從限制戒菸後肥胖以預防糖尿病至探討P2X7在脂肪細胞分化過程的角色

Abstract

近年已知第二型糖尿病會促成肝癌與不良之肝臟預後。要預防糖尿病，維持健康之體位與生活型態乃基本功。然而，戒菸後之體重增加，若未有適當之配套介入措施，反而會在短期內增加第二型糖尿病的風險。除了臨床研究，探索脂肪細胞分化時的調節機制之也是重要的肥胖研究領域。其中，P2X7接受器在脂肪細胞分化期間的角色尚有許多未知。 本論文的第一部份為臨床研究，探討整合戒菸治療及體重控制之介入模式，是否能改善糖尿病前期抽菸者之長期血糖預後。在此醫病共享決策概念分派之非隨機對照試驗，參加介入之糖尿病前期抽菸者接受FIT2模式，為同時整合完整16周之戒菸治療服務，及戒菸後體重控制之營養與運動指導；對照組之糖尿病前期抽菸者僅接受一般照護。本計畫在2013年至2017年建立之前瞻性糖尿病前期抽菸者世代，收納共589位民眾，279位民眾加入介入組。歷經平均1316天的追蹤後，217名（36.8%）發生第二型糖尿病，68名（11.5%）恢復至正常血糖。589位受試者皆進行意向分析，發現FIT2模式介入組相較於一般照護組，有較低的第二型糖尿病發生風險（HR：0.58；95% CI：0.40–0.84），與較高的機會恢復至正常血糖（HR：1.91；95% CI：1.04–3.53）。依據532名受試者於六個月時實際戒菸成果進行之時間變動分析，仍發現參與FIT2模式之成功戒菸者相較於對照組，保有較低的第二型糖尿病發生風險（HR：0.63；95% CI：0.44–0.92），與較高恢復至正常血糖的機會（HR：1.83；95% CI：1.01–3.30）。 本論文的第二部份為基礎研究，探討P2X7接受器在脂肪細胞分化期間的調控角色。本研究是採用體外3T3-L1脂肪細胞株傳統的體外兩階段分化模式。實驗過程分別使用P2X7促進劑BzATP與P2X7抑制劑A438079來了解P2X7之功能。結果發現在脂肪細胞分化第1天至第4天，P2X7的蛋白質量與細胞內脂質堆積皆逐漸增加。在分化過程第0天與第2天加入BzATP會增加脂質堆積量，反之在同樣時間點加入A438079會減少脂質堆積量。PPARγ與C/EBPα的蛋白質量在脂肪細胞分化第4天皆增加，但在分化第0天與第2天不管是加BzATP或A438079，都不會影響PPARγ與C/EBPα之基因表現。同時，BzATP也會增加分化第4天時細胞內之三酸甘油酯含量與細胞外之甘油分泌量，A438079則會減少細胞內之三酸甘油酯含量與細胞外之甘油分泌量。當只在脂肪細胞分化的第二階段加藥時，BzATP仍會增加分化第4天細胞內之脂質堆積及三酸甘油酯含量，與細胞外之甘油之分泌量；一致地，在此過程BzATP不會改變PPARγ與C/EBPα原本在分化第4天增加的基因表現量。然而，若在脂肪細胞分化的第4天才加BzATP或A438079，皆不影響已分化脂肪細胞第7天之細胞內三酸甘油酯含量與細胞外甘油之分泌量。值得注意的是，在脂肪細胞分化的第二階段投以BzATP，會抑制分化第4天時與脂肪棕化反應相關PRDM16、PGC-1α與UCP-1的基因表現量，而此抑制隨BzATP劑量加大而更明顯。此外，在分化第0天與第2天加BzATP會抑制sirtuin-3與sirtuin-5在分化第4天的基因表現量。僅在脂肪細胞分化的第二階段投以BzATP，也同樣會抑制sirtuin-3與sirtuin-5被胰島素誘導後上升的mRNA量。 綜上所述，本論文之臨床研究發現FIT2模式介入組相較於一般照護組，長期追蹤後的第二型糖尿病發生風險較低。本團隊建議應整合戒菸治療與戒菸後體重控制之營養與運動指導，以預防第二型糖尿病之發生。本論文在脂肪細胞的研究進一步發現，P2X7接受器活化後會藉由抑制sirtuin-3、sirtuin-5與棕色脂肪基因在脂肪細胞分化過程的表現量，而促進脂質堆積。本論文的系列成果將對臨床與基礎肥胖研究帶來更多啟發。

Type 2 diabetes (T2D) contributes to liver cancer and poor liver outcomes. Maintaining a healthy weight and lifestyle is the cornerstone of diabetes prevention. Post-cessation weight gain (PCWG) facilitates short-term T2D risk in prediabetic smokers in the absence of complementary measures. Stepping into regulatory mechanisms involved in adipocyte differentiation is also imperative in obesity research. Little has investigated the role of the P2X7 receptor during adipogenesis. In the first part of this dissertation, we aimed to investigate whether long-term glycemic outcomes would be improved by an organized program fighting both smoking and PCWG in prediabetic smokers. In this shared decision-making-based non-randomized controlled trial, prediabetic smokers joined the Fight Tobacco and Stay Fit (FIT2) program or received usual care. The 16-week FIT2 program combined smoking cessation therapy with individualized coaching in diet and physical activity strategies for PCWG restriction. During a mean follow-up period of 1316 days, 217 participants (36.8%) developed T2D, and 68 (11.5%) regressed to normoglycemia. In the intention-to-treat analysis (n = 589), the FIT2 program was associated with a reduced T2D risk (HR, 0.58; 95% CI, 0.40–0.84) and a higher probability of regression to normoglycemia (HR, 1.91; 95% CI, 1.04–3.53) compared with usual care. The post-program quitters were at lower T2D risk (HR, 0.63; 95% CI, 0.44–0.92) and were more likely to regress to normoglycemia (HR, 1.83; 95% CI, 1.01–3.30) compared with the controls in the time-varying analysis (n = 532). In the second part of this dissertation, we sought to explore the regulatory roles of P2X7 in adipocytes. This study utilized the in vitro 3T3-L1 differentiation model under the treatment with two-stage differentiation agents. Lipid accumulation, intracellular triglyceride, and extracellular glycerol were determined. The selective P2X7 agonist BzATP and antagonist A438079 were administered to investigate the functions of P2X7. We found that the expression of P2X7 and the lipid accumulation increased during adipocyte differentiation from D0 to D4. When administered at D0/D2, BzATP enhanced, while A438079 attenuated the degree of lipid accumulation during adipocyte differentiation. Neither did BzATP and A438079 administration affect the expression of PPARγ and C/EBPα genes that increased at D4. In addition, both intracellular triglyceride and extracellular glycerol levels at D4 were enhanced by BzATP administration and reduced by A438079 treatment. When administered at stage 2 of adipocyte differentiation, BzATP consistently enhanced lipid accumulation and intracellular triglyceride and extracellular glycerol levels without affecting mRNA and protein levels of PPARγ and C/EBPα that increased at D4. However, treating BzATP or A438079 at D4 did not affect intracellular triglyceride formation and extracellular glycerol release in differentiated adipocytes at D7. Notably, BzATP administration at stage 2 exerted a concentration-dependent inhibition on the enhanced expression of browning associated PRDM16, PGC-1α, and UCP-1 genes at D4. Furthermore, BzATP administration at D0/D2 inhibited the protein and mRNA levels of sirtuin-3/5 at D4. BzATP treatment at stage 2 also suppressed the mRNA levels of sirtuin-3/5 genes upregulated by insulin. In conclusion, our clinical study indicated that the FIT2 program was negatively associated with long-term T2D risk compared with usual care. To prevent T2D development, we recommend simultaneously promoting smoking abstinence and lifestyle coaching for PCWG restriction. In addition, our in vitro study in adipocytes demonstrated that P2X7 enhances lipid accumulation during adipogenesis by suppressing the expression of sirtuin-3/5 and the browning genes. Our serial findings will spark more advanced clinical and basic obesity research.