Secretin Receptor Agonists to Treat Diseases or Disorders of Energy Homeostasis

Abstract

The present invention relates to a secretin receptor modulator for use in the prevention and/or treatment of a disease or disorder of energy homeostasis, wherein (a) said secretin receptor modulator is a secretin receptor agonist and said disease or disorder is obesity, dyslipidemia, diabetes, insulin resistance, hyperglycemia, high blood pressure or metabolic syndrome, whereby the secretin receptor agonist increases non-shivering thermogenesis in brown adipocytes and/or increases the expression of uncoupling protein 1 (UCP1) in brown adipocytes and/or decreases food intake in a UCP1-dependent manner resulting in the prevention and/or treatment of said disease or disorder; or (b) said secretin receptor modulator is a secretin receptor antagonist and said disease or disorder is cachexia. The invention further relates to a method of increasing non-shivering thermogenesis in brown adipocytes and/or increasing the expression of uncoupling protein 1 (UCP1) in brown adipocytes, to a method of decreasing non-shivering thermogenesis in brown adipocytes, to a method of identifying a secretin receptor agonist capable of increasing non-shivering thermogenesis in brown adipocytes and/or increasing the expression of uncoupling protein 1 (UCP1) in brown adipocytes, to a method of identifying a secretin receptor antagonist capable of decreasing non-shivering thermogenesis in brown adipocytes, to the use of the secretin receptor for screening (a) for secretin receptor agonists that increase non-shivering thermogenesis in brown adipocytes and/or increase the expression of uncoupling protein 1 (UCP1) in brown adipocytes and/or decrease food intake in a UCP1-dependent manner; and/or (b) for secretin receptor antagonists that decrease non-shivering thermogenesis in brown adipocytes, and to the use of a secretin receptor agonist to activate non-shivering thermogenesis in brown adipocytes and/or to increase the expression of uncoupling protein 1 (UCP1) in brown adipocytes and/or to decrease food intake in a UCP1-dependent manner for reducing body weight for cosmetic purposes as well as to the use of a secretin receptor antagonist to decrease thermogenesis in brown adipocytes for increasing body weight for cosmetic purposes.

Claims

1. A modulator of the secretin receptor for use in a method for the prevention and/or treatment of a disease or disorder of energy homeostasis, wherein (a) said modulator is a secretin receptor agonist and said disease or disorder is obesity, dyslipidemia, diabetes, insulin resistance, hyperglycemia, high blood pressure or metabolic syndrome, whereby the secretin receptor agonist increases non-shivering thermogenesis in brown adipocytes and/or increases the expression of uncoupling protein 1 (UCP1) in brown adipocytes and/or decreases food intake in a UCP1-dependent manner resulting in the prevention and/or treatment of said disease or disorder; or (b) said modulator is a secretin receptor antagonist and said disease or disorder is cachexia.

2. The modulator of the secretin receptor for use according to claim 1, wherein the modulator of the secretin receptor is comprised in a pharmaceutical composition.

3. The modulator of the secretin receptor for use according to claim 1 or 2, wherein said modulator of the secretin receptor is to be co-administered with at least one other pharmaceutically active agent.

4. The modulator of the secretin receptor for use according to claim 3, wherein in the case of the modulator being a secretin receptor agonist said at least one other pharmaceutically active agent is selected from the group consisting of direct or indirect sympathomimetics, atrial natriuretic peptide and ANP/BNP receptor agonists.

5. A method of increasing non-shivering thermogenesis in brown adipocytes and/or increasing the expression of uncoupling protein 1 (UCP1) in brown adipocytes, comprising the step of contacting brown adipocytes with a secretin receptor agonist, thereby increasing non-shivering thermogenesis in brown adipocytes and/or increasing the expression of uncoupling protein 1 (UCP1) in brown adipocytes.

6. A method of decreasing non-shivering thermogenesis in brown adipocytes comprising the step of contacting brown adipocytes with a secretin receptor antagonist, thereby decreasing non-shivering thermogenesis in brown adipocytes.

7. A method of identifying a secretin receptor agonist capable of increasing non-shivering thermogenesis in brown adipocytes and/or increasing the expression of uncoupling protein 1 (UCP1) in brown adipocytes, comprising the steps of: (a) determining the level of non-shivering thermogenesis in brown adipocytes and/or the expression level of uncoupling protein 1 (UCP1) in brown adipocytes or obtaining established standard values of the level of non-shivering thermogenesis and/or the of the expression of uncoupling protein 1 (UCP1); (b) contacting said adipocytes of step (a) or other brown adipocytes with a test compound; (c) determining the level of non-shivering thermogenesis in said brown adipocytes of step (b) and/or the expression level of uncoupling protein 1 (UCP1) in said brown adipocytes of step (b) after contacting with the test compound; and (d) comparing the level of non-shivering thermogenesis in said brown adipocytes and/or the expression level of uncoupling protein 1 (UCP1) in said brown adipocytes determined in step (c) with the level of non-shivering thermogenesis in said brown adipocytes and/or the expression level of uncoupling protein 1 (UCP1) in said brown adipocytes determined in step (a) or to standard values of the level of the level of non-shivering thermogenesis and/or the of the expression of uncoupling protein 1 (UCP1) obtained in step (a), wherein an increase in the level of non-shivering thermogenesis in said brown adipocytes determined in step (c) as compared to said level determined or obtained in step (a) and/or an increase in the expression level of uncoupling protein 1 (UCP1) in said brown adipocytes determined in step (c) as compared to said level determined or obtained in step (a) indicates that said test compound is a secretin receptor agonist capable of increasing non-shivering thermogenesis in brown adipocytes and/or increasing the expression of uncoupling protein 1 (UCP1) in brown adipocytes.

8. A method of identifying a secretin receptor antagonist capable of decreasing non-shivering thermogenesis in brown adipocytes, comprising the steps of: (a) determining the level of non-shivering thermogenesis in brown adipocytes; (b) contacting said adipocytes of step (a) or other brown adipocytes with a test compound; (c) determining the level of non-shivering thermogenesis in said brown adipocytes of step (b) after contacting with the test compound; and (d) comparing the level of non-shivering thermogenesis in said brown adipocytes determined in step (c) with the level of non-shivering thermogenesis in said brown adipocytes determined in step (a), wherein a decrease in the level of non-shivering thermogenesis in said brown adipocytes determined in step (c) as compared to said level determined in step (a) indicates that said test compound is a secretin receptor antagonist capable of decreasing non-shivering thermogenesis in brown adipocytes.

9. The method of claim 7 or 8, further comprising a step of determining prior to, simultaneously with or after any one of the preceding steps (a) to (d) whether said test compound modulates the secretin receptor.

10. Use of the secretin receptor for screening (a) for secretin receptor agonists that increase non-shivering thermogenesis in brown adipocytes and/or increase the expression of uncoupling protein 1 (UCP1) in brown adipocytes and/or decrease food intake in a UCP1-dependent manner; and/or (b) for secretin receptor antagonists that decrease non-shivering thermogenesis in brown adipocytes.

11. Use of the secretin receptor agonist to increase non-shivering thermogenesis in brown adipocytes and/or to increase the expression of uncoupling protein 1 (UCP1) in brown adipocytes and/or to decrease food intake in a UCP1-dependent manner for reducing body weight for cosmetic purposes.

12. Use of the secretin receptor antagonist to decrease thermogenesis in brown adipocytes for increasing body weight for cosmetic purposes.

13. The modulator of the secretin receptor for use according to any one of claims 1 to 4, the method of claim 5, or the use of claim 10 or 11, wherein the secretin receptor agonist is selected from the group consisting of a small molecule, an antibody or a fragment or derivative thereof, an antibody mimetic, an aptamer, an siRNA, an shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, secretin or a fragment or derivative thereof, a secretin analogue, or a stimulant of secretin release.

14. The modulator of the secretin receptor for use according to any one of claims 1 to 4, or the method of claim 7, or the use of claim 10 or 12, wherein the secretin receptor antagonist is selected from the group consisting of an antibody or a fragment or derivative thereof, an antibody mimetic, an aptamer, an siRNA, an shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, or a small molecule.

Description

[0150] The figures show:

[0151] FIG. 1: Secretin receptor expression in mouse adipose tissues (A) and in differentiated primary adipocytes (B).

[0152] A: Relative expression of secretin receptor (Sctr) in mouse brown adipose tissue (BAT), subcutaneous inguinal (iWAT) and intra abdominal epidydimal (eWAT) white adipose tissue depots normalized to transcription factor TFIIB for N=5. Statistics were conducted using one-way ANOVA and since normality test (Shapiro-Wilk) failed (P<0.050) a Kruskal-Wallis one-way analysis of variance on ranks was performed (p=0.021). The * indicates p<0.05.

[0153] B: Expression of secretin receptor in primary adipocytes was also normalized by TFIIB-expression. Data are presented as mean of technical triplicates of N=1.

[0154] FIG. 2: Secretin induced oxygen consumption rates (OCR) in brown adipocytes.

[0155] Cell respiration measurements were conducted on the seventh day of differentiation.

[0156] A: One representative measurement is shown in technical replicates of 6-7; Dots represent meansSD. Microplate wells with differentiated brown adipocytes were injected subsequently with oligomycin (Oligo), secretin/isoproterenol (Sct/Iso), FCCP and antimycin A (Anti A).

[0157] B: Percentage fold increase above basal respiration (p=0.712).

[0158] C: Percentage attainment of maximal uncoupled FCCP respiration (p=0.946). In B and C values of three individual measurements with technical replicates of 6-8 per group were used and are represented as dots. Significance was verified by t-test.

[0159] FIG. 3: Secretin induced oxygen consumption in brown adipocytes of UCP1 knockout mice.

[0160] Measurement of oxygen consumption rates (OCR) was conducted at the seventh day of differentiation. Microplate wells with differentiated brown adipocytes were injected with oligomycin (Oligo), secretin/isoproterenol (Sct/Iso), FCCP and antimycin A (Anti A). Dots represent meansSD of 6-16 technical replicates.

[0161] A: treatment with secretin (Sct)

[0162] B: treatment with isoproterenol (Iso).

[0163] FIG. 4: Secretin induced UCP1 expression in primary adipocytes.

[0164] Differentiated primary adipocytes from inguinal white adipose tissue (A) or brown adipose tissue (B) were stimulated with 0.1 M secretin or 0.5 M isoproterenol for 6 hours. Expression of UCP1 mRNA was measured by qRT-PCR and normalized to TFIIB expression. Results were standardized to PBS treated condition. Bars represent means (N=3) and dots denote single measurements, each consisting of technical triplicates. For statistical analysis one-way ANOVA was conducted (WAT: PBS vs Sct p<0.001; PBS vs Iso p<0.001; Sct vs Iso p=0.066; BAT: PBS vs Sct p=0.008; PBS vs Iso p=0.018; Sct vs Iso p=0.291).

[0165] FIG. 5: Secretin induced respiration in wildtype 129/S6 mice.

[0166] Mice received 0.5 mg/kg secretin solved in PBS or an equal volume of PBS via i.p. injection.

[0167] A: Measurement was performed at 27 C. The mean of N=7 mice per group is shown for heat production (HP). The Mean of basal HP measured at 30 C. is depicted as grey background).

[0168] B: AUC for heat production for 45 minutes of measurement (p=0.001). For statistical analysis the basal heat production was subtracted for every mouse individually. Statistical analysis was performed using t-test.

[0169] FIG. 6: Secretin induced heat production in UCP1 knockout mice.

[0170] Mice received 0.5 mg/kg secretin solved in PBS or equal volume of PBS via i.p. injection. Measurement was performed at 27 C. In (A) the mean of N=9 wildtype mice per group and in (B) the mean of N=5-6 knockout mice are shown for heat production (mean of basal HP measured at 30 C. depicted as grey background). In (C) the AUC for heat production for 60 minutes of measurement is depicted. For statistical analysis the basal heat production was subtracted for every mouse individually. Statistics were conducted using two-way ANOVA. Equal variance test failed (P<0.050) and therefore a pairwise multiple comparison was conducted using Tukey test (genotype: p=0.005; treatment: p=0.013; genotypetreatment: p=0.128).

[0171] There was a significant difference for the treatment within wildtype mice (p=0.002; indicated by **) but not within knockout mice (p=0.482) and furthermore a significant difference for the genotype within secretin treatment (p=0.005), but not within PBS treatment (p=0.275), which is not illustrated in the graph.

[0172] FIG. 7: Effect of secretin on refeeding of fasted mice.

[0173] Mice fasted for 18 h were injected with 5 nmol secretin before the start of refeeding. Control mice were injected with PBS. Food intake is shown for 4 hours after injection. Data are presented as meanSD (N=11-12 per group). Statistical analysis were conducted for the first two hours using linear mixed-effects model with TIBICO Spotfire SPlus software (Treatment: p=0.0181; Time: p<0.0001).

[0174] FIG. 8: Effect of secretin on refeeding of fasted wildtype and Ucp1 knockout mice.

[0175] Wildtype (WT) and Ucp1 knockout (KO) mice fasted for 18 h were injected with 5 nmol secretin (SCT) before start of refeeding. Control mice of both genotypes were injected with PBS. Cumulative food intake was monitored during 4 hours of refeeding. Data are presented as mean of N=6-7 per group.

[0176] A: amount of food intake over time;

[0177] B: AUC of food intake.

[0178] FIG. 9: Sctr gene expression by transcriptome analysis (RNA-SEQ).

[0179] A: Secretin receptor (SctR), a Gs coupled GPCR, is highly expressed in BAT. Gene expression of receptors for gastrointestinal tract-related peptides obtained from RNA sequencing of murine brown adipose tissue. Data are shown in RPKM (reads per kilobase per million mapped reads). Expression of secretin receptor (SctR) is significantly higher compared to gastric inhibitory polypeptide receptor (Gipr), vasoactive intestinal peptide receptor1/2 (Vipr1/2), cholecystokinin A/B receptor (Ccka/br), ghrelin receptor (Ghsr), glucagon-like peptide 1 receptor (Glp1r), neuropeptide Y receptor type 2 (Npy2r) (p<0.0001).

[0180] B: Secretin receptor gene expression in interscapular BAT, inguinal WAT and gonadal WAT of mice.

[0181] C: Secretin receptor gene expression in primary cultured adipocytes from interscapular BAT, inguinal WAT and gonadal WAT of mice.

[0182] FIG. 10: Respiration measurements in primary brown adipocytes of wildtype and UCP1/ mice.

[0183] A: Wildtype data: Isoproterenol (500 nM) and Secretin (10 nM) activate UCP1-dependent thermogenesis in primary brown adipocytes cultured from wildtype mice. Data are from five independent experiments (N=5).

[0184] B: Knock-out data Isoproterenol (500 nM) and Secretin (10 nM) fail to activate thermogenesis in primary brown adipocytes cultured from UCP1.sup./ mice. Data are from five independent experiments (N=5).

[0185] C: Secretin activates UCP1-dependent thermogenesis in primary murine brown adipocytes. Comparison of isoproterenol (ISO) and secretin (SCT) stimulated respiration in primary brown adipocytes from UCP1.sup.+/+ and UCP1.sup./ mice. UCP1-dependent thermogenesis is expressed as fold increase above basal leak respiration. Microplate respirometry of primary brown adipocytes was conducted following the subsequent protocol. After assessment of basal oxygen consumption oligo (oligomycin) was injected to determine basal leak respiration. Next, either ISO or SCT was added to investigate UCP1 (uncoupling protein 1)-dependant uncoupled respiration. By the addition of FCCP maximal leak respiration was determined. Lastly, non-mitochondrial oxygen consumption was assessed by injecting Anti A (antimycin A).

[0186] D: SCT induced thermogenesis in brown adipocytes does not depend on beta-adrenergic receptor signaling. SCT- and ISO-stimulated respiration after 1 h pretreatment with different concentrations propranolol, a non-selective blocker of adrenergic -receptors.

[0187] E: Thermogenic effect of SCT depends on secretin receptor expression. SCT- and ISO stimulated respiration in primary brown adipocytes after siRNA-mediated knockdown of the secretin receptor (SCTR) in comparison to a non-targeting control (NC).

[0188] F: Thermogenic effect of SCT depends on Protein Kinase A activity. Fold increase of basal leak respiration after stimulation with ISO, SCT and vehicle (assay medium) w/o proteinkinase A inhibitor H89 (50 M). Inhibitor was injected together with olgio prior to addition of stimulators.

[0189] FIG. 11: Plasma SCTR levels are regulated by fasting and refeeding.

[0190] Secretin plasma levels were determined in mice after 18 h fasting, and mice fasted for 17 h and refed for 1 h. Control mice were fed ad libitum.

[0191] The examples illustrate the invention:

EXAMPLE 1

[0192] Material and Methods:

[0193] Animals

[0194] Male mice (Mus musculus, Sv129S6/J, Sv129S1/SvImJ and C56BL/6J) were housed at an ambient temperature of 22 C. and a humidity of 50-60% at the animal facility of TUM in Weihenstephan. The mice were maintained in groups of two to five mice in type-II, long cages (360 m.sup.2) with a light-dark cycle of 12 hours each. If not stated otherwise mice had ad libitum access to water and food. The chow diet for breeding and maintenance consisted of 58% carbohydrate, 33% protein and 9% fat, given as % of total energy content (17 kJ/g). UCP1-KO mice with C56BL/6J and 129/SvImJ genetic background have been kindly provided by Leslie Kozak from the Pennington Biomedical Research Center (PBRC), Baton Rouge, L .70808.

[0195] Primary Adipocytes

[0196] For culturing primary adipocytes male 129S1/SvImJ mice (wildtypes or UCP1-KO mice and wildtype littermates) at the age of 5-6 weeks were used for the dissection of inguinal and epidydimal white adipose tissue and interscapular brown adipose tissue depots. The tissue was carefully minced and treated with collagenase for 45 min at 37 C. The homogenate was filtered through a 250 m nylon mesh and centrifuged at 500 g to collect the stromal vascular fraction (SVF). The SVF cell pellet was rinsed and seeded into XF96 V3-PS cell culture microplate (Seahorse Bioscience). After reaching confluency, induction medium containing 10% fetal bovine serum (FBS), 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 1 mM dexamethasone, 850 nM insulin and 1 nM T3 was added. After 2 days of induction, cells were maintained in differentiation media (10% FBS, 850 nM insulin and 1 nM T3). Medium was changed every two days. Respiration was measured on day 7 of differentiation. For determination of mRNA levels cells were stimulated at the 7.sup.th day of differentiation for 6 hours with secretin (rat, Tocris, Art Nr 1919) or isoproterenol (Sigma-Aldrich, Art Nr. 16504-100MG) and harvested subsequently.

[0197] Respirometry

[0198] Oxygen consumption rate (OCR) was measured at 37 C. using a microplate respirometer (XF96 extracellular flux analyzer, Seahorse Bioscience). At day 7, the differentiation medium was replaced with prewarmed, unbuffered assay medium (DMEM basal medium supplemented with 25 mM glucose, 2 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax and 15 mg/l phenol red, pH 7.4) containing 2% of essentially fatty acid free bovine serum albumin (BSA), and incubated at 37 C. in a room air incubator for 1 h. Basal respiration was measured in untreated cells. Coupled respiration was inhibited by oligomycin treatment (5 M). UCP1 mediated uncoupled respiration was determined after stimulation with 0.5 M secretin (rat, Tocris, Art Nr 1919) or 0.5 M isoproterenol (Sigma-Aldrich, Art Nr. I6504-100 MG). Maximum respiratory capacity was assessed after FCCP (Sigma-Aldrich) addition (1 M). Finally, mitochondrial respiration was blocked by antimycin A (Sigma-Aldrich) (5 M) treatment and the residual OCR was considered to be due to non-mitochondrial reactions. Oxygen consumption rates were calculated using the Seahorse XF-96 software. Data were exported and reconstructed in GraphPad Prism 4.0 software.

[0199] For the presentation of the fold increase above basal and maximal uncoupled OCR values were calculated as follows:

[0200] For each condition the lowest OCR after antimycin A addition was subtracted from all other values. Respiration rates following oligomycin addition were defined as basal OCR. For induction of OCR by isoproterenol, secretin or FCCP the highest values measured after addition of the respective molecule was used. The OCR in % of basal OCR and OCR in % of FCCP OCR were calculated as follows:

[00001]$\mathrm{OCR}.Math..Math.\%.Math..Math.\mathrm{basal}.Math..Math.\mathrm{OCR}=\frac{100*\left(\mathrm{Sct}-\mathrm{antiA}\right)}{\left(\mathrm{basal}-\mathrm{antiA}\right)}.Math..Math.\mathrm{or}.Math..Math.\frac{100*\left(\mathrm{Iso}-\mathrm{antiA}\right)}{\left(\mathrm{basal}-\mathrm{antiA}\right)}$$\mathrm{OCR}.Math..Math.\%.Math..Math.\mathrm{FCCP}.Math..Math.\mathrm{OCR}=\frac{100*\left(\mathrm{Sct}-\mathrm{antiA}\right)}{\left(\mathrm{FCCP}-\mathrm{antiA}\right)}.Math..Math.\mathrm{or}.Math..Math.\frac{100*\left(\mathrm{Iso}-\mathrm{antiA}\right)}{\left(\mathrm{FCCP}-\mathrm{antiA}\right)}$$\mathrm{OCR}.Math..Math.\%.Math..Math.\mathrm{basal}.Math..Math.\mathrm{OCR}=\frac{100*\left(\mathrm{Sct}-\mathrm{antiA}\right)}{\left(\mathrm{basal}-\mathrm{antiA}\right)}.Math..Math.\mathrm{or}.Math..Math.\frac{100*\left(\mathrm{Iso}-\mathrm{antiA}\right)}{\left(\mathrm{basal}-\mathrm{antiA}\right)}$$\mathrm{OCR}.Math..Math.\%.Math..Math.\mathrm{FCCP}.Math..Math.\mathrm{OCR}=\frac{100*\left(\mathrm{Sct}-\mathrm{antiA}\right)}{\left(\mathrm{FCCP}-\mathrm{antiA}\right)}.Math..Math.\mathrm{or}.Math..Math.\frac{100*\left(\mathrm{Iso}-\mathrm{antiA}\right)}{\left(\mathrm{FCCP}-\mathrm{antiA}\right)}$

[0201] Quantitative PCR

[0202] Frozen tissue samples [interscapular brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and intra-abdominal gonadal white adipose tissue (gWAT)] or primary adipocytes harvested from cell culture were homogenized in TRIsure (Bioline, London/UK) according to the manufacturer's instructions. Precipitated RNA was transferred on spin columns (SV Total RNA Isolation System, Promega, Madison WI/USA) and centrifuged for 15 sec at 8,000 g at room temperature. The residual volume was added to the column and centrifuged for 1 min at 12,000 g at room temperature. Further processing was performed according to the manufacturer's protocol. RNA was eluted in 50 l nuclease-free water and RNA concentration was determined spectrophotometrically (Infinite 200 PRO NanoQuant, Tecan, Mnnedorf/Switzerland). Reverse transcription into cDNA was performed with 500 ng RNA in a final volume of 10 l (Quantitect Reverse Transcription Kit, Quiagen, Hilden/Germany). qRT-PCR was conducted on 384 well plates in a total volume of 12.5 l comprising 6.25 l SensiMix SYBR No-ROX (Bioline, London/UK), 250 nM forward and reverse primers, and 1 l template cDNA (LightCycler 480 System, Roche Diagnostics, Rotkreuz/Switzerland). Transcript levels of target genes were normalized to transcription factor 2b (TFIIB) expression. qRT-PCR primers were produced by Eurofins MWG Operon (Ebersberg/Germany).

[0203] Primers

TABLE-US-00001 UCP1 for5-GTACACCAAGGAAGGACCGA-3 (SEQIDNO:2) rev5-TTTATTCGTGGTCTCCCAGC-3 (SEQIDNO:3) Secretinreceptor for5-ATGCACCTGTTTGTGTCCTT-3 (SEQIDNO:4) rev5-TAGTTGGCCATGATGCAGTA-3 (SEQIDNO:5) TFIIB for5-TGGAGATTTGTCCACCATGA-3 (SEQIDNO:6) rev5-GAATTGCCAAACTCATCAAAACT-3 (SEQIDNO:7)

[0204] Food Intake Measurements

[0205] For the measurement of food intake, a feeding-drinking-activity (FDA) device was used (TSE Systems, Bad Homburg/Germany). During the measurement mice were single-housed in type-III cages. Mice were habituated to the new cage environment and received every morning at 10 a.m. a single daily intraperitoneal (i.p.) PBS-injection for four days ahead of the experiment start. The body weight of the mice was measured every day. On day four day of habituation the food was removed from the cages at 5 p.m., and the mice were food deprived over night with ad libitum access to water. On the next morning, mice were i.p. injected with either PBS as control or secretin (rat, Tocris, Art Nr 1919). The latter was given in different doses. Subsequently the mice were refed ad libitum while monitoring food intake during the following 72 hours. During this refeeding period mice were only disturbed for daily body weight measurements. The values for food intake were cropped in intervals of 5 minutes by automatic weighing of the feeder. Food intake was calculated from the decrease in feeder weight. The results were exported into a spreadsheet calculation table (Excel, Microsoft) and analyzed with a software package for statistics and graphics (GraphPad Prism 4.0, GraphPad Software, San Diego, USA).

[0206] Indirect Calorimetric Measurements

[0207] Indirect calorimetric measurements in mice were performed using an open flow respirometry system (Phenomaster, TSE Systems, Bad Homburg/Germany). All animals were kept at room temperature or cold-acclimated at 4 C. for 4 days as indicated in the results section. For the gas exchange measurement (O.sub.2 consumption and CO.sub.2 production) mice were placed individually in metabolic cages (type I, 3 liter volume) without food and water and transferred to a climate cabinet (TPK 600, Feutron, Greiz/Germany). The air was continuously pulled through the cages at a flow rate of 33 l/h. For gas analysis a subsample was dried in a cooling trap and analyzed for gas exchange. Mice in the cabinet were preconditioned to 30 C. and the basal metabolic rate (BMR) in the post-absorptive state was measured between 8:00 a.m.-12:00 p.m. The O.sub.2 consumption and CO.sub.2 production of each mouse was analyzed every 5 min over a period of 1 min. After BMR measurements, the cages were removed from the climate cabinet and the cabinet temperature was lowered to 27 C. within 20 min. Mice were injected intraperitoneally with 0.5 mg/kg secretin (rat, Tocris, Art Nr 1919) or PBS as a control. After injection the O.sub.2 consumption and CO.sub.2 production rate of each mouse was recorded for 45-60 min with high-resolution recordings at 10 sec intervals. BMR [ml O.sub.2/h] was defined as the lowest mean of three consecutive oxygen consumption values, which had a coefficient of variation less than 5%. Respiratory exchange ratios (RER) and heat production (HP) were calculated from the following formula: (RER=CO.sub.2 production/O.sub.2 consumption) and (HP[mW]=(4.44+1.43*respiratory exchange ratio)*oxygen consumption [ml/h]).

EXAMPLE 2

Secretin Receptor Expression In Vivo and In Vitro

[0208] Wildtype Sv129S6/J mice were killed for the sampling of brown adipose tissue BAT, inguinal white adipose tissue (iWAT) and intra-abdominal gonadal white adipose tissue (gWAT) depots. The secretin receptor expression was measured by quantitative RT-PCR. The mean expression level of the receptor was higher in BAT compared to iWAT and gWAT. This difference was significant for the comparison of BAT and iWAT, but not gWAT (FIG. 1A), which is in line with data from BIO-GPS (http://biogps.org/) showing second highest expression for the secretin receptor in BAT amongst all analyzed mouse tissues (see GeneAtlas MOE430, gcrma, Probeset 1443454_at: neuroblast [43.9]>adipose, brown [16.3]>placenta [12.5]>stomach [10.6]> . . . >adipose, white [5.2]).

[0209] The expression of the secretin receptor was also analyzed in primary adipocytes. Isolated cells of the stromal vascular fraction from three different mouse adipose tissue depots (BAT, iWAT and gWAT) were cultured and fully differentiated. The quantification of the secretin receptor by qRT-PCR revealed a pattern in line with the expression analysis in tissues. In differentiated primary adipocytes the expression of the secretin receptor was much higher in primary adipocytes grown from BAT as compared to iWAT or gWAT (FIG. 1B).

EXAMPLE 3

Secretin Stimulates Oxygen Consumption Rate in Primary Brown Adipocytes

[0210] The main function of brown adipocytes is heat production by uncoupled respiration. Since the secretin receptor is preferentially expressed in brown adipocytes (see example 1) the effects of secretin on their thermogenic function were investigated by measurement of oxygen consumption rates (OCR). Brown adipocytes were subsequently treated with oligomycin (Oligo which inhibits coupled respiration), secretin (Sct) or isoproterenol (Iso), FCCP (fully uncoupled respiration) and antimycin A (Anti A; non-mitochondrial OCR) after fully differentiated in a microplate respirometer plate (XF-96 flux analyzer, Seahorse Bioscience). Secretin stimulated OCR was comparable to the stimulation in response to the pan--adrenergic receptor agonist isoproterenol, which is an established activator of UCP1-mediated thermogenesis (FIG. 2A). Secretin and isoproterenol both increased OCR about 200% above basal OCR (FIG. 2B). These uncoupled OCRs correspond to about 75% of maximal uncoupled OCR induced by FCCP (FIG. 2C). There was no statistical difference between the secretin and isoproterenol stimulated OCRs when expressed in relation to basal (p=0.712) or maximal respiration (p=0.946). Both compounds were used in the same concentration. The potency of secretin to induce mitochondrial respiration in vitro can be considered equal to the classical pan--adrenergic receptor agonist isoproterenol.

EXAMPLE 4

Primary Brown Adipocytes from UCP1 Knockout Mice Lack Secretin Induced Oxygen Consumption

[0211] Secretin is able to induce respiration in brown fat cells. To investigate the dependency of the induction of mitochondrial respiration (see example 3) on the presence of UCP1 the respiration measurement was repeated with primary brown adipocytes from UCP1 knockout and wildtype control mice (129/S1-background). For both, secretin (FIG. 3A) and isoproterenol (FIG. 3B) an increase in oxygen consumption could be found in cells from wildtype mice (FIG. 3). At the same time the induction of respiration by both, secretin and isoprotrenol was lacking in cells from UCP1 knockout mice. It must be mentioned that the cells from UCP1 knockout mice also exhibited a much smaller fully uncoupled respiration by FCCP than the wildtype controls. The results of this experiment clearly point out that the induction of respiration in brown adipocytes by -adrenergic or secretin stimulation is dependent upon UCP1. The potential for direct activation of UCP1 is established for isoproterenol. In this experiment it was also shown for the peptide hormone secretin.

EXAMPLE 5

Secretin Stimulates UCP1 Expression in White and Brown Adipocytes

[0212] Differentiated inguinal white and interscapular brown primary adipocytes were stimulated with secretin or isoproterenol. Expression of mRNA was determined with quantitative PCR. Secretin increased the expression of UCP1 in white adipocytes (FIG. 4A). The effect was highly significant and comparable to the induction of UCP1 expression by isoproterenol (PBS vs. Sct: p<0.001; PBS vs. Iso: p<0.001; Sct vs. Iso: p=0.066). This shows that secretin is equally potent as isoproterenol to induce browning in cultured primary white adipocytes. Likewise, in primary brown adipocytes secretin significantly increased the expression of the UCP1 gene compared to the unstimulated PBS control (FIG. 4B; PBS vs Sct p=0.008; PBS vs Iso p=0.018; Sct vs Iso p=0.291). Stimulation of the cells with isoproterenol was used as a positive control and induced UCP1 expression to a similar level as secretin. These results demonstrate that secretin cannot only acutely activate UCP1-mediated respiration in brown adipocytes. It can also increase UCP1 expression in these cells and thereby enhance their thermogenic potential. It should be emphasized that for this experiment secretin was used in a fivefold lower concentration than isoproterenol.

EXAMPLE 6

Acute Effect of Secretin on Respiration in Wildtype Mice

[0213] For the measurement of oxygen consumption wildtype mice were injected either with PBS or with secretin at a dose of 0.5 mg/kg body weight. Prior to injection basal metabolic respiration (BMR) was determined at 30 C. for 3 hours. All BMR values were highly comparable between the two groups. Irrespective of the treatment, all mice increased heat production during the first five minutes after injection (FIG. 5A). This peak most likely reflects arousal elicited by injection and a subsequent elevated excitement which lasted a few minutes after mice were released back into their cage. After ten to fifteen minutes the mice in both groups started to calm down and move less, which has been noted by continuous observation through a window in the climate chamber. The sedation is also displayed in the declining levels of heat production (A). The PBS group almost reached basal metabolic rate levels (indicated by grey background) at about 20 minutes after injection until the end of the measurement. In contrast, mice injected with secretin maintained a distinct level of elevated heat production until the end of the recording. The area under the curve (AUC) was calculated for the entire 45 minutes of the recording for both groups (B). Basal metabolic rates were subtracted for every mouse individually (mean shown in grey in figure A). Secretin stimulated heat production as demonstrated by the significant increase in the AUC (p=0.001) (FIG. 5B).

EXAMPLE 7

Secretin Fails to Stimulate Heat Production in UCP1 Knockout Mice

[0214] Mice show increased heat production in response to secretin injection (see example 6). To test whether this thermogenic effect is dependent on the presence of UCP1 in brown adipocytes, like already demonstrated in vitro (see example 4 and FIG. 3), calorimetric measurements were conducted in UCP1 knockout mice. Mice (wt and ko) of the 129/S1-UCP1 knockout strain were individually measured at 30 C. to determine BMR. Wildtype and knockout mice showed a comparable bodyweight (p=0.851) over all groups and no difference was detected in RER and HP during the BMR measurements. Statistical analysis was performed by two-way ANOVA.

[0215] After completion of BMR measurements, the thermogenic effect of secretin and PBS were determined (FIG. 6). After the first 15 minutes of arousal observed in all mice, wildtype mice injected with secretin maintained a higher rate of heat production as compared to PBS injected controls (FIG. 6A). The area under the curve (AUC) for the whole recording was highly significant different between the two treatment groups, with higher AUC in wildtype mice treated with secretin as compared to PBS (FIG. 6C; p=0.002). This result reproduced the data for 129/S6-wildtype mice after secretin injection (example 6, FIG. 5). In the UCP1 knockout mice there was no difference for heat production between secretin and PBS treated mice during the course of recording (FIG. 6B), or for the AUC of heat production (FIG. 6C; p=0.482).

[0216] This result clearly demonstrates that the thermogenic effect of secretin depends on the activation of UCP1-mediated heat production in brown adipose tissue.

EXAMPLE 8

Suppression of Food Intake in Mice after Intraperitoneal Injection of Secretin

[0217] Next to thermogenesis, brown fat has been proposed to function in the thermoregulatory control of feeding. An increase in body core temperature caused by BAT after initiation of food intake may trigger the termination of food intake (Himms-Hagen, 1995). In extension of this hypothesis secretin may be part of a novel endocrine gutbrown fat axis controlling meal patterns. Therefore, the effects of secretin on food intake in mice fasted overnight for 18 hours were tested. Just before refeeding, mice were intra-peritoneally injected with 5 nmol secretin solved in PBS, or PBS as the vehicle control, and food intake was monitored for the following three days.

[0218] Regarding the immediate refeeding responses (FIG. 7), the PBS injected control group showed a steep rise in cumulative food intake during the first hour, stopped eating during the second hour of refeeding, and recommenced eating thereafter. This trajectory was altered by secretin. Secretin treatment caused a significant inhibition of food intake during the first two hours of refeeding (p=0.0181, FIG. 7). Specifically, in secretin injected mice the initial steep rise in cumulative food intake during the first hour of refeeding was blunted, suggesting an acute and transient anorexigenic effect of this gut hormone.

[0219] Based on the hypothesis of thermoregulatory feeding, it was assessed whether the anorexigenic effect of secretin is mediated by the thermogenic activation of brown fat. To directly test this, the effect of secretin on refeeding in Ucp1 knockout mice was determined (FIG. 8). Notably, secretin did not inhibit refeeding in Ucp1 knockout mice (KO SCT) as compared to wildtype (WT SCT). This observation proves that Ucp1-mediated thermogenesis in brown adipocytes is required for the anorexigenic action of secretin. This observation identifies secretin as a new player in energy balance regulation defining a novel endocrine gut-brown fat-brain axis in the control of energy expenditure and food intake.

EXAMPLE 9

Sctr Gene Expression by Transcriptome Analysis (RNA-SEQ)

[0220] Method: For the RNA-sequencing method total RNA isolated from brown adipose tissue (BAT) samples of mice (n=4) housed under room temperature (23 C.) was subjected to transcriptome analysis by next generation sequencing (RNA-Seq) using Illumina HiSeq 2000 platform (Illumina). Sequencing libraries were prepared using the TruSeq RNA Sample Prep kit v2 (Illumina). Libraries from 4 samples were pooled into one sequencing lane and sequenced using a 50-cycle TruSeq SBS Kit v3-HS (Illumina), resulting in a depth of >25 million reads/sample. Sequenced tags were aligned to the Ensembl 75 transcriptome annotation (NCBI38/mm10 mouse genome) using Genomatix Software Suite. All genes and transcripts were assigned relative coverage rates as measured in RPKM units (reads per kilobase per million mapped reads). In the search for gut hormones that directly activate brown adipose tissue (BAT), gene expression of gut hormone receptors was profiled by inquiring transcriptome data obtained from interscapular BAT tissue of mice. Among the detectable transcripts of gut hormone receptor genes in BAT, the secretin receptor (Sctr) gene stood out with the highest abundance expressed as reads per kilobase per million mapped reads (RPKM; FIG. 9A).

[0221] Result: Gene expression of receptors for gastrointestinal tract-related peptides obtained from RNA sequencing of murine brown adipose tissue. Data are shown as RPKM (reads per kilobase per million mapped reads). Expression of secretin receptor (Sctr) is significantly higher compared to gastric inhibitory polypeptide receptor (Gipr), vasoactive intestinal peptide receptor1/2 (Vipr1/2), cholecystokinin A/B receptor (Ccka/br), ghrelin receptor (Ghsr), glucagon-like peptide 1 receptor (Glp1r), and the PYY.sub.3-36 receptor (Npy2r)

[0222] Method: RNA was extracted from cultured cells or frozen tissue samples using TRIsure, purified with SV Total RNA Isolation System, Promega and reverse transcribed using SensiFAST cDNA Synthesis Kit (BIOLINE). The resultant cDNA was analysed by qRT-PCR. Briefly, 1 L of 1:10 diuluted cDNA and 400 nmol of each primer were mixed with SensiMix SYBR Master Mix No-ROX (Bioline). Reactions were performed in 384-well format using a lightcycler II instrument (Roche). Standard reactions containing serial diluted pooled cDNA of all samples (Pure, 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64) as a template were used to establish a standard curve, from which gene expression levels of samples were calculated. The RNA abundance of each gene was normalized to a housekeeping gene. The following primers were used:

TABLE-US-00002 Sctr F: 5-ATGCACCTGTTTGTGTCCTT-3, (SEQIDNO:12) R: 5-TAGTTGGCCATGATGCAGTA-3; (SEQIDNO:13) Gtf2b F: 5-TGGAGATTTGTCCACCATGA-3, (SEQIDNO:14) R: 5-GAATTGCCAAACTCATCAAAACT-3; (SEQIDNO:15)

[0223] Result: Among different fat depots, our qPCR analyses revealed that Sctr is highly expressed in BAT compared to white fat depots in the inguinal (iWAT) and epididymal (eWAT) region (FIG. 9B). Preferential Sctr expression in brown adipocytes was also observed in primary cultures derived from these fat depots (FIG. 9C).

EXAMPLE 10

Respiration Measurement in Primary Brown Adipocytes of Wildtype and UCP1/ Mice

[0224] Method: To test whether SCT can activate thermogenesis in brown adipocytes, we measured UCP1-mediated uncoupled respiration in cultured adherent intact primary brown adipocytes, according to a protocol recently established in our lab (Li Y, Fromme T, Schweizer S, Schttl T, Klingenspor M. EMBO Rep. 2014 October; 15(10):1069-76. doi: 10.15252/embr.201438775; Li Y, Fromme T, Klingenspor M. Meaningful respirometric measurements of UCP1-mediated thermogenesis. Biochimie. 2017 March; 134:56-61. doi: 10.1016/j.biochi.2016.12.005.). The cellular oxygen consumption rate (OCR) of primary adipocytes was determined using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Briefly, primary adipocytes were cultured and differentiated in XF96 microplates. At day 7 of differentiation, cells were washed once with prewarmed, unbuffered assay medium (DMEM basal medium supplemented with 25 mM glucose, 31 mM NaCl, 2 mM GlutaMax and 15 mg/l phenol red, pH 7.4) (basal assay medium) and then the medium was replaced with basal assay medium containing 1-2% essentially fatty acid free bovine serum albumin (BSA), and incubated at 37 C. in a room air incubator for 1 h. The drug injections ports of the sensor cartridges were loaded with the assay reagents at 10 in basal assay medium (no BSA).

[0225] All respiration assays were performed in the presence of bovine serum albumin to buffer free fatty acid levels in the respiration medium. Basal respiration was measured in untreated cells. Coupled respiration was inhibited by oligomycin treatment (5 M). UCP1 mediated uncoupled respiration was determined after isoproterenol or secretin stimulation. Maximum respiratory capacity was assessed after FCCP (Sigma-Aldrich) addition (1 M). Finally, mitochondrial respiration was blocked by antimycin A (Sigma-Aldrich) (5 M) treatment and the residual OCR was considered non-mitochondrial respiration. Oxygen consumption rates were automatically calculated by the Seahorse XF-96 software. Data were exported and reconstructed in GraphPad Prism 6.0 software.

[0226] Result: It was observed that SCT (10 nM) is as potent as ISO (500 nM), a -adrenergic receptor agonist, in stimulating UCP1-mediated thermogenesis, even at a 50-fold lower concentration (FIG. 10A). Primary brown adipocytes from UCP1 knockout mice did not respond to SCT (10 nM) or ISO (500 nM) (FIG. 10B). This is further demonstrated by quantification of SCT and ISO induced leak respiration (FIG. 10C).

[0227] Cells were pretreated with propranolol (1 M) before bioenergetic profiling. The thermogenic effect of SCT was independent on -adrenergic receptor signaling, since pretreatment of cells with propranolol, a nonselective -adrenergic receptor antagonist, did not attenuate SCT stimulated respiration while blocking the effect of ISO, a beta-adrenergic receptor agonist, in a dose dependent manner (FIG. 10D).

[0228] Cells were reverse transfected with small interfering RNAs (siRNAs) targeting SctR (#1CCUGCUGAUCCCUCUCUUU (SEQ ID NO: 8); #2CCCUGUCCAACUUCAUCAA (SEQ ID NO: 9); #3CCAUCGUGAUCAAUUUCAU (SEQ ID NO: 10)) and non-targeting control siRNA (UUUGUAAUCGUC GAUACCC (SEQ ID NO: 11)) as described previously (Li Y, Fromme T, Klingenspor M. Meaningful respirometric measurements of UCP1-mediated thermogenesis. Biochimie. 2017 March; 134:56-61. doi: 10.1016/j.biochi.2016.12.005.) before bioenergetic profiling. The thermogenic effect of SCT depends on SCTR, as siRNA-mediated downregulation of receptor expression, blunts the effects of SCT-induced increase in oxygen consumption (FIG. 10E).

[0229] Cells were pretreated with H89 (50 M) before bioenergetic profiling. Pretreatment of cells with H89, a selective inhibitor of Protein Kinase A, completely blocked the thermogenic effect of SCT (FIG. 10F), demonstrating that SCT stimulated UCP1-dependent thermogenesis acts through activation of lipolysis mediated by the canonical cAMP-PKA pathway.

EXAMPLE 11

Plasma Secretin Levels in Response to Fating and Refeeding

[0230] Blood was collected at the time the mouse was killed. Plasma SCT levels were determined by ELISA using a kit system (Cloud-Clone CEB075Mu) following the manufacturer's instructions. Concentrations were calculated using a standard curve generated by SCT standards included in the kit. The primary stimulus for periprandial SCT release into circulation is gastric acid delivered into the duodenal lumen. Accordingly, SCT release is sensitive to the nutritional status of laboratory mice. We found that plasma secretin levels were decreased by fasting (18 hours) and increased significantly within 1 hour after refeeding (FIG. 11).