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Adipose tissue lipoprotein lipase (LPL) is a necessary enzyme for storage of VLDL-TG, but whether it is a rate determining step is unknown. To test this hypothesis we included 10 upper-body obese (UBO), 11 lower-body obese (LBO) and 8 lean women. We infused ex-vivo labeled VLDL-14C-TG and then performed adipose tissue biopsies to understand the relationship between VLDL-TG storage and LPL-activity in femoral and upper-body subcutaneous fat. Both fractional tracer storage and rate of storage of the VLDL-TG tracer were evaluated. VLDL-TG storage was also examined as a function of regional adipose tissue blood flow (ATBF), insulin, VLDL-TG turnover, regional fat mass, FFM, and fat cell size. LPL-activity per adipocyte was significantly greater in obese than lean women but not significantly different per gram lipid. Both VLDL-TG fractional tracer storage per kg lipid and VLDL-TG storage rate per kg lipid were similar in abdominal and femoral fat in all three groups and not significantly different between groups. Multiple regression analysis identified FFM and femoral fat mass as significant independent predictors of VLDL-TG fractional tracer storage and insulin as a significant predictor of VLDL-TG fatty acid storage rate. LPL-activity, ATBF, and VLDL-TG turnover did not predict VLDL-TG storage. We conclude that lower FFM and greater plasma insulin is associated with greater VLDL-TG deposition in abdominal subcutaneous and femoral fat. Greater femoral fat mass signals greater femoral VLDL-TG storage. We suggest that the differences in VLDL-TG storage in abdominal and femoral fat that occur with progressive obesity are regulated through mechanisms other than LPL-activity.
Preferential upper body/visceral fat accumulation is strongly related to the development of type 2 diabetes mellitus, hypertriglyceridemia, and hypertension(1, 2, 3), whereas lower body obesity (LBO) is not(4, 5, 6). The adverse metabolic consequences of obesity are probably the result of an unfavorable balance between lipid oxidizing and lipid accumulating tissues. However, the mechanisms that determine differences in adipose tissue accumulation are not characterized in detail(7).
Although studies have described abnormalities of systemic and regional FFA release that can contribute to explain the excess FFA observed in upper-body obese (UBO) they have not provided sufficient evidence to suggest that differences in regional FFA release, as opposed to fatty acid storage, can account for differences in obesity phenotypes(8, 9). Thus, even though regional lipolysis may be important for the pathophysiological and metabolic abnormalities associated with obesity it is also clear that regional adipose tissue fatty acid storage must be of equal importance for development of unhealthy adipose depots. There are, however, only few studies that have studied regional TG fatty acid storage in humans. Moreover, most studies have studied meal fatty acid (FA) storage. In women meal fat storage into abdominal fat has been reported to be greater(10) or similar(11, 12) when compared with femoral fat storage. In response to a high-fat meal women showed a more efficient storage into femoral fat compared with men(11). In a recent study of women with a wide range of body weight and composition there was, however, no difference in meal fat taken up by abdominal or femoral adipose tissue(12). Mårin et al. found continued accumulation of dietary FA in subcutaneous fat up to one week after consumption of the test meal(10). The fact that tracer storage continued after all chylomicron TG with the tracer was cleared supports the notion that an ongoing redistribution of fatty acids occurs between tissue depots and raises the question whether FFA redistribution plays a significant role in fat accumulation in different anatomic regions in obesity. There are, however, no published data regarding postabsorptive regional VLDL-TG adipose tissue storage in humans, probably due to lack of robust in-vivo methods.
Lipoprotein lipase (LPL) is a key enzyme in the metabolism of plasma VLDL-TG and chylomicrons(13). Active adipose tissue LPL, attached to heparan sulphate proteoglycan at the luminal surface of the endothelium, hydrolyses TG so that fatty acids are available for uptake into the fat cells with some possible escape into the circulation (14). It has been suggested that LPL is the essential regulator of triglyceride fatty acid uptake into fat cells(15). Adipose tissue LPL-activity is reported to be increased in obesity(16). Moreover, both significant(11) and non-significant(10, 12) relationships between LPL-activity and the amount of meal fat uptake into adipose tissue have been reported. However, a number of substrates and hormones have an effect on LPL-activity, especially postprandially. We hypothesized that postabsorptive VLDL-TG trafficking into regional fat stores is related to regional LPL-activity in a direct way, which, if true could indicate a role for LPL in controlling body fat distribution. We used a recently developed method for ex-vivo labeling of VLDL-TG followed by autolog re-infusion and adipose tissue biopsies to concurrently measure plasma VLDL-TG specific activity (SA) and regional fat storage(17). The primary objective was to assess the relationship between quantitative storage of VLDL-TG and LPL-activity in upper-body subcutaneous and femoral adipose tissue in UBO, LBO, and lean women. As a secondary objective we assessed the impact of fat cell size, body composition, VLDL-turnover, and adipose tissue blood flow on VLDL-TG storage and its relationship with LPL-activity.
The study was approved by the local ethics committee and informed, written content was obtained from all participants.
Twenty-nine healthy, premenopausal women (10 UBO, 11 LBO, and 8 lean) who participated in an ongoing study of VLDL-TG metabolism entered the study. The kinetic data has been published previously(18). The present manuscript describes aspects of adipocyte function. All participants had been recruited through newspaper advertisements. Upper and lower body obesity was defined as a BMI >28 kg/m2 in combination with a waist-to-hip ratio (WHR)>0.85 (UBO) or a WHR<0.8 (LBO). Lean subjects were defined as a BMI<25 kg/m2. All participants were normotensive, non-smokers, used no medication except oral contraceptives, and had a normal blood count and chemistry panel documented before participation. Subjects with a fasting plasma TG > 2.3 mmol/l were not included. All were studied in the luteal phase. If there was uncertainty regarding their status as premenopausal (age>45 years), a blood sample was taken for determination of FSH and estradiol and subjects were excluded if FSH>10 IE/L and estradiol<0.5 μmol/L. Two lean women had to be excluded because of incomplete data collection.
One week before the examination day a fasting 80 ml blood sample was obtained for VLDL-TG labeling as described beneath. Body composition and fat distribution were measured using dual-energy x-ray absorptiometry (DEXA) and abdominal computed tomography scanning. Weight maintaining meals (30% fat, 55% carbohydrate, 15% protein) were designed by a clinical dietician and provided from the hospital kitchen for 3 days before the study to assure consistent nutrient and energy intake. All volunteers maintained their usual level of physical activity and were asked not to participate in heavy exercise the last 3 days before the study. Throughout the examination day, the participants remained in bed wearing light hospital clothing in a room of ambient temperature of 22–24°C.
Participants were admitted to the research laboratory at 22.00 h the day before the study. After an overnight fast two intravenous catheters were inserted - one in a dorsal hand vein and one in an antecubital vein. The hand was placed in a heated box for collection of arterialized blood. The obese volunteers had a solution of 1 MBq 133Xe (0.1 ml) injected subcutaneously in periumbilical and anterior femoral sites and a NaI detector was placed to measure activity washout throughout the day. Unfortunately, after having completed studies in the obese women with only lean subjects remaining, 131Xe was no longer available from the supplier. Thus, we were forced to give up measurements of 131Xe washout in all the remaining lean women.
At 0800 h (time 0) baseline blood samples were obtained and followed by an intravenous bolus infusion of 14C-labelled VLDL-TG over 5 min using a calibrated pump. Blood samples were drawn at times 0, 30, 60, 120, 180, 240, and 300 min, and analyzed for 14C-VLDL-TG SA and concentration, FFA, and insulin. REE was measured between 60–90 min by indirect calorimetry (Deltatrac monitor; Datex Instrumentarium, Helsinki, Finland). From 300–420 min a hyperinsulinemic euglycaemic clamp was performed. Insulin was infused at a rate of 0.6 mU × kg−1 × min−1. Plasma glucose was measured every 10 min using a Beckman analyzer (Beckmann Instruments, Palo Alto, California, USA) and clamped at ~5 mmol/L by infusion of glucose 20%. Glucose infusion rate (M-value) was used as a measure of insulin sensitivity and have been published previously(18).
Four and a half h after the 14C-VLDL-TG infusion adipose tissue biopsies were taken using a liposuction technique under local anesthesia contra lateral to the Xenon injection sites from both the subcutaneous abdominal and the anterior femoral regions. A portion of the sample was quick-frozen in liquid nitrogen and stored at −80° for later measurement of LPL-activity. The remainder was used to measure fat cell size and adipose tissue lipid SA.
After completion of the study all catheters were removed. After stabilization of blood glucose was secured the participants were dismissed.
Labeling of VLDL-TG with [1-14C]triolein (14C at carbon 1 of fatty acids) was performed as previously described(17). In short, a fasting 80 ml venous blood sample was obtained aseptically from the participant. The plasma was transferred to a sterile glass tube containing 40 μCi [1-14C]triolein and sonicated at 37°C for 6 hours. Hereafter, the VLDL fraction was separated from plasma by ultracentrifugation (40.000 rpm in 18h at 10°C, using a sterile 1.006 g × cm−3 saline solution). The ex-vivo 14C-labelled VLDL-TG sample was kept at 5°C until the examination day, where it was re-infused into the participant. Purity of the VLDL-TG solution was ensured by culture before infusion. We have previously shown that this procedure results in VLDL-TG particles that are indistinguishable from endogenously produced VLDL-TG particles(17). Thus, in each individual the total amount of VLDL-TG tracer infused (F) results from on the amount of VLDL-TG obtained for labeling and the efficiency of the labeling process.
Adipose tissue SA was measured after lipid extraction as previously described (10). Fat cell size was determined immediately after the biopsy-procedure. An adipose sample was digested using a HEPES/collagenase solution and the cells were stained with methylene blue to visualize the nuclei according to the method described by Di Girolamo et al.(19). Digital photographs of ~200 cells pr sample were obtained using Olympus BX80 microscope and Olympus DP10 camera. The cell diameter was measured using a software program Analysis28 (Olympus) using a grid at the same magnification as used by the cell digital photographs.
Heparin-releasable LPL-activity was measured in adipose tissue biopsy samples of 20–70 mg by the glycerol-stabilized method described by Nilsson-Ehle and Schotz (20) and expressed as μmol FFA per hour per gram lipid or as μmol FFA per hour pr 109 adipocytes. In short, the tissue was defrosted and incubated in 5 U/ml heparin elution buffer (15% BSA; Phosphate Buffered Saline 1X w/Ca & Mg, Celox Laboratories PBS-1000; Heparin 1000 U/ml; PBS, Sigma H-0777) for 45 min at room temperature. Afterwards the eluent was incubated in 3H-triolein containing substrate (0.5 mCi/ml, NET-431 Perkin Elmer) for 2h at 37°C (duplicate determinations). The reaction was stopped using methanol:chloroform:heptane (34:38:28), and after centrifugation the supernatant was transferred to scintillation vials and counted on scintillation counter (2×2 determination). The inter-assay coefficient of variation as determined from 10 repeated measurements was 11.0%.
Total body fat, leg fat, fat percent, and fat free mass were examined by DEXA (QDR-2000). Upper-body and visceral fat was assessed using the combination of a single-slice computed tomography (CT) scan at the L2–L3 interspace in combination with DEXA abdominal fat measurement (21). Upper body subcutaneous fat was taken as upper body fat (DXA) minus visceral fat. The abdominal scans were technically insufficient in two lean subjects. Leg fat was measured using the region of interest program with the DXA instrument. Subcutaneous blood flow was measured in the obese subjects using the 133Xenon washout method(2). The 133Xenon wash-out slope and a partitioning coefficient between blood, and adipose tissue of 10 ml × g−1 was used for these calculations. Due to technical difficulties the measurement of VLDL-TG production was unsuccessful in one LBO woman and the result of the abdominal CT scan was lost in one UBO woman.
Plasma insulin was measured by a two-site immunospecific insulin ELISA.
All data are presented as mean ± SD or median (range). VLDL-TG production rate was calculated using a biexponential fit to the plasma tracer decay curve as previously described(18). VLDL-TG FA storage was calculated both as the net fraction stored over the 270 min and as a timed storage rate. The fraction of the infused VLDL-TG tracer stored per kg fat over the 270 min was calculated as the regional adipose tissue SA (i.e. dpm per kg lipid) divided by the amount of VLDL-TG tracer (F) infused (dpm). We present the fractional storage in upper- and lower-body adipose tissue as a way to consider how the body partitions the storage of VLDL-TG into fat independent of production rates. The storage rate VLDL-TG FA into adipose tissue (mg × kg fat−1 × hour−1) was calculated as the fractional tracer storage x VLDL production rate using the molar weight of oleate ~282 mg × mmol−1. This approach provides information regarding the rate at which VLDL-TG fatty acids are stored in regional adipose tissue that takes into account possible differences in production rates between obese and non-obese participants. Both fractional tracer storage and VLDL-TG FA storage rate were used as measures of VLDL-TG storage and considered independently in the statistical analysis. For comparison between groups ANOVA was used for normal distributed data and Kruskal-Wallis for non-normal distributed data. Correlations were evaluated by Pearson’s r or Spearman’s rho. Stepwise multivariate linear regressions analysis was used to evaluate potential predictors of adipose tissue VLDL-TG tracer storage. Different models were tested using log transformed adipose tissue storage as dependent variable and combinations of the following independent variables: group-index, LPL-activity, fat cell size, insulin, VLDL-TG production rate, regional or total fat mass, and FFM or REE. Each variable was explored alone and in combination with group index in order to indentify variables most likely to predict the dependent variable. Models included no more than 3 independent variables and were carefully tested to avoid multicolinearity to ensure normal distribution of residuals. To ensure that group index, LPL-activity, and regional fat mass were not prematurely excluded from consideration, they were included separately in a non-stepwise model that also included the variables found to be significant in the stepwise model.
Subject characteristics are given in Table 1. Obese subjects had greater insulin concentrations compared with lean subjects. UBO women had significantly greater VLDL-TG concentrations compared with lean women (p<0.05) but not compared with LBO women.
LPL-activity in both abdominal and in femoral adipose tissue (μmol FFA per hour per g lipid) tended to be greater in the obese subjects compared with lean subjects, although not significantly (Table 2). However, LPL-activity per 109 adipocytes was significantly greater in obese than lean women. There was no significant difference between abdominal and femoral adipose tissue LPL-activity in any of the groups. In the combined group of women, but not in the individual groups, fasting insulin concentrations correlated positively with LPL-activity per 109 adipocytes in abdominal fat (r=0.39, p<0.05) but not in femoral fat.
Fat cell size was greater in obese women than lean women in abdominal and (p=0.06) femoral (p<0.001) subcutaneous fat (Table 2). However, there was no significant difference in fat cell size between the UBO and LBO subjects in either region. In LBO women femoral fat cells were significantly larger than abdominal fat cells. No difference between abdominal and femoral fat cell size was observed in UBO or lean subjects. Fat cell size in abdominal fat correlated significantly with abdominal LPL-activity per 109 adipocytes (r=0.45; p<0.02). Similarly, femoral fat cell size correlated significantly femoral LPL-activity per 109 adipocytes (r=0.54; p<0.01). No significant relationship was found between fat cell size and LPL-activity per gram lipid.
In both the UBO and LBO subjects significantly greater blood flow was found in femoral than in abdominal adipose tissue (Table 2). However, there was no significant difference between the two groups in abdominal or femoral ATBF.
Overall, F was significantly associated with the storage of VLDL-TG tracer per kg lipid in both adipose tissue depots (Figure 1). At time 300 min the residual plasma [1-14C]VLDL-TG SA as percentage of activity at 0 min was not significantly different between the three groups: UBO: 22±18% vs. LBO 15±11% vs. lean 8±7%. The total amount of VLDL-TG FA tracer stored in abdominal and femoral fat in each of the three groups were 5.9, 9.6, and 5.3% in abdominal subcutaneous fat in UBO, LBO and lean women, and 5.6, 5.9, and 2.0% in femoral fat in UBO, LBO, and lean non-obese women, which has been published recently (18). In brief, LBO women deposited a significantly larger amount of VLDL-TG in their femoral fat depots than lean women (both fractional and rate). Abdominal fat storage rate was significantly greater in UBO women than in lean women, whereas fractional storage was significantly greater in both UBO and LBO women compared with lean women(18).
The fraction of VLDL tracer and the rate of VLDL-TG FA storage per kg lipid in abdominal and femoral fat were not significantly different between the three groups (Figure 2 and and3).3). Moreover, VLDL-TG FA and tracer storage per kg lipid were not significantly different between abdominal fat and femoral fat in any of the three groups. Thus, partitioning of VLDL-TG storage into adipose tissue did not differ between upper and lower body fat or between women with different body fat amounts and fat distribution.
There was no significant univariate correlation between fractional tracer storage per kg lipid and LPL-activity per gram lipid, LPL-activity per 109 adipocytes, or fat cell size in any of the three groups in either abdominal or femoral adipose tissue. In addition, no significant correlation was found between VLDL-TG storage per kg lipid and ATBF in the obese groups in abdominal or femoral adipose tissue. In UBO women a significant inverse relationship was found between fractional tracer storage per kg lipid and both abdominal subcutaneous fat mass (r=−0.70, p 0.036) and femoral fat mass (r=−0.70, p=0.024) (Figure 4). Such relationships were not noted in LBO and lean women.
In a multiple linear regression analysis abdominal fractional tracer storage per kg lipid was significantly determined by FFM (inversely) (Table 3). In femoral fat fractional tracer storage per kg lipid was significantly determined by FFM and femoral fat mass. Conversely, VLDL-TG FA storage rate per kg lipid was significantly determined by fasting plasma insulin in both abdominal and femoral fat (Table 4). Group index and LPL-activity did not significantly predict fat storage in any of the tested models.
This is the first study to place quantitative, abdominal and leg adipose tissue VLDL-TG storage in-vivo in the context of recognized, potential regulators of storage. We used a newly developed method whereby autologous ex-vivo labeled 1-14C[VLDL-TG] is used to measure postabsorptive regional VLDL-TG storage (17). We found that there was no significant difference in the fractional tracer storage per kg lipid or VLDL-TG FA storage rate per kg lipid into abdominal vs. femoral adipose tissue in UBO, LBO, and lean women and that the regional storage was similar in the three groups. It follows, therefore, that since storage rates per kg lipid were not different, VLDL-TG storage in the different depots increases as fat mass increases, both as a percent of VLDL-TG disposal and as absolute disposal. In accordance with previous studies, we found greater LPL-activity per fat cell in obese compared with lean women but no difference between abdominal and femoral fat. However we found no indication of a direct relationship between LPL-activity and regional storage of VLDL-TG associated FA.
Early in-vivo and in-vitro animal studies(22, 23, 24, 25) reported a significant correlation between TG storage and LPL-activity and lead authors to denote LPL as the rate limiting enzyme in the storage of VLDL-TG associated FA. Adipose tissue LPL-activity is greater in obese than in lean subjects and increases further after weight loss (26, 27). Some (11, 24), but not all studies (10, 12), have shown good correlation between meal fat storage and adipose tissue LPL-activity. Although the adipose tissue LPL-activity is considered the rate limiting step for VLDL-TG storage there have been no humans studies addressing the relationship between postabsorptive adipose tissue LPL-activity and the quantitative regional storage of VLDL-TG in-vivo. In the present study, we did not find a robust, significant relationship between LPL-activity and VLDL-TG storage in regional fat depots in lean or obese women. The evidence from the multiple regression analysis refutes our hypothesis that LPL-activity would be correlated with VLDL-TG fatty acid storage, which we would have taken as supportive evidence that LPL is a rate limiting step in the deposition of postabsorptive VLDL-TG in adipose tissue. Importantly, the relationship between VLDL-TG FA storage rate and insulin, but not with LPL activity, could not be ascribed to multicolinearity between insulin and LPL activity in the multiple regression analysis. We suggest that adipose tissue LPL may be present in excess of what is needed to regulate VLDL-TG storage and that other steps in the process become rate limiting under postabsorptive conditions.
Insulin is a well known stimulator of both hepatic VLDL-TG production and adipose tissue LPL-activity and therefore it is not unexpected that insulin proved to be significantly related with VLDL-TG FA storage rate. However, to our surprise FFM was an independent, negative predictor of VLDL-TG fractional tracer storage. We recently reported VLDL-TG FAs serve as a significant oxidative substrate (~20% of REE) and a significant relationship between FFM and VLDL-TG turnover(28). In the present study VLDL-TG turnover was significantly and similarly correlated with both FFM and REE (data not shown). We speculate that individuals with more FFM oxidize more VLDL-TG fatty acids, leaving proportionately less circulating VLDL-TG available for deposition in adipose tissue.
Substrate availability is another factor likely to be involved in VLDL-TG storage in fat cells. It is well established that postabsorptive ATBF is reduced in obese as compared with lean individuals, and that a greater ATBF may facilitate VLDL-TG delivery to adipose tissue(2). Romanski et al. found a positive correlation between ATBF and meal fat storage in abdominal fat in lean women but not in lean men or in women in femoral fat(29). In the present study, ATBF (ml/100 ml tissue) was significantly greater in femoral than in abdominal fat in both UBO and LBO women, but storage of VLDL-TG was not different between upper and lower body fat. This argues against ATBF as a rate limiting step for postabsorptive VLDL-TG storage in UBO and LBO women. Unfortunately, we were unable to measure ATBF in lean women.
We observed a significant, inverse relationship between fat mass and VLDL-TG tracer storage per kg lipid in UBO women, both for abdominal and femoral depots. In contrast, a similar relationship was absent in LBO and lean women (Figure 4). Moreover, it has previously been reported that meal fat storage in femoral fat shows a different relationship with fat mass and fat cell size compared with abdominal subcutaneous and visceral fat(12). An inverse relationship between abdominal fat mass and VLDL storage per gram lipid, as noted in the UBO women, is expected if a VLDL-TG tracer is merely distributed in different volumes of body fat. However, the positive relationships between VLDL-TG storage and femoral fat mass, as demonstrated in our multiple regression analysis indicates that lower body fat stores become more efficient in taking up VLDL-TG as fat mass increases. Thus, our results suggest that the postabsorptive deposition of VLDL-TG in women involves different rate limiting steps in the UBO phenotype.
The limitations of this study are the number of participants, and it will be necessary to confirm these results in more studies, preferably in larger study populations. Another limitation is potential loss of LPL activity during freezing-thawing. Previous studies have found preserved(30, 31) and decreased(32) adipose tissue LPL activity after storage to at −70° to −80°C. However, higher elution and assay temperature (37ºC) confounded the ability of the authors(32) to find similar LPL activity in fresh and frozen samples. If LPL was lost during freezing-thawing in our study it could abolish significant relationships between LPL activity and other parameters. However, the level of LPL activity reported in the presents study is comparable to the level reported in a large body of other reports and the expected relationship with fat cell size was also noted. We therefore believe that the LPL activity reported in our study is representative of the heparin-releasable LPL activity present at the time of the biopsy. Moreover, we are unaware of information regarding day-to-day variability in VLDL-TG storage. This could be addressed by repeated studies using different triolein tracers (e.g. 14C [triolein] and 3H[triolein]). However, we believe that the careful selection of our distinct study groups strengthens the results of our study. Finally, we acknowledge that measurement of ATBF would have been helpful as discussed above.
The main findings of this study are that there is no clear evidence of a direct relationship between postabsorptive VLDL-TG FA storage and LPL-activity and that VLDL-TG FA storage is more closely determined by factors related to regional fat mass, fat cell size, FFM, and plasma insulin concentrations. Moreover, UBO women show a different relationship between VLDL-TG storage and regional fat mass compared with LBO and lean women. In conclusion: we found no evidence that variations in LPL-activity determine rates of VLDL-TG storage in a direct quantitative manner. The differences in VLDL-TG storage that occur with progressive obesity in women in different obesity phenotypes may be mediated through mechanisms related to FFM, insulin, and lower body fat mass.
We wish to thank Ms. Lene Ring, Ms. Lone Svensen, and Ms. Susanne Sørensen for excellent technical assistance. This work was supported by NIH grant DK45343 (to M.D.J.) and grants from the Danish Medical Research Council, the Novo Nordic Foundation, and the Danish Diabetes Foundation (to S.N.). This data have previously been presented in abstract form at the annual meeting of the American Diabetes Association annual meeting, Chicago 2007.
ClinicalTrials.gov id: NCT00646698
The authors have no conflicts of interests