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integrative Physiology

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Intestinal Permeability Is Associated With Visceral Adiposity in Healthy Women

Anders Gummesson1,2, Lena M.S. Carlsson1, Len H. Storlien2,3, Fredrik Bckhed1, Pl Lundin2, Lars Lfgren2, Kaj Stenlf4, Yan Y. Lam3, Bjrn Fagerberg1 and Bjrn Carlsson1,2
Increased visceral fat, as opposed to subcutaneous/gluteal, most strongly relates to key metabolic dysfunctions including insulin resistance, hepatic steatosis, and inflammation. Mesenteric fat hypertrophy in patients with Crohns disease and in experimental rodent models of gut inflammation suggest that impaired gut barrier function with increased leakage of gut-derived antigens may drive visceral lipid deposition. The aim of this study was to determine whether increased intestinal permeability is associated with visceral adiposity in healthy humans. Normal to overweight female subjects were recruited from a population-based cohort. Intestinal permeability was assessed using the ratio of urinary excretion of orally ingested sucralose to mannitol (S/M). In study 1 (n = 67), we found a positive correlation between waist circumference and S/M excretion within a time frame of urine collection consistent with permeability of the lower gastrointestinal tract (69 hours post-ingestion; P = 0.022). These results were followed up in study 2 (n = 55) in which we used computed tomography and dual energy X-ray absorptiometry to measure visceral and subcutaneous fat areas of the abdomen, liver fat content, and total body fat of the same women. The S/M ratio from the 612 h urine sample correlated with visceral fat area (P = 0.0003) and liver fat content (P = 0.004), but not with subcutaneous or total body fat. This novel finding of an association between intestinal permeability and visceral adiposity and liver fat content in healthy humans suggests that impaired gut barrier function should be further explored as a possible mediator of excess visceral fat accumulation and metabolic dysfunction.
Obesity (2011) 19, 22802282. doi:10.1038/oby.2011.251

Accumulation of visceral adipose tissue is a major risk factor for diabetes and cardiovascular disease. The deleterious effect of visceral fat, as opposed to subcutaneous/gluteal, may be a consequence of the fat depot (mainly the omentum and mesentery) being more metabolically active and expressing higher levels of cytokines, immunoglobulins, and complement factors (13). It is not clear, however, why some individuals preferentially store fat within their abdominal cavity. Evidence is now persuasive, and has recently been reviewed (4), suggesting that the gastrointestinal tract may play an important role in visceral inflammation and lipid deposition. Briefly, hypertrophy of mesenteric fat is common in patients with Crohns disease (5), a pathological condition characterized by inflammation and increased permeability of the gut. Furthermore, rats with experimental colitis have 35% more mesenteric fat mass as compared to controls, an effect specific to that fat depot (6). As visceral adipose tissue is located close to the gastrointestinal tract, an impaired gut barrier function may expose this fat depot to various environmental agents, in particular bacterial antigens and immune factors (4). This in turn may stimulate, inter alia,
1

adipogenesis. For example, localized lipopolysaccharide stimulation induces adipocyte hyperplasia in rats (7) and immunoglobin receptors have been shown to stimulate lipogenesis in adipocytes in vitro (1). Whether similar interactions between the gastrointestinal tract and visceral adipose tissue exists in humans, and more importantly, if this is confined to pathological conditions is largely unknown. The aim of this study, therefore, was to investigate the relationship between intestinal permeability and visceral adiposity in healthy humans.
Methods and Procedures The study was approved by the regional ethical review board at Gteborg University, Sweden, and conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent. Subjects were recruited from a population-based cohort of Swedish women (8), using stratified random selection to include healthy participants with various degrees of abdominal adiposity. Exclusion criteria included diabetes type 1, insulin treatment, ongoing infection, pancreatic disease, impaired renal function, heart failure, any serious systemic disease, alcohol or substance abuse, history of bariatric surgery, postsurgical adhesions, history of or active gastrointestinal disorders, weight loss drug therapy and, gastroenteritis within 1 month before examination. Sixty-

Sahlgrenska Center for Cardiovascular and Metabolic Research, Department of Molecular and Clinical Medicine, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; 2AstraZeneca R&D, Mlndal, Sweden; 3Boden Institute of Obesity, Nutrition, Exercise and Eating Disorders, University of Sydney, Sydney, New South Wales, Australia; 4Department for Obesity, Sahlgrenska University Hospital, Gothenburg, Sweden. Correspondence: Anders Gummesson (anders.gummesson@astrazeneca.com) Received 21 November 2010; accepted 30 June 2011; published online 18 August 2011. doi:10.1038/oby.2011.251
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short communications
integrative Physiology
seven women were included in study 1 in which intestinal permeability, height, weight, and waist circumference were measured. Intestinal permeability was estimated by measuring the urinary excretion of orally ingested nonmetabolizable sucralose and mannitol as described previously (9). Briefly, subjects were fasted overnight and instructed not to use artificial sweeteners on the day before the visit. The subjects arrived at between 8 and 9 am. No sucralose or mannitol was detected in the baseline urine sample taken upon arrival. The subjects drank a solution containing 1.0 g sucralose and 5.0 g mannitol and urine was collected during 9 h post-ingestion. A standard hospital cafeteria lunch sandwich was eaten 3 h post-sugar ingestion. Urine concentrations of sucralose and mannitol were measured using gas chromatography-mass spectrometry. Intestinal permeability was expressed as the ratio of urine excretion of sucralose to mannitol (S/M). The use of probes of different molecular sizes is to account for pre- and postmucosal factors which may affect their excretion rates (10). Thirteen months after study 1, 55 of the 67 subjects agreed to participate in study 2, in which the intestinal permeability measurement was repeated with a urine collection extended to 12 h post-ingestion. As before, height, weight, and waist circumference were measured and the same procedure for assessment of gut permeability was undertaken. Additional measurements included fat depot imaging and fasting blood samples. Plasma was analyzed for glucose, insulin, high-density lipoprotein cholesterol, triglyceride, and C-reactive protein and all were in the normal range (glucose 5.3 0.6 mmol/l; insulin 7.6 3.4 mU/l; high-density lipoprotein cholesterol 1.9 0.5 mmol/l, triglycerides 1.4 0.8 mmol/l, and C-reactive protein 4.2 8.8 mg/l). The subcutaneous and visceral fat areas of the abdomen were determined using computed tomography scans (GE Hi-Speed Advantage; General Electric, Fairfield, CT) at the level of the lumbar four vertebrae. Liver fat content was estimated by computed tomography attenuation values. Total body fat was assessed using dual energy X-ray absorptiometry with a Lunar DPX-L scanner (Lunar, Madison, WI). Visceral, subcutaneous and total fat content were adjusted for total body mass. Biochemical analyses were performed using commercially available kits. Information on alcohol consumption, smoking, concomitant medication, psychological stress, and irritable bowel syndrome symptoms were obtained using structured interviews, the Shirom Melamed Burnout Questionnaire (11) and the Gastrointestinal Symptom Rating ScaleIBS version (12). Relations of measurements of intestinal permeability and continuous variables were assessed with Pearson correlations. Positively skewed data were log-transformed before statistical analysis. When normal distribution was not achievable, Spearman correlations were used. Relations with yes/no variables such as smoking and use of anti-inflammatory drugs were assessed using independent samples t-test. P < 0.05 (twotailed) was considered as statistically significant.
results
2.5 Visceral fat area per kg of body mass (cm2/kg) #

*
2.0

1.5

1.0

0.5

1st

2nd

3rd

4th

Quartile of intestinal permeability, based on sucralose/mannitol in the 612-h urine sampling

Figure 1 Relative size of the visceral fat area (mean s.e.m.) for each quartile of intestinal permeability (lowest to highest) in healthy females (n = 55). Visceral fat area was measured using computed tomography. Intestinal permeability was estimated by measuring the sucralose/ mannitol ratio in the 612 h urine samples. #P = 0.010 for trend across groups (ANOVA). *P = 0.023 between the 1st and 4th quartiles (t-test).

In study 1, we observed a positive association between gut permeability (as assessed by the S/M ratio in urinary excretion) and waist circumference but only significant for the 69 h urine sample (r = 0.28, P = 0.022), a time frame that mainly reflects the barrier function of the lower gastrointestinal tract. No association was observed between S/M ratio of the 69 h sample and BMI (r = 0.06, P = 0.65) nor for variables that may interfere with intestinal permeability, such as anti-inflammatory drugs, psychological stress, alcohol consumption, smoking, and irritable bowel syndrome symptoms (P values 0.83, 0.47, 0.86, and 0.36, respectively). Consistent with study 1, S/M ratio of the late urine sample (612 h) of the 55 subjects (age 6770 years; BMI 25.6 2.1 kg/ m2; waist circumference 88.5 9.0 cm) in study 2 was positively correlated with waist circumference (r = 0.29, P = 0.035). Despite the studies being conducted 13 months apart, there was
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a clear within-subject correlation between the S/M ratio from the two visits (r = 0.38, P = 0.0056). Both mannitol and sucralose were detected in the 06 h and 612 h sampling periods (S: 0.76 and 0.36%; M: 16.88 and 5.09% of oral dose, respectively). We analyzed the S/M ratio of the total (012 h), early (06 h) and late (612 h) samples in relation to the fat content of liver, visceral and subcutaneous depots, total body fat, and BMI. S/M ratio correlated only with visceral fat area (r = 0.30; P = 0.032) and liver fat (r = 0.32; P = 0.019) in the 012 h period. The early (06 h) samples did not correlate with any of the above mentioned measurements (r = 0.21, 0.16, 0.05. 0.05, and 0.06, respectively, all nonsignificant), whereas S/M ratio in the 612 h samples was positively correlated with visceral fat area (r = 0.48; P = 0.0003) and liver fat (r = 0.39; P = 0.004) but not with subcutaneous fat area (r = 0.17; P = 0.23), total body fat (r = 0.24; P = 0.08), or BMI (r = 0.05; P = 0.71). When subjects were grouped according to quartiles of permeability of the lower gastrointestinal tract, visceral fat area in subjects in the highest quartile was 56% larger than those in the lowest quartile (P = 0.023; Figure 1).
discussion

The major finding is that intestinal permeability is positively associated with visceral adiposity, and with liver fat content. These observations are consistent with the association of impaired gut barrier function with visceral adipose expansion and liver fat accumulation reported in patients with Crohns disease (5), with nonalcoholic fatty liver disease (13) and in mice with induced bowel inflammation (5). The present study is the first to show that similar relationships also exist in healthy humans. The significant overall correlation between S/M ratio (012 h) and visceral fat area showed a relationship between whole gut permeability and visceral adiposity. Breaking that analysis
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down to early (06 h) and late (612 h) samples allow a crude separation of the effect into upper vs. lower intestine. Significant correlations were found only with the intestinal permeability measurements in the late urine samples which are thought to reflect colonic permeability (9). This suggests the particular involvement of the lower gastrointestinal tract in visceral adiposity and hepatic lipid deposition. In addition, the correlation between intestinal permeability and waist circumference could only be explained by variations in the visceral but not subcutaneous or total body fat content. The methodology used allows only a basic differentiation of small intestine vs. colonic permeability. Because the proximal and distal colon are both structurally and functionally distinct, tests which might be able to distinguish barrier function of different regions of the colon would be an important future direction. When gut barrier function is impaired, the leakage of gutderived antigens to the adjacent visceral adipose tissue may lead to hyperplasia, inflammation, and other metabolic dysfunctions of the fat depot. Lipopolysaccharide, a major bacterial endotoxin, has been shown to induce hyperplasia (7), insulin resistance, and the expression of inflammatory mediators in adipocytes (14). Impaired gut barrier function also increases the delivery of gut bacterial products to the liver via the portal vein which may subsequently promote hepatic lipid deposition. A 4-week infusion of lipopolysaccharide has been shown to increase liver weight and triglyceride concentration in mice, an effect attributed to the endotoxemia-induced insulin resistance and inflammation (15). Our subjects had no history of, and were free from, gastrointestinal disorders during the course of the studies. Despite this, lower bowel permeability (612 h) displayed a fivefold range over the study group (S/M ratio range 0.030.15). Therefore, the current findings suggest that even without pathologically comprised gut function, intestinal permeability still appears to play a role in visceral adipose and liver fat accumulation. A note of caution is necessary on the methodology. Confounding factors such as intestinal transit time, surface area of villi, altered renal function, etc. are always possible with an indirect measurement. However, the study group were one sex, healthy and had a tight range of age and BMI which would hopefully minimize such concerns, particularly as there was no difference in BMI between the quartiles with the lowest (mean BMI 25.29) and highest (mean BMI 25.25) visceral fat area. Whereas this is a strength of the study in relation to confounding variables it is, of course, also a limitation and further studies to determine the generalizability of these results across a more diverse population are needed. Altered gut microbiota composition has recently been associated with metabolic dysfunction and some gut microbes are known to regulate gut barrier function (16). Our data suggests that intestinal permeability may be an important part of the link between diet, gut microbial balance, inflammation, and metabolic disorders. The present findings are consistent with the emerging role of gut in metabolic health (4). The results point to the importance of future investigations in this promising area.
acknowledgMents
We thank Marie-Louise Ekholm, Eva Bergelin, Lena Strid, Matty Hllqvist, Rahil Hezaveh, Carita Fagerlund, Ulrica Prahl Abrahamsson, Lillemor Mattsson Hultn, and Gunnel stergren Lundn for excellent research assistance, and Thomas Ljung, Jarl S. Torgerson, John Brandberg, and Rosie Perkins for important advice.

disclosure
A number of authors as indicated by the author affiliation listing are, or have been, employed by AstraZeneca.
2011 The Obesity Society

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