Additive effects of nicotine and high-fat diet on hepatocellular apoptosis in mice: involvement of caspase 2 and inducible nitric oxide synthase-mediated intrinsic pathway signaling

Abstract

Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to nonalcoholic fatty liver disease (NAFLD). The health risk associated with smoking is exaggerated by obesity and is the leading causes of morbidity and mortality worldwide. We recently demonstrated that combined treatment with nicotine and a high-fat diet (HFD) triggers greater oxidative stress, activates hepatocellular apoptosis, and exacerbates HFD-induced hepatic steatosis. Given that hepatocellular apoptosis plays a pivotal role in the pathogenesis of NAFLD, using this model of exacerbated hepatic steatosis, we elucidated the signal transduction pathways involved in HFD plus nicotine-induced liver cell death. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice daily IP injections of 0.75 mg/kg BW of nicotine or saline for 10 weeks. High-resolution light microscopy revealed markedly higher lipid accumulation in hepatocytes from mice received HFD plus nicotine, compared to mice on HFD alone. Addition of nicotine to HFD further resulted in an increase in the incidence of hepatocellular apoptosis and was associated with activation of caspase 2, induction of inducible nitric oxide synthase (iNOS), and perturbation of the BAX/BCL-2 ratio. Together, our data indicate the involvement of caspase 2 and iNOS-mediated apoptotic signaling in nicotine plus HFD-induced hepatocellular apoptosis. Targeting the caspase 2-mediated death pathway may have a protective role in development and progression of NAFLD.

Fig. 1

Representative light microscopic images of glutaraldehyde-fixed, osmium tetroxide post-fixed, epoxy-embedded, and toluidine-blue stained liver sections from mice fed with NCD in the absence (a) or presence (b) of nicotine or HFD without (c) or with (d) nicotine. Note a marked increase in lipid accumulation of varying sizes in the liver after combined treatments with nicotine and HFD (d). e. In situ detection of hepatocyte apoptosis detected by TUNEL in control mice fed NCD (panel I), HFD in the absence (panel II) or presence (arrow; panel III) of nicotine. Scale bar = 20 μm. f. Quantitation of hepatocellular apoptosis. Apoptotic rate was expressed as the percentage of TUNEL-positive nuclei per total nuclei (apoptotic plus non-apoptotic nuclei) counted in a unit reference area. Values are means ± SEM (n=5). Means with unlike superscripts are significantly (p<0.05) different.

Fig. 2

a. Western blot analysis shows increased expression of hepatic active caspase 2 in the combined treatment group as compared to mice fed NCD or HFD alone. GAPDH in the immunoblot is shown as a loading control. b. Quantitation of band intensities. Data for active caspase 2 were normalized to GAPDH. Values are means ± SEM (n=5). Means with unlike superscripts are significantly (p<0.05) different. c. Visualization of caspase 2 activation in hepatocytes by immunohistochemistry in mice fed NCD (panel I) or HFD in the absence (panel II) or presence (panel III) of nicotine. d. iNOS induction in the hepatocytes from mice fed NCD (panel I) or HFD in the absence (panel II) or presence (panel III) of nicotine. f. Double immunofluorescence staining shows TUNEL (green), iNOS (red), and colocalization of TUNEL and iNOS (yellow) in apoptotic hepatocytes. Scale bar=20 μm.

Fig. 3

Key signaling pathways involved in nicotine plus HFD-induced hepatocellular apoptosis. Nicotine when combined with a HFD induces oxidative stress and results in activation of caspase 2 and JNK and induction of iNOS. It is likely that induction of iNOS, through increased nitric oxide production, perturbs BAX/BCL-2 ratio and triggers cytochrome c release from mitochondria, resulting in activation of caspases 9 and 3 and hepatocellular apoptosis [17, 22].