DOI: 10.5061/dryad.g1jwstqx7
title: Data from: Hepatic steatosis induced by nicotine plus Coca-Cola™ is prevented by nicotinamide riboside (NR)

description: 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.
description: 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
 <sup>o</sup>C) and photoperiod (12-h light and 12-h dark
 cycle) with food and water <em>ad libitum</em>. 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<sup>+</sup> 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. 
description: # Electron microcopy images Nicotine & Coke
 [https://doi.org/10.5061/dryad.g1jwstqx7](https://doi.org/10.5061/dryad.g1jwstqx7) Adult male (10-week-old) C57BL/6J mice (22–24 g BW) were housed (4–5 per cage) in a standard animal facility under controlled temperature (22 <sup>o</sup>C) and photoperiod (12-h light and 12-h dark cycle) with food and water *ad libitum*. Mice received twice-daily intraperitoneal (IP) injections of nicotine (0.75 mg/kg body weight) or saline (vehicle) for 10 weeks. Additionally, mice in both groups received Coca-Cola containing High Fructose Corn Syrup (Coke), which is a special run of Coke containing sugar and absolutely no High Fructose Corn Syrup, sugar Coke (S-Coke), or water in drinking bottles. To prevent hepatic damage and increase NAD<sup>+</sup> levels, additional groups of mice were supplemented with twice daily IP injection of NR in saline solution (200 mg/Kg BW/day). The groups were: 1. Saline + Water (Sal+Water). 2. Nicotine + Water (Nic+Water). 3. Saline + Coke (Sal+Coke). 4. Nicotine + S-Coke (Nic+S-Coke). 5. Nicotine + Coke (Nic+Coke). 6. Nicotine + Coke + Nicotinamide riboside (Nic+Coke+NR). Mice fasted overnight before euthanization with a lethal injection of sodium pentobarbital (200 mg/kg BW) and the liver was removed. 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). Additional liver pieces were quickly removed snap-frozen in liquid nitrogen and stored at -80 ̊C for Western blot analysis. 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). Liver pathology was evaluated using conventional histological analysis on hematoxylin and eosin (HE)-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. For EM 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 (Hitachi, Indianapolis, IN, USA).