維生素B3作為熱量限制模仿劑的潛力

The potential ofniacinas a calorie restriction mimetic

鄧凱誠(Kai-Cheng Deng)

指導教授 ： 楊乃成

中山醫學大學/健康管理學院/營養學系碩士班/碩士(2017年)

摘要

維生素B3又稱菸鹼素(niacin)，廣義的菸鹼素包括菸鹼酸(Nicotinic acid; NA)、菸鹼醯胺(nicotinamide; NAM)、菸鹼醯胺核苷(nicotinamide riboside)及其它與菸鹼醯胺有相同生物活性的衍生物。熱量限制模仿劑(calorie restriction mimetic; CRM)指化學分子或天然分子具有和熱量限制相仿的作用，但不需要透過飲食限制的方式而能發揮其生理功效，但是因為影響食物攝取而造成熱量限制模仿作用的分子應該加以排除。本實驗室先前發表的文獻證實熱量限制具有增加人類Hs68細胞內NAD+濃度、提升SIRT1活性、調升NAMPT (由NAM合成NAD+之限速酶)表現及拮抗FK866(為NAMPT酵素的抑制劑，具有模擬維生素B3缺乏，造成細胞內NAD+濃度下降的藥物)誘發細胞衰老的作用，本研究的目的之一在於利用Hs68細胞模式比較NAM、NA和NR模仿熱量限制的能力和機制。另外，肌醇六菸鹼酸(inositol hexanicotinate; IHN) 是一種合成的B3分子，它不會引起潮紅，是市面上最常見的B3補充品之一，但文獻上，至今未見IHN作為B3分子能力的報告，其吸收率及調升SIRT1活性的能力也不清楚。本研究以F344NNarl大鼠探討IHN的吸收率、吸收的型式、作為B3分子的能力，並評估IHN調控組織SIRT1活性的能力。細胞試驗的部份，細胞壽命以累積性生長曲線測定。SA-βG活性染色作為細胞衰老指標，細胞內NAD+以酸萃取結合酵素循環法測定，SIRT1活性以西方墨點法偵測p53乙醯化程度表示。動物試驗的部份，又分成短期和較長期的試驗，短期試驗是將大鼠(n=6)管餵不同濃度的IHN (0~1000 mg/Kg)，24小時後，利用高效液相層析法(High-performance liquid chromatography; HPLC)分析大鼠糞便、血漿及肝臟中的IHN濃度，並且測定紅血球及肝臟中NADx (指NAD、NADH及NADt)的濃度。長期試驗則是將IHN (0~1000 mg/Kg)摻入麵粉給予大鼠(n=5)，連續餵食三十天後，測定血漿及肝臟中的IHN濃度，並且測定紅血球及肝臟中NADx的濃度，以及分析肝、肌肉及腦中SIRT1活性及蛋白的表現。結果顯示在細胞方面，三種維生素皆能增加細胞內NAD+濃度而且呈現濃度效應。但在一般情況下(指細胞內NAD+濃度正常時)，額外給予維生素B3只有NAM可以延長Hs68細胞壽命，但NAM與GR不同，反而會抑制SIRT1的活性。而當以FK866降低細胞內NAD+濃度時，額外給予三種維生素B3皆能顯著的拮抗FK866誘發細胞衰老的作用，由此推論，在維生素B3缺乏的狀態，處理維生素素B3可以延緩B3缺乏引發細胞衰老的作用。動物方面，IHN在大鼠中具有良好吸收率(>85%)，但無論短期或長期試驗，在血漿及肝臟中皆偵測不出完整IHN分子，而紅血球中NADx有顯著增加，但IHN反而會降低肝及肌肉中SIRT1的活性，但此作可能主要因為肌醇本身降低SIRT1活性的影響。矛盾的是，組織中SIRT1活性下降，似乎反而伴隨著SIRT1蛋白表現的增加。動物試驗的結果顯示，IHN能吸收率極佳，能上升紅血球中NAD+，顯示具有作為B3分子能力(但能力似乎不是很強)，IHN吸收後，肝臟及血中並不存在完整的IHN分子，而IHN活化SIRT1活性的能力並不明顯，而且可能受到inositol的干擾，總結IHN作為熱量限制模仿劑的潛力並不高。

關鍵字

維生素B3 ； 熱量限制模仿劑 ； 肌醇六菸鹼酸 ； Hs68 細胞 ； 菸鹼酸 ； 菸鹼醯胺 ； 菸鹼醯胺核苷

並列摘要

Vitamin B3 includes several molecules, such as nicotinic acid (NA), nicotinamide (NAM), nicotinamide riboside (NR) and intositol hexanicotinate (IHN). A calorie restriction mimetic (CRM) refers to any intervention that can evoke similar effects on aging, health and lifespan to those of CR but without actual restrict caloric intake. However, the molecules that causes calorie restriction imitation by appetite reduction should be excluded. In a previous study, we used human Hs68 cell model and had demonstrated that CR could extend the lifespan of Hs68 cells, increase intracellular NAD+ concentration, increase NAMPT expression and antagonize the FK866-induced cell senescence (FK866 is an inhibitor of NAMPT enzyme, a rate-limiting enzyme of NAD+ synthesis from NAM, with an ability to stimulate a mimicking effect of vitamin B3 deficiency by decreasing in intracellular NAD+ concentration ). One of the purposes of this study was to compare the ability and mechanism of NAM, NA and NR to mimic calorie restriction using the Hs68 cell model. In addition, inositol hexanicotinate (IHN) is a synthetic B3 molecule which does not cause flushing, and is one of the most common B3 supplements on the market, although literature has not seen IHN to be demonstrated as a B3 molecule. The ability of IHN to absorb and increase SIRT1 activity is also unclear. In this study, F344NNarl rats were used to investigate the absorption rate of IHN, the absorption type, the NADx increase ability, and to assess the ability of IHN to regulate tissue SIRT1 activity. As for the cellular model, lifespan was measured by cumulative growth curve. SA-βG activity was used as cell senescence marker. Intracellular NAD+ was determined by acid extraction and enzyme cycling. SIRT1 activity was detected by western blot. on the other hand, the animal models were divided into short-term and longer-term trials. Rats in short-term trials (n = 6) were fed with different concentrations of IHN (0 to 1000 mg / kg) and 24 hours later were analyzed by high performance liquid chromatography (HPLC) to detect IHN concentration in the liver, and the concentration of NADx in erythrocytes and liver. Long-term test was performed by incorporating IHN (0 ~ 1000mg / Kg) into the flour (n = 5). After 30 days of continuous feeding, the concentration of IHN in plasma and liver, and the concentration of NADx in erythrocytes and liver were measured, along with the analysis of liver, muscle and brain SIRT1 activity and related protein expression. The results of cellular model showed that in normal condition, three vitamins B3 can increase intracellular NAD+ concentration with a concentration-response effect. Under the condition that NAD+ concentration is decreased, only NAM can extend lifespan when additional supplement to cells with vitamin B3, and unlike GR, NAM would inhibit the activity of SIRT1. When using FK866 to reduce intracellular NAD+ concentration, the additional administration of three kinds of vitamin B3 can significantly antagonize the role of FK866 induced cell senescence, showing that given the lack of vitamin B3 status, treatment of vitamin B3 can delay the senescence caused by B3 deficiency. In animal aspects, IHN has a good absorbance (> 85%) in rats, but no matter short-term or long-term trials, no complete IHN in the plasma and liver levels were detected, and NADx in erythrocytes was significantly increased. IHN would reduce the liver and muscle SIRT1 activity, but this may be mainly due to inositol itself to reduce the impact of SIRT1 activity. Contrary to this observation, tissue SIRT1 activity decreased, accompanied with the SIRT1 protein expression increase. The results of the animal test showed that IHN was able to absorb very well and could increase NAD+ in RBC, indicating the capacity as B3 molecules (but the ability seems not very strong). After IHN uptake, there was no complete IHN molecule in the blood, and the ability of IHN to activate SIRT1 activity was not significant and may be interfered by inositol, which imply that it did not have a strong potential for calorimetric mimetic.

並列關鍵字

vitamin B3 ； calorie restriction mimetic ； inositol hexanicotinate ； Hs68 cells ； nicotinic acid ； nicotinamide ； nicotinamide riboside

參考文獻

Altschul R, Hoffer AThe effect of nicotinic acid upon serum cholesterol and upon basal metabolic rate of young normal adults.Arch Biochem Biophys. 1958;73(2):420-4.

Allard JS, Perez E, Zou S, de Cabo R.Dietary activators of Sirt1.Mol Cell Endocrinol. 2009;299(1):58-63. doi: 10.1016/j.mce. Nov 1.

Baur JA, Pearson KJ, Price NL, et al., Resveratrol improves health and survival of mice on ahigh-calorie diet. Nature2006;444(7117): 337-42.

Belenky P, Racette FG, Bogan KL, McClure JM, Smith JS, Brenner C. Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+. Cell2007;129(3):473-84.

Bai P, Canto C, Brunyánszki A, Huber A, Szántó M, Cen Y, Yamamoto H, Houten SM, Kiss B, Oudart H, Gergely P, Menissier-de Murcia J, Schreiber V, Sauve AA, Auwerx J.PARP-2 regulates SIRT1 expression and whole-body energy expenditure.Cell Metab. 2011;13(4):450-60.doi:10.1016/j.cmet.2011.03.013.