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A growing body of evidence has consistently shown a correlation between obesity and chronic sub-clinical inflammation. Several studies have suggested that measures of body fat distribution, rather than overall adiposity, may be more closely associated with inflammation level.
To investigate the relationship between levels of inflammatory markers and specific measures of abdominal visceral and subcutaneous fat and thigh intermuscular and subcutaneous fat of older white and black adults.
Data of 2,651 black and white men and women aged 70-79 participating in the Health, Aging and Body Composition (Health ABC) study were used. Levels of the inflammatory markers, IL-6, CRP, and TNF-α were obtained from blood samples. The areas of abdominal visceral and subcutaneous fat and thigh intermuscular and subcutaneous fat were quantified on CT images. Linear regression analysis was used to evaluate the cross-sectional relationship between each body composition measure and serum levels of inflammatory markers in the four race/gender groups.
Abdominal visceral adiposity was most consistently associated with significantly higher IL-6 and CRP levels in all race/gender groups (p<0.05). Thigh intermuscular fat had an inconsistent but significant association with inflammation, and there was a trend toward lower inflammation level with increasing thigh subcutaneous fat in white and black women.
Despite the previously established differences in abdominal fat distribution across gender and race, visceral fat remained a significant predictor of inflammatory marker level across all four subgroups examined.
A growing body of evidence has consistently shown a correlation between obesity and chronic sub-clinical inflammation (1, 2). In particular, obesity has been associated with elevated levels of the inflammatory marker C-reactive protein (CRP), as well as high levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (1). Adipose tissue itself produces cytokines (3, 4); it has been estimated that adipose tissue is responsible for about 25% of systemic IL-6 (4). Increased levels of IL-6, CRP, and TNF-α have been associated with higher risk of heart failure (5), lower insulin sensitivity (6), and metabolic syndrome (7, 8). In animal studies, the removal of visceral adipose tissue reduced the negative metabolic effects of diabetes and lowered the expression of TNF-α in subcutaneous adipose tissue (9, 10).
Several studies have suggested that measures of body fat distribution, rather than overall adiposity, may be more closely associated with levels of IL-6, CRP, and TNF-α (1, 4, 11, 12). Visceral adiposity in particular has been shown to be uniquely correlated with the level of inflammation (11-13). However, studies on the effect of body fat distribution have been limited by a lack of detailed measurements in multiple fat depots, especially in depots outside of the abdomen.
Few studies have analyzed adipose distribution and inflammation level across racial groups (14). There is evidence that body fat distribution differs between races; it has been shown that Asians have more abdominal visceral fat than whites, and that whites have more abdominal visceral fat than blacks (14-17). Still, it is not known whether the relationship between fat distribution and inflammation is similar across all races and ethnicities.
The aim of this study is to investigate the relationship between levels of inflammatory markers and abdominal subcutaneous fat, abdominal visceral fat, thigh subcutaneous fat and thigh intermuscular fat in older black and white men and women.
Data were from the Health, Aging, and Body Composition (Health ABC) study, a longitudinal cohort study of body composition and health consisting of 3075 well-functioning, 70- to 79-year old black and white men and women. Participants were identified from a random sample of white Medicare beneficiaries and all age-eligible community-dwelling black residents in designated zip code areas surrounding Memphis, Tennessee, and Pittsburgh, Pennsylvania. Participants were eligible if they reported no difficulty in either walking one quarter of a mile, going up 10 steps without resting, or performing basic activities of daily living. Participants were excluded if they reported a history of active treatment for cancer in the prior three years, planned to move out of the study area in the next three years, or were currently participating in a randomized trial of a lifestyle intervention. Baseline data, collected between April 1997 and June 1998, included an in-person interview and a clinic-based examination, with evaluation of body composition, clinical and sub-clinical diseases, and physical functioning. Of the original Health ABC cohort of 3075 individuals, 424 individuals were excluded due to incomplete data on inflammatory markers, body composition, or covariates, leaving 2651 subjects for the present cross-sectional analyses. The 424 excluded persons had mean age 74.2±2.9 years and included 108 white men, 81 black men, 98 white women, and 137 black women.
Abdomen and thigh CT scans were obtained at the Pittsburgh site using a 9800 Advantage Scanner (General Electric, Milwaukee, WI) and either a Somatom Plus 4 (Siemens, Erlangen, Germany) or a Picker PQ 2000S (Marconi Medical Systems, Cleveland, OH) scanner at the Memphis site (18). The scans were conducted at 120 kVp, 200 to 250 mA seconds, at a slice thickness of 10 mm. Areas were calculated by multiplying the number of pixels of a given tissue type by the pixel area using ILD development software (RSI Systems, Boulder, CO). Scans of the abdomen were taken at the level of the space between the fourth and fifth lumbar vertebrae (L4–L5). The scan at mid-thigh level was performed at one half of the distance between the medial edge of the greater trochanter and the intercondyloid fossa. Visceral fat was manually distinguished from subcutaneous fat by tracing along the fascial plane defining the internal abdominal wall. In the thighs, intermuscular and visible intramuscular fat tissue were separated from subcutaneous adipose tissue by drawing a line along the deep fascial plane surrounding the thigh muscles.
Measures for the cytokines IL-6 and TNF-α and for CRP were obtained from frozen stored plasma or serum. Fasting blood samples were obtained in the morning, and after processing, the specimens were aliquoted into cryovials, frozen at -70°C, and shipped to the Health ABC Core Laboratory at the University of Vermont. Cytokines were measured in duplicate by enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN). The detectable limit was 0.10 pg/mL for IL-6 (by HS600 Quantikine Kit) and 0.18 pg/mL for TNF-α (by HSTA50 kit). Serum levels of CRP were also measured in duplicate by ELISA based on purified protein and polyclonal anti-CRP antibodies (Calbiochem, San Diego, CA). The CRP assay was standardized according to the World Health Organization First International Reference Standard with a sensitivity of 0.08 μg/mL. Assays of blind duplicates collected for 150 participants showed an average interassay coefficient of variation of 10.3% for IL-6, 8.0% for CRP, and 15.8% for TNF-α. In the entire study population, the correlation coefficients between IL-6 and CRP, CRP and TNF-α, and TNF-α and IL-6 were 0.39, 0.11, and 0.21 respectively.
Covariates were selected on the basis of previously identified associations with either obesity or inflammation. Sociodemographics included clinical site (Memphis, Pittsburgh), age, marital status (never married, previously married, married), and level of education (<12 years, 12 years, >12 years). Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m2). Smoking was included as cigarette use in pack-years. Physical activity was measured by questionnaire in which participation in the following activities was determined: gardening or yard work, housework, stair climbing, walking for exercise, other walking, aerobics/calisthenics, weight training, high-intensity exercises, moderate-intensity exercises, or work/volunteer/caregiving activities. The intensity of walking and exercise, and the frequency and duration of each activity in the previous seven days was ascertained. Weekly caloric expenditure was estimated in kcal/kg/hr through the assignment metabolic equivalent unit (MET) values to each activity (19). Presence of heart disease, cerebrovascular disease, lung disease, peripheral arterial disease, diabetes mellitus, osteoarthritis, and cancer was determined using standardized algorithms considering self-report and use of specific medications. Depressed mood was assessed with the Center for Epidemiologic Studies Depression (CES-D) scale. A cutoff score of 16 was used as a criterion for major depressive symptoms (20). Cognitive impairment was defined as a Modified Mini-Mental State Examination (3MS) score less than 78 (21)(21). Medications taken in the previous 2 weeks were recorded and coded according to the Iowa Drug Information System (22).
Analyses were performed separately for each of four race-gender groups because of known differences in the distribution of body adipose by race and gender. Linear regression analysis was used to evaluate the relationship between each body composition measure and serum levels of inflammatory markers, with inflammatory marker levels normalized by logarithmic transformation. Regression was performed using three different models; the first controlled for clinic site and age, the second additionally controlled for BMI to account for other body adipose that might be correlated with inflammation, and the third model controlled for all variables of the previous models and marital status, education level, pack years of smoking, physical activity, diseases, anti-inflammatory drugs, and oral steroid medication. Reported statistical analysis values include the standard deviation increase in normalized inflammatory marker level per standard deviation increase in fat area, and the significance. All statistical analyses were performed using SPSS, version 14.0 (SPSS Inc., Chicago, IL).
The main characteristics of the study population are presented by gender and race in Table 1. White persons had more abdominal visceral fat than black persons (p<0.01). Levels of IL-6 (p<0.01) and CRP (p<0.01) were higher in blacks than in whites and TNF-α level (p<0.01) was higher in whites than in blacks. Women had more abdominal subcutaneous fat (p<0.01) and thigh subcutaneous fat (p<0.01) than men. In both men and women, blacks had more thigh intermuscular fat (p<0.01). Black women had a higher mean BMI than white women. Pack-years of smoking were higher in men than in women (p<0.01) and in white persons than in black persons (p<0.01). Whites had higher levels of physical activity than blacks (p<0.01). Heart disease, lung disease, and peripheral artery disease were more prevalent in men than in women (p<0.01). The prevalence of osteoarthritis was higher in women (p<0.01) and a greater percentage of women had depressive symptoms than men (p=0.01). In both men and women, blacks had a higher prevalence of diabetes mellitus (p<0.01) and cognitive impairment (p<0.01) while the prevalence of osteorarthritis (p<0.01) and cancer (p<0.01) was higher among whites.
Standardized regression coefficients of inflammatory markers by body composition measure for men are displayed in Table 2. In both black and white men, visceral fat was associated with significantly higher levels of IL-6 and CRP in all three regression models. In white men, there was a significant negative association between thigh subcutaneous fat and levels of IL-6 and TNF-α in the fully adjusted model. Thigh intermuscular fat was related to significantly higher levels of IL-6 and CRP in both black and white men in model 1. After adjustment for BMI, this association remained significant for IL-6 in all men and also for CRP in black men.
Standardized regression coefficients of inflammatory markers by body composition measure for women are displayed in Table 3. In model 1, all of the body fat measures were positively and significantly associated with all three inflammatory markers in white women, and with IL-6 and CRP in black women. Many of these associations disappeared after adjustment for BMI in model 2, but abdominal visceral fat was significantly related to higher levels of IL-6, CRP and TNF-α in all three regression model in both black and white women. Thigh intermuscular fat area was significantly associated with higher levels of all three inflammatory makers in all three regression models in white women and not in black women. When the analyses in all four race/gender groups were adjusted for total percent body fat instead of BMI, the general results in both men and women do not change considerably.
Figure 1 depicts the standardized regression coefficients of the inflammatory markers according to both visceral and abdominal subcutaneous fat. In this representation, both body fat measures were added simultaneously to the fully adjusted model (model 3). Visceral fat was positively associated with levels of all three inflammatory markers in every race-gender group, and the association was significant in every case except in black men with TNF- α. In contrast, there was no consistent association between abdominal subcutaneous fat and inflammatory marker level after adjustment for abdominal visceral fat.
Figure 2 displays the standardized regression coefficients of the inflammatory markers according to both thigh intermuscular and thigh subcutaneous fat. Thigh intermuscular fat was significantly associated with higher levels of IL-6 in both groups of men, higher levels of CRP in black men, and higher levels of IL-6, CRP, and TNF-α in white women. There was a trend toward lower levels of IL-6, CRP, and TNF-α with increasing thigh subcutaneous fat, but the trend was only significant for IL-6 and TNF-α in white men.
In additional analyses, subjects were divided into an “unhealthy” group and a “healthy” group (with and without diseases) to investigate whether the higher levels of inflammatory markers that were associated with visceral fat were due to pre-existing disease. The associations between inflammatory marker level and visceral fat remained significant in both the healthy and unhealthy group (not tabulated).
Although a number of studies have investigated the association between visceral adiposity and inflammation, few have analyzed these associations across race and gender and none have investigated inflammatory marker level according to specific fat depots outside of the abdomen. The main finding of this study is that in both men and women, abdominal visceral fat area was significantly associated with higher levels of IL-6 and CRP across both races. Furthermore, the associations between visceral fat and levels of IL-6 and CRP were independent of total adiposity. Though not significant, a consistent trend toward lower levels of inflammatory markers with increasing thigh subcutaneous fat was found. Levels of inflammatory markers tended to increase with greater thigh intermuscular fat, although not significant in all race-gender groups.
The association between visceral fat and inflammation found in this study is consistent with the existing literature. Previous studies have shown that CRP level is significantly positively associated with visceral fat in South Asian, white, and Japanese men and women (15, 23). Significant correlations between visceral fat and levels of IL-6 and CRP, but not TNF-α, have been documented in postmenopausal white women (24). Similarly, a large study of elderly men and women found that IL-6 and CRP, but not TNF-α, were significantly associated with visceral fat (12). In these studies, the relationships between inflammatory marker levels and visceral fat were independent of total adiposity measured as total percent body fat or BMI. Our findings further support the growing body of evidence that visceral fat depot, rather than abdominal subcutaneous fat, is uniquely important to inflammation (25).
To our knowledge, no previous study has investigated the associations between inflammatory markers and thigh composition. Greater thigh intermuscular fat has been associated with type 2 diabetes and impaired glucose tolerance (26). The relationship between thigh intermuscular fat and higher inflammation found in the present study supports evidence that thigh intermuscular fat is associated with negative health outcomes. In addition, it has been shown that low thigh subcutaneous fat is a risk factor for a disadvantageous glucose and lipid profile (18), and that greater thigh subcutaneous fat is correlated to higher insulin sensitivity (26). Though not statistically significant, the trend toward lower inflammatory marker level with higher thigh subcutaneous fat may reflect the possibly protective nature of subcutaneous thigh fat.
Several studies have demonstrated gender and race differences in visceral fat deposition. As found in the present study, black men and women have been shown to have less visceral fat than white men and women (16, 17). Men have been shown to have more visceral fat and less subcutaneous fat than women (27), and this comparison holds true in our study within race. However, despite these considerable differences in abdominal fat distribution across gender and race, visceral fat remained a significant predictor of inflammatory marker level in all analysis groups. Asian women, but not men, have been shown to have significantly higher areas of visceral fat than whites (14, 15). Further studies should investigate whether visceral fat is similarly related to inflammatory markers in Asians and other racial and ethnic groups.
The causality of the associations between negative health outcomes, visceral fat, and intermuscular thigh fat are not yet clear. It has been shown that the relationship between diabetes and visceral fat is attenuated by levels of adiponectin, IL-6, TNF-α, and plasminogen activator inhibitor 1 (28), indicating the possibility that adipocytokines are key in the link between visceral fat and an unfavorable metabolic profile. Adipose tissue itself secretes IL-6 and expresses TNF-α (4). However, it is of note that this and other studies have found consistent visceral fat associations with levels of IL-6 and CRP, but not TNF-α (12, 24). An investigation of IL-6 and TNF-α level in 39 healthy subjects indicated that subcutaneous adipose tissue releases IL-6, but does not secrete TNF-α (4), and may explain the lack of association with TNF-α if visceral adipose tissue behaves similarly. In fact, a study of obese women found that TNF-α mRNA levels were significantly lower in visceral fat than in abdominal subcutaneous fat (29).
This study is subject to some limitations. The study population consisted of relatively healthy elderly adults, and the extrapolation of these findings to younger individuals or other elderly adults may not be appropriate. Due to the variety of medical conditions present in men and women in the study population, there is the possibility that the visceral fat associations found in the analysis were due to a classical acute phase response to illness or injury rather than endogenous or low-grade exogenous factors. To investigate this possibility, linear regression was conducted on a cohort subset in which the subjects with the highest decile of inflammatory marker values were excluded from the analysis. The visceral fat associations retained their significance and increased slightly in strength, indicating that the acute phase response may obscure rather than strengthen the relationship between visceral fat and inflammation (data not shown).
This study is unique in that it investigated the relationship between inflammation level and multiple specific fat depots, and explored these associations by race and gender group. Across all race and gender groups, visceral fat was found to be significantly associated with IL-6 and CRP. Thigh intermuscular fat had an inconsistent but significant association with inflammation in the four population groups, and there was found a trend toward lower inflammation level with increasing thigh subcutaneous fat.
This study was supported by National Institute on Aging contracts N01-AG-6-2101, N01-AG-6-2103, and N01-AG-6-2106. This research was supported (in part) by the Intramural Research program of the NIH, National Institute on Aging.