Elsevier

Journal of Hepatology

Volume 51, Issue 3, September 2009, Pages 535-547
Journal of Hepatology

Second-hand smoke stimulates lipid accumulation in the liver by modulating AMPK and SREBP-1

https://doi.org/10.1016/j.jhep.2009.03.026Get rights and content

Background/Aims

The underlying mechanisms of steatosis, the first stage of non-alcoholic fatty liver disease (NAFLD) that is characterized by the accumulation of lipids in hepatocytes, remain unclear. Our study aimed to investigate the hypothesis that cigarette smoke is known to change circulating lipid profiles and thus may also contribute to the accumulation of lipids in the liver.

Methods

Mice and cultured hepatocytes were exposed to sidestream whole smoke (SSW), a major component of “second-hand” smoke and a variety of cellular and molecular approaches were used to study the effects of cigarette smoke on lipid metabolism.

Results

SSW increases lipid accumulation in hepatocytes by modulating the activity of 5′-AMP-activated protein kinase (AMPK) and sterol response element binding protein-1 (SREBP-1), two critical molecules involved in lipid synthesis. SSW causes dephosphorylation/ inactivation of AMPK, which contributes to increased activation of SREBP-1. These changes of activity lead to accumulation of triglycerides in hepatocytes.

Conclusion

These novel findings are important because they point to another risk factor of smoking, i.e., that of contributing to NAFLD. In addition, our results showing that both AMPK and SREBP are critically involved in these effects of smoke point to the potential use of these molecules as targets for treatment of cigarette smoke-induced metabolic diseases.

Introduction

Cigarette smoke contains more than 47,000 toxic substances which significantly harm almost every organ of the body, leading to a variety of diseases and syndromes [1]. Cigarette smoke is composed of MSW (mainstream whole, “first-hand”) and SSW (side stream whole, major component of “second-hand” smoke) smokes. Smoking has been identified as one of the major risk factors for the development of atherosclerosis, the major component of cardiovascular disease [2], [3], [4] that manifests itself, among other things, by high lipid levels in the blood [5], [6]. Furthermore, increasing evidence suggests that risk for cardiovascular disease incidence is associated with non-alcoholic fatty liver diseases (NAFLD) independently of the classical risk factors and features of this metabolic syndrome [7], [8], [9], [10]. In the various stages of NAFLD, hepatic steatosis (the accumulation of lipid in the liver tissue) has become a significant public health concern because it tends to develop into more harmful hepatitis and cirrhosis. Because lipids in steatosis are stored as triglycerides in hepatocytes, understanding what causes this accumulation and how it occurs may contribute to elucidation of NAFLD [11].

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that control the expression of genes required for the biosynthesis of cholesterol, fatty acids, triglycerides, and phospholipids. The three isoforms of SREBP precursors located on the endoplasmic reticulum membrane, designated SREBP-1a, SREBP-1c, and SREBP-2 [12], have different functions and abundance in various animal tissues. The SREBP precursors are activated by a two-step cleavage process that releases the active form that then translocates to the nucleus of the cell to stimulate gene expression [13]. SREBP-1c preferentially controls the expression of genes involved in triglyceride synthesis and accumulation, such as fatty acid synthase (FAS) and acetyl coenzyme-A carboxylase (ACC), whereas SREBP-2 activity has been more closely linked to regulation of genes involved in cholesterol synthesis and uptake, such as low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) [14], [15], [16], [17], [18]. In the liver tissue, the predominant form of SREBPs is SREBP-1c [19].

Another important modulator of lipid metabolism is 5′-AMP-activated protein kinase (AMPK). AMPK was first identified as a kinase that phosphorylates and inactivates ACC, the rate-limiting enzyme in fatty acid biosynthesis [20]. AMP binds and activates AMPK primarily by causing conformational changes that allows Thr172 phosphorylation to occur by upstream kinases. Activation of AMPK in the liver, skeletal muscle, and adipose tissue improves the status of type 2 diabetes by preventing ATP depletion, increasing fatty acid oxidation, decreasing blood glucose, etc. It has also been found that AMPK activity is inhibited in alcohol-induced fatty liver disease [21].

Although both AMPK and SREBP are related to the metabolism of the cell, the relationship between the two is not clear. We hypothesize that components of tobacco smoke cause lipid accumulation in the liver tissue of mice exposed to “second-hand” smoke by modulating the activities of AMPK and that this enzyme is important in the activation of SREBP-1, the central modulator for triglyceride synthesis. The elucidation of the mechanisms of lipid accumulation in hepatocytes caused by cigarette smoke may help understand processes involved in atherogenesis and in initiation of NAFLD, and suggest possible ways of treating both metabolic diseases.

Section snippets

Smoke solution preparation

Sidestream whole (SSW) smoke solution was prepared from 1R3F research grade cigarettes (University of Kentucky, Louisville, KY). SSW smoke was bubbled into 10 mL serum free media for the duration of 30 puffs as previously described [22] using a puffer box built by the University of Kentucky. SSW smoke was collected from the burning end of the cigarette. The pH of the smoke solutions was adjusted to 7.4. The solution is aliquoted and kept at −20 °C (stable for up to 6 weeks).

Exposure of the animals to smoke

Six- to eight-week-old

SSW causes lipid accumulation in the liver

To test our hypothesis that SSW stimulates lipid synthesis and accumulation in hepatocytes, we used ApoB100 mice on a high-fat diet and exposed them to SSW as described in Section 2. These mice were chosen because under such feeding conditions they produce levels of lipid that lead to development of atherosclerosis [27], [28], [29], which in turn has been associated with NAFLD [30], [31], [32]. To detect the effects of SSW on lipid accumulation in the hepatocytes, mice were exposed to smoke for

Discussion

Cigarette smoke has long been recognized as one of the most preventable non-hereditary factors contributing to cardiovascular disease [2], [3], [4]. Evidence is mounting that there are relationships between cardiovascular disease and metabolic diseases such as NAFLD and diabetes [7], [8], [9], [10]. These diseases all exhibit high lipid levels. Therefore, it is logical to speculate that changes in liver function, the organ where lipid synthesis takes place, affect the initiation and development

Acknowledgements

We thank M. Petreaca for discussion and critical reading of the paper, F. Sladek for the CMV-β-galactosidase plasmid, and K. Chellapa for help with the Luciferase assay.

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    The underlying research reported in the study was funded by NIH (HL77448 and HL89940) and in part by the Tobacco-Related Disease Research Program TRDRP (11DT-0244). The authors who have taken part in this study declared that they do not have anything to disclose regarding funding from industry or conflict of interest with respect to this manuscript.

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