Hepatic steatosis induced by nicotine plus Coca-Cola™ is prevented by nicotinamide riboside (NR)

Introduction. Cigarettes containing nicotine (Nic) are a risk factor for the development of cardiovascular and metabolic diseases. We reported that Nic delivered via injections or e-cigarette vapor led to hepatic steatosis in mice fed with a high-fat diet. High-fructose corn syrup (HFCS) is the main sweetener in sugar-sweetened beverages (SSBs) in the US. Increased consumption of SSBs with HFCS is associated with increased risks of non-alcoholic fatty liver disease (NAFLD). Nicotinamide riboside (NR) increases mitochondrial nicotinamide adenine dinucleotide (NAD+) and protects mice against hepatic steatosis. This study evaluated if Nic plus Coca-Cola™ (Coke) with HFCS can cause hepatic steatosis and that can be protected by NR.

Methods. C57BL6 mice received twice daily intraperitoneal (IP) injections of Nic or saline and were given Coke (HFCS), or Coke with sugar, and NR supplementation for 10 weeks.

Results. Our results show that Nic+Coke caused increased caloric intake and induced hepatic steatosis, and the addition of NR prevented these changes. Western blot analysis showed lipogenesis markers were activated (increased cleavage of the sterol regulatory element-binding protein 1 [SREBP1c] and reduction of phospho-Acetyl-CoA Carboxylase [p-ACC]) in the Nic+Coke compared to the Sal+Water group. The hepatic detrimental effects of Nic+Coke were mediated by decreased NAD+ signaling, increased oxidative stress, and mitochondrial damage. NR reduced oxidative stress and prevented mitochondrial damage by restoring protein levels of Sirtuin1 (Sirt1) and peroxisome proliferator-activated receptor coactivator 1-alpha (PGC1a) signaling.

Discussion. We conclude that Nic+Coke has an additive effect on producing hepatic steatosis, and NR is protective. This study suggests concern for the development of NAFLD in subjects who consume nicotine and drink SSBs with HFCS.

Methods
Adult male (10-week-old) C57BL/6J mice (22–24 g BW) purchased from Taconic Farms (Germantown, NY, USA) were housed (4–5 per cage) in a standard animal facility under controlled temperature (22 oC) and photoperiod (12-h light and 12-h dark cycle) with food and water ad libitum. Mice received twice-daily intraperitoneal (IP) injections of Nic (0.75 mg/kg body weight) which is equivalent to smoking two-pack a day or saline (vehicle) for 10 weeks as reported previously in our laboratory. Nic was maintained in a dark container to prevent light exposure. Additionally, mice in both groups received Coca-Cola containing HFCS (Coke) (carbonate water, HFCS, caramel color, phosphoric acid, natural flavors, caffeine; 140 cal per 360 mL, 0 g fat, 45 mg sodium, 39 g carbohydrate, and 0 g protein) or sugar Coke (S-Coke) (carbonate water, cane sugar, caramel color, phosphoric acid, natural flavors, caffeine; 140 cal per 360 mL, 0 g fat, 45 mg sodium, 39 g carbohydrate, and 0 g protein), which is a special run of Coke containing sugar and absolutely no HFCS from a store in a neighborhood with a larger Jewish population prior to the holiday of Passover, or water in drinking bottles. To prevent hepatic damage and increase NAD+ levels, additional groups of mice were supplemented with twice daily IP injection of NR in saline solution (200 mg/Kg BW/day) or saline solution (vehicle) to the other groups with each injection having the same volume (200 ml). Mice fasted overnight before euthanization with a lethal injection of sodium pentobarbital (200 mg/kg BW) and the liver was removed. Animal handling and experimentation were in accordance with the recommendation of the current National Institutes of Health guidelines and were approved by the CDU and Lundquist Institute Animal Care and Use Committees (IACUC).

Pieces of the liver were either fixed in 2.5% glutaraldehyde (Sigma Aldrich, St. Louis, MO, USA) for high-resolution light microscopy and electron microscopy (EM). For routine histological studies, we used 4% paraformaldehyde (PFA) (Fisher Scientific, Hampton, NH, USA). Liver pathology was evaluated using conventional histological analysis on hematoxylin and eosin (H&E)-stained sections. Further evaluation of pathology was achieved by high-resolution light microscopy using glutaraldehyde-fixed, osmium tetroxide postfixed, epoxy-embedded, and toluidine blue-stained (TB) sections. The histological features were analyzed by light microscopy Olympus Bx51 (Olympus, Tokyo, Japan) at 10x or 40x objectives in five different fields.

For electron microscopy analysis, small pieces of glutaraldehyde-fixed livers were cut into small pieces, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Epon 812. Embedded liver tissues were cut with an LKB ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Hitachi electron microscopy.

引言。含有尼古丁（nicotine, Nic）的卷烟是心血管疾病与代谢性疾病发生的危险因素。本团队此前报道，通过注射或电子烟蒸汽递送的Nic，可使高脂膳食喂养小鼠出现肝脂肪变性（hepatic steatosis）。高果糖玉米糖浆（High-fructose corn syrup, HFCS）是美国含糖饮料（sugar-sweetened beverages, SSBs）的主要甜味剂。摄入含HFCS的SSBs量增加，与非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）发病风险升高相关。烟酰胺核糖（nicotinamide riboside, NR）可提升线粒体烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD+）水平，并保护小鼠免于肝脂肪变性。本研究旨在评估Nic联合含HFCS的可口可乐™（Coca-Cola™, Coke）是否可诱发肝脂肪变性，以及该病变是否可被NR所阻断。

研究方法。将C57BL/6小鼠每日两次腹腔注射（intraperitoneal, IP）Nic或生理盐水，同时给予含HFCS的Coke、含糖Coke或NR补充剂，干预时长共10周。

研究结果。本研究结果显示，Nic+Coke组小鼠热量摄入增加，并诱发肝脂肪变性，而补充NR可阻断上述变化。蛋白质印迹法（Western blot）分析显示，与Sal+Water组相比，Nic+Coke组的脂肪生成标志物被激活：固醇调节元件结合蛋白1（sterol regulatory element-binding protein 1, SREBP1c）的剪切体水平升高，乙酰辅酶A羧化酶磷酸化形式（phospho-Acetyl-CoA Carboxylase, p-ACC）水平降低。Nic+Coke对肝脏的损害作用，可通过降低NAD+信号通路活性、升高氧化应激水平以及造成线粒体损伤来介导。NR可通过恢复沉默信息调节因子1（Sirtuin1, Sirt1）与过氧化物酶体增殖物激活受体γ共激活因子1-α（peroxisome proliferator-activated receptor coactivator 1-alpha, PGC1a）的信号通路蛋白水平，减轻氧化应激并阻断线粒体损伤。

讨论。本研究结论为，Nic+Coke可产生协同效应诱发肝脂肪变性，而NR对此具有保护作用。本研究提示，对于同时摄入Nic与含HFCS的SSBs人群，需警惕其NAFLD的发生风险。

研究方法
10周龄成年雄性C57BL/6J小鼠（体质量22~24 g）购自美国纽约州杰曼敦市塔科尼农场公司（Taconic Farms），以每笼4~5只饲养于标准动物房，环境温度控制为22℃，光周期为12小时光照/12小时黑暗，自由摄食饮水。小鼠每日两次腹腔注射（IP）Nic（剂量为0.75 mg/kg体质量，等效于每日吸烟两包的尼古丁暴露量）或生理盐水（溶剂对照），干预时长共10周，实验方案参照本团队此前的研究。Nic溶液需避光保存于密闭容器中，以避免光照降解。此外，两组小鼠分别给予含HFCS的Coke（成分：碳酸水、HFCS、焦糖色、磷酸、天然香精、咖啡因；每360 mL含热量140 kcal、脂肪0 g、钠45 mg、碳水化合物39 g、蛋白质0 g）、含糖Coke（S-Coke，成分：碳酸水、蔗糖、焦糖色、磷酸、天然香精、咖啡因；每360 mL含热量140 kcal、脂肪0 g、钠45 mg、碳水化合物39 g、蛋白质0 g，该批次为逾越节前在犹太人口密集街区商店售卖的专属批次含糖可口可乐，完全不含HFCS），或饮用水（以饮水瓶供应）。为减轻肝脏损伤并提升NAD+水平，其余各组小鼠每日两次腹腔注射NR生理盐水溶液（200 mg/kg体质量/天）或生理盐水（溶剂对照），单次注射体积统一为200 ml。小鼠于安乐死前过夜禁食，随后以腹腔注射致死剂量戊巴比妥钠（200 mg/kg体质量）实施安乐死，采集肝脏组织。所有动物操作与实验流程均符合当前美国国立卫生研究院（National Institutes of Health, NIH）指南建议，并经CDU与伦德奎斯特研究所动物护理与使用委员会（IACUC）批准。

肝脏组织样本分为两部分：一部分以2.5%戊二醛（Sigma Aldrich，美国密苏里州圣路易斯市）固定，用于高分辨率光学显微镜与电子显微镜（electron microscopy, EM）检测；另一部分以4%多聚甲醛（paraformaldehyde, PFA，Fisher Scientific，美国新罕布什尔州汉普顿市）固定，用于常规组织学研究。肝脏病理评估采用常规苏木精-伊红（hematoxylin and eosin, H&E）染色切片的组织学分析。进一步的病理评估则通过高分辨率光学显微镜完成：样本经戊二醛固定、四氧化锇后固定、环氧树脂包埋后，以甲苯胺蓝（toluidine blue, TB）染色，使用奥林巴斯BX51光学显微镜（Olympus，日本东京），以10倍或40倍物镜随机选取5个视野进行组织学特征分析。

电子显微镜检测步骤如下：将戊二醛固定的肝脏组织块切成更小的碎片，经1%四氧化锇后固定、梯度乙醇脱水后，以Epon 812环氧树脂包埋。包埋后的肝脏组织用LKB超薄切片机切片，经醋酸铀与柠檬酸铅染色后，使用日立电子显微镜进行观察。