ZA200007689B - Method for detecting of a compound that mimics, potentiates or inhibits the physiological effect of Leptin. - Google Patents
Method for detecting of a compound that mimics, potentiates or inhibits the physiological effect of Leptin. Download PDFInfo
- Publication number
- ZA200007689B ZA200007689B ZA200007689A ZA200007689A ZA200007689B ZA 200007689 B ZA200007689 B ZA 200007689B ZA 200007689 A ZA200007689 A ZA 200007689A ZA 200007689 A ZA200007689 A ZA 200007689A ZA 200007689 B ZA200007689 B ZA 200007689B
- Authority
- ZA
- South Africa
- Prior art keywords
- leptin
- response element
- compound
- stat
- cell line
- Prior art date
Links
- 102000016267 Leptin Human genes 0.000 title claims description 167
- 108010092277 Leptin Proteins 0.000 title claims description 167
- 229940039781 leptin Drugs 0.000 title claims description 164
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 title claims description 163
- 238000000034 method Methods 0.000 title claims description 37
- 150000001875 compounds Chemical class 0.000 title claims description 36
- 230000001766 physiological effect Effects 0.000 title claims description 21
- 210000004027 cell Anatomy 0.000 claims description 76
- 230000000694 effects Effects 0.000 claims description 49
- 108091027981 Response element Proteins 0.000 claims description 40
- 230000004044 response Effects 0.000 claims description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 241000282414 Homo sapiens Species 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 14
- 238000013518 transcription Methods 0.000 claims description 13
- 230000035897 transcription Effects 0.000 claims description 13
- 108700008625 Reporter Genes Proteins 0.000 claims description 12
- 108060001084 Luciferase Proteins 0.000 claims description 10
- 239000005089 Luciferase Substances 0.000 claims description 9
- 102000000887 Transcription factor STAT Human genes 0.000 claims description 9
- 108050007918 Transcription factor STAT Proteins 0.000 claims description 9
- 210000001072 colon Anatomy 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 229920001184 polypeptide Polymers 0.000 claims description 7
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 230000000638 stimulation Effects 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 108020004414 DNA Proteins 0.000 claims description 6
- 239000012190 activator Substances 0.000 claims description 6
- 102000006601 Thymidine Kinase Human genes 0.000 claims description 5
- 108020004440 Thymidine kinase Proteins 0.000 claims description 5
- 230000002708 enhancing effect Effects 0.000 claims description 4
- 210000002919 epithelial cell Anatomy 0.000 claims description 4
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 claims description 3
- 108090000331 Firefly luciferases Proteins 0.000 claims description 3
- 241000700584 Simplexvirus Species 0.000 claims description 3
- 210000004185 liver Anatomy 0.000 claims 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims 1
- 102100031775 Leptin receptor Human genes 0.000 description 45
- 108010029485 Protein Isoforms Proteins 0.000 description 32
- 102000001708 Protein Isoforms Human genes 0.000 description 32
- 101710131677 Leptin receptor Proteins 0.000 description 31
- 210000001630 jejunum Anatomy 0.000 description 30
- 241000699670 Mus sp. Species 0.000 description 29
- 230000004913 activation Effects 0.000 description 25
- 108020004999 messenger RNA Proteins 0.000 description 24
- 230000004568 DNA-binding Effects 0.000 description 20
- 210000001519 tissue Anatomy 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 108010073614 apolipoprotein A-IV Proteins 0.000 description 16
- 210000001035 gastrointestinal tract Anatomy 0.000 description 16
- 238000001727 in vivo Methods 0.000 description 16
- 102000005962 receptors Human genes 0.000 description 15
- 108020003175 receptors Proteins 0.000 description 15
- 239000000284 extract Substances 0.000 description 14
- 108010019813 leptin receptors Proteins 0.000 description 14
- 102100027584 Protein c-Fos Human genes 0.000 description 12
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 12
- 230000006698 induction Effects 0.000 description 12
- 102000015617 Janus Kinases Human genes 0.000 description 11
- 108010024121 Janus Kinases Proteins 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 238000003757 reverse transcription PCR Methods 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 210000000813 small intestine Anatomy 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 10
- 150000002632 lipids Chemical class 0.000 description 10
- 230000009467 reduction Effects 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 108700002232 Immediate-Early Genes Proteins 0.000 description 9
- 208000008589 Obesity Diseases 0.000 description 9
- 239000007983 Tris buffer Substances 0.000 description 9
- 239000000499 gel Substances 0.000 description 9
- 235000020824 obesity Nutrition 0.000 description 9
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 8
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 7
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 7
- 230000037406 food intake Effects 0.000 description 7
- 210000003016 hypothalamus Anatomy 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 238000009739 binding Methods 0.000 description 6
- 239000005018 casein Substances 0.000 description 6
- 235000012631 food intake Nutrition 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 230000003389 potentiating effect Effects 0.000 description 5
- 239000013615 primer Substances 0.000 description 5
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 238000010253 intravenous injection Methods 0.000 description 4
- 230000003278 mimic effect Effects 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 238000011533 pre-incubation Methods 0.000 description 4
- 230000011664 signaling Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 150000003626 triacylglycerols Chemical class 0.000 description 4
- 235000002374 tyrosine Nutrition 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 108090001030 Lipoproteins Proteins 0.000 description 3
- 102000004895 Lipoproteins Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 3
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000035508 accumulation Effects 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000002337 electrophoretic mobility shift assay Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000003531 protein hydrolysate Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 108010004103 Chylomicrons Proteins 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- 101710173353 Cytotoxicity-associated immunodominant antigen Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 108010001127 Insulin Receptor Proteins 0.000 description 2
- 206010022489 Insulin Resistance Diseases 0.000 description 2
- 101150026829 JUNB gene Proteins 0.000 description 2
- 101150021395 JUND gene Proteins 0.000 description 2
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000009822 protein phosphorylation Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- SNBCLPGEMZEWLU-QXFUBDJGSA-N 2-chloro-n-[[(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methyl]acetamide Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CNC(=O)CCl)[C@@H](O)C1 SNBCLPGEMZEWLU-QXFUBDJGSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- JYCQQPHGFMYQCF-UHFFFAOYSA-N 4-tert-Octylphenol monoethoxylate Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCO)C=C1 JYCQQPHGFMYQCF-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 206010014486 Elevated triglycerides Diseases 0.000 description 1
- 108010075944 Erythropoietin Receptors Proteins 0.000 description 1
- 102100036509 Erythropoietin receptor Human genes 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001129927 Homo sapiens Leptin receptor Proteins 0.000 description 1
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 206010060378 Hyperinsulinaemia Diseases 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 102000003746 Insulin Receptor Human genes 0.000 description 1
- 102100036721 Insulin receptor Human genes 0.000 description 1
- 102000010787 Interleukin-4 Receptors Human genes 0.000 description 1
- 108010038486 Interleukin-4 Receptors Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 230000004163 JAK-STAT signaling pathway Effects 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108010044012 STAT1 Transcription Factor Proteins 0.000 description 1
- 102100029904 Signal transducer and activator of transcription 1-alpha/beta Human genes 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 1
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 1
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 description 1
- 101710112791 Tyrosine-protein kinase JAK2 Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000006583 body weight regulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 235000019577 caloric intake Nutrition 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000004913 chyme Anatomy 0.000 description 1
- -1 cjun Proteins 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- UKWLRLAKGMZXJC-QIECWBMSSA-L disodium;[4-chloro-3-[(3r,5s)-1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl]phenyl] phosphate Chemical compound [Na+].[Na+].O1OC2([C@@H]3CC4C[C@H]2CC(Cl)(C4)C3)C1(OC)C1=CC(OP([O-])([O-])=O)=CC=C1Cl UKWLRLAKGMZXJC-QIECWBMSSA-L 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000019439 energy homeostasis Effects 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000006539 extracellular acidification Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 238000012203 high throughput assay Methods 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 230000009097 homeostatic mechanism Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000035879 hyperinsulinaemia Effects 0.000 description 1
- 208000006575 hypertriglyceridemia Diseases 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- QNDVLZJODHBUFM-WFXQOWMNSA-N okadaic acid Chemical compound C([C@H](O1)[C@H](C)/C=C/[C@H]2CC[C@@]3(CC[C@H]4O[C@@H](C([C@@H](O)[C@@H]4O3)=C)[C@@H](O)C[C@H](C)[C@@H]3[C@@H](CC[C@@]4(OCCCC4)O3)C)O2)C(C)=C[C@]21O[C@H](C[C@@](C)(O)C(O)=O)CC[C@H]2O QNDVLZJODHBUFM-WFXQOWMNSA-N 0.000 description 1
- VEFJHAYOIAAXEU-UHFFFAOYSA-N okadaic acid Natural products CC(CC(O)C1OC2CCC3(CCC(O3)C=CC(C)C4CC(=CC5(OC(CC(C)(O)C(=O)O)CCC5O)O4)C)OC2C(O)C1C)C6OC7(CCCCO7)CCC6C VEFJHAYOIAAXEU-UHFFFAOYSA-N 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000003566 phosphorylation assay Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 101150059999 pro gene Proteins 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 239000002684 recombinant hormone Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 230000036186 satiety Effects 0.000 description 1
- 235000019627 satiety Nutrition 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000003384 transverse colon Anatomy 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000011680 zucker rat Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
> i. oo ¢ WO 00/00639 PCT/EP99/04501
METHOD FOR DETECTING OF A COMPOUND THAT MIMICS, POTENTIATES 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), 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 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 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 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 a-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 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 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)RAA have been identified (13) as STAT response elements. These elements bind STAT proteins in response to signalling molecules such - as cytokines.
The leptin receptor (OB-R) (14) is related to class I cytokine receptors which include gp130, 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 1s 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-Rp),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 treaimeni of weight, encigy balance, hacmatopoietic, 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 STATS DNA binding activity in +/+ and 0b/0b 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 STATS mechanism, a negative-feedback signal to 40 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.
y o - (1 WO 00/00639 PCT/EP99/04501
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 . 5 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; , 10 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 S$. : Suitably, the response element is coupled to a promoter gene, preferably a minimal promoter. 25 . A suitable response element is a nucleotide of formula TT(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), 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. 40 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:
So . H
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 (0b/0b) mice; OB-
Re (1), OB-Rd (2), OB-Rc (3), OB-Rb (4), OB-Ra (5) and finally a 100bp 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 B-actin levels and adjusted before dilutions were performed. Lower panel shows the quantitation of OB-R mRNA normalised to the B-actin mRNA, from the optimised linear regions of PCR, in +/+ and 0b/0b mice.
Figure 4: Expression of leptin responsive STAT proteins in mouse gut and human
CACO-2 cells.
A. Western blot analysis of STATI, 3 and 5 in 25ug protein lysates of jejunum from +/+ and ob/ob mice. All three STATS are readily detected in crude protein extracts.
Comparison of STATS expression in discrete sections in and +/+ and ob/ob mice.
B. Western blot analysis of STAT, 3 and 5 in CACO-2 cells. Positive controls are, human fibroblast, A431 and human endothelial cell lysates for STATI, 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 STATS DNA binding activity to the B-casein promoter STATS consensus element. Nuclear extracts were prepared and incubated with **P end-labelled probe and fractionated on a 4% native PAGE gel. Induction observed in +/+ and 0b/0b jejunal nuclear extracts in response to a leptin administration (Smg/kg) after 30 minutes.
Preincubation with anti-STATS antibodies reveal the major complex consist of STATSa.
No induction is observed in nuclear extracts of jejunum from db/db mice or control +/+ 40 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 STATS 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 45 STATI1/3 selective high affinity m67-SIE consensus element from the c-fos promoter.
t z WO 00/00639 PCT/EP99/04501
The complex is identified by preincubation with a anti-STAT3 and anti-STATI antibodies. STATS DNA binding to the B-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-STATS5b 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 meant+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 resuiting from biochemical changes in the cell. Ob-protein responsive cells upon stimulation may : 25 undergo biochemical changes that cause a small change in the extracellular acidification rate which can be detected by a silicon microphysiometer. The microphysiometer y biosensor methodology has been reviewed by McConnell, Science, 1992, 257, 1906. 2. Electrophoretic mobility shift assay (EMSA): 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 40 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 {1251}-leptin, the 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. 5S. 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 calctum 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 B-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 f3- 40 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
4 WO 00/00639 PCT/EP9%9/04501 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 promotor) 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.
N t
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 B-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 B-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.
( ¢ WO 00/0630 PCT/EP99/04501
EXPERIMENTAL SECTION
. 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. : 10 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
STATS 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 . 15 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 STATS 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.
A WO 00/00639 PCT/EP99/04501
EXPERIMENTAL PROCEDURES
Animals and cells-Six week old female wild-type and 0b/0b 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 (Smg/kg) were assessed using . 10 recombinant leptin (Peprotech, Rockyville, USA) dissolved in 10mM Tris administered by tail vein injection or 10mM 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, and stored at -80°C. Human Caucasian colon epithelial 5 15 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, 100w/ml penicillin, 100pg/ml streptomycin (all from Gibco/BRL, UK) at 37°C in humidified 5%
CO,/ 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 (4ug) was used to generate cDNA with first-strand cDNA beads (Pharmacia, Biotech) and oligo (dT),,.,s (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 AmpliTaqg (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 B-actin), as described previously (18). 40 Immunoblot analysis of tissues and cells-Fresh tissues were homogenised in ice-cold
RIPA buffer (1X 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
Hg protein was mixed with gel loading buffer (4% SDS, 100mM Tris, pH 6.8, 100mM B- 45 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, 10mM Tris pH 7.4, 100mM 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 STATS (C-17) (Santa Cruz
Biothechnology, Inc., USA) and STAT1,-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, 10mM NaMoO, and 1mM activated NaVOQ,. Cells were lysed with a hypotonic buffer (400p:1/25cm®) containing 10mM HEPES, pH 7.9, 10mM NaMoO,,
ImM NaVO,, ImM EDTA, ImM EGTA, 1mM NaF, 0.125 uM Okadaic Acid, ImM
DTT, 2ug.ml leupeptin, 2p1g/ml aprotinin, SOug/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 100p1 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, 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 assays-5ul nuclear extract was used for DNA binding studies with 100ng/reaction *Py-ATP end-labelled probe in DNA binding buffer (Tris 10mM, pH 7.5, 150mM NaCl, ImM DTT with 1pg/ml polydeoxy Inosine/Cytidine (Pharmacia, Sweden) to block non-specific binding. The binding reaction was incubated at room temperature for 20 minutes and then resclved on a 4% native PAGE gel. Supcrshift 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 atc at -3°, from the c-fos promoter and a rat $-Casein promoter element sequence, 5’-gga ctt ctt gga att aag gga-3’. Complementary oligonucleotide strands (Research Genetics, USA) were annealed in Tris 10mM, pH 8.0, 1mM EDTA, 50mM NaCl to a final concentration of 200ng/ul, 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/ul T4 Polynucleotide Kinase (Amersham, USB, UK) at 37°C for one hour with 1ul **Py-ATP (6000Ci/mmol Amersham, UK).
Expression of the APO-AIV and immediate-early gene mRNA species-Tissues and 40 CACO-2 cells treated for 30 minutes with or without leptin were immediately snap-frozen in liquid N, 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 45 (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..
RESULTS
The multiple OB-R mRNA isoforms were detected using RT-PCR in discrete sections of . the gastrointestinal tract (see Fig. 1A). 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- . 10 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. 1B).
This suggests that the OB-R mRNA expression presented in Fig. 1A is neither a result of contamination from fat tissues nor a result of illegitimate transcription. Expression of the ) 15 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. 1B). Western blot analysis of 20ug 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 f-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 B-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 STATS, but more 40 weakly STATI and STAT3 (Fig. 4A). Furthermore STATS 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 Smg/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 45 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 STATI and STATS. but binds STATS 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 B-casein promoter STAT DNA binding consensus element was used as a selective probe for STATS activity in vivo. Here, Smg/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 STATS DNA binding in nuclear extracts of jejunum (Fig. 5A). The STATS DNA binding activity induced in the +/+ and ob/ob jejunum was abolished by a preincubation with specific anti-STATSa antibody and less markedly with an anti-STATS5b antibody (see results from the 0b/0b 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 STATS 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-STAT! antibody had no effect (Fig. 6).
In addition, leptin treated CACO-2 cells exhibited an activation of DNA binding activity of STATS to the B-casein probe, and supershift studies identified STATSb as the major factor in the complex (Fig 6). Thus, leptin induces nuclear STATS DNA binding activity in both mouse jejunum and CACO-2 cells, however different isoforms of STATS 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 Smg/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, cjun, junB and junD in the serum-deprived CACO-2 cells as determined by quantitative PCR (Fig 7B). Since activation of STATS is usually not associated with c-fos induction, these results could indicate that other pathways are being activated in addition to the STATS 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 Smg/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/B-actin mRNA meantSEM: 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. 40 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 45 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 0b/0b 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 : 10 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 . 15 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 0b/ob but not db/db mouse hypothalamus (19). Nuclear extracts from hypothalamus and several peripheral tissues failed to shift the STATS-specific B- casein promoter element. We show that intravenous injection of leptin induced a STATS
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 (Smg/kg) and an optimal time 40 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 Smg/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 45 between 0.1mg/kg and Smg/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 STATS 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,
STATS 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
STATS3 and STATS 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 STATS 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 (GIn>Pro) in the extracellular domain of the OB-R (30). OB-R(fa) mediates a leptin-independent (constitutive) activation of STATI! and STAT3, whilst the activation of STATS 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 STATS 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 B-oxidation of fatty acids (36). Thus we have demonstrated a novel peripheral effect of leptin on jejunum function in vivo mediated through the OB-Rb receptor and a STATS mechanism. This may 40 represent a negative feedback signal from fat stores to the primary site of lipid handling, an adipo-enteric loop that contributes to the anti-obesity effects of leptin.
y WO 00/00639 PCT/EP99/04501
1. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L. & Friedman, J.M (1994). Nature 372, . 425-432 2. Campfield, L.A., Smith, F.J., Guisez, Y., Devos, R. & Burn, P (1995). Science 269, 546-549. 3. Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, S.L., Chait, B.T., Rabinowitz, D., Lallone, R.L., - 10 Burley, S.K. & Friedman, J.M. (1995). Science 269, 543-546 4. Chen, H,, Churfat, O., Tartaglia, L.A., Woolf, E.A., Wang, X,, Ellis, S.J., Lukey, N.D., Culpepper, J., ; Moore, K.J Breitbart, L.F., Duyk, G.M., Tepper, R.L. & Morgenstern, J.P (1996). Cell 84, 491-495. 5. Lee, G-H,, Proenca, R., Montez, J.M., Carroll, K.M., Darvishzadeh, J.G. and Lee, JI. & Friedman, oo
JM. (1996). Nature 379, 632-635. . 15 6. Pelleymounter, M.A,, Cullen, M.J., Baker, M.B., Hecht, R., Winter, D., Boone, T. & Collins, F (1995). Science 269, 540-543. 7. Ghilardi, N., Ziegler, S., Wiestner, A., Stoffel, R., Heim, M.H. & Skoda, R.C. (1996). Proc. Natl.
Acad. Sci. USA 93, 6231-6235. 8. Emilsson, V., Liu, Y-L, Cawthome, M.A., Morton, N.M. & Davenport M. (1997). Diabetes 4, 17-22. 9. Montaque, C.T., Faroogi, L.S., Whitehead, J.P., Soos, M.A., Rau, H., Wareham N.J., Sewter, C.P.,
Digby J.E., Mohammed, S.N., Hurst, J.A., Cheetham, C.H., Early, AR., Barnett, A H,, Prins, J.B. &
Orahilly, S. (1997). Nature 387, 903-908. 10. Cioffi et. al. Nature Medicine, 1996, 2(5), 585. 11. A. Ullrich & J. Schlessinger, Cell, 1990, 61, 203-212. 12. EN. Ihle etal, TIBS, 1994, 19, 222.227 13. Seidel et al., Proc. Nat. Acad. Sci. USA., 1995, 92, 3041 14. Tartaglia, L.A., Dembeki, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Richards, G.J., ) Campfield, L.A., Clark, F.T., Deeds, J., Muir, C., Sankey, S., Moriaty, A., Moore, K.J., Smutko, J.S.,
Mays, G.G., Woolf, F.A., Monroe, C.A. & Tepper, R.L.(1995). Cell 83, 1263-1271. : 30 15. Damell, LE. Jr, Kerr, LM. & Stark, G.R. (1994). Science 264, 1415-1421. 16. Ghilardi N & Skoda RC (1997). Mol Endocrinol. 11, 393-399. ) 17. Ghilardi et al, Proc. Natl. Acad. Sci. 1996, 93, 6231-6235 18. Takahashi Y, Oimura Y, Mizuno 1, lida K, Takahashi T, Kaji H., Abe H. & Chihara K. (1997). J. Biol.
Chem. 272, 12897-12900. 19. Vaisse, C., Halaas,J.L., Horvath,C.M., Damell, J.E.,Stoffe]l, M. & Friedman, JM. (1996). Nature
Genet 14, 95-97 20. Kieffer, T.J., Heller, R.S., Leech, C.A., Holz, G.G. & Habener, J.F. (1997). Diabetes 46, 1087-1093. 21. Hamann, A., Busing, B., Kausch, C., Ertl, J., Preibisch, G., Greten, H. & Matthaei, S. (1997).
Diabetologia 40, 810-815. 40 22. Liu Y-L., Emilsson, V. & Cawthorne, M.A. (1997). FEBS Letters 411, 351-355. 23. Chehab, F.F., Lim, M.E. & Lu, R. (1996). Nature Genet 12, 318-320. 24. Cioffi, J., Shafer, A.W., Zupanic, T.J., Smith-Gbur, J., Mikhail, A.A., Platika, D. & Snodgrass, H.R. (1996). Nature Med 2, 585 25. Levin, N., Nelson, C., Gurney, A., Vandlen, R. & De Sauvage, F. (1996) Proc. Natl. Acad. Sci. USA 45 93, 1726-1730 . 26. Mercer, J.G., Hoggard, N., Williams, L.M., Lawrence. C.B., Hannah, L.T., Morgan, P.J. and
Trayhum, P. (1996). J. Neuroendocrinol. 8, 733-735 27. White, D.W., Kuropatwinski. K.K., Devos. R., Baumann, H. & Tartaglia, L.A. (1997). J. Biol. Chem. 14, 4065-4071.
28. Barrachima, M.D., Matinez, V., Wei, J.Y. & Tache, Y. (1997). Am. J Physiol. 41, R1007-R1011. 29. Islam M.S., Morton N.M,, Hansson, A. & Emilsson, V. (1997). Bioch. Biophys. Res. Commun. 238, 851-855. ) 30. Streamson, C., Chua Jr., Chung, W.K., Wu-Peng, S., Zhang, Y., Liu, S-M., Tartaglia, L.A. & Leibel,
R.L (1996) Science 271, 994-996. 31. White, D.W., Wang, Y.P., Chua, S.C., Morgenstern, J.P., Leibel, R.L., Baumann, H and Tartaglia,
L.A. (1997). Proc. Nail Acad. Sci. USA 94, 10657-10662. 32. Feingold, K.R., Moser, A, Adi, S., Soved, M. & Grinfeld, C. (1990). Endocrinol. 127, 2247-2252. ) 33. Angelin, B. (1997). Current Opinion in Lipidology 8, 337-341. 34. Saladin, R., De Vos, P., Guerre-Millo, M., Leturque, A., Girard, J. Stahls, B. & Auwerx, J. (1995).
Nature 377, 527-529. ’ 35. Zhou, Y.T., Shimabukuro, M., koyama, K., Lee, Y., Wang, M.Y., Trieu, F., Newgard, C.B. & Unger,
R.H. (1997). Proc. Natl. Acad. Sci. USA 94, 6386-6390. 36. Hwa, J.J, Fawzi, A.B., Graziano, M.P., Ghibaudi, L., Williams, P., Vanheek, M., Davis, H., Rudinski,
M.,, Sybertz, E. & Strader, C.D. (1997). Am. J. Physiol. 41, R1204-R1209.
Claims (11)
- 0 WO 00/00639 PCT/EP99/04501 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 clement 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), AA, where N is any nucleotide and n is 4, 5 or 6, preferably 5. : 25
- 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. 40
- 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. Akitof 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.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9814199.7A GB9814199D0 (en) | 1998-06-30 | 1998-06-30 | Novel method |
Publications (1)
Publication Number | Publication Date |
---|---|
ZA200007689B true ZA200007689B (en) | 2001-12-12 |
Family
ID=10834721
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ZA200007689A ZA200007689B (en) | 1998-06-30 | 2000-12-20 | Method for detecting of a compound that mimics, potentiates or inhibits the physiological effect of Leptin. |
Country Status (15)
Country | Link |
---|---|
EP (1) | EP1092048A2 (en) |
KR (1) | KR20010053331A (en) |
CN (1) | CN1316015A (en) |
AR (1) | AR020100A1 (en) |
AU (1) | AU4902499A (en) |
BR (1) | BR9911643A (en) |
CA (1) | CA2335557A1 (en) |
GB (1) | GB9814199D0 (en) |
HU (1) | HUP0102809A2 (en) |
IL (1) | IL140075A0 (en) |
NO (1) | NO20006541L (en) |
PL (1) | PL345382A1 (en) |
TR (1) | TR200003887T2 (en) |
WO (1) | WO2000000639A2 (en) |
ZA (1) | ZA200007689B (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106636000A (en) * | 2016-12-23 | 2017-05-10 | 广东圣赛生物科技有限公司 | hESCs-TK cell line and building method and application thereof |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0832284A1 (en) * | 1995-05-30 | 1998-04-01 | Smithkline Beecham Plc | Method for the detection of compounds that modulate the effects of the obese protein |
GB9605808D0 (en) * | 1996-03-20 | 1996-05-22 | Isis Innovation | CFTR gene regulator |
CZ149999A3 (en) * | 1996-11-01 | 1999-10-13 | Smithkline Beecham Plc | Detection method of compounds modulating activity of ob-protein |
-
1998
- 1998-06-30 GB GBGB9814199.7A patent/GB9814199D0/en not_active Ceased
-
1999
- 1999-06-28 IL IL14007599A patent/IL140075A0/en unknown
- 1999-06-28 EP EP99932753A patent/EP1092048A2/en not_active Withdrawn
- 1999-06-28 BR BR9911643-0A patent/BR9911643A/en not_active Application Discontinuation
- 1999-06-28 CA CA002335557A patent/CA2335557A1/en not_active Abandoned
- 1999-06-28 TR TR2000/03887T patent/TR200003887T2/en unknown
- 1999-06-28 PL PL99345382A patent/PL345382A1/en not_active Application Discontinuation
- 1999-06-28 CN CN99808215A patent/CN1316015A/en active Pending
- 1999-06-28 HU HU0102809A patent/HUP0102809A2/en unknown
- 1999-06-28 KR KR1020007015081A patent/KR20010053331A/en not_active Application Discontinuation
- 1999-06-28 WO PCT/EP1999/004501 patent/WO2000000639A2/en not_active Application Discontinuation
- 1999-06-28 AU AU49024/99A patent/AU4902499A/en not_active Abandoned
- 1999-06-29 AR ARP990103134A patent/AR020100A1/en unknown
-
2000
- 2000-12-20 ZA ZA200007689A patent/ZA200007689B/en unknown
- 2000-12-21 NO NO20006541A patent/NO20006541L/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
IL140075A0 (en) | 2002-02-10 |
BR9911643A (en) | 2001-03-20 |
NO20006541D0 (en) | 2000-12-21 |
NO20006541L (en) | 2000-12-21 |
KR20010053331A (en) | 2001-06-25 |
CN1316015A (en) | 2001-10-03 |
WO2000000639A3 (en) | 2000-08-17 |
HUP0102809A2 (en) | 2001-12-28 |
EP1092048A2 (en) | 2001-04-18 |
AR020100A1 (en) | 2002-04-10 |
AU4902499A (en) | 2000-01-17 |
GB9814199D0 (en) | 1998-08-26 |
CA2335557A1 (en) | 2000-01-06 |
PL345382A1 (en) | 2001-12-17 |
TR200003887T2 (en) | 2001-06-21 |
WO2000000639A2 (en) | 2000-01-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Morton et al. | Leptin action in intestinal cells | |
Laubner et al. | Inhibition of preproinsulin gene expression by leptin induction of suppressor of cytokine signaling 3 in pancreatic β-cells | |
Rosenblum et al. | Functional STAT 1 and 3 signaling by the leptin receptor (OB-R); reduced expression of the rat fatty leptin receptor in transfected cells | |
Vander Kooi et al. | The glucose-6-phosphatase catalytic subunit gene promoter contains both positive and negative glucocorticoid response elements | |
Chassande et al. | Identification of transcripts initiated from an internal promoter in the c-erbAα locus that encode inhibitors of retinoic acid receptor-α and triiodothyronine receptor activities | |
Ghilardi et al. | Defective STAT signaling by the leptin receptor in diabetic mice. | |
Luoh et al. | Cloning and characterization of a human leptin receptor using a biologically active leptin immunoadhesin | |
Wang et al. | The phosphoenolpyruvate carboxykinase gene glucocorticoid response unit: identification of the functional domains of accessory factors HNF3β (hepatic nuclear factor-3β) and HNF4 and the necessity of proper alignment of their cognate binding sites | |
De Vos et al. | Glucocorticoids induce the expression of the leptin gene through a non‐classical mechanism of transcriptional activation | |
Hurd et al. | Dual role for mitogen-activated protein kinase (Erk) in insulin-dependent regulation of Fra-1 (fos-related antigen-1) transcription and phosphorylation | |
Bailey et al. | Characterization and functional analysis of cAMP response element modulator protein and activating transcription factor 2 (ATF2) isoforms in the human myometrium during pregnancy and labor: identification of a novel ATF2 species with potent transactivation properties | |
CA2335107C (en) | Leptin-mediated gene-induction | |
Brunetti et al. | Human diabetes associated with defects in nuclear regulatory proteins for the insulin receptor gene. | |
Finidori | Regulators of growth hormone signaling | |
AU715215B2 (en) | Method for the detection of compounds that modulate the effects of the obese protein | |
Rosenblum et al. | A rapid, quantitative functional assay for measuring leptin | |
ZA200007689B (en) | Method for detecting of a compound that mimics, potentiates or inhibits the physiological effect of Leptin. | |
Sharma et al. | Analysis of the role of E2A-encoded proteins in insulin gene transcription | |
MXPA01000149A (en) | Method for detecting of a compound that mimics, potentiates or inhibits the physiological effect of leptin | |
CZ149999A3 (en) | Detection method of compounds modulating activity of ob-protein | |
BILLESTRUP et al. | Molecular mechanism of growth hormone signalling | |
CZ20004897A3 (en) | Method for detecting a compound that mimics, potentiates or inhibits the physiological effect of leptin | |
EP1121457B1 (en) | Use of socs-2 or cis to screen for compounds enhancing growth hormone effect | |
WO1999004264A1 (en) | Method for identifying modifiers of the leptin: leptin receptor interaction | |
Fletcher | Structure, expression, and regulation of two TPA-inducible genes: TIS10 and TIS21 |