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Anti-diabetic and Anti-obesity effects of flavonoids in Premature citrus extract

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Abstract
Abstract 1:
Flavonoids are natural polyphenols widely found in citrus fruits and peel and known to possess anti-adipogenic effects. However, the detailed mechanisms for their anti-adipogenic effects remain to be fully elucidated. In the present study, we observed the anti-adipogenic effects of five major citrus flavonoids including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN) associated with AMPK activation in palmitate (PA)-treated HepG2 cells. Intracellular lipid accumulation and triglyceride (TG) content were quantified using Oil-Red O staining and a TG assay, respectively. Glucose uptake was assessed using a 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The phosphorylation levels of AMPK, ACC, and GSK3β, as well as the expression levels of SREBP-2 and HMGCR were analyzed using Western blot analysis. The potential interactions between the flavonoids and the γ-subunit of AMPK were investigated using molecular docking. Flavonoid treatment significantly reduced intracellular lipid accumulation and TG content, increased glucose uptake in an insulin-independent manner, increased the phosphorylation levels of AMPK, ACC, and GSK3β, and decreased the expression levels of SREBP-2 and HMGCR in PA-treated HepG2 cells. Molecular docking analysis revealed that the flavonoids bind to CBS domains of the regulatory γ-subunit of AMPK with high binding affinities, and may act as potential AMPK activators. Overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK.

Abstract 2:
The present study examined the relationship between the anti-diabetic effect of hesperidin (HES) and differential gene expression in HES treated high fat diet (HFD)-induced obese mice. Based on a 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) uptake assay, treatment with HES restored glucose uptake to control levels in an insulin-independent manner in palmitate (PA)-treated HepG2 cells. Western blot analysis confirmed that HES increased the insulin-stimulated phosphorylation of Akt and GSK3β in insulin-resistant PA-treated HepG2 cells. High fat diet (HFD)-induced obese mice treated with HES showed reduced serum insulin, blood glucose, and homeostatic model assessment for insulin resistance (HOMA-IR) values. In addition, both glucose tolerance and insulin tolerance were significantly improved to normal level by HES in HFD-induced obese mice. RNA sequencing disclosed that the expression levels of twelve up-regulated and six down-regulated genes related to insulin signaling and glucose metabolism were restored to normal level by HES in the liver of HFD-induced obese mice. A protein-protein interaction (PPI) network was constructed via search tool for the retrieval of interacting genes/proteins (STRING) analysis, and Eno1, Pik3cd, Hk2, Trib3, Myc, Nos3, Ppargc1a, and Igf2 were located in the functional hubs of the PPI network of glucose metabolism. Furthermore, Western blot analysis confirmed that HES improved insulin sensitivity and glucose homeostasis by normalizing the expression levels of hexokinase-II, enolase-1, and PI3 kinase p110δ to normal level. Overall, these results suggest that HES exerts a potential anti-diabetic effect by normalizing the expression levels of genes related to the insulin signaling and glucose metabolism, which were perturbed in the liver of HFD-induced obese mice.

Abstract 3:
The present study was conducted to examine the effect of premature citrus extract (PCE) on the serum parameters of beagle dogs. The body weight did not significantly differ between the groups supplemented with PCE and control group throughout the experiment. The groups supplemented with PCE showed significantly decreased serum triglyceride levels compared with the control group at week 12. Serum cholesterol analysis confirmed that the PCE-supplemented group had significantly decreased serum levels of low-density lipoprotein-cholesterol and increased serum levels of the beneficial high-density lipoprotein-cholesterol compared to those in the control group at week 12. In addition, the group supplemented with PCE for 12 weeks showed significantly decreased serum glucose levels compared with those in the control group at week 0. Furthermore, serological tests revealed that PCE did not alter functional parameters of liver, kidney, and pancreas in the serum of beagle dogs over 12 weeks. These results suggest that supplementation of PCE in the diet decreased triglyceride, low-density lipoprotein-cholesterol, and glucose levels and increased the high-density lipoprotein-cholesterol level without affecting the functional parameters of liver, kidney, and pancreas in the serum of beagle dogs.
Author(s)
라잔 프리얀카
Issued Date
2022
Awarded Date
2022-08
Type
Dissertation
URI
https://dcoll.jejunu.ac.kr/common/orgView/000000010868
Alternative Author(s)
Priyanka Rajan
Affiliation
제주대학교 대학원
Department
대학원 수의학과
Advisor
Han, Chang-Hoon
Table Of Contents
General Introduction 14
PART-I 24
Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells 24
1.1. Abstract 25
1.2. Introduction 26
1.3. Materials and Methods 29
1.3.1. Cell culture and treatment 29
1.3.2. Preparation of PA 30
1.3.3. Cell Viability Assay 30
1.3.4. Oil Red O staining 30
1.3.5. TG assay 31
1.3.6. 2-NBDG uptake assay 31
1.3.7. Preparation of cell lysates and Western blot analysis 32
1.3.8. Molecular docking 33
1.3.9. Statistical analysis 34
1.4. Results 35
1.4.1. Cell viability 35
1.4.2. Intracellular lipid accumulation and TG content 37
1.4.3. Glucose uptake 39
1.4.4. AMPK signaling pathway 41
1.4.5. Phosphorylation of GSK3β 44
1.4.6. Molecular docking analysis 45
1.5. Discussion 52
PART-II 57
Anti-diabetic effect of hesperidin on palmitate-treated HepG2 cells and high fat diet-induced obese mice 57
2.1. Abstract 58
2.2. Introduction 59
2.3. Materials and Methods 61
2.3.1. Materials and chemicals 61
2.3.2. Cell culture and treatment 61
2.3.3. Preparation of PA 62
2.3.4. 2-NBDG uptake assay 62
2.3.5. Preparation of cell lysates and Western blot analysis 63
2.3.6. Animals 63
2.3.7. Sample treatment 64
2.3.8. Intraperitoneal glucose tolerance test (IPGTT) 65
2.3.9. Intraperitoneal insulin tolerance test (IPITT) 65
2.3.10. Blood glucose, serum insulin, and HOMA-IR 65
2.3.11. RNA isolation, library preparation, sequencing, and data analysis 66
2.3.12. Statistical analysis 67
2.4. Results 68
2.4.1. Glucose uptake 68
2.4.2. Insulin signaling pathway 69
2.4.3. Glucose tolerance 71
2.4.4. Insulin sensitivity 73
2.4.5. Serum parameters 75
2.4.6. Differential gene expression 77
2.4.7. Protein expressions 82
2.5. Discussion 85
PART-III 91
Effects of premature citrus extract on serum parameters in beagle dogs 91
3.1. Abstract 92
3.2. Introduction 93
3.3. Materials and Methods 95
3.3.1. Preparation of diet containing PCE 95
3.3.2. Animals 95
3.3.3. Animal experiment 96
3.3.4. Serum TG level 97
3.3.5. Serum total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol level 97
3.3.6. Other serum parameters 98
3.3.7. Statistical analysis 99
3.4. Results 100
3.4.1. Body weight change 100
3.4.2. Serum TG level 101
3.4.3. Serum cholesterol levels 102
3.4.4. Serum glucose level 104
3.4.5. Liver function 105
3.4.6. Kidney function 107
3.4.7. Amylase 109
3.4.8. Serum proteins 110
3.5. Discussion 112
General Conclusion 116
References 121
Acknowledgements 140
Degree
Doctor
Publisher
제주대학교 대학원
Appears in Collections:
General Graduate School > Veterinary Medicine
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