WO2023240376A1 - Use of intestinal epithelial cell nuclear receptor repressor ncor as target in drug screening - Google Patents
Use of intestinal epithelial cell nuclear receptor repressor ncor as target in drug screening Download PDFInfo
- Publication number
- WO2023240376A1 WO2023240376A1 PCT/CN2022/098294 CN2022098294W WO2023240376A1 WO 2023240376 A1 WO2023240376 A1 WO 2023240376A1 CN 2022098294 W CN2022098294 W CN 2022098294W WO 2023240376 A1 WO2023240376 A1 WO 2023240376A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ncor
- intestinal epithelial
- insulin
- mice
- nuclear receptor
- Prior art date
Links
- 210000002490 intestinal epithelial cell Anatomy 0.000 title claims abstract description 60
- 102000006255 nuclear receptors Human genes 0.000 title claims abstract description 29
- 108020004017 nuclear receptors Proteins 0.000 title claims abstract description 29
- 108020005497 Nuclear hormone receptor Proteins 0.000 title claims abstract description 28
- 238000007877 drug screening Methods 0.000 title abstract 2
- 206010022489 Insulin Resistance Diseases 0.000 claims abstract description 37
- 208000008589 Obesity Diseases 0.000 claims abstract description 29
- 235000020824 obesity Nutrition 0.000 claims abstract description 29
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims abstract description 23
- 229940079593 drug Drugs 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 17
- 201000010099 disease Diseases 0.000 claims abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 12
- 238000010172 mouse model Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 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 claims description 39
- 239000008103 glucose Substances 0.000 claims description 39
- 230000000694 effects Effects 0.000 claims description 28
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 20
- 239000003613 bile acid Substances 0.000 claims description 17
- 150000002632 lipids Chemical class 0.000 claims description 17
- 230000000968 intestinal effect Effects 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 12
- 239000003124 biologic agent Substances 0.000 claims description 11
- 206010012601 diabetes mellitus Diseases 0.000 claims description 11
- 239000003112 inhibitor Substances 0.000 claims description 11
- 238000010521 absorption reaction Methods 0.000 claims description 10
- 206010060378 Hyperinsulinaemia Diseases 0.000 claims description 7
- 230000003451 hyperinsulinaemic effect Effects 0.000 claims description 7
- 201000008980 hyperinsulinism Diseases 0.000 claims description 7
- 230000037356 lipid metabolism Effects 0.000 claims description 7
- 230000028327 secretion Effects 0.000 claims description 7
- 235000012000 cholesterol Nutrition 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 5
- 230000003914 insulin secretion Effects 0.000 claims description 5
- 230000004783 oxidative metabolism Effects 0.000 claims description 5
- 230000037361 pathway Effects 0.000 claims description 5
- 208000002705 Glucose Intolerance Diseases 0.000 claims description 4
- 206010018429 Glucose tolerance impaired Diseases 0.000 claims description 4
- 208000035150 Hypercholesterolemia Diseases 0.000 claims description 4
- 208000031226 Hyperlipidaemia Diseases 0.000 claims description 4
- 239000000859 incretin Substances 0.000 claims description 4
- MGXWVYUBJRZYPE-YUGYIWNOSA-N incretin Chemical class C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=C(O)C=C1 MGXWVYUBJRZYPE-YUGYIWNOSA-N 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 150000003626 triacylglycerols Chemical class 0.000 claims description 4
- 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 claims description 3
- 238000005265 energy consumption Methods 0.000 claims description 3
- 230000029142 excretion Effects 0.000 claims description 3
- 230000004153 glucose metabolism Effects 0.000 claims description 3
- 230000003993 interaction Effects 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 2
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 claims 1
- 102100025101 GATA-type zinc finger protein 1 Human genes 0.000 claims 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims 1
- 230000031891 intestinal absorption Effects 0.000 claims 1
- 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 abstract description 52
- 102000004877 Insulin Human genes 0.000 abstract description 26
- 108090001061 Insulin Proteins 0.000 abstract description 26
- 229940125396 insulin Drugs 0.000 abstract description 26
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 206010070834 Sensitisation Diseases 0.000 abstract 1
- 230000008313 sensitization Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 81
- 235000009200 high fat diet Nutrition 0.000 description 51
- 210000004369 blood Anatomy 0.000 description 41
- 239000008280 blood Substances 0.000 description 41
- 239000000243 solution Substances 0.000 description 15
- 108010088406 Glucagon-Like Peptides Proteins 0.000 description 14
- 235000021588 free fatty acids Nutrition 0.000 description 14
- 230000037396 body weight Effects 0.000 description 12
- 210000004185 liver Anatomy 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 210000003608 fece Anatomy 0.000 description 10
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 238000011813 knockout mouse model Methods 0.000 description 6
- 210000000496 pancreas Anatomy 0.000 description 6
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 5
- 210000000577 adipose tissue Anatomy 0.000 description 5
- 230000037149 energy metabolism Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 210000000579 abdominal fat Anatomy 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 102000004311 liver X receptors Human genes 0.000 description 4
- 108090000865 liver X receptors Proteins 0.000 description 4
- 235000021590 normal diet Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- ZOBPZXTWZATXDG-UHFFFAOYSA-N 1,3-thiazolidine-2,4-dione Chemical compound O=C1CSC(=O)N1 ZOBPZXTWZATXDG-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229940122355 Insulin sensitizer Drugs 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010016731 PPAR gamma Proteins 0.000 description 3
- 102100038825 Peroxisome proliferator-activated receptor gamma Human genes 0.000 description 3
- 229940123464 Thiazolidinedione Drugs 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000037406 food intake Effects 0.000 description 3
- 235000012631 food intake Nutrition 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 210000003405 ileum Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000037323 metabolic rate Effects 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000013116 obese mouse model Methods 0.000 description 3
- 238000007410 oral glucose tolerance test Methods 0.000 description 3
- 230000036284 oxygen consumption Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- GZCGUPFRVQAUEE-NKKIJKBOSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxy-3-tritiohexanal Chemical compound O=C[C@H](O)[C@](O)([3H])[C@H](O)[C@H](O)CO GZCGUPFRVQAUEE-NKKIJKBOSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000008169 Co-Repressor Proteins Human genes 0.000 description 2
- 108010060434 Co-Repressor Proteins Proteins 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 230000001610 euglycemic effect Effects 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 230000000910 hyperinsulinemic effect Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 238000012528 insulin ELISA Methods 0.000 description 2
- 210000004731 jugular vein Anatomy 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000006372 lipid accumulation Effects 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035924 thermogenesis Effects 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- DRCWOKJLSQUJPZ-DZGCQCFKSA-N (4ar,9as)-n-ethyl-1,4,9,9a-tetrahydrofluoren-4a-amine Chemical compound C1C2=CC=CC=C2[C@]2(NCC)[C@H]1CC=CC2 DRCWOKJLSQUJPZ-DZGCQCFKSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 102100038495 Bile acid receptor Human genes 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 1
- 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 1
- 102100039994 Gastric inhibitory polypeptide Human genes 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 101000603876 Homo sapiens Bile acid receptor Proteins 0.000 description 1
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 1
- 101000886868 Homo sapiens Gastric inhibitory polypeptide Proteins 0.000 description 1
- 101001008429 Homo sapiens Nucleobindin-2 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102100027441 Nucleobindin-2 Human genes 0.000 description 1
- 102000023984 PPAR alpha Human genes 0.000 description 1
- 108010015181 PPAR delta Proteins 0.000 description 1
- 108010044210 PPAR-beta Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229940090124 dipeptidyl peptidase 4 (dpp-4) inhibitors for blood glucose lowering Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000081 effect on glucose Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 230000004136 fatty acid synthesis Effects 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000003614 peroxisome proliferator Substances 0.000 description 1
- 108091008725 peroxisome proliferator-activated receptors alpha Proteins 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 230000028503 regulation of lipid metabolic process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229920000260 silastic Polymers 0.000 description 1
- 229960004034 sitagliptin Drugs 0.000 description 1
- MFFMDFFZMYYVKS-SECBINFHSA-N sitagliptin Chemical compound C([C@H](CC(=O)N1CC=2N(C(=NN=2)C(F)(F)F)CC1)N)C1=CC(F)=C(F)C=C1F MFFMDFFZMYYVKS-SECBINFHSA-N 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 150000001467 thiazolidinediones Chemical class 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 1
- 229950004616 tribromoethanol Drugs 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 108090000195 villin Proteins 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Definitions
- the invention belongs to the field of medical technology, relates to the field of genetic engineering technology, and particularly relates to the application of a new target intestinal epithelial cell nuclear receptor inhibitor NCoR for preventing and treating insulin resistance and obesity-related diseases.
- Diabetes and obesity have become one of the major chronic diseases that threaten the health of urban and rural residents in my country. Their incidence rates are increasing year by year in China and around the world, and they have become a major public health problem. Diabetes is mainly divided into type 1 and type 2 diabetes. Type 1 diabetes is an autoimmune disease. The main reason is the necrosis of pancreatic beta cells and the absolute lack of insulin secretion. The pathogenesis and pathophysiological process of type 2 diabetes are complex and involve polygenic and multi-organ abnormalities. Insulin resistance is the most important pathophysiological defect during the pathogenesis, and obesity and related chronic inflammation are the main causes of insulin resistance. , studies in recent years have shown that intestinal flora is also one of the important influencing factors.
- TZD insulin sensitizer thiazolidinedione
- the nuclear receptor inhibitor NCoR is one of the nuclear receptor corepressor proteins.
- the nuclear receptor superfamily includes peroxisome proliferator receptor PPAR ⁇ , PPAR ⁇ / ⁇ , PPAR ⁇ , farnesoid X receptor FXR and liver X receptor. LXR, etc., are all involved in the regulation of lipid metabolism and inflammatory pathways, and the target of TZD is PPAR ⁇ .
- corepressor proteins such as NCoR bind to the upstream promoter region of nuclear receptors and inhibit their activity; when ligands bind to the ligand-binding domain LBD, nuclear receptors dissociate from NCoR, etc. , recruiting coactivators to exert their transcriptional regulatory effects.
- NCoR is widely expressed in many tissues, and its systemic knockout can be fatal. Studies on conditional knockout of NCoR in different tissues have found that although NCoR can interact with a variety of nuclear receptors, in different cells, NCoR knockout will specifically activate certain nuclear receptors to regulate gene transcription. The functions are not the same in different cells.
- NCoR In adipose tissue [5] , knockout of NCoR can significantly reduce macrophage infiltration and inflammation in adipose tissue of high-fat fed mice through activation of PPAR ⁇ , and improve insulin resistance in insulin target tissues; NCoR in macrophages [7] Conditional knockout of LXR can relieve the inhibition of LXR, activate gene expression of fatty acid synthesis pathway, increase the synthesis of anti-inflammatory fatty acids, inhibit NF- ⁇ B-dependent inflammatory response, thereby improving the overall inflammatory state and increasing insulin sensitivity; while in muscle Tissue [11] , the main effect of NCoR knockout is the activation of PPAR ⁇ and so on.
- NCoR will also show activation of a specific nuclear receptor after being knocked out in intestinal epithelial cells, and further regulate the absorption and metabolism of nutrients such as lipids, the metabolism of bile acids, and the incretin GLP-1 secretion and composition of intestinal flora, thereby regulating overall insulin sensitivity.
- the technical problem solved by the present invention is to provide the application of the intestinal epithelial cell nuclear receptor inhibitor NCoR as a target in screening or preparing drugs or biological preparations for preventing, alleviating or treating insulin resistance, obesity and related diseases, thereby providing new solutions for diabetes and obesity. Treatments such as these provide an effective solution.
- Another technical problem solved by the present invention is to provide an application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in preparing insulin-sensitizing or lipid-lowering mouse models, which is specifically verified at the animal level by knocking out mouse intestinal epithelial cells.
- NCoR can increase glucose tolerance and clearance, reduce lipid absorption, and promote lipid metabolism and clearance.
- the first aspect of the technical solution of the present invention is to provide an application of the intestinal epithelial cell nuclear receptor inhibitor NCoR as a target in screening or preparing drugs or biological agents for preventing, alleviating or treating insulin resistance, obesity and related diseases.
- the insulin resistance, obesity and related diseases are diabetes, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, obesity and glucose intolerance.
- the biological agent or the drug is used to inhibit the interaction between the nuclear receptor NCoR of intestinal epithelial cells and the nuclear receptor, change the content and composition of bile acids, and reduce the content of lipids such as intestinal triglycerides and cholesterol. Absorption, increase its excretion; promote oxidative metabolism heat production and energy consumption; promote the secretion of incretin GLP-1, regulate insulin secretion and glucose and lipid metabolism pathways.
- the above-mentioned effects of the biological agent or the drug do not depend on changes in intestinal flora.
- the nuclear receptors include Lxr, Ppar ⁇ or Fxr.
- the second aspect of the technical solution of the present invention is to provide the application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in preparing insulin-sensitizing or lipid-lowering mouse models.
- the inhibition of NCoR includes, but is not limited to, knocking out the gene by genetic recombination through the Cre-LoxP system to obtain an insulin-sensitizing or lipid-lowering mouse model with tissue-specific knockout of NCoR.
- the insulin-sensitizing or lipid-lowering mouse model with tissue-specific knockout of NCoR can increase glucose tolerance and clearance capacity, reduce lipid absorption capacity, and promote lipid metabolism and clearance capacity.
- the tissue is selected from intestinal epithelial cells.
- the third aspect of the technical solution of the present invention is the application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in the preparation of kits for screening drugs or biological agents for preventing, alleviating or treating insulin resistance, obesity and related diseases.
- the insulin resistance, obesity and related diseases are diabetes, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, obesity and glucose intolerance.
- the biological agent or the drug is used to inhibit the interaction between the nuclear receptor NCoR of intestinal epithelial cells and the nuclear receptor, change the content and composition of bile acids, and reduce the content of lipids such as intestinal triglycerides and cholesterol. Absorption, increase its excretion; promote oxidative metabolism heat production and energy consumption; promote the secretion of incretin GLP-1, regulate insulin secretion and glucose and lipid metabolism pathways.
- the above-mentioned effects of the biological agent or the drug do not depend on changes in intestinal flora. Beneficial technical effects:
- the present invention found that conditionally knocking out the NCoR gene of intestinal epithelial cells in mice to obtain NCoR tissue-specific knockout mice, and then feeding them with a high-fat diet, can significantly reverse the obesity and insulin resistance of the mice. type, reduce body weight, reduce liver and abdominal fat weight, improve oral glucose tolerance and insulin sensitivity, and use the gold standard hyperinsulinemic euglycemic clamp test to evaluate, further accurately proving the overall and target organs such as liver and adipose tissue. Insulin sensitivity was significantly improved.
- conditional knockout of NCoR in intestinal epithelial cells of mice can also significantly improve hyperinsulinemia and liver lipid accumulation in obese mice, change the content and composition of bile acids, reduce intestinal lipid absorption, and stimulate active pancreatic Secretion of glucagon-like peptide (GLP-1).
- GLP-1 glucagon-like peptide
- conditional intestinal epithelial cell NCoR knockout mice by co-raising wild-type mice with conditional intestinal epithelial cell NCoR knockout mice, it was proven that the phenotypic improvement effect of conditional intestinal epithelial cell NCoR knockout is not mediated by intestinal flora. .
- the inventors discovered for the first time that conditional knockout of NCoR in intestinal epithelial cells can improve overall insulin sensitivity and reduce body weight, indicating that it can be used as a target.
- By inhibiting the NCoR of intestinal epithelial cells it increases the secretion of the incretin hormone GLP-1, changes the content and composition of bile acids, inhibits intestinal lipid absorption, increases energy metabolic rate, etc., thereby improving lipid metabolism, insulin resistance and obesity. , helps to change the current situation of lack of clinical insulin sensitizers, and has positive significance for the prevention and treatment of type 2 diabetes and obesity.
- FIG 1 shows the effects of conditional intestinal epithelial cell NCoR knockout of the present invention on body weight and tissue weight
- A shows wild-type mice (WT mice) and intestinal epithelial cell NCoR knockout mice ( IKO mice);
- B is the average weekly food intake of WT and IKO mice during high-fat diet feeding;
- C is the main organs liver and abdominal cavity of WT and IKO mice after high-fat diet feeding The wet weight of fat (WAT) and pancreas (Pancreas);
- D is the proportion of liver, abdominal fat (WAT) and pancreas (Pancreas) in the body weight of the main organ of WT and IKO mice after feeding with high-fat diet.
- Figure 2 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on the insulin sensitivity phenotype of mice fed normal diet and high-fat diet;
- FIG 3 shows the effects of NCoR knockout in conditional intestinal epithelial cells of the present invention on hormone secretion in high-fat diet-fed mice: A. Fasting insulin levels in WT and IKO mice after high-fat diet feeding and 15-minute blood insulin levels after sugar stimulation; B. Fasting glucose-insulin-stimulating hormone-1 (GLP-1) levels in WT and IKO mice after high-fat diet feeding; C. GLP-1 levels 10 minutes after glucose stimulation in WT and IKO mice after high-fat diet feeding.
- GLP-1 glucose-insulin-stimulating hormone-1
- Figure 4 shows the results of a hyperinsulinemic euglycemic clamp experiment to accurately evaluate insulin sensitivity in high-fat diet-fed mice after NCoR knockout in conditional intestinal epithelial cells of the present invention
- a glucose infusion rate B glucose disposal rate
- D Inhibition rate of hepatic glucose output by insulin at steady state compared with basal state
- E Inhibition rate of blood free fatty acid levels by insulin at steady state compared with basal state
- F Blood insulin levels at basal and steady state.
- Figure 5 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on lipid levels in mice fed a high-fat diet; A blood triglyceride level; B blood total cholesterol level; C blood free fatty acid level; D liver glycerol Triglyceride level; E total cholesterol level in liver; F free fatty acid level in liver; G triglyceride level in feces; H total cholesterol level in feces; I free fatty acid level in feces.
- Figure 6 is a co-house experiment of conditional intestinal epithelial cell NCoR knockout mice and wild-type mice of the present invention
- A the co-housed group (CO-WT and CO-IKO) after high-fat diet feeding Oral glucose tolerance of mice and mice in separate feeding groups (WT and IKO);
- B Insulin of mice in co-raising group (CO-WT and CO-IKO) and mice in separate feeding group (WT and IKO) after high-fat diet feeding Tolerance;
- C Body weight changes of mice in the co-feeding group (CO-WT and CO-IKO) and separate feeding groups (WT and IKO) during high-fat diet feeding;
- D After high-fat diet feeding in the co-feeding group (CO- Fasting insulin levels and blood insulin levels 10 minutes after glucose stimulation in WT and CO-IKO) mice and in separate rearing groups (WT and IKO) mice.
- Figure 7 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on energy metabolism in mice fed a high-fat diet.
- Figure 8 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on the bile acid composition of mice fed a high-fat diet.
- A The composition of bile acids in the blood of WT and IKO mice after feeding with a high-fat diet
- B The composition of bile acids in the ileum of WT and IKO mice after feeding with a high-fat diet
- C The composition of bile acids in the feces of WT and IKO mice after feeding with a high-fat diet
- NCoR flox/flox mice were backcrossed with Villin Cre tool mice for several generations to obtain male homozygous mice (IKO) with conditional knockout of NCoR in intestinal epithelial cells.
- IKO male homozygous mice
- NCoR flox mice with corresponding birth dates and genders were selected.
- /flox mice served as wild-type control mice (WT). There were 10-15 mice in each group of WT and IKO groups. They were fed a high-fat diet (60% of calories came from fat, purchased from Research Diets Company), and changes in body weight and food intake were monitored weekly. After 15 weeks of high-fat diet feeding, the mice were sacrificed, and the liver, abdominal fat, and pancreas were removed and weighed and recorded.
- NCoR knockout in conditional intestinal epithelial cells has no effect on the body weight of mice fed a normal diet, but can significantly inhibit high-fat diet-induced obesity, and significantly reduce the weight of abdominal fat and liver and their respective proportions in body weight. , it can also significantly increase the proportion of pancreas to body weight; but has no significant effect on the food intake of mice.
- mice in the WT and IKO groups in Example 1 were fed with the high-fat diet, an oral glucose tolerance test was performed while still being fed with normal feed. After the mice were fasted for 6 hours, blood was taken from the tail tip to measure fasting (0 time) blood glucose, and then glucose solution (2g/kg) was administered intragastrically at 15 minutes, 30 minutes, 60 minutes and 120 minutes after glucose stimulation. Take blood to measure blood sugar level. At the 9th week of high-fat diet feeding, an oral glucose tolerance test was conducted with the same method as above. At the 10th week of high-fat diet feeding, an insulin tolerance test was conducted.
- mice After the mice were fasted for 6 hours, blood was taken from the tail tip to measure fasting (0 time) blood glucose, and then insulin (0.3 U/kg) was injected subcutaneously at the tail tip 15 minutes, 30 minutes, 60 minutes and 120 minutes after insulin injection. Take blood to measure blood sugar level.
- conditional knockout of NCoR in intestinal epithelial cells had no effect on glucose tolerance in mice fed a normal diet, but could significantly improve glucose tolerance and insulin sensitivity abnormalities caused by high-fat diet feeding.
- Example 3 Effects of NCoR knockout in conditional intestinal epithelial cells on insulin and glucoinsulin-stimulating hormone
- Example 1 In the 11th week of high-fat feeding, the WT and IKO mice in Example 1 were fasted for 6 hours. Blood was collected from the tail tip, placed on ice, and glucose solution (2g/kg) was administered orally. Collect blood from the tip of the tail and place on ice. Centrifuge at 12,000 rpm for 1 min, aspirate the supernatant, and measure blood insulin levels using Alpco mouse insulin ELISA kit.
- the WT and IKO mice in Example 1 were fasted for 6 hours, and sitagliptin (25 mg/kg, DPP4 enzyme inhibitor) was administered intragastrically at 5.25 hours after fasting.
- Glucose solution (2g/kg) was administered intragastrically every hour.
- Blood was collected from the tail tip 15 minutes after glucose stimulation and placed in a centrifuge tube with EDTA anticoagulant and protease inhibitor added in advance. Place it on ice and centrifuge at 1000g for 10 minutes at 4 degrees. , draw the supernatant, and use Alpco's mouse active GLP-1 ELISA kit to measure blood active GLP-1 levels.
- the WT and IKO mice in Example 1 were fasted for 4 hours, anesthetized with carbon dioxide, and the thorax and heart were opened to collect blood. In the blood vessel, centrifuge at 1000g and 4 degrees for 20 minutes, aspirate the supernatant, and use the Alpco mouse active GLP-1 ELISA kit to measure the blood active GLP-1 level.
- conditional intestinal epithelial cell NCoR knockout can significantly improve hyperinsulinemia caused by high-fat diet and obesity, and significantly increase the levels of active intestinal insulin hormone GLP-1 after fasting and 15 minutes of sugar stimulation, indicating that NCoR Knockout can promote insulin secretion and improve insulin sensitivity by increasing the level of GLP-1.
- Example 4 Precise evaluation of the effect of NCoR knockout on insulin sensitivity in conditional intestinal epithelial cells
- the glucose infusion rate at this time is the GIR
- the blood sample at the steady state is taken.
- use the steele formula to calculate the hepatic glucose output HGP at the basal state and steady state and calculate the inhibition rate, glucose disposal rate GDR, and the main response muscles Insulin-stimulated glucose disposal rate of glucose uptake rate IS-GDR.
- Blood free fatty acid FFA and blood insulin levels were measured at basal and steady states, and the inhibition rate of insulin on free fatty acid levels, which mainly reflects the insulin sensitivity of adipose tissue, was calculated.
- conditional intestinal epithelial cell NCoR knockout can significantly increase the glucose infusion rate GIR, glucose disposal rate GDR and exogenous insulin in the IKO group of mice compared with WT mice.
- the inhibition rate of glucose output and the inhibition rate of free fatty acids, while the insulin-stimulated glucose disposal rate IS-GDR, which reflects muscle insulin sensitivity, has an increasing trend, but there is no significant difference, indicating that conditional intestinal epithelial cell NCoR knockout It can significantly improve the insulin sensitivity of mice overall and in liver and adipose tissue.
- Example 5 Effects of conditional intestinal epithelial cell NCoR knockout on lipid levels, etc.
- the WT and IKO mice in Example 1 were sacrificed after being fed a high-fat diet for 15 weeks, and blood was taken from the heart.
- the total triglyceride (TG) and total cholesterol (TC) kits of Zhongsheng Beikong Company were used to measure the blood levels.
- the contents of TG and TC were measured using the NEFA kit from Wako Company to determine the level of free fatty acids (FFA) in the blood.
- Example 1 After the WT and IKO mice in Example 1 were fed a high-fat diet for 15 weeks, they were sacrificed and their livers were removed and cryopreserved. After grinding and homogenizing small pieces of tissue, the corresponding lipid levels were measured using the aforementioned TG, TC and FFA kits.
- each mouse was raised in a single cage, and the feces collected for 24 hours was dried at 60 degrees and methanol:chloroform (1:2, V/V ), extract at 37 degrees for 12 hours, take the supernatant, evaporate to dryness again, and redissolve the precipitate by adding 10% TritonX-100 isopropyl alcohol solution.
- the corresponding lipid levels were determined using the aforementioned TG, TC and FFA kits.
- conditional intestinal epithelial cell NCoR knockout can significantly reduce the blood triglyceride and total cholesterol levels of IKO group mice compared with the WT group of mice, but has no significant effect on blood free fatty acid levels. Influence. Liver triglyceride, total cholesterol and free fatty acid contents in the IKO group were also significantly reduced, indicating that conditional intestinal epithelial cell NCoR knockout can also significantly improve liver lipid accumulation caused by high-fat diet and obesity.
- conditional intestinal epithelial cell NCoR knockout may inhibit intestinal lipid absorption, thereby reducing weight, improve obesity, etc.
- Another batch of WT and IKO mice obtained by the method in Example 1 were divided into 4 groups from the beginning of weaning.
- the WT and IKO groups were raised separately according to genotype from the beginning of weaning after 3 weeks of age.
- the CO-WT and CO-IKO groups were mixed and raised starting from weaning at 3 weeks of age, so as to compare the phenotypic differences between different groups to determine the intestinal flora in conditional intestinal epithelial cell NCoR knockout mice. role in phenotype.
- the mice were fed a high-fat diet for 10 weeks, and an oral glucose tolerance test was performed. The method was the same as in Example 2. In the 12th week of high-fat diet feeding, an insulin tolerance test was conducted, and the method was the same as in Example 2.
- Example 7 Effects of conditional intestinal epithelial cell NCoR knockout on energy metabolism in high-fat diet-fed mice.
- Example 8 Effects of conditional intestinal epithelial cell NCoR knockout on bile acid composition in high-fat diet-fed mice.
- mice in Example 1 were fed a high-fat diet for 15 weeks, the mice were sacrificed, feces, ileal mucosal tissue and blood samples were collected. After bile acid extraction, the UPLC/Synapt G2-Si QTOF MS system was used to determine its content concentration. .
Abstract
The present invention belongs to the technical field of medicines, and relates to the technical field of genetic engineering, in particular to use of an intestinal epithelial cell nuclear receptor repressor NCoR as a new target for preventing and treating insulin resistance and obesity-related diseases. Disclosed is use of an intestinal epithelial cell nuclear receptor repressor NCoR as a target in drug screening. Specifically disclosed is use of an intestinal epithelial cell nuclear receptor repressor NCoR as a target in screening or preparing a drug or biological preparation for preventing, relieving, or treating insulin resistance, obesity, and related diseases. Also disclosed is use of the intestinal epithelial cell nuclear receptor repressor NCoR in preparing an insulin sensitization or lipid-lowering mouse model.
Description
本发明属于医药技术领域，涉及基因工程技术领域，特别是涉及一种防治胰岛素抵抗和肥胖相关疾病的新靶标肠道上皮细胞核受体抑制子NCoR的应用。The invention belongs to the field of medical technology, relates to the field of genetic engineering technology, and particularly relates to the application of a new target intestinal epithelial cell nuclear receptor inhibitor NCoR for preventing and treating insulin resistance and obesity-related diseases.
糖尿病及肥胖成为威胁我国城乡居民健康的主要慢性病之一，在中国及全球的发病率逐年攀升，已成为主要的公共卫生问题。糖尿病主要分为1型和2型糖尿病，1型糖尿病属于自身免疫性疾病，主要原因是胰岛beta细胞坏死，胰岛素分泌绝对不足。2型糖尿病的发病机制和病理生理过程复杂，涉及多基因多器官异常，在发病过程中胰岛素抵抗是其最重要的病生理缺陷，而肥胖及其相关的慢性炎症则是导致胰岛素抵抗的主要原因，近些年研究表明肠道菌群也是重要影响因素之一。Diabetes and obesity have become one of the major chronic diseases that threaten the health of urban and rural residents in my country. Their incidence rates are increasing year by year in China and around the world, and they have become a major public health problem. Diabetes is mainly divided into type 1 and type 2 diabetes. Type 1 diabetes is an autoimmune disease. The main reason is the necrosis of pancreatic beta cells and the absolute lack of insulin secretion. The pathogenesis and pathophysiological process of type 2 diabetes are complex and involve polygenic and multi-organ abnormalities. Insulin resistance is the most important pathophysiological defect during the pathogenesis, and obesity and related chronic inflammation are the main causes of insulin resistance. , studies in recent years have shown that intestinal flora is also one of the important influencing factors.
目前临床上应用的糖尿病治疗药物中明确可改善胰岛素抵抗的只有胰岛素增敏剂噻唑烷二酮(TZD)类化合物，但是TZD类药物因增重和心血管风险等副作用而被撤市或限制应用
[3]，因此，实际上目前并没有较好的胰岛素增敏剂。因此，研究开发新的糖尿病治疗靶点和 药物势在必行，有着重要的社会价值。
Among currently clinically used diabetes treatment drugs, only the insulin sensitizer thiazolidinedione (TZD) compounds are clearly able to improve insulin resistance. However, TZD drugs have been withdrawn from the market or restricted in use due to side effects such as weight gain and cardiovascular risk. [3] Therefore, in fact, there is currently no better insulin sensitizer. Therefore, it is imperative to research and develop new diabetes treatment targets and drugs, which has important social value.
核受体抑制子NCoR属于核受体共抑制蛋白之一，核受体超家族包括过氧化物酶增殖体受体PPARα，PPARβ/δ，PPARγ，法尼醇X受体FXR和肝脏X受体LXR等，都参与了脂质代谢和炎症通路的调节，TZD的作用靶点即为PPARγ。在没有配体结合的情况下，NCoR等共抑制蛋白结合在核受体上游启动子区，抑制其活性；而当配体与配体结合结构域LBD结合后，核受体与NCoR等解离，招募共激活蛋白来发挥其转录调控作用。The nuclear receptor inhibitor NCoR is one of the nuclear receptor corepressor proteins. The nuclear receptor superfamily includes peroxisome proliferator receptor PPARα, PPARβ/δ, PPARγ, farnesoid X receptor FXR and liver X receptor. LXR, etc., are all involved in the regulation of lipid metabolism and inflammatory pathways, and the target of TZD is PPARγ. In the absence of ligand binding, corepressor proteins such as NCoR bind to the upstream promoter region of nuclear receptors and inhibit their activity; when ligands bind to the ligand-binding domain LBD, nuclear receptors dissociate from NCoR, etc. , recruiting coactivators to exert their transcriptional regulatory effects.
NCoR在多组织广泛表达，其全身性敲除可致死。针对不同组织NCoR条件性敲除的研究发现，虽然NCoR可与多种核受体相互作用，但在不同细胞，NCoR敲除后会特异性的活化某种核受体来调节基因转录，其在不同细胞中功能并不相同。在脂肪组织
[5]，NCoR被敲除后通过活化PPARγ，可显著减轻高脂喂养小鼠脂肪组织巨噬细胞浸润和炎症状态，改善胰岛素靶组织的胰岛素抵抗；NCoR在巨噬细胞
[7]的条件性敲除则可解除对LXR的抑制，激活脂肪酸合成通路基因表达，增加抗炎脂肪酸的合成，抑制NF-κB依赖的炎症反应，从而改善整体炎症状态，增加胰岛素敏感性；而在肌肉组织
[11]，NCoR敲除后的主要作用则是PPARδ等的活化。因此我们认为，NCoR在肠道上皮细胞被敲除后也会表现为某种特定核受体受到活化，并进一步调节脂质等营养物质的吸收代谢、胆汁酸的代谢、肠促胰岛素GLP-1的分泌以及肠道菌群组成等，从而调节整体胰岛素敏感性。
NCoR is widely expressed in many tissues, and its systemic knockout can be fatal. Studies on conditional knockout of NCoR in different tissues have found that although NCoR can interact with a variety of nuclear receptors, in different cells, NCoR knockout will specifically activate certain nuclear receptors to regulate gene transcription. The functions are not the same in different cells. In adipose tissue [5] , knockout of NCoR can significantly reduce macrophage infiltration and inflammation in adipose tissue of high-fat fed mice through activation of PPARγ, and improve insulin resistance in insulin target tissues; NCoR in macrophages [7] Conditional knockout of LXR can relieve the inhibition of LXR, activate gene expression of fatty acid synthesis pathway, increase the synthesis of anti-inflammatory fatty acids, inhibit NF-κB-dependent inflammatory response, thereby improving the overall inflammatory state and increasing insulin sensitivity; while in muscle Tissue [11] , the main effect of NCoR knockout is the activation of PPARδ and so on. Therefore, we believe that NCoR will also show activation of a specific nuclear receptor after being knocked out in intestinal epithelial cells, and further regulate the absorption and metabolism of nutrients such as lipids, the metabolism of bile acids, and the incretin GLP-1 secretion and composition of intestinal flora, thereby regulating overall insulin sensitivity.
到目前为止，关于对肠道上皮细胞NCoR在肥胖、胰岛素抵抗和糖尿病等发生发展中的作用未见报道，关于以肠道上皮细胞NCoR为靶点治疗肥胖和糖尿病的药物和生物制剂也未见报道。So far, there are no reports on the role of intestinal epithelial cell NCoR in the development of obesity, insulin resistance, and diabetes. There are also no reports on drugs and biological agents that target intestinal epithelial cell NCoR to treat obesity and diabetes. Report.
发明内容Contents of the invention
本发明解决的技术问题是提供了肠道上皮细胞核受体抑制子NCoR作为靶标在筛选或制备预防、缓解或治疗胰岛素抵抗、肥胖及相关疾病的药物或生物制剂中的应用，从而为糖尿病和肥胖等的治疗提供一种有效的解决办法。The technical problem solved by the present invention is to provide the application of the intestinal epithelial cell nuclear receptor inhibitor NCoR as a target in screening or preparing drugs or biological preparations for preventing, alleviating or treating insulin resistance, obesity and related diseases, thereby providing new solutions for diabetes and obesity. Treatments such as these provide an effective solution.
本发明解决的另一技术问题是提供一种肠道上皮细胞核受体抑制子NCoR在制备胰岛素增敏或降脂小鼠模型中的应用，具体在动物水平验证通过敲除小鼠肠道上皮细胞NCoR的表达，能够增加葡萄糖耐受和清除能力，减少脂质吸收能力，促进脂质代谢和清除能力。Another technical problem solved by the present invention is to provide an application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in preparing insulin-sensitizing or lipid-lowering mouse models, which is specifically verified at the animal level by knocking out mouse intestinal epithelial cells. The expression of NCoR can increase glucose tolerance and clearance, reduce lipid absorption, and promote lipid metabolism and clearance.
为此，本发明提供了如下技术方案：To this end, the present invention provides the following technical solutions:
本发明技术方案的第一方面是提供了一种以肠道上皮细胞核受体抑制子NCoR作为靶标在筛选或制备预防、缓解或治疗胰岛素抵抗、肥胖及相关疾病的药物或生物制剂中的应用。The first aspect of the technical solution of the present invention is to provide an application of the intestinal epithelial cell nuclear receptor inhibitor NCoR as a target in screening or preparing drugs or biological agents for preventing, alleviating or treating insulin resistance, obesity and related diseases.
优选的是，所述胰岛素抵抗、肥胖及相关疾病为糖尿病、高胰岛血症、高血脂症、高胆固醇血症、肥胖及葡萄糖不耐性。Preferably, the insulin resistance, obesity and related diseases are diabetes, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, obesity and glucose intolerance.
优选的是，所述生物制剂或所述药物用于抑制肠道上皮细胞核受体NCoR与核受体的相互作用，改变胆汁酸的含量和组成，减少肠道甘油三酯、胆固醇等脂质的吸收，增加其排泄；促进氧化代谢产热和能量消耗；促进肠促胰岛素GLP-1的分泌，调节胰岛素分泌及糖脂代 谢通路。所述生物制剂或所述药物上述作用并不依赖于肠道菌群变化。Preferably, the biological agent or the drug is used to inhibit the interaction between the nuclear receptor NCoR of intestinal epithelial cells and the nuclear receptor, change the content and composition of bile acids, and reduce the content of lipids such as intestinal triglycerides and cholesterol. Absorption, increase its excretion; promote oxidative metabolism heat production and energy consumption; promote the secretion of incretin GLP-1, regulate insulin secretion and glucose and lipid metabolism pathways. The above-mentioned effects of the biological agent or the drug do not depend on changes in intestinal flora.
所述的核受体包括Lxr，Pparα或Fxr。The nuclear receptors include Lxr, Pparα or Fxr.
本发明技术方案的第二方面是提供一种肠道上皮细胞核受体抑制子NCoR在制备胰岛素增敏或降脂小鼠模型中的应用。The second aspect of the technical solution of the present invention is to provide the application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in preparing insulin-sensitizing or lipid-lowering mouse models.
优选的是，所述抑制NCoR是，包括但不限于，通过Cre-LoxP系统以基因重组方式敲除该基因，以获得NCoR组织特异性敲除的胰岛素增敏或降脂小鼠模型。Preferably, the inhibition of NCoR includes, but is not limited to, knocking out the gene by genetic recombination through the Cre-LoxP system to obtain an insulin-sensitizing or lipid-lowering mouse model with tissue-specific knockout of NCoR.
优选的是，所述NCoR组织特异性敲除的胰岛素增敏或降脂小鼠模型能够增加葡萄糖耐受和清除能力，减少脂质吸收能力，促进脂质代谢和清除能力。Preferably, the insulin-sensitizing or lipid-lowering mouse model with tissue-specific knockout of NCoR can increase glucose tolerance and clearance capacity, reduce lipid absorption capacity, and promote lipid metabolism and clearance capacity.
优选的是，所述的组织选自肠道上皮细胞。Preferably, the tissue is selected from intestinal epithelial cells.
本发明技术方案的第三方面一种肠道上皮细胞核受体抑制子NCoR在制备筛选预防、缓解或治疗胰岛素抵抗、肥胖及相关疾病药物或生物制剂的试剂盒中的应用。The third aspect of the technical solution of the present invention is the application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in the preparation of kits for screening drugs or biological agents for preventing, alleviating or treating insulin resistance, obesity and related diseases.
所述胰岛素抵抗、肥胖及相关疾病为糖尿病、高胰岛血症、高血脂症、高胆固醇血症、肥胖及葡萄糖不耐性。The insulin resistance, obesity and related diseases are diabetes, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, obesity and glucose intolerance.
优选的是，所述生物制剂或所述药物用于抑制肠道上皮细胞核受体NCoR与核受体的相互作用，改变胆汁酸的含量和组成，减少肠道甘油三酯、胆固醇等脂质的吸收，增加其排泄；促进氧化代谢产热和能量消耗；促进肠促胰岛素GLP-1的分泌，调节胰岛素分泌及糖脂代谢通路。所述生物制剂或所述药物上述作用并不依赖于肠道菌群变化。 有益技术效果：Preferably, the biological agent or the drug is used to inhibit the interaction between the nuclear receptor NCoR of intestinal epithelial cells and the nuclear receptor, change the content and composition of bile acids, and reduce the content of lipids such as intestinal triglycerides and cholesterol. Absorption, increase its excretion; promote oxidative metabolism heat production and energy consumption; promote the secretion of incretin GLP-1, regulate insulin secretion and glucose and lipid metabolism pathways. The above-mentioned effects of the biological agent or the drug do not depend on changes in intestinal flora. Beneficial technical effects:
本发明通过系统的研究发现：条件性敲除小鼠肠道上皮细胞NCoR基因，得到NCoR组织特异性敲除的小鼠，再进行高脂饮食喂养后，可显著逆转小鼠肥胖及胰岛素抵抗表型，降低体重，降低肝脏及腹腔脂肪重量，改善口服葡萄糖耐受性及胰岛素敏感性，采用金标准高胰岛素正常血糖钳夹实验评价后，进一步精确证明了整体和肝脏、脂肪组织等靶器官的胰岛素敏感性均得到了显著改善。同时，条件性敲除小鼠肠道上皮细胞NCoR也可显著改善肥胖小鼠的高胰岛素血症和肝脏脂质堆积，改变胆汁酸的含量和组成，减少肠道脂质吸收，并刺激活性胰高血糖素样肽(GLP-1)的分泌。采用代谢笼对小鼠的能量代谢的监控表明条件性敲除小鼠肠道上皮细胞NCoR还可增加代谢率，如产热及氧化代谢等，但并不增加小鼠的活动频率。进一步的，通过野生型小鼠与条件性肠道上皮细胞NCoR敲除小鼠共饲养的方法，证明条件性肠道上皮细胞NCoR敲除对表型的改善作用并不依赖肠道菌群介导。综上所述，发明人首次发现，通过条件性敲除肠道上皮细胞NCoR，可以改善整体的胰岛素敏感性并降低体重，说明其可以作为靶标。通过抑制肠道上皮细胞NCoR来增加肠促胰岛素激素GLP-1的分泌，改变胆汁酸的含量和组成，抑制肠道脂质吸收，增加能量代谢率等，从而改善脂质代谢，胰岛素抵抗及肥胖，有助于改变临床胰岛素增敏剂缺乏的现状，对2型糖尿病及肥胖的预防和治疗都有着积极的意义。Through systematic research, the present invention found that conditionally knocking out the NCoR gene of intestinal epithelial cells in mice to obtain NCoR tissue-specific knockout mice, and then feeding them with a high-fat diet, can significantly reverse the obesity and insulin resistance of the mice. type, reduce body weight, reduce liver and abdominal fat weight, improve oral glucose tolerance and insulin sensitivity, and use the gold standard hyperinsulinemic euglycemic clamp test to evaluate, further accurately proving the overall and target organs such as liver and adipose tissue. Insulin sensitivity was significantly improved. At the same time, conditional knockout of NCoR in intestinal epithelial cells of mice can also significantly improve hyperinsulinemia and liver lipid accumulation in obese mice, change the content and composition of bile acids, reduce intestinal lipid absorption, and stimulate active pancreatic Secretion of glucagon-like peptide (GLP-1). Monitoring the energy metabolism of mice using metabolic cages showed that conditional knockout of NCoR in intestinal epithelial cells of mice can also increase metabolic rate, such as thermogenesis and oxidative metabolism, but does not increase the activity frequency of mice. Furthermore, by co-raising wild-type mice with conditional intestinal epithelial cell NCoR knockout mice, it was proven that the phenotypic improvement effect of conditional intestinal epithelial cell NCoR knockout is not mediated by intestinal flora. . In summary, the inventors discovered for the first time that conditional knockout of NCoR in intestinal epithelial cells can improve overall insulin sensitivity and reduce body weight, indicating that it can be used as a target. By inhibiting the NCoR of intestinal epithelial cells, it increases the secretion of the incretin hormone GLP-1, changes the content and composition of bile acids, inhibits intestinal lipid absorption, increases energy metabolic rate, etc., thereby improving lipid metabolism, insulin resistance and obesity. , helps to change the current situation of lack of clinical insulin sensitizers, and has positive significance for the prevention and treatment of type 2 diabetes and obesity.
本发明的其他优点、目标和特征将部分通过下面的说明体现，部 分还将通过对本发明的研究和实践而为本领域的技术人员所理解。Other advantages, objects, and features of the present invention will be apparent in part from the description below, and in part will be understood by those skilled in the art through study and practice of the present invention.
图1为本发明条件性肠道上皮细胞NCoR敲除后对体重及组织重量的影响；A为高脂饮食喂养期间野生型小鼠(WT小鼠)和肠道上皮细胞NCoR敲除小鼠(IKO小鼠)的体重变化情况；B为高脂饮食喂养期间WT和IKO小鼠的每周平均摄食情况；C为高脂饮食喂养后WT和IKO小鼠的主要脏器肝脏(Liver)，腹腔脂肪(WAT)和胰腺(Pancreas)湿重；D为高脂饮食喂养后WT和IKO小鼠的主要脏器肝脏(Liver)，腹腔脂肪(WAT)和胰腺(Pancreas)占体重比例。Figure 1 shows the effects of conditional intestinal epithelial cell NCoR knockout of the present invention on body weight and tissue weight; A shows wild-type mice (WT mice) and intestinal epithelial cell NCoR knockout mice ( IKO mice); B is the average weekly food intake of WT and IKO mice during high-fat diet feeding; C is the main organs liver and abdominal cavity of WT and IKO mice after high-fat diet feeding The wet weight of fat (WAT) and pancreas (Pancreas); D is the proportion of liver, abdominal fat (WAT) and pancreas (Pancreas) in the body weight of the main organ of WT and IKO mice after feeding with high-fat diet.
图2为本发明条件性肠道上皮细胞NCoR敲除后对正常饮食和高脂饮食喂养小鼠胰岛素敏感性表型的影响；A正常饮食喂养下WT和IKO小鼠口服葡萄糖耐量；B高脂饮食喂养后WT和IKO小鼠口服葡萄糖耐量；C高脂饮食喂养后WT和IKO小鼠胰岛素耐量。Figure 2 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on the insulin sensitivity phenotype of mice fed normal diet and high-fat diet; A. Oral glucose tolerance of WT and IKO mice fed normal diet; B. High-fat diet Oral glucose tolerance of WT and IKO mice after diet feeding; C Insulin tolerance of WT and IKO mice after high-fat diet feeding.
图3为本发明条件性肠道上皮细胞NCoR敲除后对高脂饮食喂养小鼠分泌激素的影响：A高脂饮食喂养后WT和IKO小鼠空腹胰岛素和糖刺激后15分钟血胰岛素水平；B高脂饮食喂养后WT和IKO小鼠空腹糖促胰岛素激素-1(GLP-1)水平；C高脂饮食喂养后WT和IKO小鼠糖刺激后10分钟GLP-1水平。Figure 3 shows the effects of NCoR knockout in conditional intestinal epithelial cells of the present invention on hormone secretion in high-fat diet-fed mice: A. Fasting insulin levels in WT and IKO mice after high-fat diet feeding and 15-minute blood insulin levels after sugar stimulation; B. Fasting glucose-insulin-stimulating hormone-1 (GLP-1) levels in WT and IKO mice after high-fat diet feeding; C. GLP-1 levels 10 minutes after glucose stimulation in WT and IKO mice after high-fat diet feeding.
图4为本发明条件性肠道上皮细胞NCoR敲除后高脂饮食喂养小鼠进行高胰岛素正常血糖钳夹实验对胰岛素敏感性精确评价的结果；A葡萄糖输注速率；B葡萄糖处置速率；C胰岛素刺激的葡萄糖处置速率；D稳态时相比基础状态胰岛素对肝糖输出抑制率；E稳态时相比 基础状态胰岛素对血游离脂肪酸水平抑制率；F基础和稳态时血胰岛素水平。Figure 4 shows the results of a hyperinsulinemic euglycemic clamp experiment to accurately evaluate insulin sensitivity in high-fat diet-fed mice after NCoR knockout in conditional intestinal epithelial cells of the present invention; A glucose infusion rate; B glucose disposal rate; C Glucose disposal rate stimulated by insulin; D. Inhibition rate of hepatic glucose output by insulin at steady state compared with basal state; E. Inhibition rate of blood free fatty acid levels by insulin at steady state compared with basal state; F. Blood insulin levels at basal and steady state.
图5为本发明条件性肠道上皮细胞NCoR敲除后对高脂饮食喂养小鼠脂质水平的影响；A血甘油三酯水平；B血总胆固醇水平；C血游离脂肪酸水平；D肝脏甘油三酯水平；E肝脏总胆固醇水平；F肝脏游离脂肪酸水平；G粪便中甘油三酯水平；H粪便中总胆固醇水平；I粪便中游离脂肪酸水平。Figure 5 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on lipid levels in mice fed a high-fat diet; A blood triglyceride level; B blood total cholesterol level; C blood free fatty acid level; D liver glycerol Triglyceride level; E total cholesterol level in liver; F free fatty acid level in liver; G triglyceride level in feces; H total cholesterol level in feces; I free fatty acid level in feces.
图6为本发明条件性肠道上皮细胞NCoR敲除小鼠和野生型小鼠的共饲养(co-house)实验；A高脂饮食喂养后共饲养组(CO-WT和CO-IKO)小鼠和分开饲养组(WT和IKO)小鼠的口服葡萄糖耐量；B高脂饮食喂养后共饲养组(CO-WT和CO-IKO)小鼠和分开饲养组(WT和IKO)小鼠的胰岛素耐量；C高脂饮食喂养期间共饲养组(CO-WT和CO-IKO)小鼠和分开饲养组(WT和IKO)小鼠的体重变化情况；D高脂饮食喂养后共饲养组(CO-WT和CO-IKO)小鼠和分开饲养组(WT和IKO)小鼠的空腹胰岛素水平和糖刺激后10分钟的血胰岛素水平。Figure 6 is a co-house experiment of conditional intestinal epithelial cell NCoR knockout mice and wild-type mice of the present invention; A, the co-housed group (CO-WT and CO-IKO) after high-fat diet feeding Oral glucose tolerance of mice and mice in separate feeding groups (WT and IKO); B Insulin of mice in co-raising group (CO-WT and CO-IKO) and mice in separate feeding group (WT and IKO) after high-fat diet feeding Tolerance; C. Body weight changes of mice in the co-feeding group (CO-WT and CO-IKO) and separate feeding groups (WT and IKO) during high-fat diet feeding; D. After high-fat diet feeding in the co-feeding group (CO- Fasting insulin levels and blood insulin levels 10 minutes after glucose stimulation in WT and CO-IKO) mice and in separate rearing groups (WT and IKO) mice.
图7为本发明条件性肠道上皮细胞NCoR敲除后对高脂饮食喂养小鼠能量代谢的影响。A高脂饮食喂养后WT和IKO小鼠能量代谢率；B高脂饮食喂养后WT和IKO小鼠耗氧量；C高脂饮食喂养后WT和IKO小鼠二氧化碳产生量；D高脂饮食喂养后WT和IKO小鼠在有光照和黑暗中的活动量。Figure 7 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on energy metabolism in mice fed a high-fat diet. A. Energy metabolism rate of WT and IKO mice after feeding with high-fat diet; B. Oxygen consumption of WT and IKO mice after feeding with high-fat diet; C. Carbon dioxide production of WT and IKO mice after feeding with high-fat diet; D. Feeding with high-fat diet Activity levels of WT and IKO mice in the light and dark.
图8为本发明条件性肠道上皮细胞NCoR敲除后对高脂饮食喂养小 鼠胆汁酸组成的影响。A高脂饮食喂养后WT和IKO小鼠血液胆汁酸的组成；B高脂饮食喂养后WT和IKO小鼠回肠胆汁酸的组成；C高脂饮食喂养后WT和IKO小鼠粪便胆汁酸的组成；D高脂饮食喂养后WT和IKO小鼠回肠、粪便和血液中12α羟基甾醇型胆汁酸和非12α羟基甾醇型胆汁酸的比值。Figure 8 shows the effect of NCoR knockout in conditional intestinal epithelial cells of the present invention on the bile acid composition of mice fed a high-fat diet. A. The composition of bile acids in the blood of WT and IKO mice after feeding with a high-fat diet; B. The composition of bile acids in the ileum of WT and IKO mice after feeding with a high-fat diet; C. The composition of bile acids in the feces of WT and IKO mice after feeding with a high-fat diet ;D Ratio of 12α-hydroxysterol bile acids and non-12α-hydroxysterol bile acids in the ileum, feces and blood of WT and IKO mice after high-fat diet feeding.
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.
在不背离本发明的范围或精神的情况下，可对本发明说明书的具 体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples are intended to be illustrative only.
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.
为使本发明的目的、技术方案、优点更加清楚，下面将结合附图对本发明作进一步的详细描述。In order to make the purpose, technical solution, and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the accompanying drawings.
实施例1条件性肠道上皮细胞NCoR敲除后对体重的影响Example 1 Effect of conditional intestinal epithelial cell NCoR knockout on body weight
将NCoR flox/flox小鼠与Villin Cre工具鼠进行回交数代后得到肠道上皮细胞NCoR条件性敲除的雄性纯合子小鼠(IKO)，同时选取出生日期和性别均相对应的NCoR flox/flox小鼠作为野生型对照小鼠(WT)。WT和IKO组小鼠每组各10-15只，给予高脂饮食(60％的热量来自于脂肪，购自Research Diets公司)喂养，每周监测体重和摄食变化。高脂饮食喂养15周后处死小鼠，摘取肝脏，腹腔脂肪和胰腺称重记录。NCoR flox/flox mice were backcrossed with Villin Cre tool mice for several generations to obtain male homozygous mice (IKO) with conditional knockout of NCoR in intestinal epithelial cells. At the same time, NCoR flox mice with corresponding birth dates and genders were selected. /flox mice served as wild-type control mice (WT). There were 10-15 mice in each group of WT and IKO groups. They were fed a high-fat diet (60% of calories came from fat, purchased from Research Diets Company), and changes in body weight and food intake were monitored weekly. After 15 weeks of high-fat diet feeding, the mice were sacrificed, and the liver, abdominal fat, and pancreas were removed and weighed and recorded.
结果表明：条件性肠道上皮细胞NCoR敲除后对正常饮食喂养小鼠的体重无影响，但可显著抑制高脂饮食诱导的肥胖，并显著降低腹腔脂肪和肝脏的重量以及各自占体重的比例，同时也可显著增加胰腺占体重的比例；但对小鼠的摄食量无明显影响。The results show that NCoR knockout in conditional intestinal epithelial cells has no effect on the body weight of mice fed a normal diet, but can significantly inhibit high-fat diet-induced obesity, and significantly reduce the weight of abdominal fat and liver and their respective proportions in body weight. , it can also significantly increase the proportion of pancreas to body weight; but has no significant effect on the food intake of mice.
实施例2条件性肠道上皮细胞NCoR敲除后对胰岛素抵抗的改善作用Example 2 Improvement of insulin resistance after NCoR knockout in conditional intestinal epithelial cells
实施例1中的WT和IKO组小鼠在高脂饮食喂养之前，仍进行 正常饲料喂养时，进行口服葡萄糖耐量试验。小鼠禁食6小时后，尾尖取血测定空腹(0时)血糖，随后灌胃给予葡萄糖溶液(2g/kg)，于糖刺激后15分钟，30分钟，60分钟和120分钟时尾尖取血测定血糖值。于高脂饮食喂养第9周，进行口服葡萄糖耐量试验，方法同上。于高脂饮食喂养第10周，进行胰岛素耐量实验。小鼠禁食6小时后，尾尖取血测定空腹(0时)血糖，随后皮下注射给予胰岛素(0.3U/kg)，于胰岛素注射后15分钟，30分钟，60分钟和120分钟时尾尖取血测定血糖值。Before the mice in the WT and IKO groups in Example 1 were fed with the high-fat diet, an oral glucose tolerance test was performed while still being fed with normal feed. After the mice were fasted for 6 hours, blood was taken from the tail tip to measure fasting (0 time) blood glucose, and then glucose solution (2g/kg) was administered intragastrically at 15 minutes, 30 minutes, 60 minutes and 120 minutes after glucose stimulation. Take blood to measure blood sugar level. At the 9th week of high-fat diet feeding, an oral glucose tolerance test was conducted with the same method as above. At the 10th week of high-fat diet feeding, an insulin tolerance test was conducted. After the mice were fasted for 6 hours, blood was taken from the tail tip to measure fasting (0 time) blood glucose, and then insulin (0.3 U/kg) was injected subcutaneously at the tail tip 15 minutes, 30 minutes, 60 minutes and 120 minutes after insulin injection. Take blood to measure blood sugar level.
结果表明：肠道上皮细胞NCoR条件性敲除，对在正常饮食喂养下小鼠的葡萄糖耐受性无影响，但可显著改善高脂饮食喂养导致的葡萄糖耐受性和胰岛素敏感性异常。The results showed that conditional knockout of NCoR in intestinal epithelial cells had no effect on glucose tolerance in mice fed a normal diet, but could significantly improve glucose tolerance and insulin sensitivity abnormalities caused by high-fat diet feeding.
实施例3条件性肠道上皮细胞NCoR敲除后对胰岛素及糖促胰岛素激素的影响Example 3 Effects of NCoR knockout in conditional intestinal epithelial cells on insulin and glucoinsulin-stimulating hormone
实施例1中的WT和IKO小鼠在高脂喂养第11周，禁食6小时后，尾尖取血，置于冰上，灌胃给予葡萄糖溶液(2g/kg)，于糖刺激后15分钟尾尖取血，置于冰上。12000rpm离心1min，吸取上清，采用Alpco公司小鼠胰岛素ELISA试剂盒测定血胰岛素水平。In the 11th week of high-fat feeding, the WT and IKO mice in Example 1 were fasted for 6 hours. Blood was collected from the tail tip, placed on ice, and glucose solution (2g/kg) was administered orally. Collect blood from the tip of the tail and place on ice. Centrifuge at 12,000 rpm for 1 min, aspirate the supernatant, and measure blood insulin levels using Alpco mouse insulin ELISA kit.
实施例1中的WT和IKO小鼠在高脂喂养第15周，禁食6小时，于禁食5.25小时灌胃给予西他列汀(25mg/kg，DPP4酶抑制剂)，于禁食6小时灌胃给予葡萄糖溶液(2g/kg)，于糖刺激后15分钟尾尖取血于提前加入EDTA抗凝剂和蛋白酶抑制剂的离心管中，置于冰上，1000g，4度离心10分钟，吸取上清，采用Alpco公司小鼠活性GLP-1 ELISA试剂盒测定血活性GLP-1水平。In the 15th week of high-fat feeding, the WT and IKO mice in Example 1 were fasted for 6 hours, and sitagliptin (25 mg/kg, DPP4 enzyme inhibitor) was administered intragastrically at 5.25 hours after fasting. Glucose solution (2g/kg) was administered intragastrically every hour. Blood was collected from the tail tip 15 minutes after glucose stimulation and placed in a centrifuge tube with EDTA anticoagulant and protease inhibitor added in advance. Place it on ice and centrifuge at 1000g for 10 minutes at 4 degrees. , draw the supernatant, and use Alpco's mouse active GLP-1 ELISA kit to measure blood active GLP-1 levels.
实施例1中的WT和IKO小鼠在高脂喂养15周后，禁食4小时，采用二氧化碳麻醉，剖开胸腔心脏取血，至预先加有DPP4抑制剂和蛋白酶抑制剂的BD抗凝取血管中，1000g，4度离心20分钟，吸取上清，采用Alpco公司小鼠活性GLP-1ELISA试剂盒测定血活性GLP-1水平。After 15 weeks of high-fat feeding, the WT and IKO mice in Example 1 were fasted for 4 hours, anesthetized with carbon dioxide, and the thorax and heart were opened to collect blood. In the blood vessel, centrifuge at 1000g and 4 degrees for 20 minutes, aspirate the supernatant, and use the Alpco mouse active GLP-1 ELISA kit to measure the blood active GLP-1 level.
结果表明：条件性肠道上皮细胞NCoR敲除可显著改善高脂饮食和肥胖导致的高胰岛素血症，并显著增加空腹和糖刺激15分钟后的活性肠道胰岛素激素GLP-1水平，说明NCoR敲除后可通过增加GLP-1的水平从而促进胰岛素分泌以及改善胰岛素敏感性等。The results show that conditional intestinal epithelial cell NCoR knockout can significantly improve hyperinsulinemia caused by high-fat diet and obesity, and significantly increase the levels of active intestinal insulin hormone GLP-1 after fasting and 15 minutes of sugar stimulation, indicating that NCoR Knockout can promote insulin secretion and improve insulin sensitivity by increasing the level of GLP-1.
实施例4条件性肠道上皮细胞NCoR敲除后对胰岛素敏感性影响的精确评价Example 4 Precise evaluation of the effect of NCoR knockout on insulin sensitivity in conditional intestinal epithelial cells
采用实施例1中的方法得到的另一批次的WT和IKO小鼠，高脂饮食喂养9周后，给予三溴乙醇(250mg/kg)麻醉，颈部开口，剥离出右侧颈静脉，结扎远心端，以软管(Silastic 508-001，Dow Corning Corp.)进行颈静脉插管，并从皮下穿行至背部留置，缝合伤口，恢复3天。实验当天禁食6小时后，首先以25μCi每小时速度输注4min的初始量，然后改为以5μCi每小时的速率输注D-[3-3H]葡萄糖(购自Perkin Elmer)，90分钟(记为t＝0min)时结束并取血。随后，以5μCi/hour和20mU/kg/min恒定速率输注含D-[3-3H]葡萄糖的胰岛素溶液同时，开始输注50％的葡萄糖溶液。每隔10min尾尖取血测定血糖，根据血糖值调整葡萄糖的输注速率，当葡萄糖的输注速率在 30min内保持基本恒定，相应血糖值也稳定在120mg/dl±5mg/dl范围内时，可认为达到稳态，此时的葡萄糖输注速率即为GIR，取稳态时血样。测定基础状态和稳态时血液中3H的闪烁值，根据闪烁值测定结果，采用steele公式，计算得到基础状态及稳态时肝糖输出HGP并计算抑制率，葡萄糖处置速率GDR，以及主要反应肌肉葡萄糖摄取速率的胰岛素刺激的葡萄糖处置速率IS-GDR。测定基础状态和稳态时血游离脂肪酸FFA和血胰岛素水平，计算得到主要反映脂肪组织胰岛素敏感性的胰岛素对游离脂肪酸水平的抑制率。Another batch of WT and IKO mice obtained by the method in Example 1 were fed with a high-fat diet for 9 weeks, and then anesthetized with tribromoethanol (250 mg/kg), the neck was opened, and the right jugular vein was peeled off. The distal end was ligated, and a flexible tube (Silastic 508-001, Dow Corning Corp.) was used to intubate the jugular vein, and it was passed through the subcutaneous tissue to the back and left in place. The wound was sutured and the patient recovered for 3 days. After fasting for 6 hours on the day of the experiment, an initial amount of 25 μCi per hour was infused for 4 min, and then D-[3-3H] glucose (purchased from Perkin Elmer) was infused at a rate of 5 μCi per hour for 90 min ( It ends when t=0min) and blood is taken. Subsequently, the insulin solution containing D-[3-3H] glucose was infused at a constant rate of 5 μCi/hour and 20 mU/kg/min. Simultaneously, the infusion of 50% glucose solution was started. Take blood from the tail tip every 10 minutes to measure blood sugar, and adjust the glucose infusion rate according to the blood sugar value. When the glucose infusion rate remains basically constant within 30 minutes and the corresponding blood sugar value is also stable within the range of 120mg/dl±5mg/dl, It can be considered that a steady state has been reached, and the glucose infusion rate at this time is the GIR, and the blood sample at the steady state is taken. Determine the scintillation value of 3H in the blood at the basal state and steady state. Based on the scintillation value measurement results, use the steele formula to calculate the hepatic glucose output HGP at the basal state and steady state and calculate the inhibition rate, glucose disposal rate GDR, and the main response muscles Insulin-stimulated glucose disposal rate of glucose uptake rate IS-GDR. Blood free fatty acid FFA and blood insulin levels were measured at basal and steady states, and the inhibition rate of insulin on free fatty acid levels, which mainly reflects the insulin sensitivity of adipose tissue, was calculated.
结果表明：高脂饮食喂养后，相比WT小鼠，条件性肠道上皮细胞NCoR敲除可显著性增加IKO组小鼠的葡萄糖输注速率GIR，葡萄糖处置速率GDR及外源性胰岛素对肝糖输出的抑制率和对游离脂肪酸的抑制率，而反映肌肉胰岛素敏感性的胰岛素刺激的葡萄糖处置速率IS-GDR有增加的趋势，但无显著性差异，说明条件性肠道上皮细胞NCoR敲除可显著改善小鼠整体和肝脏及脂肪组织的胰岛素敏感性。The results show that after high-fat diet feeding, conditional intestinal epithelial cell NCoR knockout can significantly increase the glucose infusion rate GIR, glucose disposal rate GDR and exogenous insulin in the IKO group of mice compared with WT mice. The inhibition rate of glucose output and the inhibition rate of free fatty acids, while the insulin-stimulated glucose disposal rate IS-GDR, which reflects muscle insulin sensitivity, has an increasing trend, but there is no significant difference, indicating that conditional intestinal epithelial cell NCoR knockout It can significantly improve the insulin sensitivity of mice overall and in liver and adipose tissue.
实施例5条件性肠道上皮细胞NCoR敲除后对脂质水平等影响Example 5 Effects of conditional intestinal epithelial cell NCoR knockout on lipid levels, etc.
实施例1中的WT和IKO小鼠在高脂饮食喂养15周后，处死，心脏取血，采用中生北控公司的总甘油三酯(TG)和总胆固醇(TC)试剂盒测定血中TG和TC的含量，采用Wako公司的NEFA试剂盒测定血中游离脂肪酸(FFA)的水平。The WT and IKO mice in Example 1 were sacrificed after being fed a high-fat diet for 15 weeks, and blood was taken from the heart. The total triglyceride (TG) and total cholesterol (TC) kits of Zhongsheng Beikong Company were used to measure the blood levels. The contents of TG and TC were measured using the NEFA kit from Wako Company to determine the level of free fatty acids (FFA) in the blood.
实施例1中的WT和IKO小鼠高脂饮食喂养15周后，处死，摘取肝脏冻存。取小块组织研磨匀浆后，采用前述TG，TC和FFA试剂盒 测定相应脂质水平。After the WT and IKO mice in Example 1 were fed a high-fat diet for 15 weeks, they were sacrificed and their livers were removed and cryopreserved. After grinding and homogenizing small pieces of tissue, the corresponding lipid levels were measured using the aforementioned TG, TC and FFA kits.
实施例1中的WT和IKO小鼠高脂饮食喂养的第12周，每只小鼠单笼饲养，收集24h的粪便，60度烘干后，加入甲醇：氯仿(1：2,V/V)，37度萃取12小时，取上清，再度蒸干，沉淀加入含10％TritonX-100异丙醇溶液复溶。采用前述TG，TC和FFA试剂盒测定相应脂质水平。In the 12th week of the high-fat diet feeding of the WT and IKO mice in Example 1, each mouse was raised in a single cage, and the feces collected for 24 hours was dried at 60 degrees and methanol:chloroform (1:2, V/V ), extract at 37 degrees for 12 hours, take the supernatant, evaporate to dryness again, and redissolve the precipitate by adding 10% TritonX-100 isopropyl alcohol solution. The corresponding lipid levels were determined using the aforementioned TG, TC and FFA kits.
结果表明：高脂饮食喂养后，相比WT组小鼠，条件性肠道上皮细胞NCoR敲除可显著降低IKO组小鼠的血甘油三酯和总胆固醇水平，但对血游离脂肪酸水平无显著影响。IKO组的肝脏甘油三酯、总胆固醇和游离脂肪酸含量也显著降低，说明条件性肠道上皮细胞NCoR敲除也可显著改善高脂饮食和肥胖导致的肝脏脂质堆积。在粪便中，相比WT组小鼠，IKO组的粪便中甘油三酯和总胆固醇的含量明显升高，说明条件性肠道上皮细胞NCoR敲除可能会抑制肠道的脂质吸收，从而降低体重，改善肥胖等。The results show that after feeding a high-fat diet, conditional intestinal epithelial cell NCoR knockout can significantly reduce the blood triglyceride and total cholesterol levels of IKO group mice compared with the WT group of mice, but has no significant effect on blood free fatty acid levels. Influence. Liver triglyceride, total cholesterol and free fatty acid contents in the IKO group were also significantly reduced, indicating that conditional intestinal epithelial cell NCoR knockout can also significantly improve liver lipid accumulation caused by high-fat diet and obesity. In the feces, compared with the WT group of mice, the contents of triglycerides and total cholesterol in the feces of the IKO group were significantly higher, indicating that conditional intestinal epithelial cell NCoR knockout may inhibit intestinal lipid absorption, thereby reducing weight, improve obesity, etc.
实施例6条件性肠道上皮细胞NCoR敲除小鼠和野生型小鼠的共饲养(co-house)实验Example 6 Co-house experiment of conditional intestinal epithelial cell NCoR knockout mice and wild-type mice
采用实施例1中的方法得到的另一批次的WT和IKO小鼠，从离乳开始，分为4组，WT和IKO组为从3周龄后离乳开始就按基因型分开饲养，CO-WT和CO-IKO组为从3周龄后离乳开始混合饲养，从而通过比较不同组别之间表型的差异以确定肠道菌群在条件性肠道上皮细胞NCoR敲除小鼠表型中的作用。小鼠进行高脂饮食喂养第10周，进行口服葡萄糖耐量试验，方法同实施例2。高脂饮食喂养第 12周，进行胰岛素耐量试验，方法同实施例2。高脂饮食喂养第13周，小鼠禁食6小时后，尾尖取血，置于冰上，灌胃给予葡萄糖溶液(2g/kg)，于糖刺激后10分钟尾尖取血，置于冰上。12000rpm离心1min，吸取上清，采用Alpco公司小鼠胰岛素ELISA试剂盒测定血胰岛素水平。Another batch of WT and IKO mice obtained by the method in Example 1 were divided into 4 groups from the beginning of weaning. The WT and IKO groups were raised separately according to genotype from the beginning of weaning after 3 weeks of age. The CO-WT and CO-IKO groups were mixed and raised starting from weaning at 3 weeks of age, so as to compare the phenotypic differences between different groups to determine the intestinal flora in conditional intestinal epithelial cell NCoR knockout mice. role in phenotype. The mice were fed a high-fat diet for 10 weeks, and an oral glucose tolerance test was performed. The method was the same as in Example 2. In the 12th week of high-fat diet feeding, an insulin tolerance test was conducted, and the method was the same as in Example 2. In the 13th week of high-fat diet feeding, after the mice were fasted for 6 hours, blood was taken from the tail tip and placed on ice. Glucose solution (2g/kg) was administered intragastrically. Blood was taken from the tail tip 10 minutes after glucose stimulation and placed on ice. on ice. Centrifuge at 12,000 rpm for 1 min, aspirate the supernatant, and measure blood insulin levels using Alpco mouse insulin ELISA kit.
结果表明：共饲养后与单独饲养相比，CO-WT组与WT组的体重，葡萄糖耐受性与胰岛素敏感性均无明显差异，而CO-IKO组和IKO组的体重，葡萄糖耐受性与胰岛素敏感性也无明显差异；共饲养组与单独饲养组条件性肠道上皮细胞NCoR敲除后对高胰岛素血症的改善作用也无明显差异；说明肠道菌群在条件性肠道上皮细胞NCoR敲除对高脂喂养的肥胖小鼠胰岛素敏感性表型的改善作用中并没有起主要作用。The results showed that after co-raising compared with raising alone, there was no significant difference in body weight, glucose tolerance and insulin sensitivity between the CO-WT group and the WT group, while the body weight, glucose tolerance and insulin sensitivity of the CO-IKO group and the IKO group were not significantly different. There is no significant difference in insulin sensitivity; there is also no significant difference in the improvement of hyperinsulinemia after NCoR knockout in conditioned intestinal epithelial cells between the co-feeding group and the separate feeding group; indicating that the intestinal flora plays an important role in conditioned intestinal epithelial cells Cellular NCoR knockout does not play a major role in improving the insulin sensitivity phenotype of high-fat fed obese mice.
实施例7条件性肠道上皮细胞NCoR敲除对高脂饮食喂养小鼠能量代谢的影响。Example 7 Effects of conditional intestinal epithelial cell NCoR knockout on energy metabolism in high-fat diet-fed mice.
采用实施例1中的方法得到的另一批次的WT和IKO小鼠，高脂饮食喂养10周后，移入Columbus代谢笼中，单笼饲养，适应性饲养一天后，监测小鼠的耗氧量VO2，二氧化碳产生量VCO2及活动Acti量vity，并计算能量代谢率(EE，Energy Expenditure)。Another batch of WT and IKO mice obtained by the method in Example 1 were fed with a high-fat diet for 10 weeks, then moved into Columbus metabolic cages and raised in single cages. After one day of adaptive feeding, the oxygen consumption of the mice was monitored. Measure VO2, carbon dioxide production VCO2 and activity activity, and calculate the energy metabolic rate (EE, Energy Expenditure).
结果表明：高脂饮食喂养后，相比WT组，IKO组小鼠的耗氧量，二氧化碳产生量和能量代谢率均显著增加，但活动频率显著减少，说明条件性肠道上皮细胞NCoR敲除可显著增加肥胖小鼠的氧化代谢和产热等过程，从而降低体重，改善肥胖。The results showed that after feeding a high-fat diet, compared with the WT group, the oxygen consumption, carbon dioxide production and energy metabolism rate of mice in the IKO group were significantly increased, but the activity frequency was significantly reduced, indicating that conditional intestinal epithelial cell NCoR knockout It can significantly increase oxidative metabolism and thermogenesis in obese mice, thereby reducing body weight and improving obesity.
实施例8条件性肠道上皮细胞NCoR敲除对高脂饮食喂养小鼠胆汁酸组成的影响。Example 8 Effects of conditional intestinal epithelial cell NCoR knockout on bile acid composition in high-fat diet-fed mice.
实施例1中的小鼠高脂高脂饮食喂养15周后，处死小鼠，收集粪便、回肠粘膜组织和血液样本，提取胆汁酸后，采用UPLC/Synapt G2-Si QTOF MS系统测定其含量浓度。After the mice in Example 1 were fed a high-fat diet for 15 weeks, the mice were sacrificed, feces, ileal mucosal tissue and blood samples were collected. After bile acid extraction, the UPLC/Synapt G2-Si QTOF MS system was used to determine its content concentration. .
结果表明：高脂饮食喂养后，相比WT组，IKO组小鼠回肠和粪便的胆汁酸的含量组成发生了较大变化，胆汁酸总量显著降低，12α羟基甾醇型胆汁酸与非12α羟基甾醇型胆汁酸的也比例显著降低，影响了肠腔内脂质的乳化过程及后续的脂质水解过程，减少了肠道的脂质吸收。The results showed that after feeding a high-fat diet, the bile acid content and composition of the ileum and feces of mice in the IKO group changed significantly compared with the WT group, and the total bile acid content was significantly reduced. The proportion of sterol bile acids is also significantly reduced, which affects the emulsification process of lipids in the intestinal lumen and the subsequent lipid hydrolysis process, reducing intestinal lipid absorption.
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.
Claims (10)
- 一种以肠道上皮细胞核受体抑制子NCoR作为靶标在筛选或制备预防、缓解或治疗胰岛素抵抗、肥胖及相关疾病的药物或生物制剂中的应用。An application of using the intestinal epithelial cell nuclear receptor inhibitor NCoR as a target in screening or preparing drugs or biological agents for preventing, alleviating or treating insulin resistance, obesity and related diseases.
- 根据权利要求1的应用，其特征在于，所述胰岛素抵抗、肥胖及相关疾病为糖尿病、高胰岛血症、高血脂症、高胆固醇血症、肥胖及葡萄糖不耐性。The application according to claim 1, wherein the insulin resistance, obesity and related diseases are diabetes, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, obesity and glucose intolerance.
- 根据权利要求1的应用，其特征在于，所述生物制剂或所述药物用于抑制肠道上皮细胞核受体抑制子NCoR与核受体的相互作用，改变胆汁酸的含量和组成，减少肠道甘油三酯、胆固醇等脂质的吸收，增加其排泄；促进氧化代谢产热和能量消耗；促进肠促胰岛素GLP-1的分泌，调节胰岛素分泌及糖脂代谢通路；所述生物制剂或所述药物上述作用并不依赖于肠道菌群变化。The application according to claim 1, characterized in that the biological agent or the drug is used to inhibit the interaction between the intestinal epithelial cell nuclear receptor inhibitor NCoR and the nuclear receptor, change the content and composition of bile acids, and reduce intestinal Absorption of lipids such as triglycerides and cholesterol increases their excretion; promotes oxidative metabolism heat production and energy consumption; promotes the secretion of incretin GLP-1, regulates insulin secretion and glucose and lipid metabolism pathways; the biological agent or the The above-mentioned effects of the drug do not depend on changes in intestinal flora.
- 根据权利要求3的应用，其特征在于，所述的核受体包括Lxr，Pparα或Fxr。The application according to claim 3, characterized in that the nuclear receptor includes Lxr, Pparα or Fxr.
- 一种肠道上皮细胞核受体抑制子NCoR在制备胰岛素增敏或降脂小鼠模型中的应用。Application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in the preparation of insulin-sensitizing or lipid-lowering mouse models.
- 根据权利要求5的应用，其特征在于，通过Cre-LoxP系统以基因重组方式敲除该基因，以获得NCoR组织特异性敲除的胰岛素增敏或降脂小鼠模型。The application according to claim 5, characterized in that the gene is deleted by genetic recombination through the Cre-LoxP system to obtain an insulin-sensitizing or lipid-lowering mouse model with tissue-specific knockout of NCoR.
- 根据权利要求6的应用，其特征在于，所述NCoR组织特异性敲除的胰岛素增敏或降脂小鼠模型能够增加葡萄糖耐受和清除能 力，减少脂质吸收能力，促进脂质代谢和清除能力。The application according to claim 6, characterized in that the insulin-sensitizing or lipid-lowering mouse model with tissue-specific knockout of NCoR can increase glucose tolerance and clearance capacity, reduce lipid absorption capacity, and promote lipid metabolism and clearance. ability.
- 根据权利要求7的应用，其特征在于，所述的组织选自肠道上皮细胞。The application according to claim 7, characterized in that the tissue is selected from intestinal epithelial cells.
- 一种肠道上皮细胞核受体抑制子NCoR在制备筛选预防、缓解或治疗胰岛素抵抗、肥胖及相关疾病药物或生物制剂的试剂盒中的应用。Application of an intestinal epithelial cell nuclear receptor inhibitor NCoR in the preparation of a kit for screening drugs or biological agents for preventing, alleviating or treating insulin resistance, obesity and related diseases.
- 根据权利要求9的应用，其特征在于，所述胰岛素抵抗、肥胖及相关疾病为糖尿病、高胰岛血症、高血脂症、高胆固醇血症、肥胖及葡萄糖不耐性。The application according to claim 9, characterized in that the insulin resistance, obesity and related diseases are diabetes, hyperinsulinemia, hyperlipidemia, hypercholesterolemia, obesity and glucose intolerance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2022/098294 WO2023240376A1 (en) | 2022-06-12 | 2022-06-12 | Use of intestinal epithelial cell nuclear receptor repressor ncor as target in drug screening |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2022/098294 WO2023240376A1 (en) | 2022-06-12 | 2022-06-12 | Use of intestinal epithelial cell nuclear receptor repressor ncor as target in drug screening |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023240376A1 true WO2023240376A1 (en) | 2023-12-21 |
Family
ID=89192830
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/098294 WO2023240376A1 (en) | 2022-06-12 | 2022-06-12 | Use of intestinal epithelial cell nuclear receptor repressor ncor as target in drug screening |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2023240376A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0127132D0 (en) * | 2001-11-12 | 2002-01-02 | Karobio Ab | Assay |
| WO2009024550A1 (en) * | 2007-08-20 | 2009-02-26 | N.V. Organon | N-benzyl, n' -arylcarbonylpiperazine derivatives as lxr modulators |
| WO2009143705A1 (en) * | 2008-05-30 | 2009-12-03 | 国鼎生物科技股份有限公司 | Screening mehtod for liver x receptor agonists and application thereof |
| US20160376279A1 (en) * | 2014-03-13 | 2016-12-29 | Salk Institute For Biological Studies | Fxr agonists and methods for making and using |
| CN114617965A (en) * | 2020-12-10 | 2022-06-14 | 中国医学科学院药物研究所 | Application of intestinal epithelial cell nucleus receptor inhibitor NCoR as target in drug screening |
-
2022
- 2022-06-12 WO PCT/CN2022/098294 patent/WO2023240376A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0127132D0 (en) * | 2001-11-12 | 2002-01-02 | Karobio Ab | Assay |
| WO2009024550A1 (en) * | 2007-08-20 | 2009-02-26 | N.V. Organon | N-benzyl, n' -arylcarbonylpiperazine derivatives as lxr modulators |
| WO2009143705A1 (en) * | 2008-05-30 | 2009-12-03 | 国鼎生物科技股份有限公司 | Screening mehtod for liver x receptor agonists and application thereof |
| US20160376279A1 (en) * | 2014-03-13 | 2016-12-29 | Salk Institute For Biological Studies | Fxr agonists and methods for making and using |
| CN114617965A (en) * | 2020-12-10 | 2022-06-14 | 中国医学科学院药物研究所 | Application of intestinal epithelial cell nucleus receptor inhibitor NCoR as target in drug screening |
Non-Patent Citations (2)
| Title |
|---|
| PINGPING LI; WUQIANG FAN; JIANFENG XU; MIN LU; HIROYASU YAMAMOTO; JOHAN AUWERX; DOROTHYD. SEARS; SASWATA TALUKDAR; DAYOUNG OH; AI : "Adipocyte NCoR Knockout Decreases PPAR Phosphorylation and Enhances PPAR Activity and Insulin Sensitivity", CELL, ELSEVIER, AMSTERDAM NL, vol. 147, no. 4, 23 September 2011 (2011-09-23), Amsterdam NL , pages 815 - 826, XP028109267, ISSN: 0092-8674, DOI: 10.1016/j.cell.2011.09.050 * |
| SUNGSOON FANG, SUH JAE MYOUNG, REILLY SHANNON M, YU ELIZABETH, OSBORN OLIVIA, LACKEY DENISE, YOSHIHARA EIJI, PERINO ALESSIA, JACIN: "Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance", NATURE MEDICINE, NATURE PUBLISHING GROUP US, NEW YORK, vol. 21, no. 2, 5 January 2015 (2015-01-05), New York, pages 159 - 165, XP055404392, ISSN: 1078-8956, DOI: 10.1038/nm.3760 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Polyzos et al. | Fatty liver in lipodystrophy: a review with a focus on therapeutic perspectives of adiponectin and/or leptin replacement | |
| Larter et al. | Insulin resistance, adiponectin, cytokines in NASH: which is the best target to treat? | |
| Kintscher et al. | PPARγ-mediated insulin sensitization: the importance of fat versus muscle | |
| Haczeyni et al. | The selective peroxisome proliferator–activated receptor‐delta agonist seladelpar reverses nonalcoholic steatohepatitis pathology by abrogating lipotoxicity in diabetic obese mice | |
| Beylot | Effects of inulin-type fructans on lipid metabolism in man and in animal models | |
| Gastaldelli | Role of beta-cell dysfunction, ectopic fat accumulation and insulin resistance in the pathogenesis of type 2 diabetes mellitus | |
| Boden et al. | Effect of thiazolidinediones on glucose and fatty acid metabolism in patients with type 2 diabetes | |
| JP6861795B2 (en) | A pharmaceutical composition for the prevention or treatment of diabetes containing amodiaquine and an anti-diabetic drug as active ingredients. | |
| Peyrou et al. | Hepatic PTEN deficiency improves muscle insulin sensitivity and decreases adiposity in mice | |
| Arai et al. | Trehalose prevents adipocyte hypertrophy and mitigates insulin resistance in mice with established obesity | |
| KR20080106455A (en) | Combination treatment of metabolic disorders | |
| Einstein et al. | Accretion of visceral fat and hepatic insulin resistance in pregnant rats | |
| Wang et al. | Impact of NAFLD and its pharmacotherapy on lipid profile and CVD | |
| Cordoba-Chacon | Loss of hepatocyte-specific PPARγ expression ameliorates early events of steatohepatitis in mice fed the methionine and choline-deficient diet | |
| JP4386906B2 (en) | Diabetes model | |
| WO2023240376A1 (en) | Use of intestinal epithelial cell nuclear receptor repressor ncor as target in drug screening | |
| Zhang et al. | Tangduqing granules attenuate insulin resistance and abnormal lipid metabolism through the coordinated regulation of PPARγ and DGAT2 in type 2 diabetic rats | |
| CN114617965A (en) | Application of intestinal epithelial cell nucleus receptor inhibitor NCoR as target in drug screening | |
| Su et al. | Research progress on drugs for diabetes based on insulin receptor/insulin receptor substrate | |
| JP4147220B2 (en) | Arteriosclerosis preventive and therapeutic drug | |
| Pillion et al. | Paradoxical organ-specific adaptations to streptozotocin diabetes mellitus in adult rats | |
| Kang et al. | PPARδ agonists and metabolic diseases | |
| Fang et al. | Exendin-4 improves diabetic kidney disease in C57BL/6 mice independent of brown adipose tissue activation | |
| Duran-Ortiz et al. | Tissue-specific disruption of the growth hormone receptor (GHR) in mice: An update | |
| Shuai-Nan et al. | The Chinese patent medicine, Jin-tang-ning, ameliorates hyperglycemia through improving β cell function in pre-diabetic KKAy mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22946077 Country of ref document: EP Kind code of ref document: A1 |