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.

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.