Leptin and the risk of Barrett’s oesophagus
- B J Kendall1,
- G A Macdonald2,
- N K Hayward3,
- J B Prins2,
- I Brown4,
- N Walker5,
- N Pandeya1,
- A C Green1,
- P M Webb1,
- D C Whiteman1,
- for the Study of Digestive Health
- 1Division of Population Studies and Human Genetics, Queensland Institute of Medical Research, Brisbane, Australia
- 2Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Brisbane, Australia
- 3Division of Cancer and Cell Biology, Queensland Institute of Medical Research, Brisbane, Australia
- 4Sullivan and Nicolaides Pathology, Brisbane, Australia
- 5Queensland Medical Laboratories, Brisbane, Australia
- Dr D C Whiteman, c/- Cancer and Population Studies Group, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, QLD 4029, Australia;
- Accepted 29 November 2007
- Final version accepted 1 December 2007
- Published Online First 4 January 2008
Background: Obesity is associated with increased risks of Barrett’s oesophagus and oesophageal adenocarcinoma. Alterations in serum leptin and adiponectin, obesity-related cytokines, have been linked with several cancers and have been postulated as potential mediators of obesity-related carcinogenesis; however, the relationship with Barrett’s oesophagus remains unexplored.
Methods: Serum leptin and adiponectin concentrations were measured on two subsets of participants within a case–control study conducted in Brisbane, Australia. Cases were people aged 18–79 years with histologically confirmed Barrett’s oesophagus newly diagnosed between 2003 and 2006. Population controls, frequency matched by age and sex to cases, were randomly selected from the electoral roll. Phenotype and medical history data were collected through structured, self-completed questionnaires. Odds ratios (OR) and 95% CI were calculated using multivariable logistic regression analysis.
Results: In the pilot analysis (51 cases, 67 controls) risks of Barrett’s oesophagus were highest among those in the highest quartile of serum leptin (OR 4.6, 95% CI 0.6 to 33.4). No association was seen with adiponectin. In the leptin validation study (306 cases, 309 controls), there was a significant threefold increased risk of Barrett’s oesophagus among men in the highest quartile of serum leptin (OR 3.3, 95% CI 1.7 to 6.6) and this persisted after further adjustment for symptoms of gastro-oesophageal reflux (OR 2.4, 95% CI 1.1 to 5.2). In contrast, the risk of Barrett’s oesophagus among women decreased with increasing serum leptin concentrations.
Conclusions: High serum leptin is associated with an increased risk of Barrett’s oesophagus among men but not women. This association is not explained simply by higher body mass or gastro-oesophageal reflux among cases. The mechanism remains to be determined.
Until recently, oesophageal adenocarcinoma (OA) was a rare disease with an incidence of less than five per million person-years; however, the incidence of this cancer has increased sharply in the United States, Australia and Europe during the past three decades.1–4 This has occurred on a background of increasing incidence of Barrett’s oesophagus,5–8 a metaplastic change in the lining of the oesophagus, which is a precursor for nearly all OA.9
There is general acceptance that chronic reflux of gastric acid into the lower oesophagus is the principal cause of Barrett’s oesophagus and OA.10–12 Although obesity is associated with gastro-oesophageal reflux symptoms and oesophageal acid exposure13–17 it has been shown to be independently associated with the risk of OA18–20 and Barrett’s oesophagus.11 Two recent studies have shown that the association between obesity and the risk of Barrett’s oesophagus is mediated via central obesity.21 22 In these studies, adjustment for reflux symptoms did not abolish and in one study22 increased the association between central obesity and Barrett’s oesophagus, suggesting that mechanisms in addition to central obesity-mediated gastro-oesophageal reflux may be playing a role in this association.
Obesity has been associated with increased risks of a number of cancers, leading to speculation that factors secreted by adipose tissue may be contributing.23 Leptin is an adipokine secreted by adipocytes and gastric chief cells and in mice functions to maintain energy balance.24 In humans, the serum leptin concentration is closely correlated with body fat mass and obese people are typically hyperleptinaemic.25 In vitro, leptin has been shown to be mitogenic and angiogenic and induces proliferation in a variety of human cell types including oesophageal cancer cell lines.26 27 Epidemiological studies have demonstrated an association between serum leptin concentration and cancer risk for a number of malignancies including cancer of the colon, breast and prostate.28 29
Adiponectin is another adipokine secreted predominately by adipose tissue. Adiponectin functions as an insulin sensitiser and also has cardioprotective, immunomodulatory and anti-inflammatory actions. In contradistinction to most other adipokines, the serum concentration of adiponectin decreases with increasing obesity. Hypoadiponectinaemia has been linked to carcinogenesis in a range of tissues,30 31 including carcinoma of the upper stomach.32
Given the epidemiological evidence linking obesity with the risk of OA and central obesity with the risk of Barrett’s oesophagus, further investigation of plausible biological mechanisms is warranted. Here, we describe the findings of a case–control study with an initial pilot analysis comparing serum leptin and adiponectin concentrations between people with and without Barrett’s oesophagus and a subsequent validation study of the association between serum leptin and Barrett’s oesophagus.
PARTICIPANTS AND METHODS
We conducted a laboratory analysis using serum samples from participants in a population-based case–control study of Barrett’s oesophagus. Approval to undertake the study was obtained from the human research ethics committees of the Queensland Institute of Medical Research, the University of Queensland and the following teaching hospitals in Brisbane, Australia: Royal Brisbane and Women’s Hospital, Princess Alexandra Hospital, Mater Misericordiae Hospital, Prince Charles Hospital and Greenslopes Private Hospital. We obtained written informed consent from case patients and control participants to take part. Those who did not speak English or were too ill to participate were excluded.
People aged 18–79 years newly diagnosed with histologically confirmed Barrett’s oesophagus between 1 February 2003 and 30 June 2006. Barrett’s oesophagus was defined as the presence of intestinal metaplasia (columnar epithelium with goblet cells) in a biopsy taken from the tubular oesophagus by upper gastrointestinal endoscopy, regardless of the extent of involved mucosa.33
All patients diagnosed with Barrett’s oesophagus at the two major private pathology laboratories and the single public pathology laboratory serving metropolitan Brisbane (population 1.5 million) during the period between 1 February 2003 and 30 June 2006 were identified. To comply with Australian privacy laws, pathology laboratories were able to release patient contact details to study investigators only after first obtaining written permission from the patients concerned. For all patients diagnosed through the private pathology laboratories, a notice explaining the study was automatically generated in the computerised report to the treating doctor. If no objection was forthcoming from the treating doctor, the pathology laboratory wrote to the patients requesting permission to release their contact details to the investigators. A second letter was sent in the event of non-response. For patients diagnosed through the public laboratory, a letter signed by the Chief Health Officer for Queensland was mailed to each potential case participant. If no contact was made after two mail-outs, then these potential cases were deemed “non-responders” and no further attempts were made to contact them. Patients who had died, were too ill, mentally incompetent or unable to complete an English language questionnaire were excluded. In total, 1714 patients with Barrett’s oesophagus diagnosed by the pathology laboratories were approached. No response was received from 410 (24%) patients, eight patients had died in the interim, 200 (12%) declined and 1096 (64%) agreed to be contacted by study investigators. Of those who agreed to be contacted, 487 patients were excluded because they had a previous diagnosis of Barrett’s oesophagus and 130 patients did not meet inclusion criteria (primary residence out of study area, too ill, did not speak English, unwilling to give blood sample) and were excluded. To confirm the tubular oesophageal origin of the index biopsy, the pathology report, pathology request form and endoscopy report relating to the index biopsy were reviewed by two investigators. Following this review, a further 86 patients with only intestinal metaplasia of the gastro-oesophageal junction were excluded, leaving 393 participants meeting the case definition. The date of diagnosis of Barrett’s oesophagus and the presence or absence of dysplasia were recorded for these cases.
Control participants from the same geographical region were randomly selected from the Australian Electoral Roll (enrolment is compulsory by law in Australia), broadly matched by age (in five-year age groups) and sex to the case series. Control participants were contacted in a similar manner to cases, except that the initial approach came directly from the study investigators. Invitations were mailed to 1554 potentially eligible controls selected from the electoral roll, of whom 30 (2%) were excluded (deceased 2, too ill 4, no English 17, other exclusion 7), 404 (26%) could not be contacted or did not respond, 372 (24%) declined and 748 accepted the invitation (48% of all potential controls selected from the roll, 67% of those able to be contacted). Of these 748, 646 returned a completed questionnaire.
Data were collected from participants through structured, self-completed questionnaires, followed by standard telephone interviews conducted by trained research nurses. We collected information about height and weight (current and heaviest ever). Items on the questionnaire asking about recent gastrointestinal symptoms were derived from prevalence surveys in Australian populations;34 35 items asking about historical reflux exposures were based on those used in previous case–control studies of OA.10 36 Participants were asked if they had ever experienced acid reflux, defined as “a sour taste from acid or bile rising up into the mouth or throat”. If so, they were asked to report their age when these symptoms were first experienced, as well as the frequency of episodes in the past year (or year before diagnosis for cases). Participants were also asked to report reflux frequency at each of four periods (ages 10–19 years, 20–29 years, 30–49 years and 50–79 years, as applicable).
Non-fasting samples of whole blood were collected in plain tubes from study participants and transported overnight to the laboratory. Serum was stored at −20°C in 0.5 ml aliquots. Samples for analysis were thawed once only.
Non-fasting samples of whole blood were collected in plain tubes from 91% of participating cases and 85% of participating controls and transported overnight to the laboratory. Serum was stored at −20°C in 0.5 ml aliquots. Samples for analysis were thawed once only. We conducted a pilot study of the association between the two adipokines, leptin and adiponectin, and Barrett’s oesophagus in August 2005 using sera from 118 participants (67 controls, 51 cases), sampled from among study participants within disparate strata of body mass index (BMI) (<25 kg/m2; >30 kg/m2) and frequency matched by age and sex. Following the results of the pilot study, we subsequently conducted a validation study of the association between serum leptin and Barrett’s oesophagus in September 2006 using sera from the remaining cases who had useable serum aliquots (n = 306) and a subset of controls (n = 309) matched by sex and five-year age group to the cases. There was no difference in the frequency of reflux symptoms or BMI distribution between those with and without leptin data for either cases or controls.
Leptin assays were performed using human leptin radio-immunoassay (LINCO Research, St Charles, Missouri, USA), whereas total serum adiponectin (including all multimeric isoforms) was measured using human adiponectin radio-immunoassay (LINCO Research). Assays were performed according to the manufacturer’s instructions using 100 μl thawed sera. The between-run precision of the leptin assay was based on 13 runs and determined at two levels: level 1, mean 3.5 ng/ml, coefficient of variation 11.3% and level 2, mean 24.2 ng/ml, coefficient of variation 6.9%. To assess the repeatability of serum leptin measures, we performed two separate assays on 20 participants (10 controls, 10 cases) using two different aliquots of serum derived from the same blood draw. The intraclass correlation coefficient for the two measures was 0.78 (95% confidence interval (CI) 0.52 to 0.91), indicating very high levels of agreement between assays.
The primary aim of the analysis of the validation study was to assess the association between serum leptin concentrations and the risk of Barrett’s oesophagus after accounting for the potentially confounding effects of age, sex, BMI and gastro-oesophageal reflux. Because the distribution of leptin deviated from the normal within some strata of sex and BMI, we used medians and interquartile ranges to describe measures of central tendency and used the Wilcoxon rank sum test to test for differences in leptin distribution between cases and controls. To estimate the relative risk of Barrett’s oesophagus associated with serum leptin concentration, we calculated the odds ratio (OR) and 95% CI by unconditional multivariable logistic regression analysis in SAS version 9.1 (SAS Institute Inc, Cary, North Carolina, USA). Our approach was to fit models that contained terms for leptin as a categorical variable, adjusted for exact age in years, sex and BMI. We fitted further models that adjusted for the frequency of symptoms of gastro-oesophageal reflux, as well as terms for aspirin use in the past five years and ever use of H2 antagonists, proton-pump inhibitors or other acid-suppressant medications. We categorised leptin separately for men and women according to quartile cutpoints in the control distribution and used the lowest quartile as the reference category in all models. BMI at current age was calculated by dividing weight in kilograms by the square of height in meters. We used standard BMI categories for analysis (<25 kg/m2, 25–29.9 kg/m2, 30.0–34.9 kg/m2, ⩾35.0 kg/m2). To test for trend, categorical variables were included in the model as continuous data (with category values taking the mean of the category) and the Wald test was used as an approximation of the Mantel extension χ2 with one degree of freedom. Statistical significance was determined at α = 0.05 and all tests for statistical significance were two-sided.
The characteristics of case patients and control participants in the pilot and validation studies are presented in table 1. Within the pilot and validation studies, the distributions of cases and controls were similar for age and sex as a consequence of the frequency matching. The majority of study participants were born in Australia, New Zealand or the British Isles, as is typical of the Australian resident population. Barrett’s oesophagus cases reported higher prevalences of smoking, overweight and obesity and symptoms of gastro-oesophageal reflux than controls.
As expected, serum leptin concentrations were higher among female controls (n = 33, median 23.7 ng/ml) than male controls (n = 34, median 7.6 ng/ml, p<0.01). Female cases with Barrett’s oesophagus also had higher serum leptin concentrations (n = 25, mean 26.8 ng/ml) than male cases (n = 26, mean 13.5 ng/ml, p<0.01) and within each sex, cases had higher concentrations than controls. When categorised according to the quartile serum leptin distribution among controls, we found that Barrett’s oesophagus cases were substantially more likely than controls to have high serum leptin concentrations (table 2).
We compared serum adiponectin concentrations between controls and Barrett’s oesophagus cases among women (control median 11.22 μg/ml; case median 9.68 μg/ml, p = 0.86) and men (control median 8.69 μg/ml; case median 8.09 μg/ml, p = 0.54). We categorised participants according to sex-specific quartile cutpoints from the control adiponectin distribution and conducted logistic regression analyses controlling for the effect of sex, age and BMI (table 2). We found no evidence that Barrett’s oesophagus cases had systematically higher or lower serum adiponectin concentrations than controls. On the basis of these exploratory findings, we pursued leptin as a potential risk factor for Barrett’s oesophagus in a validation study.
Leptin assays were performed on 309 controls and 306 cases in the validation study. We first measured the association between BMI and Barrett’s oesophagus in this sample of participants. Among men, we found modestly increased age-adjusted risks of Barrett’s oesophagus associated with BMI 25.0–29.9 kg/m2 (OR 1.5, 95% CI 0.9 to 2.3) and BMI greater than 30.0 kg/m2 (OR 1.7, 95% CI 1.0 to 3.1) compared with the referent category (BMI <25 kg/m2). Risks of Barrett’s oesophagus were attenuated after further adjusting for the frequency of acid reflux symptoms (BMI 25.0–29.9 kg/m2 OR 1.3, 95% CI 0.8 to 2.1; BMI >30.0 kg/m2 OR 1.3, 95% CI 0.7 to 2.6). BMI was not associated with the risk of Barrett’s oesophagus among women in this sample (BMI 25.0–29.9 kg/m2 OR 1.2, 95% CI 0.6 to 2.6; BMI >30.0 kg/m2 OR 0.8, 95% CI 0.3 to 1.9).
In cases and controls, the serum leptin concentration was two to three times higher in women than men. Serum leptin concentrations were correlated with BMI among controls (women r = 0.72, men r = 0.52) and cases (women r = 0.55, men r = 0.57) and we observed stepwise increases in median serum leptin concentrations observed for each successive category of BMI among women and men (table 3). In women, the serum leptin concentration was slightly higher in controls than cases in all BMI categories. In contrast, in men, serum leptin concentrations were higher in cases than controls in all BMI categories, this difference being greatest among those with a BMI of 35 kg/m2 or greater. In this BMI category, median serum leptin concentrations in male cases were nearly two times higher than those of controls and approached those observed for female Barrett’s oesophagus cases in the same BMI category.
In age and BMI-adjusted logistic regression analyses stratified by sex, we found a significant threefold increased risk of Barrett’s oesophagus among men in the highest quartile of serum leptin (OR 3.3, 95% CI 1.7 to 6.6; table 4). Further adjustment for the frequency of gastro-oesophageal reflux symptoms attenuated the association somewhat, although a significantly elevated risk persisted (OR 2.4, 95% CI 1.1 to 5.2; table 4). We fitted further models including terms for aspirin use in the past five years and ever use of H2 antagonists, proton-pump inhibitors or other acid-suppressant medications to observe for changes in the risk estimates associated with leptin category but these had no significant effect on risk estimates (not shown).
In contrast to the positive associations between serum leptin and Barrett’s oesophagus risk among men, we found suggestive evidence for a negative association between serum leptin concentration and the risk of Barrett’s oesophagus in women. To determine whether the association in women might differ by menopausal status, we repeated the analyses in women stratified by age (<50 years; ⩾50 years; table 5). In this analysis, there was no evidence that the serum leptin concentration was associated with increased risks of Barrett’s oesophagus in postmenopausal women; indeed our data suggest the contrary. There were too few women aged less than 50 years to permit meaningful interpretation of the data.
We have found that the serum leptin concentration is strongly associated with the risk of Barrett’s oesophagus in men, but not in women. In men, the serum leptin concentration was highest in cases with a BMI greater than 35 kg/m2, in which the serum concentrations were nearly two times those of BMI-matched controls and approached the magnitude of concentrations observed among female cases. Although associations between obesity and central obesity and the risk of Barrett’s oesophagus have been described previously,11 21 22 37 38 we are not aware of earlier studies investigating leptin and Barrett’s oesophagus.
The strengths of this study include the prospective, population-based ascertainment of patients newly diagnosed with Barrett’s oesophagus and the use of population controls. Our two-stage approach, in which the findings of a small pilot study were confirmed by statistically significant associations in a larger validation study, makes chance an unlikely explanation for the observations. Although BMI was self-reported, we believe that bias arising from systematic differences in reporting between cases and controls is unlikely to account for the findings as participants were unaware of the hypotheses being tested; the instrument used to derive these data was the same for both groups and there was a strong correlation between reported BMI and serum leptin concentration in cases and controls. Assays were performed blind to participant data, thus there is no reason to suspect that our findings were the result of systematic differences in laboratory analysis.
Three factors that potentially confound the association between serum leptin concentration and the risk of Barrett’s oesophagus are sex, obesity and gastro-oesophageal reflux. We approached this issue in several ways. First, we stratified the analyses by sex and found strikingly different patterns of association. Second, we adjusted for obesity by stratifying the analyses by BMI and found that within each stratum of BMI, male cases with Barrett’s oesophagus had consistently higher mean serum leptin concentrations than male controls, indicating that the association in men was not explained simply by confounding for obesity. Among women, there was no evidence that leptin concentrations were higher among cases than controls. Third, we adjusted for the effects of BMI and gastro-oesophageal reflux in multivariable logistic regression models to obtain deconfounded risk estimates and the association persisted. Whereas some residual confounding by these factors may be anticipated, it is unlikely to explain the observed association entirely.
What are the potential explanations for our findings? Recent studies by Corley et al21 and Edelstein et al22 have reported strong associations between central obesity and the risk of Barrett’s oesophagus. Although central obesity has been shown to increase gastro-oesophageal reflux13 15 most likely by its effects on intra-abdominal pressure39 40 and lower oesophageal sphincter integrity,40 41 in both of the recent studies the association between central obesity and the risk of Barrett’s oesophagus persisted despite adjustment for reflux symptoms. Indeed, in the study by Edelstein et al22 the association was moderately strengthened. These authors and others have speculated that a proportion of the effect of obesity on the risk of Barrett’s oesophagus is likely to be through non-gastro-oesophageal reflux disease pathways including leptin.22 42
Leptin, in vitro, is mitogenic and angiogenic and induces proliferation in a variety of human tissues.26 43 Studies of OA cell lines have shown that leptin stimulates cell proliferation in a dose-dependent manner and inhibits apoptosis27 and that these effects occur synergistically with acid44 via Akt activation.45 A recent study has shown that the leptin receptor is highly expressed in Barrett’s mucosa.46 It could be postulated that in addition to gastro-oesophageal reflux initiating mucosal injury and intestinal metaplasia, leptin acting via the leptin receptors on the Barrett’s mucosa may act synergistically with acid in the maintenance of the metaplastic change and promote the development and persistence of genetic abnormalities via its proliferative and anti-apoptotic effects.
Why is leptin not associated with the risk of Barrett’s oesophagus in women? In obese women, peripheral adipocytes secrete significantly more leptin than omental adipocytes.47 In contrast, in obese men, the leptin secretion by adipocytes is similar at both anatomical sites.47 Given these gender differences in leptin secretion, women with central obesity would be expected to have lower serum leptin concentrations than women without central obesity, assuming the same BMI. The serum leptin concentration would then be negatively associated with central obesity. If so, then this would explain the inverse association that we observed between the serum leptin concentration and the risk of Barrett’s oesophagus among women.
In summary, this study has shown a strong association between serum leptin concentrations and the risk of Barrett’s oesophagus in men. This association is not explained simply by higher body mass or gastro-oesophageal reflux among cases and our findings warrant further study of potential metabolic factors that may underlie this association.
The authors express their thanks to Sullivan and Nicolaides Pathology, Queensland Medical Laboratories and the Queensland Health Pathology Service for identifying participants for this study. They are also grateful to Peter Schultz, Lauren Aoude, Loralie Parsonson, Stephen Walsh, Mitchell Stark, John Cardinal and Herlina Handoko for technical support.
Study of Digestive Health Investigators
Queensland Institute of Medical Research, Brisbane, Australia: David C Whiteman MBBS, PhD; Adele C Green MBBS PhD; Nicholas K Hayward PhD; Peter G Parsons PhD; Sandra J Pavey PhD; David M Purdie PhD; Penelope M Webb D Phil.
University of Queensland, Brisbane, Australia: David Gotley FRACS; B Mark Smithers FRACS.
The University of Adelaide, Adelaide, Australia: Glyn G Jamieson FRACS.
Flinders University, Adelaide, Australia: Paul Drew PhD; David I Watson FRACS.
Envoi Pathology, Brisbane, Australia: Andrew Clouston PhD, FRCPA.
Funding: This research was supported by grant number CA 001833-03 from the United States National Cancer Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. PMW and DCW are Senior Research Fellows of the National Health and Medical Research Council of Australia. NP is supported by a PhD scholarship from the National Health and Medical Research Council of Australia. The funding bodies played no role in the design or conduct of the study, the collection, management, analysis, or interpretation of the data, or the preparation, review or approval of the manuscript.
Ethics approval: Ethics approval was obtained.