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Glucose independent pleiotropic effects of a dipeptidyl peptidase-4 inhibitor on diabetic complications
DPP-4 억제제의 당뇨 합병증에서다양한 혈당 비의존성 기전 연구

DC Field Value Language
dc.contributor.advisor조명행-
dc.contributor.author정은수-
dc.date.accessioned2017-07-13T16:44:47Z-
dc.date.available2017-07-13T16:44:47Z-
dc.date.issued2016-02-
dc.identifier.other000000132192-
dc.identifier.urihttps://hdl.handle.net/10371/120230-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 수의과대학 수의학과, 2016. 2. 조명행.-
dc.description.abstractDipeptidyl peptidase-4 (DPP-4) inhibitors are widely used as anti-diabetic agents in clinical practice. Gemigliptin, a new and selective DPP-4 inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic complications have not yet been reported.
This study evaluated the inhibitory effects of gemigliptin, a highly selective dipeptidyl peptidase-4 inhibitor, on the formation of advanced glycation end products (AGEs) and AGE cross-links with proteins in in vitro as well as in type 2 diabetic db/db mice. In in vitro assay, gemigliptin dose-dependently inhibited methylglyoxal-modified AGE-bovine serum albumin (BSA) formation (IC50 = 11.69 mM). AGE-collagen cross-linking assays showed that gemigliptin had a potent inhibitory effect (IC50 = 1.39 mM) on AGE-BSA cross-links to rat tail tendon collagen, and its activity was stronger than aminoguanidine (IC50 = 26.4 mM). In addition, gemigliptin directly trapped methylglyoxal in a concentration-dependent manner in vitro. To determine whether gemigliptin inhibits the in vivo glycation processes, gemigliptin (100 mg/kg/day) was orally administered into type 2 diabetic db/db mice for 12 weeks. Elevated serum levels of AGEs in db/db mice were suppressed by the administration of gemigliptin. These inhibitory effects of gemigliptin on the glycation process in both in vitro and in vivo suggest its therapeutic potential for ameliorating AGE-related diabetic complications.
Podocytes participate in the formation and regulation of the glomerular filtration barrier. Loss of podocytes occurs during the early stages of diabetic nephropathy and impairs glomerular filtration. Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used as anti-diabetic agents in clinical practice. In this study, we showed that gemigliptin, a novel DPP-4 inhibitor, reduced podocyte apoptosis in type 2 diabetic db/db mice without reducing hyperglycemia. Gemigliptin (100 mg/kg/day) was administered orally for 12 weeks in db/db mice. Blood glucose levels and albuminuria were measured. The renal cortex was collected for histological examination, and molecular assays were used to detect 8-hydroxydeoxyguanosine, advanced oxidation protein products (AOPP), the receptor for advanced glycation end products (RAGE), and integrin-linked kinase (ILK). Type 2 diabetic db/db mice exhibited albuminuria, renal histopathological changes, and podocyte loss. Administration of gemigliptin to db/db mice suppressed albuminuria, enzyme activity and expression of DPP-4, and podocyte apoptosis. The effect of gemigliptin on diabetes-induced podocyte loss was associated with the suppression of oxidative damage, AOPP accumulation, RAGE expression, and ILK expression. These results indicate the possible benefits of using gemigliptin in diabetes patients to treat renal impairment without affecting glycemic control.
Retinal pericyte loss and neovascularization are characteristic features of diabetic retinopathy. Gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic retinopathy have not yet been reported. We evaluated the efficacy of gemigliptin on retinal vascular leakage in db/db mice, which is an animal model for type 2 diabetes, and neovascularization in oxygen-induced retinopathy (OIR) mice, which is an animal model for ischemic proliferative retinopathy.
Gemigliptin (100mg/kg/day) was orally administered to the db/db mice for 12 weeks. C57BL/6 mice on postnatal day 7 (P7) were exposed to 75% hyperoxia for 5 days, followed by exposure to room air from P12 to P17 to induce OIR. Gemigliptin (50 mg/kg/day) was intraperitoneally injected daily from P12 to P17. Retinal neovascularization was analyzed in flat-mounted retinas on P17. We determined the efficacy and possible mechanism of gemigliptin on high glucose-induced apoptosis of primary human retinal pericytes. The oral administration of gemigliptin for 4 months significantly ameliorated retinal pericyte apoptosis and vascular leakage in the db/db mice. Gemigliptin also ameliorated retinal neovascularization in the OIR mice. Gemigliptin attenuated the overexpression of plasminogen activator inhibitor-1 (PAI-1) in the retinas of diabetic and OIR mice. Gemigliptin and PAI-1 siRNA significantly inhibited pericyte apoptosis by inhibiting the overexpression of PAI-1, which is induced by high glucose. Our results suggest that gemigliptin has potent anti-angiogenic and anti-apoptotic activities via suppressing DPP-4 and PAI-1, and the results support the direct retinoprotective action of gemigliptin.
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dc.description.tableofcontentsGeneral Introduction 1
1. Diabetes mellitus 1
2. Complications of diabetes mellitus 1
2.1. Diabetic nephropathy 2
2.2. Diabetic retinopath 4
3. Dipeptidyl peptidase 4 (DPP-4) inhibitor 6
3.1. Mode of actions of DPP-4 inhibitor 7
3.2. Pleiotropic effect of DPP-4 inhibitors 8
4. Aim of the stud 12
References 14

Part I. Gemigliptin, a novel dipeptidyl peptidase-IV inhibitor, exhibits potent anti-glycation properties in vitro and in vivo 23
Abstract 24
1. Introduction 25
2. Materials and Methods 27
2.1 In vitro tests 27
2.1.1 Inhibitory activity on AGEs formation 27
2.1.2 AGE cross-linking assay 27
2.1.3 Scavenging of carbonyl intermediates of AGE formation 28
2.2 In vivo tests 28
2.2.1 Animals 28
2.2.2 Quantification of serum AGEs levels 28
2.2.3 RBC-IgG assay 29
2.3 Statistical analysis 29
3. Results 30
3.1 Inhibitory effect of DPP-4 inhibitors on AGEs formation in vitro 30
3.2 Inhibitory effect of DPP-4 inhibitors on cross-linking of preformed AGE-BSA with rat tail tendon collagen in vitro 32
3.3 Methylglyoxal scavenging effect of DPP-4 inhibitors 34
3.4 Gemigliptin inhibits AGEs formation and AGE cross-links in vivo 36
4. Discussion 39
5. References 43

Part II. Gemigliptin improves renal function and attenuates podocyte injury in mice with diabetic nephropathy 48
Abstract 49
1. Introduction 50
2. Materials and Methods 52
2.1 Animals and experimental design 52
2.2 Measurement of albuminuria and histological analysis 52
2.3 Detection of 8-hydroxydeoxyguanosine and nephrin in urine samples 53
2.4 Renal DPP-4 enzymatic activity assay 53
2.5 Renal advanced oxidation protein products assay 53
2.6 Immunostainin 54
2.7 Double immunofluorescence staining 54
2.8 Western blotting analysis 55
2.9 Statistical analysis 55
3. Results 57
3.1 Body weight and blood glucose 57
3.2 Gemigliptin inhibits diabetes-induced podocyte injury 59
3.3 Gemigliptin inhibits renal DPP-4 activity and protein expression 62
3.4 Gemigliptin prevents the accumulation of AOPP and expression of its receptor in renal tissues 64
3.5 Gemigliptin inhibits ILK expression 68
4. Discussion 71
5. References 77

Part III. Gemigliptin, a dipeptidyl peptidase-4 inhibitor, inhibits retinal pericyte injury in db/db mice and retinal neovascularization in mice with ischemic retinopath 86
Abstract 87
1. Introduction 88
2. Materials and Methods 91
2.1. Animals 91
2.2. Type 2 diabetic db/ db mice 91
2.3. OIR mice and analysis of retinal neovascularization 91
2.4. Trypsin digest preparation to isolate retinal vascular beds 92
2.5. Determination of the endothelial cells/pericytes (E/P) ratio 92
2.6. TUNEL assay and immunofluorescence staining 93
2.7. Retinal fluorescein isothiocyanate-dextran leakage 93
2.8. Immunofluorescence staining for occludin in retinal whole mounts 94
2.9. Protein array 94
2.10. DPP-4 enzymatic activity assay 95
2.11. Primary human retinal pericyte culture 95
2.12. Apoptosis assay using flow cytometry 95
2.13. Western blot analysis 96
2.14. Statistical analysis 96
3. Results 97
3.1. Gemigliptin inhibits diabetes-induced retinal pericyte injury 97
3.2 Gemigliptin decreases diabetes-induced BRB disruption 102
3.3 Gemigliptin attenuates retinal neovascularization in OIR mice 105
3.4 Gemigliptin regulates the expression of angiogenesis-related factors 108
3.5 Gemigliptin inhibits high glucose-induced pericyte apoptosis in vitro 111
3.6 High glucose-induced pericyte apoptosis is synergistically suppressed by a combination treatment using gemigliptin and DPP-4 siRNA 113
3.7 Gemigliptin suppressed PAI-1 expression, which inhibited high glucose-induced pericyte apoptosis 117
4. Discussion 121
5. References 127

국문 초록 136
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dc.formatapplication/pdf-
dc.format.extent5432107 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectDiabetic mellitus-
dc.subjectDipeptidyl peptidase-4 inhibitor-
dc.subjectDiabetic complications-
dc.subjectDiabetic nephropathy-
dc.subjectDiabetic retinopathy-
dc.subject.ddc636-
dc.titleGlucose independent pleiotropic effects of a dipeptidyl peptidase-4 inhibitor on diabetic complications-
dc.title.alternativeDPP-4 억제제의 당뇨 합병증에서다양한 혈당 비의존성 기전 연구-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages137-
dc.contributor.affiliation수의과대학 수의학과-
dc.date.awarded2016-02-
Appears in Collections:
College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Theses (Ph.D. / Sc.D._수의학과)
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