VLDL과 VLDL 수용체를 통한 지방조직 염증반응 및 열생산 조절 연구

학위논문 (박사) -- 서울대학교 대학원 : 자연과학대학 생명과학부, 2021. 2. 김재범.Triglyceride is a key metabolite for cell growth, division, and being used as an energy source. In plasma, lipid metabolites, including triglycerides, are circulated in the form of lipoproteins. Among various lipoproteins, very low density lipoprotein (VLDL) contains large portion of triglyceride, and VLDL receptor (VLDLR) is involved in uptake and storage of circulating VLDL. VLDLR is highly expressed in adipose tissue. It has been reported that VLDLR KO mice exhibit reduced adipose tissue mass compared to control mice. Although VLDLR appears to play key roles in VLDL uptake and lipid homeostasis in fat tissues, it is largely unknown whether adipose tissue VLDLR could contribute to systemic energy homeostasis under metabolic alterations. In the chapter one, I have demonstrated that elevated VLDLR in white adipose tissue (WAT) could aggravate adipose tissue inflammation and glucose intolerance in obese mice. Moreover, VLDL administration upregulated intracellular levels of C16:0 ceramides in a VLDLR-dependent manner, potentially leading to pro-inflammatory responses by promoting M1-like macrophage polarization. Adoptive transfer of VLDLR KO bone marrow to wild type (WT) mice relieved inflammatory responses and improved glucose intolerance in diet-induced obese (DIO) mice. These data suggest that increased VLDL-VLDLR signaling in WAT is responsible for aggravation of adipose tissue inflammation and insulin resistance in obesity. In the chapter two, I have shown that VLDLR-mediated VLDL uptake in brown adipose tissue (BAT) would contribute to adaptive thermogenesis upon cold exposure. By blocking VLDL secretion, WT mice became cold-intolerant. In VLDLR KO mice, thermogenic capacity was impaired in BAT. In brown adipocytes, VLDL treatment enhanced thermogenic gene expression as well as simultaneously potentiated mitochondrial oxygen consumption in a VLDLR-dependent manner. In VLDLR KO mice, the expression levels of PPAR target genes were downregulated in BAT, while administration of PPAR agonist restored thermogenic abnormality and mitochondrial dysfunction. Moreover, VLDL-dependent thermogenic activation was not affected in brown adipocyte-specific PPAR KO mice. These data indicate that VLDL-VLDLR axis in BAT is crucial for thermogenic regulation via PPAR activation during cold acclimation. Taken together, these data suggest that adipose tissue VLDLR could mediate VLDL uptake, leading to modulation of not only lipid metabolism but also the manipulation of cellular signaling. Furthermore, VLDLR in WAT would aggravate pro-inflammatory responses upon obesity and VLDLR in BAT could facilitate adaptive thermogenesis under cold stimulation.국문 초록 체내 중성지방은 세포 성장 및 분열에 필요한 주요 대사산물이며, 전신 에너지항상성에 필수적인 호르몬의 전구체로 사용된다. 혈장에서 중성지방과 같은 지질대사물은 VLDL (very low density lipoprotein)이라고 불리는 지질단백질의 형태로 순환한다. VLDL 수용체(VLDLR)는 혈중 VLDL의 흡수 및 저장에 관여한다. VLDLR는 다른 조직에 비해 지방조직에서 많은 량 발현하고 있다. VLDLR 결핍 생쥐는 대조군 생쥐와 비교하여 지방조직 질량이 감소된 형태를 보였으며, 이는 지방조직 VLDLR가 지질대사에 밀접하게 관여되어 있음을 시사한다. 현재까지 VLDLR는 VLDL 흡수 및 지방조직 질량 감소와 관련됨이 보고되었지만, 지방조직 VLDLR가 다양한 환경상태에서 전신 에너지항상성에 기여할 수 있는지 여부는 알려지지 않았다. 본 학위논문 1 장에서는 백색지방조직(WAT)의 VLDLR 발현 증가로 인해 비만 생쥐에서 지방조직 염증반응과 체내 인슐린 저항성을 촉진시킴을 규명했다. 더욱이, VLDL 처리는 VLDLR 의존적으로 C16:0 세라마이드의 축적을 유도하였으며, 이는 백색지방조직 내 전염증반응(pro-inflammatory responses) 및 M1 유사 대식세포 유도를 촉진한다는 실험결과를 얻었다. 뿐만 아니라, VLDLR 결핍 골수를 정상 생쥐로 이식하였을 때, 고지방식이 의존적 비만 유도 염증반응이 완화되고 인슐린 저항성이 개선됨을 관찰했다. 이를 통해 백색지방조직 VLDL-VLDLR 축이 비만에서 지방조직 염증반응 및 인슐린 저항성의 악화를 야기하는 원인일 수 있음을 규명했다. 본 학위 논문 2 장에서는 저온자극 의존적 열생산 과정에 갈색지방조직 VLDLR가 매개될 수 있음을 밝히고자 했다. VLDLR의 혈중 리간드로 알려진 VLDL의 분비가 억제된 야생형 생쥐는 저온자극 동안 열생산 능력이 감소되어 있었다. 또한, VLDLR 결핍 생쥐에서 열생산 능력이 손상되어 있음을 관찰했다. 갈색지방세포에서 VLDL 처리는 열생산 유전자 발현을 향상시키는 동시에 VLDLR 의존적 미토콘드리아 산소소비를 촉진하는 한다는 사실도 발견했다. 또한, VLDLR 결핍 생쥐에서 PPAR delta 표적 유전자의 발현 수준이 갈색지방조직에서 감소 되어있는 반면, PPAR delta agonist의 투여는 열생산이상과 미토콘드리아 기능장애를 회복시킨다는 실험결과를 얻었다. 더욱이, VLDL 의존적 열생산능은 갈색지방세포 특이적 PPAR delta 결핍 생쥐 (PPAR delta BKO)에서 약화되어 있음을 관찰했다. 이러한 데이터는 갈색지방조직 VLDL-VLDLR 축이 저온노출 동안 PPAR delta 활성화를 유도하며 열생산에 관여할 수 있음을 시사한다. 본 연구를 통하여, 지방조직 VLDLR가 VLDL 흡수를 매개하며 지질대사뿐만 아니라 지방조직 신호 전달과정을 조절할 수 있음을 규명했다. 이를 통한 지방조직 염증반응 및 열생산 조절은, 지방조직 VLDLR가 대사환경 변화에 대한 적응 및 전신 에너지항상성을 조절할 수 있는 새로운 인자일 수 있음을 제안한다.TABLE OF CONTENTS ABSTRACT.i TABLE OF CONTENTSiv LIST OF FIGURESvii LIST OF TABLESxi BACKGROUND 1 1. Adipose tissue as an energy storage organ. 1 1) White adipose tissue on energy storage 1 2) Obesity-induced adipose tissue inflammation & insulin resistance. 2 3) Adipose tissue macrophages & M1/M2 polarization 3 2. Adipose tissue as a thermal regulator. 5 1) Brown adipose tissue and thermogenesis. 5 2) Thermogenic brown adipose tissue & fuel utilization. 6 3. Lipoprotein as a triglyceride carrier in blood 8 1) Triglyceride & Lipoproteins. 8 2) Lipoprotein receptors & VLDLR 9 3) VLDLR in adipose tissue 11 4. Purposes. 12 CHAPTER ONE: In white adipose tissue, VLDLR mediates obesity-induced insulin resistance with adipose tissue inflammation 14 1. Abstract. 15 2. Introduction. 16 3. Materials and methods 20 4. Results 25 5. Discussion. 58 CHAPTER TWO: In brown adipose tissue, VLDLR regulates adaptive thermogenesis via facilitating PPAR delta signaling. 64 1. Abstract. 65 2. Introduction. 66 3. Materials and methods 79 4. Results 72 CONCLUSION. 97 1. VLDLR as a stress responder 97 2. VLDLR-mediated lipid metabolites as cellular signaling messengers 98 3. VLDLR as a therapeutic target. 100 ABSTRACT IN KOREAN. 101 REFERENCES 104 LIST OF FIGURES Figure 1. Obesity induces adipose tissue inflammation with macrophages recruitments 4 Figure 2. In brown adipose tissue, cold exposure requires energy substrates for adaptive thermogenesis 7 Figure 3. Lipoprotein and LDL receptor family. 10 Figure 4. VLDLR expression is elevated in WAT from obese mice 26 Figure 5. The level of VLDLR mRNA shows a positive correlation with body mass index in the human fat tissue 27 Figure 6. VLDLR expression is elevated in obese ATMs. 28 Figure 7. The protein level of VLDLR is increased in obese ATMs. 30 Figure 8. The expression level of VLDLR is increased in obese macrophages 31 Figure 9. VLDLR is overexpressed in macrophages. 32 Figure 10. Macrophages overexpressing VLDLR accumulate triglycerides 33 Figure 11. Macrophages overexpressing VLDLR induce expression of pro-inflammatory genes in the presence of VLDL 34 Figure 12. Macrophages of VLDLR deficiency reduce intracellular triglyceride accumulation in the presence of VLDL 36 Figure 13. Macrophage LPL is not crucial to modulate intracellular triglycerides contents with or without VLDL 37 Figure 14. Pro-inflammatory genes are less increased in VLDLR deficient macrophages upon VLDL 39 Figure 15. Insulin sensitivity is enhanced by condition media from VLDLR deficient macrophage treatment. 40 Figure 16. M1-like macrophage polarization accelerates VLDL uptake in a VLDLR dependent manner. 42 Figure 17. VLDLR accelerates M1-like macrophage polarization with VLDL. 44 Figure 18. The level of ceramides is elevated through VLDL-VLDLR axis in macrophages. 46 Figure 19. The level of C:16 ceramides is elevated through VLDL-VLDLR axis in macrophages. 47 Figure 20. MAPK pathway is involved in VLDL-induced inflammatory responses through ceramides 50 Figure 21. Bone marrow transplantation is performed using WT and VLDLR deficient mice. 52 Figure 22. BMT from VLDLR-deficient is not altered in body weight and metabolic organs weight upon HFD. 53 Figure 23. HFD-fed KO BMT mice ameliorate insulin resistance. 54 Figure 24. Adipose tissue inflammation is reduced in HFD-fed KO BMT mice. 56 Figure 25. Graphical abstract of chapter 1. 57 Figure 26. The expression level of VLDLR is elevated in cold-stimulated BAT. 73 Figure 27. The level of VLDL is elevated in cold-exposed primary brown adipocytes . 74 Figure 28. adrenergic activation induces the expression of VLDLR and uptake of VLDL in brown adipocytes 76 Figure 29. Circulating VLDL is downregulated after VLDL-secretion blocking . 77 Figure 30. Circulating VLDL is required for adaptive thermogenesis upon cold exposure. 78 Figure 31. In cold stimulated BAT, thermogenic genes are reduced after blocking of VLDL secretion. 80 Figure 32. VLDLR KO mice are impaired thermogenic activity under cold stimulation 81 Figure 33. The mRNA levels of thermogenic genes are reduced in BAT of cold-stimulated VLDLR KO mice. 82 Figure 34. In brown adipocytes, degree of mitochondrial oxygen consumption rate is downregulated by VLDLR deficiency 84 Figure 35. PPARs signaling are associated with cold-stimulated BAT 85 Figure 36. The mRNA levels of PPAR target genes are downregulated in cold-stimulated BAT of VLDLR KO mice.. 87 Figure 37. In brown adipocytes, PPAR is involved in VLDL/VLDLR-dependent thermogenic gene regulation and oxygen consumption under adrenergic stimulation 88 Figure 38. GW501516 restores thermogenic activity in VLDLR KO mice 90 Figure 39. Brown adipocytes specific PPAR KO (PPAR BKO) mice are generated using UCP1-Cre mice. 91 Figure 40. PPAR BKO mice are impaired adaptive thermogenesis upon cold exposure 93 Figure 41. Upon cold exposure, the mRNA levels of thermogenic genes are downregulated in BAT from PPAR BKO mice 94 Figure 42. VLDL-induced thermogenic activity is diminished in PPAR BKO mice. 95 Figure 43. Graphical abstract of chapter 2 96 LIST OF TABLE Table. PCR primer sequences 24Docto

An Efficient Multi-Dimensional Handover Scheme for Next-Generation Networks

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차세대 무선 네트워크를 위한 핸드오버 기능 구조 제안 (Handover Functional Architecture for Next Generation Wireless Networks)

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