg protein lysates of jejunum from +/+ and ob/ob mice. All three STATs are readily detected in crude protein extracts. Comparison of STAT5 expression in discrete sections in and +/+ and ob/ob mice. B. Western blot analysis of STAT 1, 3 and 5 in CACO-2 cells. Positive controls are, human fibroblast, A431 and human endothelial cell lysates for STAT1, 3 and 5, respectively. STATs 2 and 6 were also detected (data not shown). Figure 5: Gel mobility shift analysis of STAT DNA binding in vivo in response to leptin. An induction of STAT5 DNA binding activity to the β-casein promoter STAT5 consensus element. Nuclear extracts were prepared and incubated with 32 P end-labelled probe and fractionated on a 4% native PAGE gel. Induction observed in +/+ and ob/ob jejunal nuclear extracts in response to a leptin administration (5mg/kg) after 30 minutes. Preincubation with anti-STAT5 antibodies reveal the major complex consist of STAT5a. No induction is observed in nuclear extracts of jejunum from db/db mice or control +/+ and ob/ob mice. Figure 6 Gel mobility shift analysis of STAT DNA binding in CACO-2 cells in response to leptin. An induction of STAT3 and STAT5 DNA binding activity in response to leptin (20-200nM, 15 minutes) is shown. A dose response relationship is observed in CACO-2 nuclear extracts after leptin treatment in STAT3 activation as assessed by the STAT 1/3 selective high affinity m67-SIE consensus element from the c-fos promoter. The complex is identified by preincubation with a anti-STAT3 and anti-STATl antibodies. STAT5 DNA binding to the β-casein probe is also observed in nuclear extracts of CACO-2 cells after leptin treatment. The major protein complex was identified by preincubation with anti-STAT5b specific antibody. Figure 7 Leptin induction of immediate-early gene transcription in mouse jejunum and in the human CACO-2 cells. A. Northern blot of total RNA from ob/ob jejunum from Tris control and intravenous leptin (5mg/kg) treated animals. 5-fold inductions in c-fos mRNA levels are apparent 30 minutes after leptin administration. B. Quantitative PCR was performed as described (18) on total RNA from CACO- 2 cell monolayers treated with leptin (200nM) for 30 minutes. Serial dilution of cDNA from control and leptin treated cell was used to obtain optimised linear region of PCR. Figure 8 Leptin administration reduces APO-AIV mRNA levels after high fat load. Leptin (5mg/kg) treatment results in a significant 2-fold reduction in APO-AIV mRNA levels in jejunum of ob/ob mice. Mice were fasted for 5 hours and given leptin intravenously 15 minutes before administration of fat load (0.75ml vegetable oil). 90 minutes later mice were sacrificed and the jejunum removed for RNA extraction. Expression of APO-AIV mRNA was normalised to 28S rRNA and results expressed as mean±SEM (n=3 and *p<0.05). The methodology by which the CaCo-2 cell line was identified as a leptin responsive cell line is described below. General methodology for identification of leptin responsive cell lines is known and for example includes the following :- 1. Microphysiometer: This method detects small changes in pH resulting from biochemical changes in the cell. Ob-protein responsive cells upon stimulation may undergo biochemical changes that cause a small change in the extracellular acidification rate which can be detected by a silicon microphysiometer. The microphysiometer biosensor methodology has been reviewed by McConnell, Science, 1992, 257, 1906. 2. Electrophoretic mobility shift assay (EMS A): Nuclear extracts from cells after treatment with ob-protein are mixed with radiolabeled oligonucleotides containing a promiscuous or specific STAT response element DNA sequence. Extracts from cells that respond to the ob-protein may cause a gel shift of the oligonucleotide for the STAT response element. References: Book "Recombinant DNA", 2nd Edition, Watson et al. , 1992, Page 158; Lamb et al., Blood, 1994, 83, 2063; 3. Measurement of protein phosphorylation assay: The coupling of receptor activation to the final response through tyrosine phosphorylation of intracellular proteins may be assayed by the use of antibodies recognising phosphorylated tyrosines. More specifically since the leptin receptor may stimulate tyrosine phosphorylation of the JAK STAT pathway this method provides a method of detecting leptin response cell lines. Specific JAK/ STAT antibodies may be used alongside antibodies for tyrosine phosphorylation to detect leptin activation in a leptin responsive cell line. Inhibition as well as stimulation of protein phosphorylation may occur. In particular, inhibition by the ob-protein of insulin stimulated phosphorylation of the insulin receptor and insulin receptor substrate- 1 has been shown in rat-1 fibroblasts over expressing insulin receptors (Kroder et. al 1996, Exp. Clin. Endocrinol. Diabetes, 104, suppl 2, p66) 4. Displacement binding: After incubation of cell lines with radiolabelled leptin, for example [l2 τ]_ι e p tmj me non-specific binding versus specific binding of leptin can studied by the addition of unlabelled leptin. A high specific to non-specific ratio binding suggests that the cell line may contain the leptin receptor. 5. Detection of the protein for a functional form, preferably a functional long form, of the ob-receptor by use of selective antibodies. 6. Detection of mRNA for a functional form, preferably a functional long form, of the ob-receptor by Northern, RT-PCR or "slot blot" analysis. 7. Detection of increased c-fos mRNA after treatment with leptin. C-fos mRNA may be detected by Northern, RT-PCR or "slot blot" analysis. The response element, the reporter, and preferably the promoter, are suitably incorporated into a vector capable of transfecting the ob-responsive cell line. Suitable vectors are commercially available vectors, such as pGL2-basic luciferase vector (Promega). A suitable configuration of the vector is the STAT DNA response element upstream of a promoter and a reporter gene. A more suitable configuration of the vector is the STAT DNA response element in multiple tandem repeats (2-10) upstream of a thymidine kinase promoter and a luciferase reporter gene Vectors are constructed containing a reporter gene for example firefly luciferase or chloramphenicol acetyltransferase enzyme linked to a minimal promoter for example the herpes simplex virus thymidine kinase or SV40 promoter. The DNA fragments for the STAT response element are inserted into the vector using appropriate restriction enzyme sites upstream of the minimal promoter. The response element, the reporter and the promoter, as required, are incorporated into the vector using conventional expression techniques, for example the DNA fragments for the response element may be inserted into the vector using appropriate restriction enzyme sites upstream of the minimal promoter. STAT response element-luciferase enzyme reporter systems can be constructed as described by Lamb et al., Blood, 1994, 8, 2063 and Seidel et al., Proc. Nat. Acad. Sci. USA., 1995, 92, 3041. Leptin-responsive cells are transfected with the STAT response element-minimal promoter-luciferase reporter constructs using standard methodology for example the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, the cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a period of transfection (12- 24 hours) the cells are treated with varying concentrations of compound and then harvested and lysed. The lysates are assayed for luciferase, and if appropriate β- galactosidase, activity. Potentiation or antagonist activity can be assayed by pre- or co- addition of an appropriate concentration of leptin to the compound under evaluation and measuring the potentiation or reduction in luciferase response relative to that of leptin alone. Standard methods exist for assaying luciferase enzyme activity for example Ow et al, Science, 1986, 234, 856 and de Wet et al., Mol. Cell Biol, 1987, 7, 725. as well as several commercial kits. Stable cell lines can be generated by transfecting an leptin-responsive cell line with the reporter construct and a selectable marker. Selectable markers are routinely used to generate stable cell lines as described in Recombinant DNA, 2nd edition, J.D. Watson et. al., 1992, page 216. These stably transfected cell lines can be used to generate high throughput assays for compounds that mimic, potentiate or block the physiological effects of the leptin. The invention also extends to a compound that mimics, potentiates or inhibits the physiological effect of the leptin, when identified by the method disclosed herein. The invention also extends to a kit of parts adapted for use in the method disclosed herein. The invention also extends to the use of native or engineered CACO-2 cells to detect compounds that mimic, potentiate or inhibit the physiological effects of leptin by measuring other responses that leptin elicits in such cells. The invention also extends to the use of native or engineered CACO-2 cells to measure the functional activity of preparations of leptin mimetics , potentiators or inhibitors. When used herein 'a compound which mimics the physiological effects of leptin' refers to a compound which is capable of acting in the absence of the leptin to either stimulate leptin receptor to provide substantially the same physiological effect as the ob protein or to activate a response down stream of this receptor (post-receptor). When used herein 'a compound that potentiates the physiological effect of leptin' refers to a compound which enhances the potency and/or maximal physiological effect of leptin. When used herein 'a compound that inhibits the physiological effect of the leptin' refers to a compound which 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, (eg a viral pro mo tor) or a regulatable promoter to optimise the expression of the polypeptide for the identification of agonists or antagonists as necessary. Alternatively, the response element and the reporter are expressed in a cell line, wherein a constitutive or regulatable promoter has been engineered into a position upstream of the chromosomally encoded gene for the leptin receptor by the method of homologous recombination. Such methods are reviewed by Waldman, Critical Reviews in Oncology/Hematology, 1992, 12, 49 and a particular example is given in the Riele et al, Proceedings of the National Academy of Sciences, 1992, 89, 5128. The following examples illustrate the invention but do not limit it in any way. Example General Procedure: The CaCo-2 cells are transfected with a reporter plasmid containing a STAT response element, in multiple tandem copies upstream of a minimal promoter for example herpes simplex thymidine kinase and a luciferase gene reporter construct using standard methodology for example the calcium phosphate method (Graham and Van Der Eb, Virology, 1973, 52, 456). To correct for differences in transfection efficiency, the cells can be co-transfected with a reference plasmid expressing β-galactosidase activity. After a period of transfection (12-24 hours) the cells are treated with varying concentrations of compound and then harvested and lysed. The lysates are assayed for luciferase, and if appropriate β-galactosidase, activity. Antagonist activity can be assayed by pre- or co- addition of an appropriate concentration of leptin to the compound under evaluation and measuring the reduction in luciferase response relative to that of leptin alone. Standard methods exist for assaying luciferase enzyme activity for example Ow et al., Science, 1986, 234, 856 and de Wet et al., 1987, 7, 725. as well as several commercial kits. EXPERIMENTAL SECTION SUMMARY The adipocyte hormone, leptin, activates STAT3 in the hypothalamus mediating increased satiety and increased energy expenditure. To date, leptin mediated activation of the STAT pathway in vivo has not been established in tissues other than hypothalamus. We now describe leptin receptor expression and in vivo signalling in discrete regions of the mouse gut associated with lipid handling. Expression of the functional isoform, OB- Rb, is restricted to the jejunum. Intravenous injection of leptin rapidly induced nuclear STAT5 DNA binding activity in jejunum of +/+ and obese (ob/ob) mice, but had no effect in the diabetic (db/db) mouse that lacks the OB-Rb isoform. In addition, an induction of the immediate-early gene c-fos is observed in vivo. Leptin-mediated induction of a number of immediate-early genes and activation of STAT3 and STAT5 in a human model of small intestine epithelium CACO-2 cells corroborate this effect. Furthermore, intravenous leptin administration caused a significant 2-fold reduction in the APO-AIV transcript levels in jejunum 90 minutes after a fat load. Our results suggest that jejunum is a direct target of leptin action and this activity is dependent on the presence of OB-Rb. Lack of leptin or resistance to leptin action in this site may contribute to obesity and its related syndromes by directly affecting lipid handling. The abbreviations used herein are: NIDDM, non-insulin dependent diabetes; OB-R, Leptin-receptor; RT-PCR, reverse transcription polymerase chain reaction; JAK, janus kinase; STAT, signal transducers and activators of transcription; CACO-2, Human colon epithelial cell line; APO-AIV, apolipoprotein AIV. EMEM, Earles minimal essential medium. EXPERIMENTAL PROCEDURES Animals and cells-Six week old female wild-type and ob/ob mice of the Aston strain were bred in house and were kept on a 12h light: 12h dark cycle with routine access to water and standard laboratory chow (Beekay rat and mouse toxicology diet, Bantin and Kingman, Hull, UK). The in vivo effects of leptin (5mg/kg) were assessed using recombinant leptin (Peprotech, Rockyville, USA) dissolved in lOmM Tris administered by tail vein injection or lOmM Tris vehicle to controls. After the indicated times, mice were sacrificed by cervical dislocation and tissues dissected, cleaned and washed in ice- cold saline. Tissues were taken fresh for extraction of nuclear proteins as described below or snap-frozen in liquid N 2 and stored at -80°C. Human Caucasian colon epithelial cells, CACO-2 (European animal cell culture, Porton Down, UK), were received at confluency and cultivated in Earles minimal essential medium (EMEM) including 1% non essential amino acids (NEAA), 2mM L-glutamine, 10% foetal calf serum, lOOu/ml penicillin, lOOμg/ml streptomycin (all from Gibco/BRL, UK) at 37°C in humidified 5% CO 2 / air cabinet, for 3-4 days before experiments. Cells were pre-incubated for 24 hours in serum-free supplemented EMEM with 0.1% BSA. Cells were then incubated in basal EMEM with 0.1% BSA with or without leptin at given concentrations or the Tris control vehicle. Leptin receptor mRNA expression-Total RNA was isolated from tissues and cells using RNaid plus kit (BIO 101, Inc. USA) and treated immediately with DNase I (Gibco/BRL, UK). Approximately RNA (4μg) was used to generate cDNA with first-strand cDNA beads (Pharmacia, Biotech) and oligo (dT) 12 . 18 (Invitrogen). The integrity and loading of the RNA was studied by detection scanning of ribosomal rRNA bands (28S and 18S) in agarose gels. Potential contamination from fat was studied using leptin specific primers (Table I) in an RT-PCR amplification. RT-PCR detection of the multiple OB-R transcript isoforms in the mouse gut as well as the OB-Rb transcript in the CACO-2 cells, was performed using oligonucleotide primers listed in Table I. All PCR amplifications were 2 performed using AmpiiTaq (PerkinElmer) at 95°C, 45sec denaturation, 57°C, 45 sec annealing, 72°C, 45 sec extension (except that for the long OB-Rb isoform extension was for 1 minute) in a Progene thermal cycler (Techne/Cambridge). PCR products were then cloned directly into a pCR-TRAP cloning system (GeneHunter Corp., USA) and the identity of PCR products confirmed by sequencing using ThermoSequenase terminator cycle sequencing kit (Amersham Life Sciences, UK). For quantitative PCR, cDNA samples from +/+ and ob/ob mice were serially diluted and PCR performed at a fixed number of cycles (37 for OB-R and 30 for β-actin), as described previously (18). Immiinoblot analysis of tissues and cells-Fresh, tissues were homogenised in ice-cold RIPA buffer (IX PBS, 1% IGEPAL, 0.5% sodiumdeoxycholate) containing protease inhibitor cocktail (Boehringer Mannheim, Germany) and the protein lysates used for a protein assay (Sigma Diagnostics, UK) and standard western analysis. Approximately 20 μg protein was mixed with gel loading buffer (4% SDS, lOOmM Tris, pH 6.8, lOOmM β- mercaptoethanol) and resolved on 10% SDS-PAGE. For monolayers of CACO-2 cells, protein was extracted by addition of gel loading buffer to the flasks. Proteins were transferred to PVDF (Millipore, UK) membranes by electrotransfer and immunoblots performed by blocking in 2% BSA, lOmM Tris pH 7.4, lOOmM NaCl, 0.1% Tween 20 and the OB-R was detected with M-18 or N-20 antibodies (Santa Cruz Biothechnology, Inc., USA), STAT proteins were detected with STAT5 (C-17) (Santa Cruz Bio hechnology, Inc., USA) and STATl,-2,-3, and -6 (Transduction Laboratories, USA) antibodies. Bands were visualised using an ECL kit according to instructions (Amersham, UK). Nuclear extracts-CACO-2 cells were immediately placed on ice after treatment and the medium removed. Cells were quickly washed with ice cold PBS and then PBS with 5mM NaF, lOmM NaMoO 4 and ImM activated NaVO 3 . Cells were lysed with a hypotonic buffer (400μl/25cm 2 ) containing lOmM HEPES, pH 7.9, lOmM NaMoO 4 , ImM NaVO 3 , ImM EDTA, ImM EGTA, ImM NaF, 0.125 μM Okadaic Acid, ImM DTT, 2μg.ml leupeptin, 2μg/ml aprotinin, 50μg/ml AEBSF and 0.2% IGEPAL CA-630 detergent (Sigma, UK) by cell scraping and gentle trituration. The lysate was incubated on ice for 5 minutes and the nuclei pelleted by microcentrifugation for 20 seconds at 14000rpm. The supernatant was discarded and the nuclei gently resuspended in lOOμl high salt buffer (hypotonic buffer plus 200mM NaCl, 20% glycerol). Nuclear protein was extracted by rotating the tubes for 30 minutes at 4°C and the debris pelleted by microcentrifugation at 14000rpm for 20 minutes. Nuclear extracts were aliquoted then snap-frozen in liquid N 2 before gel-mobility shift assay (see below). Tissues were dissected, washed in ice-cold saline, minced and then homogenised in 1 :10 w/v hypotonic lysis buffer and nuclear extracts obtained essentially as described for CACO-2 cells. Gel mobility shift assaysSμl nuclear extract was used for DNA binding studies with lOOng/reaction 32 Pγ-ATP end-labelled probe in DNA binding buffer (Tris lOmM, pH 7.5, 150mM NaCl, ImM DTT with lμg/ml polydeoxy Inosine/Cytidine (Pharmacia, Sweden) to block non-specific binding. The binding reaction was incubated at room temperature for 20 minutes and then resolved on a 4% native PAGE gel. Supershift experiments involved a further 15 minutes incubation with the appropriate antibody. The oligonucleotide probes were the mutated high affinity human Serum Inducible Element (m67 SIE), 5 '-cat ttc ccg taa ate at -3', from the c-fos promoter and a rat β-Casein promoter element sequence, 5'-gga ctt ctt gga art aag gga-3'. Complementary oligonucleotide strands (Research Genetics, USA) were annealed in Tris lOmM, pH 8.0, ImM EDTA, 50mM NaCl to a final concentration of 200ng/μl, by heating the mixture to 90°C for 10 minutes and then allowing it to cool slowly to room temperature. Probes were labelled with 3U/μl T4 Polynucleotide Kinase (Amersham, USB, UK) at 37°C for one hour with lμl 3 Pγ-ATP (6000Ci/mmol Amersham, UK). Expression of the APO-AIV and immediate-early gene mRNA species-Tissues and CACO-2 cells treated for 30 minutes with or without leptin were immediately snap-frozen in liquid N 2 and RNA extracted as described above. Standard northern blot hybridisation was performed using digoxygenin-labelled cDNA probes to determine c-fos and APO- AIV mRNA levels in vivo. Bound probes were detected by the anti-digoxygenin detection system using a polyclonal antibody conjugate to alkaline phosphatase (Boehringer Mannheim, Germany) with the chemiluminescent substrate CDP-Star (Tropix, USA), and finally bands were quantitated by scanning laser densitometry. Expression of the immediate-early genes c-fos, c-jun, junB and junD in response to leptin was established by quantitative PCR, using primers listed in Table I.. - H- RESULTS The multiple OB-R mRNA isoforms were detected using RT-PCR in discrete sections of the gastrointestinal tract (see Fig. 1 A). The different short isoforms, OB-Ra, OB-Rc, OB- Rd and OB-Re are expressed throughout the gut. In contrast, the functional long OB-Rb mRNA is found to be predominantly expressed in jejunum (Fig. 1A). Using leptin- specific primers, RT-PCR amplification of cDNA samples from jejunum of +/+ and ob/ob mice resulted in negative detection, whilst PCR amplification of a common extracellular domain of the OB-R in the same samples was readily obtained (Fig. IB). This suggests that the OB-R mRNA expression presented in Fig. 1 A is neither a result of contamination from fat tissues nor a result of illegitimate transcription. Expression of the functional leptin receptor OB-Rb mRNA was readily detected in the human model of small intestine epithelium CACO-2 by the use of RT-PCR (Fig. IB). Western blot analysis of 20μg crude protein lysates using antibodies raised against the carboxy terminal sequence of the short isoform OB-Ra, a predominant isoform in many tissues (18), results in a band with a predicted size of approximately 120 kDa protein, that is predominantly expressed in the small intestine of both +/+ and ob/ob mice (Fig. 2A). The discrepancy between the results obtained using RT-PCR or western analysis to detect OB- R expression, reflects the difference in detection limit of these two methods. Accordingly, the western analysis indicates a difference in the OB-R abundance in different sections of the gut (see Fig. 2A). Antibodies raised against the amino terminal of the common human OB-R receptor detected a -120 kDa protein in the CACO-2 cells which presumably accounts for the predominant short OB-R isoform (Fig. 2B). Relative expression levels of the total OB-R transcript normalised to endogenously expressed β-actin were then measured in discrete sections of the gastrointestinal tract using primers that recognise a sequence common to all isoforms encoding the amino terminal of the receptor, using the quantitative PCR assay described previously (22). Here the ratios of OB-R and β-actin PCR products from optimised linear regions of PCR condition, generated from equal volumes of cDNA were compared (Fig 3A). No differences in OB-R mRNA levels were detected between +/+ and ob/ob mouse tissues, however the OB-R transcript is 2-3 fold more abundant in small intestine than in stomach and transverse colon (Fig. 3B). These results agree well with the results observed using the western analysis of OB-Ra distribution as shown above (see Fig 2A). To address leptin-signalling mechanisms in the region of the small intestine expressing the OB-Rb, we looked for the presence of the STAT proteins known to transmit the leptin signal. Western blot analysis of +/+ and ob/ob jejunum readily detects STAT5, but more weakly STAT1 and STAT3 (Fig. 4A). Furthermore STAT5 is also well expressed in colon and duodenum (Fig. 4A). CACO-2 cells also express leptin responsive STAT isoforms (Fig 4B). Overnight fasted +/+ and ob/ob mice were administered 5mg/kg recombinant murine leptin by tail vein injection. 30 minutes after injection, mice were sacrificed and the jejunum removed for preparation of nuclear extracts. We used two selective probes to assess STAT activation by EMSA analysis. The m67-SIE mutated high affinity STAT consensus binding element from the c-fos promoter preferentially binds STAT1 and STAT3, but binds STAT5 with low affinity. This probe did not reveal an induction of STAT DNA binding activity in response to leptin treatment in vivo in the mouse gut (data not shown). The rat β-casein promoter STAT DNA binding consensus element was used as a selective probe for STAT5 activity in vivo. Here, 5mg/kg leptin treatment induced a marked activation of DNA binding activity in both +/+ and ob/ob mice and was not observed in control animals treated with Tris only (Fig.5A). In contrast, the same dose of leptin injected into the db/db mice, which lack the OB-Rb isoform, caused no activation of STAT5 DNA binding in nuclear extracts of jejunum (Fig. 5 A). The STAT5 DNA binding activity induced in the +/+ and ob/ob jejunum was abolished by a preincubation with specific anti-STAT5a antibody and less markedly with an anti-STAT5b antibody (see results from the ob/ob jejunum in Fig. 5B). These results suggest that jejunum is a direct target of leptin action, and this activity is mediated through OB-Rb and a STAT5 mechanism. A 15 minute leptin exposure resulted in a dose-dependent activation of STAT DNA binding to the m67-SIE probe in serum-deprived CACO-2 cells. This activity was abolished by an anti-STAT3 antibody whilst anti-STATl antibody had no effect (Fig. 6). In addition, leptin treated CACO-2 cells exhibited an activation of DNA binding activity of STAT5 to the β-casein probe, and supershift studies identified STAT5b as the major factor in the complex (Fig 6). Thus, leptin induces nuclear STAT5 DNA binding activity in both mouse jejunum and CACO-2 cells, however different isoforms of STAT5 seem to form the protein-DNA complex in these two systems. Leptin administration also produced an induction of the immediate early gene c-fos in the jejunum of ob/ob mice after 30 minutes (Fig. 7A). Intravenous injection of 5mg/kg leptin resulted in a 5-fold increase in c-fos mRNA levels as detected by northern blot analysis. Similarly, leptin treatment for 30 minutes caused a dramatic increase in expression of the immediate early genes, c-fos, c-jun, junB and junD in the serum-deprived CACO-2 cells as determined by quantitative PCR (Fig 7B). Since activation of STAT5 is usually not associated with c-fos induction, these results could indicate that other pathways are being activated in addition to the STAT5 pathway in vivo. We then investigated whether leptin could modulate transcription of genes involved in one of the major functions of jejunum, that is, lipid handling, by measuring the effect of leptin on the APO-AIV mRNA levels. Injection of 5mg/kg leptin into ob/ob mice fed on a high fat diet resulted in a significant 2-fold reduction in APO-AIV mRNA levels; APO- AIV mRNA/β-actin mRNA mean±SEM: control vehicle=2.23±0.16 and leptin treated=1.04±0.16 (p<0.05, n=3) 90 min after high fat load (Fig 8). These results, suggest that leptin could act as an immediate brake on fat accumulation by reducing transport of dietary triglycerides into plasma. DISCUSSION The adipocyte hormone, leptin, signals to the hypothalamus to inhibit food intake and increase energy expenditure (1). Leptin has also been reported to play a role in peripheral tissue biology (8, 20, 23, 24). Leptin treatment of +/+ and ob/ob mice cause a reduction in body weight and fat mass in excess of that resulting from inhibition of food intake only (25). This would suggest that leptin can affect fat accumulation, metabolism and energy homeostasis that is in part independent of the regulation of food intake. We now demonstrate the presence of the full-length leptin receptor OB-Rb, in a distinct region of small intestine associated with lipid uptake. Administration of leptin caused activation of the STATS signalling pathway in jejunum after 30 minutes in +/+ and ob/ob mice, but not in db/db mice which lack OB-Rb. Leptin also caused a reduction in the APO-AIV mRNA 90 minutes after administration of high fat load. The immediate and direct effect of leptin on jejunal lipid handling could represent a front-line mechanism against fat accumulation. The short OB-R isoforms are widely expressed in tissues whilst the long OB-Rb isoform is more restricted in tissue distribution (7). OB-Rb is relatively well expressed in hypothalamus, particularly in regions associated with the regulation of body weight homeostasis (26). However, we and others have identified expression of OB-Rb in peripheral tissues such as pancreatic islets, lymph nodes and hematopoietic stem cells (7,8,24). Current views hold, that only the OB-Rb receptor isoform can activate the JAK/STAT cascade to mediate the biological effects of leptin. This is based on a number of in vivo and in vitro experiments showing that OB-Rb is required to affect food intake, insulin secretion or stimulate cell proliferation (8,16,19). In these studies the short receptor isoforms, which predominate in most tissues, are unable to mediate such a response to leptin, and no biological function has been assigned to these isoforms to date. It has been suggested that the shorter OB-R isoforms can modify the activity of OB-Rb during homodimerization and subsequent aggregation of the cytosolic domain (7). This effect of the shorter isoforms seem weak, however, according to the recent data of White et al. (27) who demonstrate that the signalling capacity of OB-Rb is quite resistant to repression by the shorter receptor isoforms. In the present study, we demonstrate that the multiple short isoforms are expressed throughout the gastrointestinal tract whilst the functional OB-Rb is predominantly expressed in the jejunum and more weakly in ileum, the two major sites that are involved in lipid handling. Furthermore, both western blot and quantitative-PCR analysis show that OB-R is more abundant in the small intestine than in either stomach or colon. In fact a recent study on the effects of leptin on gastric emptying imply that leptin does not control the delivery rate of the chyme to the small intestine (27). This is in agreement with the lack of OB-Rb expression found in this site in current study. The results on OB-R expression suggested to us that small intestine, particularly jejunum, is an important target of leptin action. Previous studies in vivo have shown that intravenous injection of leptin induced a STAT3 DNA binding activity in ob/ob but not db/db mouse hypothalamus (19). Nuclear extracts from hypothalamus and several peripheral tissues failed to shift the STAT5-specific β- casein promoter element. We show that intravenous injection of leptin induced a STAT5 DNA binding activity in jejunum of +/+ and ob/ob mice, but had no effect in the db/db mouse. Here, we chose a sub-maximal leptin concentration (5mg/kg) and an optimal time frame of 30 min based on previous in vivo reports on STAT activation (19). We do therefore not rule out activation of other leptin mechanisms that do not appreciably overlap this time-frame in jejunum. The 5mg/kg dose is somewhat higher than the physiological concentration of leptin, however the activity of recombinant leptin from different sources varies significantly. Hence literature reports show that doses of leptin between O. lmg/kg and 5mg/kg cause a 50% reduction in food intake. This suggests that the recombinant leptin may be considerably less potent than endogenous leptin The rapid activation of STAT5 DNA binding and immediate-early gene transcription is consistent with a direct effect of leptin on the gut. Our in vivo results show that the effect of leptin in jejunum requires the presence of the long OB-Rb isoform. In addition, STAT5 activation and immediate-early gene induction in response to leptin in the human model of small intestinal epithelium, the CACO-2 cell, may be interpreted as corroboration of a direct effect of leptin on enterocytes. That the associated activation of STAT3 and STAT5 in this cell line appears broader than the primary gut tissue is also a recognised phenomenon of in vitro systems (7). The requirement for depriving cells of serum before and during leptin treatment and the use of high concentrations of leptin for the demonstrated activation of the STAT DNA binding in CACO-2 cells has been observed previously in other cell-lines (29). The importance of STAT5 in mediating the effect of leptin on body weight homeostasis is further supported by in vitro evidence from the obese (fa/fa) Zucker rat, which contain a missense mutation (Gln->Pro) in the extracellular domain of the OB-R (30). OB-R(fa) mediates a leptin-independent (constitutive) activation of STAT1 and STAT3, whilst the activation of STAT5 is completely abolished (31). These animal models, together with the leptin deficient (ob/ob) and OB-Rb deficient (db/db) mice, all develop early onset obesity and we believe that a lack of leptin effect in small intestine of these animal models could in part contribute to the obesity phenotype. The small intestine forms the primary interface between ingested nutrient and the carefully regulated internal environment of an organism. During conditions of obesity, endogenous intestinal triglyceride production is increased (32), contributing to elevated plasma triglyceride levels. Our results show for the first time that leptin can cause a rapid activation of STAT5 in jejunum which is associated with a reduction of the APO-AIV transcript levels 90 minutes after ingestion of a fatty meal. This suggests that leptin may play a physiological role in lipid handling at this site in vivo. The APO-AIV system serves as a conduit for transport of triglycerides as chylomicrons in the circulation and their transfer to acceptor membranes in various tissues. De-regulation of this function could be a part of the mechanism that leads to obesity and elevated levels of lipoproteins. Furthermore, hypertriglyceridaemia and hyperlipoproteinaemia are risk factors for cardiovascular disease and atherosclerosis (33). In physiological conditions, however, postprandial rises in plasma leptin (34), by reducing APO-AIV could function as a buffer system to reduce the chylomicron triglyceride levels. It is also possible that leptin may serve as a tonic inhibitory mechanism on the APO-AIV system to reduce the levels of secreted triglycerides. It is also known that leptin can induce enzymes of fatty acid oxidation (35) and thus promote a switch in fuel metabolism to β-oxidation of fatty acids (36). 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Claims: Claims: 1. A method for the detection of a compound that mimics, potentiates or inhibits the physiological effect of leptin, which method comprises: (a) for a compound which mimics the physiological effect of the leptin, assessing the effect of the compound upon a leptin activated signal transducer and activator of transcription (STAT) DNA response element coupled to a reporter gene; or (b) for a compound which potentiates or inhibits the physiological effect of leptin, assessing the effect of the compound upon the response provided by leptin upon a leptin activated STAT DNA response element coupled to a reporter gene; wherein, the response element and the reporter are expressed in the human Caucasian colon epithelial cell line (CACO-2) or the response element and the reporter are expressed in CACO-2 cells, which cell line (the engineered cell line) is also transfected with a polypeptide which is capable of mediating or enhancing the stimulation by leptin of an leptin activated STAT DNA response element and contains the appropriate STAT proteins. 2. A method according to claim 1, wherein the response element is coupled to a promoter gene, preferably a minimal promoter. 3. A method according to claim 2, wherein the response element is a nucleotide of formula TT(N) n AA, where N is any nucleotide and n is 4, 5 or 6, preferably 5. 4. A method according to claim 2, wherein the response element is a nucleotide of formula TTCCCGGAA. 5. A method according to claim 1 , wherein the reporter gene is firefly luciferase or chloramphenicol acetyltransferase enzyme. 6. A method according to claim 1 , wherein the promoter is the herpes simplex virus thymidine kinase or SV40 promoter. 7. A method according to claim 1, wherein the ob-responsive cell line is a liver or liver hepatoma derived cell line. 8. A method according to claim 1, wherein the response element, the reporter, and the promoter, are incorporated into a vector capable of transfecting the ob-responsive cell line. 9. A method according to claim 8, wherein the vectors pGL2-basic luciferase vector (Promega). 10. A method according to claim 8 or claim 9, wherein the configuration of the vector is such that the STAT DNA response element is upstream of the promoter and reporter gene. 11. A kit of parts adapted for use in the method for the detection of a compound that mimics, potentiates or inhibits the physiological effect of leptin, which method comprises: (a) for a compound which mimics the physiological effect of the leptin, assessing the effect of the compound upon a leptin activated signal transducer and activator of transcription (STAT) DNA response element coupled to a reporter gene; or (b) for a compound which potentiates or inhibits the physiological effect of leptin, assessing the effect of the compound upon the response provided by leptin upon a leptin activated STAT DNA response element coupled to a reporter gene; wherein, the response element and the reporter are expressed in the human Caucasian colon epithelial cell line (CACO-2) or the response element and the reporter are expressed in CACO-2 cells, which cell line (the engineered cell line) is also transfected with a polypeptide which is capable of mediating or enhancing the stimulation by leptin of an leptin activated STAT DNA response element and contains the appropriate STAT proteins.

Inventors: CAWTHORNE MICHAEL ANTHONY, EMILSSON VALUR, MORTON NICHOLAS MICHAEL, CAWTHORNE, MICHAEL, ANTHONY, EMILSSON, VALUR, MORTON, NICHOLAS, MICHAEL
Applicants: CAWTHORNE MICHAEL ANTHONY, EMILSSON VALUR, MORTON NICHOLAS MICHAEL, UNIV BUCKINGHAM, CAWTHORNE, MICHAEL, ANTHONY, EMILSSON, VALUR, MORTON, NICHOLAS, MICHAEL, THE UNIVERSITY OF BUCKINGHAM
Application year: 1999
Main classification: C12Q1
IPC codes: C12Q1