To explore possible associations between adipose tissue (AT) blood flow (ATBF), AT depot sizes and adipocyte-derived hormones (adipokines) in women.
In all, 43 healthy women were divided into four groups: normal-weight (n=11) and obese (n=11) pre-menopausal women and normal-weight (n=10) and obese (n=11) post-menopausal women.
Fasting levels of adipokines were obtained, and a single-slice computed tomography scan at the level of L4–L5 was used to estimate fat depot sizes. ATBF was assessed by xenon washout while in a fasting state and after oral glucose load. We also measured glucose, insulin and non-esterified fatty acids.
Total, subcutaneous and visceral AT areas strongly correlated with ATBF (all P<0.001). Circulating leptin levels strongly and inversely correlated with ATBF (P=0.001), but this association did not remain after adjustment for body mass index. Adiponectin was not associated with blood flow.
ATBF is closely linked to subcutaneous and visceral AT size. Further analyses are needed to determine possible mediators of this association, including mechanistic studies to assess a putative role for leptin as a significant modulator of blood flow.
AT blood flow (ATBF) is an important regulator of AT metabolism, because this tissue is highly vascularised and variations in blood flow facilitate storage and removal of lipids when needed (for example, stress, exercise and fasting).1, 2 Furthermore, post-prandial ATBF may facilitate signalling between AT and other tissues, such as the liver and skeletal muscle.3 The dynamics of ATBF is related to the degree of obesity, and obese subjects respond less to a mixed meal than lean subjects.4, 5 Notably, an attenuated post-prandial ATBF may decrease glucose and triglyceride (TG) uptake, leading to post-prandial hyperglycaemia, hyperinsulinemia and hyperlipidemia. In line with this, McQuaid et al.6 recently demonstrated that obese subjects have a post-prandial reduction in chylomicron-TG-derived fatty acid storage. This provides a possible pathophysiological basis for ectopic fat deposition and lipotoxicity and support for ATBF being a key player in the metabolic disturbances seen in obesity and obesity-related diseases. A putative regulator of ATBF is AT distribution. Regional metabolic differences in lipolysis and fatty acid uptake between upper- and lower-body fat have been described, as well as differences between subcutaneous and visceral fat depots.7, 8 It has been suggested that subcutaneous AT may act as a buffer for dietary lipids, that is a ‘metabolic sink’, protecting other tissues from a lipid overflow with associated lipotoxicity.7 Visceral adipocytes are phenotypically different from subcutaneous adipocytes,9 and increased metabolic/cardiovascular risk is linked to visceral AT accumulation.
We recently found that ATBF is influenced by nitric oxide activity and autonomic nerve balance.10 Putative modulators of a link between obesity, ATBF, nitric oxide activity and autonomic activity include the adipokines leptin and adiponectin, which are secreted into the peripheral circulation from AT.2, 11, 12, 13 These adipokines predict risk for type 2 diabetes and cardiovascular disease,14, 15, 16 and in vitro studies have demonstrated the nitric oxide-mediated vasorelaxation effects of both leptin and adiponectin.2, 11, 12, 13
With menopause, women change their fat distribution to a more central (android) location.17 On the basis of the metabolic differences between subcutaneous and visceral fat, and our recent demonstration of dysregulation of ABTF in post-menopausal overweight women,10 we hypothesised that ATBF is closely related to the size of the abdominal fat depot. We also hypothesised that adipokines are potential independent regulators of ATBF.
Subjects and methods
Details of the subjects included in this study were described previously.10 Briefly, we recruited 43 healthy women and divided them into four groups: normal-weight (n=11) and obese (n=11) pre-menopausal women, and normal-weight (n=10) and obese (n=11) post-menopausal women. Post-menopausal status was defined as the lack of menstrual periods for at least 12 continuous months. Basal characteristics are shown in Table 1. Exclusion criteria were pregnancy, thyroid disease, hypertension, known diabetes, cancer, previous stroke, known heart disease, psychiatric disorder, alcohol abuse, electrolyte disturbances and treatment with glucocorticoids, systemic estrogens or lipid-lowering agents. The pre-menopausal women were all studied in the follicular phase. The regional ethics committee for Northern Sweden approved the study, and written informed consent was obtained from all participants.
Clinical measurements were made at rest between 08:00 and 13:00 hours after an overnight fast. Baseline anthropometry (weight, waist, hip and blood pressure) was measured and an oral glucose tolerance test was performed as previously described.10 A computed tomography scan was performed on a separate day, within a month from the other measurements.
ATBF was measured by 133Xe washout.18 A dose of 1–2 MBq 133Xe was injected subcutaneously into the para-umbilical area. Calculation of fasting ATBF was based on two independent readings taken 15 and 10 min before the oral glucose tolerance test, and post-prandial blood flow was calculated at 15, 30, 45, 60, 90 and 120 min after glucose loading, as previously described.19, 20
Blood sampling and analyses
A cannula (Optiva 2 18–20; G Johnson & Johnson, New Brunswick, Canada) was inserted retrogradely into a distal forearm vein and kept patent by a continuous slow infusion of saline. The lower part of the forearm was heated in a chamber (Biomedical Engineering Department, Huddinge University Hospital, Stockholm, Sweden) to provide arterialised blood samples. A 10 ml aliquot of arterialised venous blood was collected twice before and then 15, 30, 45, 60, 90 and 120 min after glucose intake (Figure 1). At each time point, 1.5 ml of blood was placed in an ethylenediaminetetraacetic acid tube, immediately put on ice and centrifuged at 4 °C. The plasma was collected and stored at −80 °C. The remaining blood volume was centrifuged and the serum was stored at −80 °C for later analyses.
Leptin and adiponectin levels were analysed with double-antibody radioimmunoassays (Linco Research, St Louis, MO, USA). The total coefficient of variation was 4.7% at both low (2–4 ng ml−1) and high (10–15 ng ml−1) levels for leptin, and for adiponectin it was 15.2% at low levels (2–4 μg ml−1) and 8.8% at high levels (26–54 μg ml−1). Non-esterified fatty acids were analysed using reagent non-esterified fatty acids C (Wako Chemicals GmbH, Neuss, Germany) on Hitachi 912 (Roche, Basel, Switzerland). Insulin was analysed by a direct chemiluminiscent technique using an insulin reagent kit with a Modular E 170 immunoanalyzer (Roche). High-sensitivity C-reactive protein and IL-6 solid chemiluminescent immunometric phase assays were performed using Immulite (Siemens, Munich, Germany). All other parameters were analysed by dry chemistry on a Vitros 950 (Ortho-Clinical Diagnostics, Raritan, NJ, USA).
All participants underwent a computed tomography examination with a General Electric LightSpeed 4 Channel computed tomography scanner (GE, Milwaukee, WI, USA). A previously described method was used.21, 22 Image data were acquired as 5 mm slices at the L4–L5 level (120 mAs, 50 cm field of view). Anterior and lateral scanograms were used to identify the correct slice positions. The image data were used for calculation of total, subcutaneous and visceral AT areas by using defined levels of Hounsfield units.
Blood flow calculations were performed in an excel sheet for Windows 2007 (Microsoft, Redmond, WA, USA). The remaining statistical analyses were performed using SPSS Statistics 18 (SPSS, Chicago, IL, USA). P<0.05 was considered to be statistically significant. The Kruskal–Wallis non-parametric test was used to test for differences between groups, followed by the Mann–Whitney post-hoc test if significant differences were found. For bivariate correlation analyses, we used natural logarithm values to achieve approximate normality. Partial correlation analyses were used to assess the relation of ATBF to other parameters after adjustments were made as described in the text. Area under the curve (AUC) responses after glucose loads were estimated according to the trapezoid rule. Tertiles of AT areas were calculated using group-specific cut-off values for pre- and post-menopausal women separately. The homoeostatic model assessment index was calculated as (fasting glucose (mmol l−1) × fasting insulin (μU ml−1))/22.5.23
Pre- and post-menopausal obese women had significantly higher body mass index (BMI), waist circumferences, waist-to-hip ratios and intra-abdominal AT areas compared with pre-menopausal normal-weight women (Table 1). Anthropometric data were reported previously for this cohort.10 Notably, both post-menopausal normal-weight and post-menopausal obese women had an approximately twofold larger intra-abdominal AT area compared with their pre-menopausal counterparts, despite similar BMI. The post-menopausal obese group had higher levels of glucose (AUC) and free fatty acids (AUC) compared with the pre-menopausal groups. Fasting levels of glucose, insulin and free fatty acids were published previously.10 Insulin resistance, expressed as a homoeostatic model assessment index, was highest in the post-menopausal obese group. Five individuals (two post-menopausal normal-weight and three post-menopausal obese) had 2 h glucose levels 11.1, thus fulfilling the criteria of diabetes mellitus, despite normal fasting glucose levels.
The obese groups had higher leptin levels, and levels of adiponectin were lowest in obese pre-menopausal women. The ATBF (AUC) response to glucose was significantly lower in both obese groups compared with normal-weight pre-menopausal women. Post-menopausal obese women had significantly lower ATBF (AUC) than their normal weight counterparts.
ATBF and association with measures of obesity, biomarkers and adipokines
ATBF (AUC) was significantly associated with all measures of obesity (BMI, waist, waist-to-hip ratio, total AT area, subcutaneous AT area and intra-abdominal AT area). These associations remained after adjustment for menopausal status and age (Table 2). ATBF was not associated with glucose and insulin levels. Associations were found between ATBF and free fatty acids, and between ATBF and homoeostatic model assessment index; however, only the homoeostatic model assessment index association remained significant after adjustment for menopausal status and age. Leptin was strongly inversely associated with ATBF; the association remained after adjustment for menopausal status and age, but not after adjustment for BMI. By contrast, no association was seen between blood flow and adiponectin. Because baseline levels for ATBF differed among the groups, we also recalculated all data using centred cumulative response, but this did not significantly change the results. The highly significant negative association between leptin and ATBF, and the non-significant association between adiponectin and ATBF are visualised with scatter plots in Figures 2a and b.
AT blood during oral glucose tolerance test
ATBF was significantly lower at baseline in both obese groups compared with normal-weight pre- and post-menopausal women (P<0.05) (Figure 3). After glucose loading, at all time points, post-menopausal obese women had lower ATBF than pre-menopausal normal-weight women (P<0.05). The same pattern was seen between the two post-menopausal groups, except that the difference was non-significant at 60 and 90 min. Obese pre-menopausal women had significantly lower ATBF than their normal weight counterparts throughout the test (P<0.05). There were no significant differences in blood flow between the two obese groups. The post-menopausal normal-weight women had lower ATBF than pre-menopausal normal-weight women at 60 and 120 min (P<0.05).
ATBF and association with total, subcutaneous and visceral AT
We studied ATBF according to the amount of AT in each depot (expressed as tertiles with group-specific cut-offs) (Figures 4a–c). In each depot, we found highly significant differences in ATBF between the lowest and medium tertiles (P<0.01), and between the lowest and highest tertiles (P<0.01). We also found significant differences in ATBF between the medium and highest tertiles (P<0.05) for total and intra-abdominal AT. In the subcutaneous depot, there was a trend (non-significant) towards a blood flow difference between the medium and highest tertile of the AT area.
We found a strong association between ATBF and subcutaneous, as well as visceral, AT areas. This suggests that total AT mass, rather than specific sites of fat accumulation, is linked to dysregulation of ATBF. It was previously shown that both fasting ATBF and ATBF responsiveness to nutrients5 are reduced in obesity, but the relationship of ATBF with different depots of abdominal fat had, to our knowledge, not been explored. The importance of our findings is underscored by recent data from obese study participants showing a reduced ability to store fat in AT after meals.6 The absence of a difference between AT accumulation and ATBF in different fat depots suggests that subcutaneous AT accumulation has a relatively greater role than visceral AT in ectopic fat deposition and lipotoxicity, due to its larger volume. Our data therefore support the hypothesis of subcutaneous fat acting as the primary metabolic ‘sink,’ protecting other organs from excessive post-prandial lipid levels.
Visceral fat depot size has been strongly linked to metabolic and cardiovascular diseases;24 differences in insulin action, gene expression, metabolic responses and adipokine secretion25, 26 among fat depots could explain this difference. It has also been proposed that extrinsic factors, including depot-specific blood flow and/or innervation,27 could contribute to distinct gene expression patterns and metabolic profiles in adipocytes in different anatomical regions. It is possible that lipid and glucose uptake in AT depots is regulated differently in people with obesity. Studies using positron emission tomography have elegantly demonstrated differences in perfusion and insulin-stimulated glucose uptake between visceral and abdominal subcutaneous AT in obese people.28, 29 Women are more protected from cardiovascular events than men, until their body fat distribution changes with menopause and takes on a more android (male) distribution.30, 31 If the menopausal transition also has differential effects on lipid and glucose uptake in different fat depots, this would clearly be an area of interest for further studies.
Notably, total perfusion through both abdominal subcutaneous and visceral depots can be up to 900 ml min−1 (∼18% of average cardiac output) in obese people.28 Thus, ATBF not only has an important impact on cardiac work load, but is also a powerful regulator of AT metabolism. Although the ATBF response to nutrient intake is thought to be of importance in the regulation of metabolism by facilitating signalling between AT and other tissues, such as skeletal muscle and liver,3 the physiological significance of and mechanisms behind post-prandial hyperaemia are not fully understood. Importantly, the rapid post-prandial increase in ATBF and the rapid insulin-mediated suppression of adipocyte lipolysis change the direction of free fatty acids flux in tissues.32 Related to this, we found an inverse correlation between insulin resistance and ATBF.
We also found a strong inverse association between circulating levels of leptin and ATBF. Hyperleptinemia has been linked to an increased risk of later development of type 2 diabetes and cardiovascular disease, including myocardial infarction and stroke.33, 34, 35 Several mechanisms have been suggested to be behind these associations, including mediation of platelet aggregation, increased sympathetic activity and stimulation of inflammation.36, 37 The regulatory effects of leptin on vascular endothelial cells in rodents are also well established,2, 11, 38 but less is known about putative effects on human endothelial function and blood flow regulation in vivo. The non-significant results after adjustments for BMI in this study should be interpreted with caution, because leptin and measures of obesity are strongly correlated and it is debatable whether models exploring the effect of leptin should be adjusted for obesity. On the other hand we cannot rule out that the association between fat mass and ATBF could be determined by other factors than leptin. Furthermore, the links between leptin and later risk of diabetes, as well as cardiovascular outcomes, have been found mainly in males.33, 34, 35 Studies are thus needed on possible differences between males and females regarding the vascular effects of leptin. This includes detailed mechanistic studies.
We found no association between adiponectin and ATBF. This suggests that adiponectin does not have a major role in ATBF regulation. In contrast, hypoadiponectinemia is linked to endothelial dysfunction in peripheral arteries,39 and plasma total adiponectin concentrations are inversely related to the risk of myocardial infarction.
Our study has some limitations. We found a very high correlation between the size of subcutaneous and intra-abdominal depots (R=0.77, P<0.001), in line with previous studies,40, 41 making it difficult to separate the effects of the different depots. Another weakness is the cross-sectional design, which makes it impossible to explore causality.
In summary, we found highly significant correlations between ATBF and total, visceral and subcutaneous AT areas. We also show a strong inverse association between circulating levels of leptin and ATBF, although this was non-significant after adjustment for BMI. Further mechanistic studies are needed to assess the clinical significance of our findings.
This study was supported by the Swedish Research Council, the Swedish Heart and Lung Foundation, the Heart Foundation of Northern Sweden, Northern Sweden County Council, Västerbotten County Council, and the Faculty of Medicine, Umeå University. We gratefully acknowledge nurses Inger Arnesjö and Veronica Sjöberg for their technical assistance in this study.
About this article
Hormone Molecular Biology and Clinical Investigation (2014)