|Home | About | Journals | Submit | Contact Us | Français|
Smokers weigh less and have less body fat than nonsmokers. Increased body fat and weight gain are observed following smoking cessation. To assess a possible molecular mechanism underlying the inverse association between smoking and body weight, we hypothesized that smoking may induce the expression of a fat depleting gene in the airway epithelium, the cell population that takes the brunt of the stress of cigarette smoke.
To assess if smoking up-regulates expression in the airway epithelium of genes associated with weight loss, microarray analysis was used to evaluate genes associated with fat-depletion in large airway epithelial samples obtained by fiberoptic bronchoscopy from normal smokers and normal nonsmokers. As a candidate gene we further evaluated the expression of alpha2-zinc-glycoprotein1 (AZGP1), a soluble protein that stimulates lipolysis, induces a reduction in body fat in mice, is associated with the cachexia related to cancer, and is known to be expressed in secretory cells of lung epithelium. AZGP1 protein expression was assessed by Western analysis and localization in the large airway epithelium by immunohistochemistry.
Both microarray and TaqMan analysis demonstrated that AZGP1 mRNA levels were higher in the large airway epithelium of normal smokers compared to normal nonsmokers (p<0.05, all comparisons). Western analysis of airway biopsies of smokers compared with non-smokers demonstrated upregulation of AZGP1 at the protein level, and immunohistochemical analysis demonstrated upregulation of AZGP1 in secretory as well as neuroendocrine cells of smokers.
In the context that AZGP1 is involved in lipolysis and fat loss, its overexpression in the airway epithelium of chronic smokers may represent one mechanism for the weight difference in smokers vs nonsmokers.
Cigarette smoking is associated with lower body mass index and cessation of smoking is associated with weight gain1,2. Cross-sectional studies show that smokers weigh less than age-matched nonsmokers1,3–7, while longitudinal data show that most smokers gain weight after smoking cessation2,8–16. The mechanisms underlying the lower weight of smokers are undoubtedly complex, and a variety of studies have linked it to decreased food intake, increased metabolic rate, increased physical activity, and the metabolic effects of nicotine1,17–27.
In the present study we propose an additional mechanism that may contribute to the smoking-associated weight loss, based on the hypothesis that smoking may induce the up-regulation in the respiratory epithelium of genes that code for proteins associated with weight loss28,29. To evaluate this concept, we assessed our microarray data of airway epithelial gene expression in healthy smokers and healthy nonsmokers for genes that have been reported to be associated with mediating weight loss28,29. Strikingly, the data showed that smoking markedly up-regulated the airway epithelial expression of alpha2-zinc-glycoprotein 1 (AZGP1), a gene associated with the up-regulation of uncoupling proteins that have been implicated in regulating energy balance and body weight28–32.
AZGP1, a 38 to 41 kDa peptide normally found in body fluids, functions as a lipid mobilizing factor29,33–36. AZGP1 is found in the urine of cancer patients with cachexia36, is overexpressed in carcinomas associated with fat loss34,37, and mice treated with AZGP1 have a significant decrease in body fat, without a change in food or water intake35. AZGP1 is known to be normally expressed in the secretory epithelia of the liver, breast, gastrointestinal tract, sweat glands, and of interest to the present study, the lung38,39. The name AZGP1 is based on the knowledge that it precipitates with zinc salts and has electrophoretic mobility similar to that of alpha2 globulins33. The mechanisms of AZGP1 are not fully understood, but it is believed to regulate lipid degradation through activation of guanosine triphosphate-dependent adenylate cyclase activity mediated through the β3-adrenoreceptor35,40.
Based on the knowledge that increased body fat and weight gain are observed following smoking cessation, lower body mass index is associated with cigarette smoking, adipose tissue metabolism appears to be altered in smokers, and our preliminary observations, we further investigated whether cigarette smokers upregulated the expression of AZGP1 in the human airway epithelium? The analysis included assessment of AZGP1 gene expression of large airway epithelium obtained by fiberoptic bronchoscopy and brushings from healthy nonsmokers and healthy smokers using microarrays with TaqMan PCR confirmation. The data demonstrate that the expression of AZGP1 is significantly upregulated in healthy smokers. Western analysis of large airway biopsies confirmed the upregulation of AZGP1 in smokers compared to non-smokers at the protein level. Interestingly, anti-AZGP1 immunofluorescence assessment of large airway bronchial biopsies and brushed large airway epithelium showed that, in addition to being upregulated in secretory cells of smokers, AZGP1 is expressed in neuroendocrine cells of smokers. In the context that AZGP1 is linked to weight loss, the upregulation of AZGP1 in the airway epithelium of healthy smokers may represent a pathway contributing to the weight loss associated with smoking.
Healthy nonsmokers and healthy current cigarette smokers were evaluated at the Weill Cornell NIH General Clinical Research Center and Department of Genetic Medicine Clinical Research Facility under protocols approved by the Weill Cornell Medical College Institutional Review Board. Written informed consent was obtained from each individual before enrollment in the study. Nonsmokers and smokers were determined to be phenotypically healthy on the basis of clinical history, physical examination, routine blood screening tests, urinalysis, chest X-ray, electrocardiogram, and pulmonary function testing. No individual in either study group had any evidence of a malignancy. Current smoking status was confirmed by history, venous carboxyhemoglobin levels, and urinalysis for nicotine levels and its derivative cotinine. Individuals who met the inclusion criteria underwent fiberoptic bronchoscopy with brushing and/or endobronchial biopsy.
Epithelial cells from the large airways were collected using flexible bronchoscopy. Smokers were asked not to smoke the evening prior to the procedure. After achieving mild sedation and anesthesia of vocal cords, a flexible bronchoscope (Pentax, EB-1530T3) was advanced to the desired bronchus. Large airway epithelial samples were collected by gentle brushing of the 3rd to 4th order bronchi. The epithelial cells were subsequently collected in 5 ml of 4°C LHC8 medium (GIBO, Grand Island, NY). An aliquot of this was used for cytology and differential cell count and the remainder was processed immediately for RNA extraction. Total cell counts were obtained using a hemocytometer while differential cell counts were determined on sedimented cells prepared by centrifugation (Cytospin 11, Shandon Instruments, Pittsburgh, PA) and stained with DiffQuik (Baxter Healthcare, Miami, FL).
Analyses were done using three different Affymetrix (Santa Clara, CA) microarrays, including HuGeneFL (7,000 probe sets), HG-U133A (22,000 probe sets) and HG-U133 Plus 2.0 (54,000 probe sets). The protocols used were as described by the manufacturer. Total RNA was extracted from epithelial cells using TRIzol (Invitrogen, Carlsbad, CA) with further cleanup by RNeasy (Qiagen, Valencia, CA). This process yielded 2 to 4 μg RNA per 106 cells. Samples were processed as previously described using kits and methods from Affymetrix41–44. Hybridizations to test chips and to the microarrays were done according to Affymetrix protocols, and microarrays were processed by the Affymetrix fluidics station and scanned with an Affymetrix GeneArray 2500 (HuGeneFL) and the Affymetrix GeneChip Scanner 3000 7G (HG-U133A and HG-U133 Plus 2.0). To maintain quality, only samples hybridized to test chips with a GAPDH of 3′ to 5′ ratio of <3 were deemed satisfactory.
Captured images were analyzed using Microarray Suite version 5.0 algorithm (Affymetrix). These data were normalized using GeneSpring version 7.2 software (Agilent Technologies, Palo Alto, CA) as follows: (1) per array, by dividing raw data by the 50th percentile of all measurements; and (2) per gene, by dividing the raw data by the median expression level for each gene across all arrays in a data set.
TaqMan real-time reverse transcription-PCR (RT-PCR) was done on RNA samples from the large airways of 17 normal nonsmokers and 15 normal smokers that had also been assessed with the HG-U133 Plus 2.0 array. cDNA was synthesized from 2 μg RNA in a 100 μl reaction volume, using the TaqMan Reverse Transcriptase Reaction kit (Applied Biosystems, Foster City, CA), with random hexamers as primers. Dilutions of 1:10 and 1:100 were made from each sample and triplicate wells were run for each dilution. TaqMan PCR reactions were carried out using pre-made gene expression assays for the AZGP1 gene from Applied Biosystems and 2 μl cDNA were used in each 25 μl reaction volume. The endogenous control was 18S rRNA and relative expression levels were determined using the ΔΔCt method (Applied Biosystems) with the average value for the nonsmokers as the calibrator. The PCR reactions were run in an Applied Biosystems Sequence Detection System 7500.
To assess which airway epithelial cells express AZGP1, bronchial biopsies were obtained from the large airways of healthy nonsmokers and healthy smokers using conventional methods. Immunohistochemistry was subsequently done on paraffin-embedded endobronchial biopsies. Sections were deparaffinized and rehydrated through a series of xylenes and alcohol. To enhance staining, an antigen recovery step was carried out by microwave treatment at 100°C, 15 min in citrate buffer solution (Labvision, Fremont, CA) followed by cooling at 23°C, 20 min. Endogenous peroxidase activity was quenched using 0.3% H2O2 and normal goat serum was used to reduce background staining. Samples were incubated with the primary antibody rabbit polyclonal anti-AZGP1 (1/5000 dilution, Biovendor, Candler, NC) at 4°C overnight. Rabbit IgG (Jackson Immunoresearch) was used as the isotype control. Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and chromogenic substrate 3-amino-9-ethyl-carbazole (AEC; Dako, Carpinteria, CA, USA) were used to detect antibody binding. The sections were counterstained with Mayer’s hematoxylin (Polyscientific, bayshore, NY) and mounted using GVA mounting medium (Zymed, San Francisco, CA). Brightfield microscopy was performed using a Nikon Microphot microscope equipped with a Plan 40X N.A. 0.70 objective lens. Images were captured with an Olympus DP70 CCD camera.
Colocalizations of AZGP1 with a neuroendocrine cell marker (chromogranin A) and a secretory cell marker (mucin5AC) were also performed with cytospin preparations of large airway epithelium brushings. The following antibodies were used: AZGP1, rabbit polyclonal anti-AZGP1 (1/5000 dilution, Biovendor, Candler, NC), with rabbit IgG (Jackson Immunoresearch) as the control; for chromogranin A, mouse monoclonal (LK2H10+PHE5) anti-human chromogranin (1/500 dilution; Thermo Scientific, Waltham, MA) and mouse IgG as the isotype control (Sigma, St Louis, MO); for mucin 5AC, mouse monoclonal (CLH2) anti-human mucin 5AC (1/50; Vector Laboratories, Burlingame, CA) and mouse IgG as the isotype control (Sigma, St Louis, MO). Following incubation with the primary antibodies, goat anti-rabbit Cy5 (Jackson ImmunoResearch) was used as a secondary antibody for AZGP1 and goat anti-mouse Cy3 (Jackson ImmunoResearch) was used as a secondary antibody for chromogranin A and mucin 5AC. Nuclei were counter stained with 4′,6-diamidino-2-phenylindole (DAPI, 1/20,00 dilution, Molecular Probes). A total of nine slides were used for each airway epithelial cell sample: (1) AZGP1 alone, (2) rabbit IgG control for AZGP1, (3) chromogranin A alone, (4) mucin 5AC alone, (5) mouse IgG control for chromogranin and mucin 5AC, (6) AZGP1 and chromogranin A colocalization, (7) rabbit IgG control for AZGP1 and mouse IgG control for chromogranin A, (8) AZGP1 and mucin 5AC colocalization, and (9) rabbit IgG control for AZGP1 and mouse IgG control for mucin 5AC. Images were captured using an Olympus IX 70 fluorescence microscope with 60-fold magnification. Images were analyzed using MetaMorph software (Universal Imaging Corporation, Downingtown, PA). Pseudocolor images were formed by encoding Cy5 fluorescence in the green channel, Cy3 fluorescence in the red channel.
In order to compare the frequency of AZGP1 positive cells between healthy nonsmokers and healthy smokers, cytospin preparations of large airway epithelial cells from 5 nonsmokers and 6 smokers were stained with rabbit anti-human AZGP1 or rabbit IgG as control. Goat anti-rabbit Cy3 was used as a secondary antibody. Nuclei were counter stained with DAPI. The percentage of AZGP1 positive cells was calculated as (AZGP1 positive cells per field/total number of nuclei per field) in 10 random 60x fields for each subject using Olympus IX 70 fluorescence microscope. Mean AZGP1 positive fractions in ten fields for each subject of the nonsmoking and smoking groups were compared by t test.
Western analysis was used to quantitatively assess AZGP1 protein expression in large airway brushing samples from healthy nonsmokers and healthy smokers. Brushed large airway epithelial cells were obtained as described. Initially, the cells were centrifuged at 600 × g, 5 min, 4°C. The whole cells were lysed with red cell lysis buffer (Cell Lytic Mammalian Tissue Lysis/Extraction reagent, Sigma-Aldrich) followed by whole cell lysis buffer (ACK lysing buffer, Invitrogen), and protease inhibitor (Sigma-Aldrich) was added to the sample. The sample was centrifuged at 10,000 × g and the protein-containing supernatant collected. The protein concentrations were assessed using a bicinchoninic acid (BCA) protein concentration kit (Pierce, Rockford, IL). Equal concentration of protein (10 μg) mixed with SDS Sample Loading Buffer (Invitrogen, Carlsbad, CA) and reducing agent was loaded on Tris-glycine gels (Invitrogen). Protein electrophoresis was carried out at 100V, 2 hr, 23°C. Sample proteins were transferred (25V, 1hr, 4°C) to a 0.45 μm thick PVDF membrane (Invitrogen) using a powerpack 300 power source (Bio-Rad, Hercules, CA) and Tris-glycine transfer buffer (Bio-Rad). After transfer the membranes were blocked with 5% milk in PBS for 1 hr, 23°C. The membranes were incubated with primary rabbit polyclonal anti-AZGP1 antibody (Biovendor, Candler NC) at 1:1000 dilution, 2 hr, 4°C. Recombinant AZGP1 protein (Biovendor) was used as a positive control. Detection was performed using horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000 dilution, Santa Cruz Biotechnology) and the Enhanced Chemiluminescent reagent (ECL) system (GE, Healthcare, Pittsburgh, PA) using Hyperfilm ECL (GE Healthcare). To assess the Western analyses quantitatively, the film was digitally imaged, maintaining exposure within the linear range of detection. The contrast was inverted, the pixel intensity of each band determined, and the background pixel intensity for a negative area of the film of identical size subtracted using MetaMorph image analysis software (Universal Imaging, Downingtown, PA). The membrane was subsequently stripped and reincubated with horseradish peroxidase-conjugated anti-β actin antibody (Santa Cruz Biotechnology) as a control for equal protein concentration.
Average expression values for AZGP1 in large airway samples were calculated from normalized expression levels for healthy nonsmokers and healthy smokers and p values for all comparisons were calculated using the two sample unequal variance Welch t-test without correction for multiple testing.
All data has been deposited in the Gene Expression Omnibus site, (http://www.ncbi.nlm.nih.gov/geo), which is curated by the National Center for Bioinformatics. The accession number is GSE10135.
The funding source of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report or the decision to submit this report for publication.
A total of 92 individuals, 37 healthy nonsmokers and 55 healthy smokers, were included in microarray assessment of expression profiles of the large airway epithelium. These included 9 healthy nonsmokers and 13 healthy smokers from the HuGeneFL data set, 5 healthy nonsmokers and 6 healthy smokers from the HG-U133A data set, and 23 healthy nonsmokers and 36 healthy smokers from the HG-U133 Plus 2.0 data set (Table I, Figure 1,Supplemental Figure 1). All individuals had normal general physical examination and no significant prior medical history. There were no differences between groups with regard to gender, race or age (p>0.05). All individuals were HIV negative with blood and urine parameters within normal ranges (p>0.05 for all comparisons). The mean (± standard deviation) body mass index of the 37 healthy nonsmokers and 55 healthy smokers was 25.3 ± 3.6 kg/m2 and 27.7 ± 6.1 kg/m2, respectively, and the values were not significantly different (p> 0.05). Healthy smokers had an average history of smoking of 27 ± 2 pack-yr and venous blood carboxyhemoglobin levels and urine nicotine and cotinine confirmed smoking status of these individuals. Pulmonary function testing revealed normal lung function in healthy nonsmokers and healthy smokers. (Table I). The number of airway epithelial cells recovered ranged from 6.9 to 9.4 × 106 (Table I). In all cases, >96% of cells recovered were epithelial cells. The various categories of airway epithelial cells were as expected for large airways44.
Using the criteria of Affymetrix Detection Call of Present (P call) in ≥50%, AZGP1 was significantly upregulated in the large airway epithelium of healthy smokers as compared to healthy nonsmokers in every data set (Figure 1,Supplemental Figure 1). Assessment of GEO deposited microarray data from the independent data set of Spira et al45 confirmed the significant expression level of AZGP1 in the large airway epithelium smokers (Figure 2).
To confirm the results obtained from microarray studies, TaqMan RT-PCR was carried out on RNA samples from the large airways of 17 healthy nonsmokers and 15 healthy smokers (Figure 3). The TaqMan data confirmed the upregulation of AZGP1 mRNA expression in healthy smokers compared to healthy nonsmokers (1.9 ± 0.34 -fold increase, p<0.05).
Western analysis was carried out on large airway samples from a total of 10 healthy non-smokers and 10 healthy smokers to quantitatively assess AZGP1 expression. Overall, healthy smokers had increased AZGP1 expression when compared to nonsmokers (Figure 4A). Analysis of the digitally imaged film with MetaMorph image analysis software revealed significantly increased AZGP1 protein expression in healthy smokers compared to healthy nonsmokers (Figure 4B; p<0.02).
Immunohistochemistry of large airway epithelial biopsy specimens obtained from healthy nonsmokers and healthy smokers was used to assess the cell specific expression of AZGP1 (Figure 5). Positive staining for AZGP1 was observed in the large airway epithelial cells in both nonsmokers and smokers. Qualitatively, healthy smokers demonstrated stronger staining and in more cells.
To detect the cell type that expressing AZGP1, dual immunofluorescence was applied to large airway epithelial cells from a non-smoker prepared by cytospin using cell type specific marker mucin 5AC for secretory cells and chromogranin A for neuroendocrine cells (Figure 6A–O). Secretory cells expressing mucin5AC were readily detected and a subset of these also expressed AZGP1 (Figure 6A–F). Ciliated cells visible in the same fields never expressed AZGP1. The distinct subcellular distribution of AZGP1 and mucin5AC in conjunction with non-specific antibody controls (not shown) confirmed the specificity of each antibody. However, not all secretory cells expressed AZGP1 (Figure 6 G–I). Chromogranin A positive cells were also observed in the cytospin preparation of large airway epithelial cells and all of these expressed AZGP1(Figure 6 J–O). Controls were performed with matched IgG isotypes to show that the observed signals are attributable to the specific proteins with no cross bleeding between the channels used for detection (not shown).
To quantify the AZGP1 positive cells in nonsmokers and smokers, the fraction of AZGP1 positive cells in representative fields of immunofluorescently stained cytospin preparations from 5 nonsmokers and 6 smokers was assessed. Representative low–power views of slides used in the quantitative analysis were shown (Figure 7A–D). The percentage positive cells in healthy non-smokers (6.9 ± 0.7) was significantly lower than in healthy smokers (9.9 ± 0.9, p<0.05) (Figure 7E).
Cigarette smoking is linked with decreased body weight in smokers, and when smokers stop smoking, they often gain weight. Based on the knowledge that the airway epithelium takes the brunt of the stress of cigarette smoke46, we asked the question: does smoking alter the expression of a gene whose product is linked to weight loss? Using microarray analysis to compare gene expression of the airway epithelium in healthy smokers vs nonsmokers, we identified that AZGP1, a gene linked to weight loss29,33–36,47, was upregulated in the airway epithelium of smokers. The microarray data was confirmed at the mRNA level by quantitative TaqMan PCR, and at the protein level by immunohistochemistry and Western analysis. We do not have data demonstrating increased levels of AZGP1 in biologic fluids, and thus we cannot prove that smoking-induced increases in AZGP1 gene expression in airway epithelium is sufficient to mediate weight loss. However, with the background knowledge that smoking is associated with weight loss1–27, smoking cessation results in significant weight gain from increased body fat11–16, AZGP1 stimulates lipolysis in vitro and in vivo29,35,48, high systemic levels of AZGP1 are linked to cachexia34, and administration of AZGP1 to experimental animals is associated with weight loss29,35, the finding that AZGP1 expression is up-regulated in the airway epithelium in healthy smokers provides another mechanism explaining the weight change that occurs in cigarette smokers.
Cigarette smokers of the same age and gender weigh less in comparison to nonsmokers and anorexia is associated with cigarette smoking1–7,25,27. Weight gain is a known deterrent to smoking cessation with an average weight gain of 2.4 to 5 kg12–16. A primary reason smokers give for not trying to quit smoking and for relapsing after cessation is weight gain16,49, and the increase in the prevalence of overeating and obesity in the United States has been attributed in part to smoking cessation50. Weight gain after smoking cessation is largely because of increased body fat12–16. Mechanisms that have been investigated include increased energy intake, decreased resting metabolic rate, and decreased physical activity8,14,19,23,51,52. Studies have not consistently shown increased caloric intake as an explanation for the weight gain following smoking cessation18,53. Nicotine, the major addictive component of tobacco, mediates decreased body weight and food intake in experimental animals and induces lipolysis in smokers22,24,27. This lipolytic effect of smoking is attributed to the effect of nicotine on release of catecholamine, that in turn, mediates lipolysis in adipocytes1,22,54,55. Some studies have shown that nicotine, as a potent secretagogue in some cell types, mediates the release of peptides that regulate food intake and energy expenditure such as leptin, neuropeptide Y and orexins25,56,57.
AZGP1 was first isolated from human plasma33, and later found to be expressed in secretory epithelial cells of the lung, liver, breast, gastrointestinal tract and sweat glands38. Consistent with its production by secretory epithelium, AZGP1 is present in most body secretions58,59. Several types of malignant tumors over-express AZGP134,60–64, and it has been proposed as a cancer marker34,60–64.
Although the biological functions of AZGP1 have not been fully elucidated, it has been demonstrated to act as a lipid mobilizing factor and is associated with the dramatic weight loss seen in many cancer patients29,34–36. AZGP1 is identified as an adipokine since it is secreted by adipocytes37,65. AZGP1 contains a class I major histocompatibility complex (MHC) fold and is the sole soluble member of this superfamily of molecules66–68. Uncoupling proteins, members of the mitochondrial carrier family which are postulated to be involved in the control of energy metabolism and body fat, are induced by AZGP1 and cigarette smoke27,28,30–32,69–71. Treatment with AZGP1 stimulates lipolysis in isolated mouse and human adipocytes, and induces a rapid and selective reduction in body fat both in normal and ob/ob mice35,37. This lipolytic action is mediated via the β3-adrenoreceptor on adipocytes with up-regulation of cAMP40. Finally, AZGP1 deficient mice have increased body weight when subjected to a standard or high fat diet when compared to wild-type mice47.
Based on the knowledge that smoking is associated with weight loss, smoking cessation results in weight gain largely from increased body fat, and AZGP1 stimulates lipolysis in vitro and in vivo, the finding that AZGP1 expression is upregulated in healthy smokers may reflect one mechanism contributing to the weight changes that occur in cigarette smokers. AZGP1 has been known to be expressed in normal lung tissue as evidenced by immunohistochemical staining demonstrating AZGP1 expression in airway secretory cells38. A study assessing AZGP1 mRNA levels in lung tissue of patients with primary lung cancer and lung metastases revealed no significant difference in AZGP1 levels among smokers and nonsmokers39, but the airway epithelium was not assessed directly, and thus the relationships among lung cancer, smoking and the expression of AZGP1 in airway epithelium is unclear. The blood AZGP1 was identified in the urine of cancer patients with cachexia34,35, and there are several studies supporting a role for AZGP1 in cancer72,73. In the present study, the data demonstrates that AZGP1 is significantly upregulated in the large airway epithelium of healthy cigarette smokers. Interestingly, the immunohistochemistry demonstrated not only expression of AZGPI in the secretory cells, but also in neuroendocrine cells of smokers. The observation that AZGP1 is expressed in neuroendocrine cells may be important in the context that hormonal effects resulting in lipolysis are known to be neurohormonally regulated74.
We thank A. Heguy for helpful discussions; and N Mohamed for help in preparing this manuscript.
Conflict of Interest: For all authors, there is no conflict of interest
*These studies were supported, in part, by NIH R01 HL074326, P50 HL084936, UL1-RR024996; and the Will Rogers Memorial Fund, Los Angeles, CA.