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IntroductionCigarettes 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.MethodsC57BL/6J 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.ResultsOur 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 (PGC1) signaling.ConclusionWe 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.

Electronic cigarettes (e-cigarettes) are a prevalent alternative to conventional nicotine cigarettes among smokers and people who have never smoked. Increased concentrations of serum free fatty acids (FFAs) are crucial in generating lipotoxicity. We studied the effects of acipimox, an antilipolytic drug, on e-cigarette-induced cardiac dysfunction. C57BL/6J wild-type mice on high fat diet were treated with saline, e-cigarette with 2.4% nicotine [e-cigarette (2.4%)], and e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction were diminished in mice exposed to e-cigarettes (2.4%) compared with saline and acipimox-treated mice. Mice exposed to e-cigarette (2.4%) had increased circulating levels of inflammatory cytokines and FFAs, which were diminished by acipimox. Gene Set Enrichment Analysis revealed that e-cigarette (2.4%)-treated mice had gene expression changes in the G2/M DNA damage checkpoint pathway that was normalized by acipimox. Accordingly, we showed that acipimox suppressed the nuclear localization of phospho-p53 induced by e-cigarette (2.4%). Additionally, e-cigarette (2.4%) increased the apurinic/apyrimidinic sites, a marker of oxidative DNA damage which was normalized by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1 protein levels and 4-hydroxynonenal (4-HNE). These markers of oxidative stress were decreased by acipimox. Therefore, inhibiting lipolysis with acipimox normalizes the physiological changes induced by e-cigarettes and the associated increase in inflammatory cytokines, oxidative stress, and DNA damage.

CARF (CDKN2AIP) regulates cellular fate in response to various stresses. However, its role in metabolic stress is unknown. We found that fatty livers from mice exhibit low CARF expression. Similarly, overloaded palmitate inhibited CARF expression in HepG2 cells, suggesting that excess fat-induced stress downregulates hepatic CARF. In agreement with this, silencing and overexpressing CARF resulted in higher and lower fat accumulation in HepG2 cells, respectively. Furthermore, CARF overexpression lowered the ectopic palmitate accumulation in HepG2 cells. We were interested in understanding the role of hepatic CARF and underlying mechanisms in the development of NAFLD. Mechanistically, transcriptome analysis revealed that endoplasmic reticulum (ER) stress and oxidative stress pathway genes significantly altered in the absence of CARF. IRE1α, GRP78, and CHOP, markers of ER stress, were increased, and the treatment with TUDCA, an ER stress inhibitor, attenuated fat accumulation in CARF-deficient cells. Moreover, silencing CARF caused a reduction of GPX3 and TRXND3, leading to oxidative stress and apoptotic cell death. Intriguingly, CARF overexpression in HFD-fed mice significantly decreased hepatic steatosis. Furthermore, overexpression of CARF ameliorated the aberrant ER function and oxidative stress caused by fat accumulation. Our results further demonstrated that overexpression of CARF alleviates HFD-induced insulin resistance assessed with ITT and GTT assay. Altogether, we conclude that excess fat-induced reduction of CARF dysregulates ER functions and lipid metabolism leading to hepatic steatosis.

IntroductionCigarettes 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. MethodsC57BL/6J 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. ResultsOur 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 (PGC1) signaling. ConclusionWe 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.

Abstract Electronic cigarettes (e-cigarettes) are a prevalent alternative to conventional nicotine cigarettes among smokers and people who have never smoked. Increased concentrations of serum free fatty acids (FFAs) are crucial in generating lipotoxicity. We studied the effects of acipimox, an antilipolytic drug, on e-cigarette-induced cardiac dysfunction. C57BL/6J wild-type mice on high fat diet were treated with saline, e-cigarette with 2.4% nicotine [e-cigarette (2.4%)], and e-cigarette (2.4%) plus acipimox for 12 weeks. Fractional shortening and ejection fraction were diminished in mice exposed to e-cigarettes (2.4%) compared with saline and acipimox-treated mice. Mice exposed to e-cigarette (2.4%) had increased circulating levels of inflammatory cytokines and FFAs, which were diminished by acipimox. Gene Set Enrichment Analysis revealed that e-cigarette (2.4%)-treated mice had gene expression changes in the G2/M DNA damage checkpoint pathway that was normalized by acipimox. Accordingly, we showed that acipimox suppressed the nuclear localization of phospho-p53 induced by e-cigarette (2.4%). Additionally, e-cigarette (2.4%) increased the apurinic/apyrimidinic sites, a marker of oxidative DNA damage which was normalized by acipimox. Mice exposed to e-cigarette (2.4%) had increased cardiac Heme oxygenase 1 protein levels and 4-hydroxynonenal (4-HNE). These markers of oxidative stress were decreased by acipimox. Therefore, inhibiting lipolysis with acipimox normalizes the physiological changes induced by e-cigarettes and the associated increase in inflammatory cytokines, oxidative stress, and DNA damage.

Electronic cigarettes or e-cigarettes are the most frequently used tobacco product among adolescents. Despite the widespread use of e-cigarettes and the known detrimental cardiac consequences of nicotine, the effects of e-cigarettes on the cardiovascular system are not well-known. Several in vitro and in vivo studies delineating the mechanisms of the impact of e-cigarettes on the cardiovascular system have been published. These include mechanisms associated with nicotine or other components of the aerosol or thermal degradation products of e-cigarettes. The increased hyperlipidemia, sympathetic dominance, endothelial dysfunction, DNA damage, and macrophage activation are prominent effects of e-cigarettes. Additionally, oxidative stress and inflammation are unifying mechanisms at many levels of the cardiovascular impairment induced by e-cigarette exposure. This review outlines the contribution of e-cigarettes in the development of cardiovascular diseases and their molecular underpinnings.

Epidemiological studies have shown an increased risk of cardiovascular diseases in children born to mothers who smoked during pregnancy. The cardiovascular risk in the offspring associated with in utero nicotine exposure is further exaggerated by maternal obesity. The consumption of electronic cigarettes (e-cigarettes) is alarmingly increasing among adolescents and young adults without the knowledge of their harmful health effects. There has also been a substantial increase in e-cigarette use by women of reproductive age. This study investigates the detrimental effects of gestational exposure of e-cigarette and a high-fat diet (HFD) on neonatal hearts. Time-mated pregnant mice were fed a HFD and exposed to saline or e-cigarette aerosol with 2.4% nicotine from embryonic day 4 (E4) to E20. We demonstrated that in utero exposure of e-cigarettes and HFD from E4 to E20 triggers cardiomyocyte (CM) apoptosis in the offspring at postnatal day1 (PND1), PND3, and PND14. Induction of CM apoptosis following gestational exposure of e-cigarettes and HFD was associated with inactivation of AMP-activated protein kinase (AMPK), increased cardiac oxidative stress coupled with perturbation of cardiac BAX/BCL-2 ratio and activation of caspase 3 at PND 14. Electron microscopy further revealed that left ventricles of pups at PND14 after e-cigarette exposure exhibited apoptotic nuclei, convoluted nuclear membranes, myofibrillar derangement, and enlarged mitochondria occasionally showing signs of crystolysis, indicative of cardiomyopathy and cardiac dysfunction. Our results show profound adverse effects of prenatal exposure of e-cigarette plus HFD in neonatal hearts that may lead to long-term adverse cardiac consequences in the adult.

The study of the intestinal or gut microbiome is a newer field that is rapidly gaining attention. Bidirectional communication between gut microbes and the host can impact numerous biological systems regulating immunity and metabolism to either promote or negatively impact the host's health. Habitual routines, dietary choices, socioeconomic status, education, host genetics, medical care and environmental factors can all contribute to the composition of an individual's microbiome. A key environmental factor that may cause negative outcomes is the consumption of nicotine products. The effects of nicotine on the host can be exacerbated by poor dietary choices and together can impact the composition of the gut microbiota to promote the development of metabolic disease including non-alcoholic fatty liver disease. This review explores the contribution of nicotine, poor dietary choices and other unhealthy lifestyle factors to gut dysbiosis.

The use of electronic nicotine delivery systems (ENDS), also known as e-cigarettes, with a variety of e-liquids/e-juices, is increasing at an alarming rate among adolescents who do not realize the potential harmful health effects. This study examines the harmful effects of ENDS on the liver. Apolipoprotein E null (ApoE-/-) mice on a western diet (WD) were exposed to saline or ENDS with 2.4% nicotine aerosol for 12 weeks using our mouse ENDS exposure model system, which delivers nicotine to mice and leads to equivalent serum cotinine levels found in human cigarette users. ApoE-/- mice on a WD exposed to ENDS exhibited a marked increase in hepatic lipid accumulation compared with ApoE-/- on a similar diet exposed to saline aerosol. The detrimental effects of ENDS on hepatic steatosis were associated with significantly greater oxidative stress, increased hepatic triglyceride levels, and increased hepatocyte apoptosis, independent of adenosine monophosphate-activated protein kinase signaling. In addition, hepatic RNA sequencing analysis revealed that 433 genes were differentially expressed in ENDS-exposed mice on WD compared with saline-exposed mice. Functional analysis indicates that genes associated with lipid metabolism, cholesterol biosynthesis, and circadian rhythm were most significantly altered in the liver in response to ENDS. Conclusion: These results demonstrate profound adverse effects of ENDS on the liver. This is important information for regulatory agencies as they regulate ENDS.

In spite of the widespread use of electronic cigarettes, also known as e-cigarettes, and the proposed adverse cardiac effects of nicotine, the detrimental effects of e-cigarettes on the heart are not well known. This study examines the detrimental effects of e-cigarettes with nicotine at doses that yield circulating nicotine and cotinine in the ranges similar to the levels found in habitual smokers, and a high fat diet (HFD) on cardiac structure and function in a commonly used model of diet-induced obesity (DIO). C57BL/6J mice on an HFD were exposed to e-cigarette in the presence (2.4% nicotine) or absence (0% nicotine) of nicotine and saline aerosol for 12 weeks. Echocardiographic data demonstrated a decrease in left ventricular (LV) fractional shortening, LV ejection fraction, and velocity of circumferential fiber shortening (VCF) in mice treated with e-cigarette (2.4% nicotine) compared to e-cigarette (0% nicotine) or saline exposed mice. Cardiomyocytes (CMs) of mice treated with e-cigarette (2.4% nicotine) exhibited LV abnormalities, including lipid accumulation (ventricular steatosis), myofibrillar derangement and destruction, and mitochondrial hypertrophy, as revealed by transmission electron microscopy. The detrimental effects of e-cigarettes (2.4% nicotine) on cardiac structure and function was accompanied by increased oxidative stress, plasma free fatty acid levels, CM apoptosis, and inactivation of AMP-activated protein kinase and activation of its downstream target, acetyl-CoA-carboxylase. Our results indicate profound adverse effects of e-cigarettes (2.4% nicotine) on the heart in obese mice and raise questions about the safety of the nicotine e-cigarettes use.

α7-Nicotinic acetylcholine receptor (α7nAChR) agonists confer protection against a wide variety of cytotoxic insults and suppress oxidative stress and apoptosis in various cell systems, including hepatocytes. We recently demonstrated that nicotine, when combined with a high-fat diet (HFD), triggers oxidative stress, activates hepatocyte apoptosis, and exacerbates HFD-induced hepatic steatosis in male mice. This study evaluates whether PNU-282987 (PNU), a specific α7nAChR agonist, is effective in preventing nicotine plus HFD-induced hepatic steatosis. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice-daily intraperitoneal injections of 0.75 mg/kg body weight (BW) of nicotine, PNU (0.26 mg/kg BW), PNU plus nicotine, or saline for 10 weeks. PNU treatment was effective in attenuating nicotine plus HFD-induced increase in hepatic triglyceride levels, hepatocyte apoptosis, and hepatic steatosis. The preventive effects of PNU on nicotine plus HFD-induced hepatic steatosis were mediated by suppression of oxidative stress and activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) together with inhibition of its downstream target sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), and acetyl-coenzyme A-carboxylase (ACC). We conclude that the α7nAChR agonist PNU protects against nicotine plus HFD-induced hepatic steatosis in obese mice. PNU appears to work at various steps of signaling pathways involving suppression of oxidative stress, activation of AMPK, and inhibition of SREBP1c, FAS, and ACC. α7nAChR agonists may be an effective therapeutic strategy for ameliorating fatty liver disease, especially in obese smokers.

Electronic cigarettes (E-cig) use is increasing rapidly, particularly among youths. Animal models for E-cig exposure with pharmacokinetics resembling human E-cig users are lacking. We developed an E-cig aerosol exposure system for rodents and a chronic intermittent delivery method that simulates E-cig users who vape episodically during wakefulness and abstain during sleep. Mice were exposed to E-cig in a programmed schedule at very low, low, medium, or high doses defined by duration of each puff, number of puffs per delivery episode and frequency of episodes in the dark phase of a 12/12-h circadian cycle for 9 consecutive days. The plasma nicotine/cotinine levels and their time courses were determined using LC/MS-MS. We assessed the body weight, food intake and locomotor activity of Apolipoprotein E null (ApoE-/-) mice exposed to chronic intermittent E-cig aerosol. Plasma nicotine and cotinine levels were positively correlated with exposure doses. Nicotine and cotinine levels showed a circadian variation as they increased with time up to the maximum nicotine level of 21.8 ± 7.1 ng/mL during the daily intermittent E-cig exposure in the 12-h dark phase and then declined during the light phase when there was no E-cig delivery. Chronic E-cig exposure to ApoE-/- mice decreased body weight, food intake and increased locomotion. Our rodent E-cig exposure system and chronic intermittent exposure method yield clinically relevant nicotine pharmacokinetics associated with behavioral and metabolic changes. The methodologies are essential tools for in vivo studies of the health impacts of E-cig exposure on CNS, cardiovascular, pulmonary, hepatic systems, metabolism and carcinogenesis.

Electronic cigarettes (e-cigarettes), also known as electronic nicotine delivery systems, are a popular alternative to conventional nicotine cigarettes, both among smokers and those who have never smoked. In spite of the widespread use of e-cigarettes and the proposed detrimental cardiac and atherosclerotic effects of nicotine, the effects of e-cigarettes on these systems are not known. In this study, we investigated the cardiovascular and cardiac effects of e-cigarettes with and without nicotine in apolipoprotein-E knockout (ApoE-/-) mice. We developed an e-cigarette exposure model that delivers nicotine in a manner similar to that of human e-cigarettes users. Using commercially available e-cigarettes, bluCig PLUS, ApoE-/- mice were exposed to saline, e-cigarette without nicotine [e-cigarette (0%)], and e-cigarette with 2.4% nicotine [e-cigarette (2.4%)] aerosol for 12 wk. Echocardiographic data show that mice treated with e-cigarette (2.4%) had decreased left ventricular fractional shortening and ejection fraction compared with e-cigarette (0%) and saline. Ventricular transcriptomic analysis revealed changes in genes associated with metabolism, circadian rhythm, and inflammation in e-cigarette (2.4%)-treated ApoE-/- mice. Transmission electron microscopy revealed that cardiomyocytes of mice treated with e-cigarette (2.4%) exhibited ultrastructural abnormalities indicative of cardiomyopathy. Additionally, we observed increased oxidative stress and mitochondrial DNA mutations in mice treated with e-cigarette (2.4%). ApoE-/- mice on e-cigarette (2.4%) had also increased atherosclerotic lesions compared with saline aerosol-treated mice. These results demonstrate adverse effects of e-cigarettes on cardiac function in mice.NEW & NOTEWORTHY The present study is the first to show that mice exposed to nicotine electronic cigarettes (e-cigarettes) have decreased cardiac fractional shortening and ejection fraction in comparison with controls. RNA-seq analysis reveals a proinflammatory phenotype induced by e-cigarettes with nicotine. We also found increased atherosclerosis in the aortic root of mice treated with e-cigarettes with nicotine. Our results show that e-cigarettes with nicotine lead to detrimental effects on the heart that should serve as a warning to e-cigarette users and agencies that regulate them.

The prevalence of electronic cigarette (e-cigarettes) use has rapidly increased worldwide. Use of tobacco products has been associated with DNA damage and metabolic syndrome. Using Apolipoprotein E knockout (ApoE-/-) mice on a western diet (WD), a mouse model of non-alcoholic fatty liver disease (NAFLD), we recently demonstrated that nicotine in e-cigarettes activates hepatocyte apoptosis, and causes hepatic steatosis. This study examines the harmful effects of e-cigarettes on the liver with a special emphasis on DNA damage and mitochondrial dysfunction. ApoE-/- mice were exposed to saline, e-cigarettes without nicotine or e-cigarettes with 2.4% nicotine for 12 weeks using our newly developed mouse e-cigarette exposure model system that delivers nicotine to mice leading to equivalent serum cotinine levels found in human cigarette users. Mice exposed to e-cigarette (2.4% nicotine) had increased apurinic/apyrimidinic (AP) sites, a manifestation of DNA damage. Additionally, e-cigarette (2.4% nicotine) produced a decrease in NAD+/NADH ratio and increased oxidative stress in hepatic cells, in comparison with saline and e-cigarette (0%). Western blot analysis showed that mice treated with e-cigarette (2.4% nicotine) had increased poly (ADP ribose) polymerase (PARP1) activity associated with reduced levels of Sirtuin 1 (SIRT1). Furthermore, mitochondrial DNA mutations and PTEN-induced kinase 1 (PINK1) were increased in mice treated with e-cigarette (2.4% nicotine). Transmission electron microscopy revealed that hepatocytes of mice treated with e-cigarette (2.4% nicotine) exhibited increased vacuolization of the mitochondria and a reduction in cellular organelles. These results demonstrate the adverse effects of e-cigarettes exposure leading to NAD+ deficiency which may suggest a mechanistic link between e-cigarette-induced hepatic DNA damage and mitochondrial dysfunction.

Cigarette smoking is an important risk factor for diabetes, cardiovascular disease and non-alcoholic fatty liver disease. The health risk associated with smoking can be aggravated by obesity. Smoking might also trigger cardiomyocyte (CM) apoptosis. Given that CM apoptosis has been implicated as a potential mechanism in the development of cardiomyopathy and heart failure, we characterize the key signaling pathways in nicotine plus high-fat diet (HFD)-induced CM apoptosis. Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily intraperitoneal (IP) injections of nicotine (0.75 mg/kg body weight [BW]) or saline for 16 weeks. An additional group of nicotine-treated mice on HFD received twice-daily IP injections of mecamylamine (1 mg/kg BW), a non-selective nicotinic acetylcholine receptor antagonist, for 16 weeks. Nicotine when combined with HFD led to a massive increase in CM apoptosis that was fully prevented by mecamylamine treatment. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase-2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P

Non-alcoholic fatty liver disease (NAFLD) poses a serious health hazard affecting 20-40% of adults in the general population in the USA and over 70% of the obese and extremely obese people. In addition to obesity, nicotine is recognized as a risk factor for NAFLD, and it has been reported that nicotine can exaggerate obesity-induced hepatic steatosis. The development of NAFLD has serious clinical complications because of its potential progression from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. Multiple mechanisms can be involved in nicotine plus high-fat diet-induced (HFD) hepatic steatosis. Emerging evidence now suggests that nicotine exacerbates hepatic steatosis triggered by HFD, through increased oxidative stress and hepatocellular apoptosis, decreased phosphorylation (inactivation) of adenosine-5-monophosphate-activated protein kinase and, in turn, up-regulation of sterol response-element binding protein 1-c, fatty acid synthase, and activation of acetyl-coenzyme A-carboxylase, leading to increased hepatic lipogenesis. There is also growing evidence that chronic endoplasmic reticulum stress through regulation of several pathways leading to oxidative stress, inflammation, perturbed hepatic lipid homeostasis, apoptosis, and autophagy can induce hepatic steatosis and its progression to NASH. Evidence also suggests a central role of the gut microbiota in obesity and its related disorders, including NAFLD. This review explores the contribution of nicotine and obesity to the development of NAFLD and its molecular underpinning.

Chromogranin A (CgA) is widely expressed in endocrine and neuroendocrine tissues as well as in the central nervous system. We observed CgA expression (mRNA and protein) in the gastrocnemius (GAS) muscle and found that performance of CgA-deficient Chga-KO mice in treadmill exercise was impaired. Supplementation with CgA in Chga-KO mice restored exercise ability suggesting a novel role for endogenous CgA in skeletal muscle function. Chga-KO mice display (i) lack of exercise-induced stimulation of pAKT, pTBC1D1 and phospho-p38 kinase signaling, (ii) loss of GAS muscle mass, (iii) extensive formation of tubular aggregates (TA), (iv) disorganized cristae architecture in mitochondria, (v) increased expression of the inflammatory cytokines Tnfα, Il6 and Ifnγ, and fibrosis. The impaired maximum running speed and endurance in the treadmill exercise in Chga-KO mice correlated with decreased glucose uptake and glycolysis, defects in glucose oxidation and decreased mitochondrial cytochrome C oxidase activity. The lack of adaptation to endurance training correlated with the lack of stimulation of p38MAPK that is known to mediate the response to tissue damage. As CgA sorts proteins to the regulated secretory pathway, we speculate that lack of CgA could cause misfolding of membrane proteins inducing aggregation of sarcoplasmic reticulum (SR) membranes and formation of tubular aggregates that is observed in Chga-KO mice. In conclusion, CgA deficiency renders the muscle energy deficient, impairs performance in treadmill exercise and prevents regeneration after exercise-induced tissue damage.

CARF (CDKN2AIP) regulates cellular fate in response to various stresses. However, its role in metabolic stress is unknown. We found that fatty livers from mice exhibit low CARF expression. Similarly, overloaded palmitate inhibited CARF expression in HepG2 cells, suggesting that excess fat-induced stress downregulates hepatic CARF. In agreement with this, silencing and overexpressing CARF resulted in higher and lower fat accumulation in HepG2 cells, respectively. Furthermore, CARF overexpression lowered the ectopic palmitate accumulation in HepG2 cells. We were interested in understanding the role of hepatic CARF and underlying mechanisms in the development of NAFLD. Mechanistically, transcriptome analysis revealed that endoplasmic reticulum (ER) stress and oxidative stress pathway genes significantly altered in the absence of CARF. IRE1α, GRP78, and CHOP, markers of ER stress, were increased, and the treatment with TUDCA, an ER stress inhibitor, attenuated fat accumulation in CARF-deficient cells. Moreover, silencing CARF caused a reduction of GPX3 and TRXND3, leading to oxidative stress and apoptotic cell death. Intriguingly, CARF overexpression in HFD-fed mice significantly decreased hepatic steatosis. Furthermore, overexpression of CARF ameliorated the aberrant ER function and oxidative stress caused by fat accumulation. Our results further demonstrated that overexpression of CARF alleviates HFD-induced insulin resistance assessed with ITT and GTT assay. Altogether, we conclude that excess fat-induced reduction of CARF dysregulates ER functions and lipid metabolism leading to hepatic steatosis.

Electronic cigarettes or e-cigarettes are the most frequently used tobacco product among adolescents. Despite the widespread use of e-cigarettes and the known detrimental cardiac consequences of nicotine, the effects of e-cigarettes on the cardiovascular system are not well-known. Several in vitro and in vivo studies delineating the mechanisms of the impact of e-cigarettes on the cardiovascular system have been published. These include mechanisms associated with nicotine or other components of the aerosol or thermal degradation products of e-cigarettes. The increased hyperlipidemia, sympathetic dominance, endothelial dysfunction, DNA damage, and macrophage activation are prominent effects of e-cigarettes. Additionally, oxidative stress and inflammation are unifying mechanisms at many levels of the cardiovascular impairment induced by e-cigarette exposure. This review outlines the contribution of e-cigarettes in the development of cardiovascular diseases and their molecular underpinnings.

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.

Low vitamin D levels are associated with minority subjects, the metabolic syndrome, and inflammation. The effect of vitamin D supplementation on markers of inflammation has not been well studied. The aim of the study was to evaluate the effects of high doses of vitamin D supplementation for 1 year on serum biomarkers of inflammation in Latino and African-American subjects with pre-diabetes and hypovitaminosis D. Latino (n=69) and African-American (n=11) subjects who had both pre-diabetes and hypovitaminosis D with a mean age of 52.0 years, a BMI of 32.7 kg/m(2), and 70% of whom were females, were randomized to receive weekly doses (mean±SD) of vitamin D (85 300 IU±16 000) or placebo oil for 1 year. Serum levels of interleukin-6, tumor necrosis factor, highly sensitive C-reactive protein), plasminogen activator inhibitor 1, and insulin-like growth factor-1 were measured at baseline, 6, and 12 months. Serum 25-OH vitamin D levels of 22 ng/ml at baseline quickly rose to nearly 70 ng/ml in subjects receiving vitamin D and did not change in the placebo group. Two-way repeated measures ANOVA showed no differences between the 2 groups in any of the 5 selected parameters. High dose vitamin D supplementation for 1 year in minority subjects with pre-diabetes and hypovitaminosis D failed to affect serum biomarkers of inflammation.Clinical trial reg. no.: NCT00876928, clinicaltrials.gov.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver pathologies and is associated with obesity and the metabolic syndrome. Here, we investigated the molecular mechanisms by which a novel cystine based glutathione precursor with added selenomethionine (F1) prevents hepatic steatosis in a moderate high fat dietary model of NAFLD. Adult (8 weeks old), male apolipoprotein E (ApoE)−/− mice were fed with a normal diet (ND) or high fat diet (HFD), consisting of 21% fat and 0.21% cholesterol, with or without dietary supplementation of F1 (3 g/kg food) for 16 weeks. Compared with ApoE−/− mice fed with ND with or without F1, ApoE−/− mice fed with HFD exhibited significant weight gain, hepatomegaly, and increased serum cholesterol and triglycerides levels with no change in serum albumin levels. High resolution light and electron microscopy revealed micro-and macro-vesicular steatosis in ApoE−/− mice fed on a HFD. HFD- induced obesity also led to increased lipogenesis, oxidative stress, activation of c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), perturbation of the BAX/BCL-2 rheostat, hepatocyte apoptosis, and activation of caspases 9 and 3. F1 fully prevented the adverse effects of HFD on serum triglyceride levels, body and liver weights, and hepatic steatosis and substantially attenuated HFD-induced increase in lipogenesis, oxidative stress, kinase activation, apoptotic signaling, and hepatocyte ultrastructural abnormalities. These results demonstrate that administration of F1, a glutathione precursor, ameliorates HFD-induced hepatic steatosis in ApoE−/− mice and emphasizes the role of oxidative stress in diet-induced obesity and hepatic steatosis.

The study of the intestinal or gut microbiome is a newer field that is rapidly gaining attention. Bidirectional communication between gut microbes and the host can impact numerous biological systems regulating immunity and metabolism to either promote or negatively impact the host’s health. Habitual routines, dietary choices, socioeconomic status, education, host genetics, medical care and environmental factors can all contribute to the composition of an individual’s microbiome. A key environmental factor that may cause negative outcomes is the consumption of nicotine products. The effects of nicotine on the host can be exacerbated by poor dietary choices and together can impact the composition of the gut microbiota to promote the development of metabolic disease including non-alcoholic fatty liver disease. This review explores the contribution of nicotine, poor dietary choices and other unhealthy lifestyle factors to gut dysbiosis.

In spite of the widespread use of electronic cigarettes, also known as e-cigarettes, and the proposed adverse cardiac effects of nicotine, the detrimental effects of e-cigarettes on the heart are not well known. This study examines the detrimental effects of e-cigarettes with nicotine at doses that yield circulating nicotine and cotinine in the ranges similar to the levels found in habitual smokers, and a high fat diet (HFD) on cardiac structure and function in a commonly used model of diet-induced obesity (DIO). C57BL/6J mice on an HFD were exposed to e-cigarette in the presence (2.4% nicotine) or absence (0% nicotine) of nicotine and saline aerosol for 12 weeks. Echocardiographic data demonstrated a decrease in left ventricular (LV) fractional shortening, LV ejection fraction, and velocity of circumferential fiber shortening (VCF) in mice treated with e-cigarette (2.4% nicotine) compared to e-cigarette (0% nicotine) or saline exposed mice. Cardiomyocytes (CMs) of mice treated with e-cigarette (2.4% nicotine) exhibited LV abnormalities, including lipid accumulation (ventricular steatosis), myofibrillar derangement and destruction, and mitochondrial hypertrophy, as revealed by transmission electron microscopy. The detrimental effects of e-cigarettes (2.4% nicotine) on cardiac structure and function was accompanied by increased oxidative stress, plasma free fatty acid levels, CM apoptosis, and inactivation of AMP-activated protein kinase and activation of its downstream target, acetyl-CoA-carboxylase. Our results indicate profound adverse effects of e-cigarettes (2.4% nicotine) on the heart in obese mice and raise questions about the safety of the nicotine e-cigarettes use.

Breast cancer results from a complex interplay of genetics and environment that alters immune and inflammatory systems to promote tumorigenesis. Obesity and cigarette smoking are well-known risk factors associated breast cancer development. Nicotine known to decrease inflammatory signals also modulates immune responses that favor breast cancer development. However, the mechanisms by which nicotine and obesity contribute to breast cancer remain poorly understood. In this study, we examined potential mechanisms by which nicotine (NIC) and high-fat diet (HFD) promote growth of HCC70 and HCC1806 xenografts from African American (AA) triple negative (TN) breast cancer cells. Immunodeficient mice fed on HFD and treated with NIC generated larger HCC70 and HCC1806 tumors when compared to NIC or HFD alone. Increased xenograft growth in the presence of NIC and HFD was accompanied by higher levels of tissue-resident macrophage markers and anti-inflammatory cytokines including IL4, IL13, and IL10. We further validated the involvement of these players by in vitro and ex vivo experiments. We found a proinflammatory milieu with increased expression of IL6 and IL12 in xenografts with HFD. In addition, nicotine or nicotine plus HFD increased a subset of mammary cancer stem cells (MCSCs) and key adipose browning markers CD137 and TMEM26. Interestingly, there was upregulation of stress-induced pp38 MAPK and pERK1/2 in xenografts exposed to HFD alone or nicotine plus HFD. Scratch-wound assay showed marked reduction in proliferation/migration of nicotine and palmitate-treated breast cancer cells with mecamylamine (MEC), a nicotine acetylcholine receptor (nAchR) antagonist. Furthermore, xenograft development in immune-deficient mice, fed HFD plus nicotine, was reduced upon cotreatment with MEC and SB 203580, a pp38MAPK inhibitor. Our study demonstrates the presence of nicotine and HFD in facilitating an anti-inflammatory tumor microenvironment that influences breast tumor growth. This study also shows potential efficacy of combination therapy in obese breast cancer patients who smoke.

The prevalence of electronic cigarette (e-cigarettes) use has rapidly increased worldwide. Use of tobacco products has been associated with DNA damage and metabolic syndrome. Using Apolipoprotein E knockout (ApoE−/−) mice on a western diet (WD), a mouse model of non-alcoholic fatty liver disease (NAFLD), we recently demonstrated that nicotine in e-cigarettes activates hepatocyte apoptosis, and causes hepatic steatosis. This study examines the harmful effects of e-cigarettes on the liver with a special emphasis on DNA damage and mitochondrial dysfunction. ApoE−/− mice were exposed to saline, e-cigarettes without nicotine or e-cigarettes with 2.4% nicotine for 12 weeks using our newly developed mouse e-cigarette exposure model system that delivers nicotine to mice leading to equivalent serum cotinine levels found in human cigarette users. Mice exposed to e-cigarette (2.4% nicotine) had increased apurinic/apyrimidinic (AP) sites, a manifestation of DNA damage. Additionally, e-cigarette (2.4% nicotine) produced a decrease in NAD+/NADH ratio and increased oxidative stress in hepatic cells, in comparison with saline and e-cigarette (0%). Western blot analysis showed that mice treated with e-cigarette (2.4% nicotine) had increased poly (ADP ribose) polymerase (PARP1) activity associated with reduced levels of Sirtuin 1 (SIRT1). Furthermore, mitochondrial DNA mutations and PTEN-induced kinase 1 (PINK1) were increased in mice treated with e-cigarette (2.4% nicotine). Transmission electron microscopy revealed that hepatocytes of mice treated with e-cigarette (2.4% nicotine) exhibited increased vacuolization of the mitochondria and a reduction in cellular organelles. These results demonstrate the adverse effects of e-cigarettes exposure leading to NAD+ deficiency which may suggest a mechanistic link between e-cigarette-induced hepatic DNA damage and mitochondrial dysfunction.