A Multicompartmental Model of In Vivo Adipose Tissue Glycerol Kinetics and Capillary Permeability in Lean and Obese Humans
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糖尿病学杂志 2005年第7期
1 Diabetes & Metabolic Medicine, St. Bartholomew’s and The London School of Medicine, London, U.K
2 Shriners’s Hospital for Children, University of Texas Medical Branch, Galveston, Texas
3 Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota
4 Clinical Nutrition Research Unit, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
Lipolysis of adipose tissue triglycerides releases glycerol. Twenty-four volunteers, of whom 6 were obese and 13 were women, received a primed-constant infusion of 2H5-glycerol for 120 min during postabsorptive steady-state conditions. Arterial, abdominal venous, and interstitial (microdialysis) samples were taken, and a four-compartment model was applied to assess subcutaneous abdominal adipose tissue glycerol kinetics. Adipose tissue blood flow was measured using 133Xe washout. Venous glycerol concentrations (median 230 eol/l [interquartile range 210eC268]) were consistently greater than those of arterial blood (69.1 eol/l [56.5eC85.5]), while glycerol isotopic enrichments (tracer-to-tracee ratio) were greater in arterial blood (8.34% [7.44eC10.1]) than venous blood (2.34% [1.71eC2.69], P < 0.01). Microdialysate glycerol enrichment was 1.44% (1.11eC1.79), indicating incomplete permeability of glycerol between capillary blood and interstitium. Calculated interstitial glycerol concentrations were between 270 eol/l (256eC350) and 332 eol/l (281eC371) (examining different boundary conditions). The calculated capillary diffusion capacity (ps) was between 2.21 ml · 100 g tissueeC1 · mineC1 (1.31eC3.13) and 3.09 ml · 100 g tissueeC1 · mineC1 (1.52eC4.90) and correlated inversely with adiposity (Rs eC0.45, P < 0.05). Our results support previous estimates of interstitial glycerol concentration within adipose tissue and reveal capillary diffusion capacity is reduced in obesity.
Triglycerides in adipose tissue represent the body’s major source of endogenous fuel and are mobilized when energy requirements exceed exogenous energy supply, such as during exercise (1) and fasting (2). However, excessive body fat, particularly increased abdominal fat mass, is associated with increased rates of systemic lipolysis (3) and excessive release of free fatty acids into the circulation, contributing to insulin resistance, diabetes, and dyslipidemia (3,4). Therefore, dysregulation of lipolysis has important physiological and clinical implications.
The breakdown of endogenous fat involves the conversion of triglyceride to fatty acids and glycerol; complete hydrolysis of 1 mol triglyceride releases 3 mol fatty acids and 1 mol glycerol. Therefore, the rate of release of glycerol can be used to assess the lipolytic rate. Several methods have been used to measure glycerol kinetics in vivo in human subjects. Whole-body adipose tissue glycerol kinetics can be studied with intravenously infused isotopically labeled glycerol (5eC7). However, interpretation of results from this technique is complicated by the contribution of glycerol derived from lipolysis of circulating triglyceride-rich lipoproteins (such as chylomicrons) by lipoprotein lipase (LPL). Regional abdominal adipose tissue glycerol kinetics can be studied by two methods that rely on arteriovenous balance principles (8). One method involves placing a small catheter in a superficial abdominal vein to determine glycerol concentration in venous effluent from subcutaneous abdominal adipose tissue (7,9eC12). The other more widely used approach places thin microdialysis tubes into subcutaneous adipose tissue, allowing the estimation of glycerol concentration in interstitial adipose tissue fluid (10eC19). Correct interpretation of microdialysis results depends upon knowledge of the permeability of the capillary endothelium to glycerol diffusion, which is needed to convert interstitial glycerol concentrations to venous concentrations (15,18,19). Changes in capillary permeability regulate glycerol release, but this issue has previously been little considered, perhaps in part because there has been no viable method of measuring it in vivo. Capillary permeability is determined by capillary diffusion capacity, which is measured as the product of permeability and surface area (ps) (20eC22). Recently, Gudbjrnsde畉tir et al. (22) published the first in vivo data for the ps for glucose and insulin in human muscle capillaries, using a combination of arteriovenous difference microdialysis and mathematical modeling methods.
The combined use of glycerol tracers, abdominal vein catheterization, and microdialysis probes, in conjunction with a mathematical modeling approach (23), provides a novel potential approach for evaluating adipose tissue glycerol kinetics in vivo in human subjects and for estimating the ps for glycerol from in vivo data. This article reports the first application of this approach.
RESEARCH DESIGN AND METHODS
Twenty-four subjects participated in this study (Table 1). All subjects were weight stable for at least 2 months before the study and were considered to be in good health after completing a comprehensive medical evaluation, including history and physical examination, blood tests, and electrocardiogram. Six subjects were obese (BMI >30 kg/m2). In 15 subjects (12 female), body composition was measured by using dual-energy X-ray absorptiometry (Lunar Instruments, Madison, WI) within 2 weeks before the study. None of the subjects were taking regular medication, and premenstrual female subjects were studied during the follicular phase of their cycle. All obese subjects had normal glucose tolerance confirmed by a standard oral glucose tolerance test. The study was approved by the Human Studies Committee and the Clinical Research Center Scientific Advisory Board, and all subjects gave informed written consent. Other aspects of these studies have been previously reported (24).
Subjects were admitted to the Clinical Research Center in the afternoon before the study. At 1800, subjects ingested a meal containing 12 kcal/kg body wt for lean subjects and 12 kcal/kg adjusted body wt for obese subjects (adjusted body weight = ideal body weight [IBW] + [actual body weight eC IBW] x 0.25). At 2000, subjects ingested a defined liquid formula snack containing 250 kcal, 40 g carbohydrate, 6.1 g fat, and 8.8 g protein (Ensure; Ross Laboratories, Columbus, OH). After this snack, all subjects fasted until completion of the study the following day.
The following morning, 20-gauge catheters were inserted into a forearm vein for isotope infusion and into a radial artery for arterial blood sampling. A superficial abdominal vein was cannulated with a 10- to 20-cm, 22-gauge polyurethane catheter (Hydrocath; Viggo-Spectramed, Oxnard, CA) (7,9,12,24). Blood obtained from this site represents effluent from adipose tissue and overlying skin. All vascular catheters were kept patent by continuous saline infusion.
Three or four microdialysis probes (CMA, Acton, MA) were placed percutaneously without anesthesia into subcutaneous abdominal adipose tissue. Each probe consisted of dialysis tubing (10 x 0.5 mm, 20,000 MW cutoff) and was perfused overnight with Ringer’s lactate solution (0.3 e蘬/min) before insertion to ensure the elimination of any glycerol from the catheter itself. Perfusion of the probe permits the equilibration of adipose tissue interstitial glycerol with perfusate, which can be collected. All probes were placed within 10 cm of the midline and were at least 3 cm apart from each other. Each was continuously perfused with lactated Ringer’s solution at a rate of 0.1 e蘬/min by using a syringe infusion pump (Harvard Apparatus, South Natick, MA). Both venous and microdialysis catheters and the 133Xe depot were positioned so as to sample the more superficial subcutaneous tissue as described by Enevoldsen et al. (25).
Subjects remained supine throughout the study, and room temperature was kept constant at 23°C during the entire study. Baseline arterial and abdominal venous blood samples were obtained 60 min after catheters were placed. Adipose tissue interstitial fluid samples were collected 60 min after probe insertion. The first 60-min fraction of the dialysate effluent was discarded to eliminate the influence of the initial trauma caused by probe insertion on glycerol measurements. Previous studies (11,12) have shown a transient rise in interstitial fluid ATP, an index of tissue damage, during the first 15 min after probe insertion. After baseline, samples were collected and a primed (3.6 eol/kg)-continuous (0.24 eol · kgeC1 · mineC1) infusion of 1,1,2,3,3-[2H5]glycerol (Tracer Technologies, Somerville, MA) was started and maintained for 120 min using a syringe infusion pump (Harvard Apparatus, South Natick, MA). Arterial and abdominal venous blood samples were taken at 45, 60, 75, 90, 105, 110, 115, and 120 min. Dialysate from the microdialysis probes was collected for 60 min, between 60 and 120 min of isotope infusion, and the fluid from all probes were pooled to ensure recovery of adequate amounts of glycerol to permit accurate measurement of isotopic enrichment.
Abdominal subcutaneous adipose tissue blood flow (ATBF) was evaluated using the 133Xe washout technique (7,11,12,19,25). Immediately after starting the isotope infusion, 120eC150 mCi of 133Xe dissolved in 0.1 ml saline was injected over 60 s into the subcutaneous abdominal adipose tissue space. The decline in 133Xe activity was monitored continuously from 60 to 120 min after injection with a sodium iodide scintillation detector (Canberra Industries, Meriden, CT) placed 40 cm from the 133Xe depot and coupled with a multichannel analyzer (ND 600; Schaumburg, IL) set to measure the 81 keV 133Xe photopeak.
Analyses.
Arterial and venous plasma triglyceride concentrations were measured enzymatically (26). Arterial and venous blood concentrations of glycerol were determined enzymatically with an automated analyzer (Technicon, Tarrytown, NY). Isotopic enrichment (tracer-to-tracee ratio [TTR]) of glycerol in plasma and in microdialysate fluid was determined by gas chromatographyeCmass spectrometry by using an MSD 5971 system (Hewlett-Packard, Palo Alto, CA) with a 12 m x 0.2eCmm HP-1 fused silica capillary column (Hewlett-Packard). Plasma was deproteinized with barium hydroxide and zinc sulfate and then cetrifuged to isolate the protein-free supernatant. The microdialysate samples and the supernatant of the plasma samples were passed through stacked cation (Dowex AG-50W-X8) and anion (Dowex AG-1-X8) exchange columns. A trimethylsilyl derivative of glycerol was formed and injected into the gas chromatographyeCmass spectrometer. Ions, produced by electron impact ionization, were selectively monitored at mass-to-charge ratios 205.1, 206.1, and 208.1.
Calculations.
ATBF was calculated from 133Xe clearance as previously described (7,19,26), assuming an adipose tissueeCtoeCblood partition coefficient for xenon of 10 ml/100 g for all subjects (17eC19,27). Whole-body glycerol rate of appearance, Ra(systemic) (eol · kgeC1 · mineC1), in blood was calculated by using Steele’s equation for steady-state conditions modified for use with stable isotopes (28,29): Ra(systemic) = I/TTRart, where I is the isotope infusion rate in eol · kgeC1 · mineC1 and TTRart is the TTR of glycerol in arterial plasma at isotopic equilibrium.
Regional subcutaneous abdominal adipose tissue net glycerol release rate [Ra(local)], in ng · l00 g adipose tissueeC1 · mineC1, was calculated by using standard principles of arteriovenous balance and blood glycerol concentrations (29): Ra(local) = ATBF x (Glyvein eC Glyart), where ATBF is the rate of subcutaneous ATBF in ml · 100 g adipose tissueeC1 · mineC1 and Glyart and Glyvein are arterial and venous blood glycerol concentrations in eol/l.
Ra(local) (eol · 100 g tissueeC1 · mineC1) was also calculated by using standard arteriovenous balance methodology in conjunction with isotope tracer enrichment data (29): Ra(local) = ATBF x Glyart x ([TTRart/TTRvein] eC 1).
Model of adipose tissue glycerol kinetics.
We adapted the approach of Biolo et al. (23) to produce a four-compartment model for adipose tissue glycerol metabolism (Fig. 1) that contains several components. First, glycerol enters adipose tissue interstitial fluid after it is released by hormone-sensitive lipaseeCmediated lipolysis of adipocyte triglyceride. Interstitial glycerol can enter the venous blood compartment (FVI) by a process that depends on the permeability of capillary endothelium. We assumed that permeability for glycerol efflux is the same as that of glycerol influx in adipose tissue. Second, arterial delivery of glycerol to adipose tissue [F(in)] is determined by the product of Glyart and blood flow. Glycerol present in the arteriolar compartment can pass directly into the vein by functional shunting (FVA) or into adipose tissue interstitium (FIA). Third, glycerol released into adipose tissue venous blood by LPL action on circulating triglycerides (FLPL) was considered to originate on the intraluminal side of the endothelial barrier. This glycerol can enter the interstitial compartment (FIL) or be delivered to the systemic circulation by directly entering veins that drain adipose tissue (FVL).
Our model is not able to determine the exact proportion of LPL-derived glycerol directed to the vein or adipose tissue interstitium. However, we can examine two boundary conditions: 1) when all LPL-derived glycerol enters the interstitium and none enters the vein directly (boundary condition P) and 2) when all LPL-derived glycerol enters the vein directly and none enters the interstitium (boundary condition Q). For any individual subject, the true value of each model parameter estimate lies between the two boundary conditions.
Boundary condition P.
By definition, FVL = 0 and FIL = F(LPL). The relationship between arterial, abdominal venous, and interstitial glycerol is calculated as (20,21) F(out) = FVI + FVA;
The fluxes F(out), FVI, and FVA between compartments are indicated in Fig. 1. TTRart, TTRvein, and TTRinterstitial are glycerol TTR in arterial blood, venous blood, and interstitial fluid, respectively. This equation can be rearranged to (TTRart eC TTRinterstitial) x FVI = (TTRart eC TTRvein)F(out);
Boundary condition Q.
By definition, FVL = 0 and FIL = F(LPL). The relationships between arterial, abdominal venous, and interstitial glycerol are calculated as FVA + FVI + FLPL = F(out);
Therefore,
F(out) is calculated as the product of glycerol concentration in the vein, and ATBF and FLPL are the product of plasma flow and the arteriovenous difference in triglyceride concentration. Summers et al. (12) considered that glycerol released by LPL would not enter the interstitium in significant amounts, i.e., boundary condition Q is physiologically more plausible.
For both boundary conditions, we used the parameters from our model in conjunction with single-pass unidirectional extraction (E) principles (21) to estimate the capillary diffusion capacity (ps), which is a measure of the capillary surface area and the permeability of the capillaries to glycerol. The single-pass unidirectional extraction, which is the amount of glycerol arriving in the arterial blood that is transported into the tissue, is estimated by the model E = FIA/F(in), where FIA = F(in) eC FVA.
However, E is also fundamentally related to capillary diffusion capacity (21): 1 eC E = e(eCps/ATBF), where ps is the capillary diffusion capacity expressed in ml · 100 g tissueeC1 · mineC1. These equations can be combined to the following: ps = eCATBF 1ogn (l eC E).
Having calculated ps and E allows determination of interstitial glycerol concentration Glyinterstitial, because Glyinterstitial = (Glyvein eC Glyart x [1 eC E])/E;
Data from previous studies allowed us to expect additional constraints on the model. First, the data from previous studies (7,19) demonstrated no metabolism of labeled glycerol by adipose tissue. Therefore, the model does not need to include uptake of plasma-free glycerol into adipose tissue. Second, we assumed that the incorporation of labeled glycerol into triglyceride-rich lipoproteins was negligible during the short duration of the tracer infusion study. In similar studies, we found that isotopic enrichment of glycerol within triglyceride-rich lipoprotein was <3% plasma glycerol enrichment within this time of infusion (B.W.P., S.K., unpublished observations).
Statistics.
The glycerol concentration and enrichment data were normally distributed. To assess for steady-state conditions, we undertook an ANOVA of the plasma concentrations and enrichments, seeking an effect of time. Some model parameters were not normally distributed; therefore, nonparametric statistics were used when possible. Data are expressed as median (interquartile range), and Wilcoxon’s and Spearman’s (Rs) tests were used to analyze the data. A P value of 0.05 was considered to be statistically significant.
The model parameters for glycerol kinetics are expressed as the median of the parameters from the model run on the 24 subjects’ observational data (i.e., median of the models), which is shown in Tables 2 and 3. However, as an example of what individual data looked like, Figs. 2A and B show the parameter values obtained by entering the median observational data (concentrations and specific enrichments) from 24 subjects into the model (i.e., the model of the median data).
RESULTS
Glycerol concentrations and TTR.
The measured glycerol TTR in artery, abdominal vein, and abdominal interstitial fluid and concentration in artery and abdominal vein are shown in Fig. 3. The time course for circulating glycerol concentration and TTR demonstrate the presence of physiologic and isotopic steady-state conditions; ANOVA showed no significant changes with time. Arterial blood glycerol concentration (median [interquartile range] was 69.1 eol/l [56.5eC85.5], significantly lower than abdominal venous blood glycerol (230 eol/l [210eC268], P < 0.01). The model-derived values (using both boundary conditions) of adipose tissue interstitial glycerol concentration (Table 2) were greater than measured abdominal venous blood glycerol concentration (P < 0.001 for both).
Glycerol TTR was consistently greater in arterial plasma (8.34% [7.44eC10.1]) than abdominal venous plasma (2.34% [1.71eC2.69]) (P < 0.001), which was consistently greater than glycerol TTR in abdominal interstitial fluid (1.44% [1.11eC1.79]) (P < 0.001 for all comparisons), indicating flux of glycerol from adipocytes to interstitial fluid to the vascular compartment (Fig. 3). There was no significant uptake of glycerol tracer across adipose tissue; labeled blood glycerol content was 2.4% (eC3.7 to 5.0) lower in abdominal venous than arterial samples, not statistically significantly different from zero.
Triglyceride concentration.
Triglyceride concentrations were consistently lower in abdominal venous plasma (731 eol/l [647eC1,133]) than arterial plasma (784 eol/l [678eC1,188], P < 0.001).
ATBF.
Median abdominal subcutaneous ATBF was 3.07 ml · 100 g tissueeC1 · mineC1 (2.13eC4.06). ATBF correlated with ps (for both boundary conditions (Rs 0.55, P < 0.01) (Fig. 4), and blood flow was lower in obese than lean subjects (e.g., Rs with BMI = eC0.625, P < 0.001).
Glycerol release rates.
Systemic glycerol Ra was 2.11 eol · kgeC1 · mineC1 (1.71eC2.31). Local adipose tissue glycerol release rate calculated by arteriovenous balance was 483 nmol · 100 g tissueeC1 · mineC1 (339eC630). Local glycerol release calculated from isotope balance was not significantly different. Glycerol release by LPL action contributed 18.3% (15.0eC21.2) of the total local glycerol release. The proportion of local glycerol release that came from LPL action decreased with BMI and percentage IBW (Rs > 0.46, P < 0.05 for both).
Compartmental model parameters.
The results of the compartmental modeling of boundary conditions P and Q are shown in Table 2. Figs. 2A and B give an example of the parameter estimates from a single dataset. Table 3 shows parameters for lean (BMI <25 kg/m2) and obese (BMI >30 kg/m2) subjects.
Boundary condition P.
The proportion of arterial glycerol that entered the interstitial space, Fia/Fin, was 60.1% (47.8eC81.0) in boundary condition P. This proportion also represents the single-pass unidirectional extraction value. The boundary condition P values for capillary diffusion capacity (ps) and interstitial glycerol concentration are shown in Table 2. Neither ps nor interstitial glycerol concentration was significantly different between male and female subjects. Age was related to ps (Rs = 0.43, P < 0.05) but not to interstitial glycerol concentration. There was an inverse relationship between ps value and adiposity (correlation ps with BMI Rs = eC0.49, ps with percentage IBW Rs = eC0.45, ps with percent body fat Rs = eC0.54, P < 0.05 for all). There were positive correlations between interstitial glycerol concentration and adiposity (correlation with BMI Rs = 0.50, with percentage IBW Rs = 0.52, with percent body fat Rs = 0.52, P < 0.05 for all).
Boundary condition Q.
In boundary condition Q, by definition, all glycerol released by LPL action entered the venous blood without traversing the interstitial space. Each of the glycerol model parameters, FIA, FVA, and FVI, calculated for boundary condition Q were different from the parameters calculated for boundary condition P in all subjects (P < 0.001 for all model parameters).
The proportion of arterial glycerol that entered the interstitial space, Fia/Fin, was 52.8% (40.8eC65.3), which was consistently less than that seen in boundary condition P (P < 0.001). This proportion also represents the single-pass unidirectional extraction value.
The calculated values for capillary diffusion capacity (ps) and interstitial glycerol concentration in boundary condition Q are shown in Table 2. Neither ps nor interstitial glycerol concentration was significantly different between male and female subjects. Permeability surface area (ps) was related to age (Rs = 0.42, P < 0.05), but interstitial glycerol concentration was not. There was significance between ps value and adiposity (correlation with BMI Rs = eC0.58, with percentage IBW Rs = eC0.49, with percent body fat Rs = eC0.68, P < 0.05 for all). There were positive correlations between interstitial glycerol concentration and adiposity (correlation with BMI Rs = 0.41, with percentage IBW Rs = 0.41, with percent body fat Rs = 0.52, P < 0.05 for all).
DISCUSSION
In the present study, we adopted a novel approach to study postabsorptive regional abdominal subcutaneous adipose tissue glycerol kinetics in human subjects. Our data demonstrate that glycerol released from subcutaneous adipocytes travels along a concentration gradient from adipose tissue interstitial space to local veins before entering the systemic circulation. Of the glycerol appearing in the venous drainage from adipose tissue (median 732 nmol · 100 g tissueeC1 · mineC1), 52% (383 nmol · 100 g tissueeC1 · mineC1) was derived from lipolysis of adipocyte triglycerides and 14% (99 nmol · 100 g tissueeC1 · mineC1) from lipolysis of circulating plasma triglycerides. Between 8 and 11% (58eC82 nmol · 100 g tissueeC1 · mineC1, for boundary condition P and Q, respectively) of venous glycerol had been functionally shunted directly from arterial to venous blood without uptake into the interstitium. The remainder of glycerol that appeared in adipose tissue venous drainage was comprised of arterial glycerol that passed into the interstitium before reappearing in the local venous circulation.
Our approach also allowed calculation of the ps index of the capillary perfusion capacity for glycerol in subcutaneous abdominal human adipose tissue. Unfortunately, ps is difficult to measure, and other groups have suggested values ranging from 3.0 to 5.0 ml · 100 g tissueeC1 · mineC1 (11,17eC19,30), based in vitro methods (21,31eC34). Our calculated values for ps were based on measurements made in vivo and were generally lower than ps values assumed in studies (11,15,17eC19,30). In our study, ps values declined with increasing adiposity, consistent with the histological changes in adipose tissue observed in obese subjects (35,36). Our data suggest that studies of regional lipolytic activity that depend on interstitial glycerol concentration measurements should assume different ps values in lean and obese subjects. It is not known whether different adipose tissue beds have different capillary diffusion capacities, although data from a recent study (22) have shown that ps in human muscle changes in response to physiological stimuli. In our study, variation in ps was significantly related to obesity and age rather than sex, but further work would be necessary to examine whether other factors (e.g., insulin resistance or abnormal LPL activity) are more closely linked to variation in ps. In our study, ps was also related to ATBF, which itself is known to be reduced in obesity (17,18,27). It is likely that both ATBF and ps are reduced by structural alterations that occur in adipose tissue with increased adiposity (i.e., increased distance between capillary and adipocyte, and decreased capillary density); however, the association with blood flow does make it difficult to resolve the independent effects on ps of obesity and of low blood flow.
The ps for glycerol is probably related to the ps for other substances within the same tissue, because glycerol appears to diffuse in and out of tissues without any active transport processes. The methods we have used would allow future exploration of the factors that change the capillary diffusion capacity of human adipose tissue. The consequence of assuming an erroneous value for ps on local glycerol release rate is underscored in Table 3. Assuming a ps value of 5, rather than using the values calculated from our observations, caused an artifactual increase in local glycerol release rate but did not change the comparison between lean and obese subjects.
The validity of our calculations of ps depends upon the limitations and assumptions of our methods. These assumptions include the following. 1) There was no metabolism of glycerol by adipose tissue, 2) a true steady state was achieved in all sampling sites, 3) microdialysate samples were in equilibrium with interstitial glycerol with no isotope effect, 4) samples obtained by microdialysis were representative of adipose tissue interstitial fluid that was drained by the abdominal vein cannula, and 5) LPL-induced clearance of plasma triglycerides produces glycerol in equimolar amounts with little generation of mono- or diglycerides (37). We also assumed that there was no significant glycerol tracer recycling from adipocyte or plasma triglycerides. The possibility of tracer recycling is unlikely because the adipocyte triglyceride pool is very large, and any labeled glycerol that found its way there would be infinitely diluted. Moreover, there was little incorporation of labeled glycerol into plasma triglyceride during the course of this study.
In the present study, there was no significant glycerol uptake by adipose tissue, which is consistent with previous studies (7,19) involving labeled glycerol, and the absence of significant glycerol kinase activity in adipose tissue (38). In contrast, Lee et al. (39) reported acylation of glycerol in several tissues and Kurpad et al. (40) reported significant glycerol-isotope uptake by subcutaneous adipose tissue. However, the Kurpad et al. study may not have achieved steady-state conditions, glycerol balance being assessed after only 60 min of labeled glycerol infusion. In the present study, labeled glycerol was infused for 120 min, and the time-course data (Fig. 3 and ANOVA) for plasma glycerol concentration and TTR suggest that steady-state conditions were achieved. Although the other assumptions cannot be specifically tested, they have been accepted in previous studies.
Many results seen in this study were similar to previous values. Thus, whole-body glycerol Ra was similar to previous reports (5eC7), as were arterial and abdominal venous blood glycerol concentrations (7,11,12,27). The values of calculated interstitial glycerol concentration in the current study are similar to those reported previously and, as expected, show a direct correlation with increasing adiposity (17eC18). However, our calculated interstitial glycerol concentrations were higher than measured abdominal venous blood glycerol. In contrast, in a previous study (12) that measured interstitial glycerol concentration directly by microdialysis sampling, the value for adipose tissue interstitial glycerol concentration was lower than venous plasma glycerol concentration. The reason for this discrepancy between studies may be related to the technical difficulty in accurately measuring adipose tissue interstitial glycerol concentration directly, which have at times (e.g., Maggs et al. [41]) yielded results markedly discrepant with other studies (13) and the values we report. Early studies (14eC17) that measured adipose tissue interstitial glycerol concentration by using microdialysis involved a system that had poor glycerol recovery and required time-consuming calibrations. Our calculated values for adipose tissue interstitial glycerol concentration did not require probe calibration or an estimate of probe glycerol recovery. Current probes have >90% glycerol recovery when the perfusion rate is very slow (42).
The action of LPL on intravascular circulating triglycerides can release glycerol into the circulation. This study and previous studies (7,12,27,43) show significant clearance of circulating triglyceride by adipose tissue. In this study, the proportion of local glycerol release from LPL action declined significantly with obesity in accordance with previous comparisons of lean and obese subjects (27,44).
Some LPL-generated glycerol could diffuse into the interstitium before later release into venous plasma, while another fraction of LPL-generated glycerol would go to the venous blood without traversing the interstitium. Because we did not know the fraction of the LPL glycerol following these two paths, we considered the boundary conditions where 100% of the LPL-generated glycerol goes into the vein directly and none goes into the interstitium (boundary condition Q) or the converse (boundary condition P). The true situation lies between these two extremes, but one can draw inferences from the similarities of the two boundary conditions. LPL action on triglyceride-rich lipoproteins occurs within the capillary lumen (44) so it is plausible that most LPL-generated glycerol is "swept away" directly into the venous blood rather than accessing the interstitial fluid glycerol pool, as argued by Summers et al. (12). However, some LPL-generated glycerol might be taken up into the interstitial space without mixing with venous blood. Unstirred water layers exist at the boundary of most cell surfaces (45) and such local conditions could theoretically allow LPL-generated glycerol to cross the capillary barrier and enter the interstitial space.
The findings of the present study may not represent glycerol kinetics in other fat depots. In vitro studies of isolated adipocytes demonstrate regional differences in lipolytic activity and hormonal regulation of lipolysis. For example, abdominal adipose tissue is more lipolytic, more sensitive to -adrenergic stimulation, and more resistant to insulin-mediated antilipolysis than gluteal or femoral fat depots (46eC48). Such heterogeneity of adipose tissue emphasizes that results from a single depot such as the one studied here should be extrapolated to whole-body fat metabolism with caution.
Our results also confirm some limitations of using systemic glycerol Ra as a measure of whole-body adipose tissue lipolysis. We found that glycerol released during lipolysis of plasma triglycerides could have accounted for 15eC20% of total glycerol added to the bloodstream as it passes through abdominal adipose tissue. In addition, systemic glycerol Ra could miss 10eC20% of whole-body adipose tissue lipolysis because of the inability to detect lipolysis of intraperitoneal fat, when glycerol released into the portal vein is cleared by the liver (49). Therefore, measuring systemic glycerol Ra by infusing a labeled glycerol tracer probably provides a reasonable estimate of whole-body adipose tissue lipolytic activity under postabsorptive conditions that simultaneously overestimates (glycerol released by lipolysis of plasma triglycerides) and underestimates (glycerol released by lipolysis of intraperitoneal triglycerides) selected areas of regional adipose tissue glycerol release.
In summary, the data from this study show that glycerol released during lipolysis of adipose tissue triglycerides moves along a concentration gradient from interstitial space, to local veins, and into the systemic circulation. The calculated capillary diffusion capacity (ps) for glycerol increased with increasing adiposity and was lower than values previously proposed by assessments made in vitro.
ACKNOWLEDGMENTS
This study was supported by National Institutes of Health Grants DK37948, DK56341 (Clinical Nutrition Research Unit), RR00036 (General Clinical Research Center), and RR00594 (Biomedical Mass Spectrometry Resource), The Special Trustees of St. Bartholomew’s and The London School of Medicine, and The Wellcome Trust.
We thank the staff of the Clinical Research Centre for help with the studies and the volunteer subjects for participation.
ATBF, adipose tissue blood flow; IBW, ideal body weight; LPL, lipoprotein lipase; TTR, tracer-to-tracee ratio
REFERENCES
Klein S, Coyle EF, Wolfe RR: Fat metabolism during low-intensity exercise in endurance-trained and untrained men. Am J Physiol267 :E934 eCE940,1994
Klein S, Sakurai Y, Romijn JA, Carroll RM: Progressive alterations in lipid and glucose metabolism during short-term fasting in young adult men. Am J Physiol265 :E801 eCE806,1993
Jensen MD: Regulation of forearm lipolysis in different types of obesity: in vivo evidence for adipocyte heterogeneity. J Clin Invest87 :187 eC193,1991
Krotkiewski M, Bjrntorp P, Sjstrm L, Smith U: Impact of obesity on metabolism in men and women. J Clin Invest72 :1150 eC1162,1983
Klein S, Young VR, Blackburn GL, Bistrian BR, Wolfe RR: Palmitate and glycerol kinetics during brief starvation in normal weight young adult and elderly subjects. J Clin Invest78 :928 eC933,1986
Judd RL, Nelson R, Klein S, Jensen MD, Miles JM: Measurement of plasma glycerol specific activity by high performance liquid chromatography to determine glycerol flux. J Lipid Res39 :1106 eC1110,1988
Coppack SW, Persson M, Judd RL, Miles JM: Glycerol and non-esterified fatty acid metabolism in human muscle and adipose tissue in vivo. Am J Physiol276 :E233 eCE240,1999
Zierler KL: Theory of the use of arteriovenous concentration differences for measuring metabolism in steady and non-steady states. J Clin Invest40 :2111 eC2115,1961
Frayn KN, Coppack SW, Humphreys SM, Whyte PL: Metabolic characteristics of human adipose tissue in vivo. Clin Sci76 :509 eC516,1989
Arner P, Beow J: Assessment of adipose tissue metabolism in man: comparison of Fick and microdialysis techniques. Clin Sci85 :247 eC256,1993
Simonsen L, Beow J, Madsen J: Adipose tissue metabolism in humans determined by vein catheterization and microdialysis techniques. Am J Physiol266 :E357 eCE365,1994
Summers LKM, Arner P, Ilic V, Clark ML, Humphreys SM, Frayn KN: Adipose tissue metabolism in the postprandial period: microdialysis and arteriovenous techniques compared. Am J Physiol274 :E651 eCE655,1998
Samra JS, Ravell CL, Giles SL, Arner P, Frayn KN: Interstitial glycerol concentration in human skeletal muscle and adipose tissue is close to the concentration in blood. Clin Sci90 :453 eC456,1996
Arner P, Bolinder J, Eliasson A, Lundin A, Ungerstedt U: Microdialysis of adipose tissue and blood for in vivo lipolysis studies. Am J Physiol255 :E737 eCE742,1988
Wahrenberg H, Lonnqvist F, Arner P: Mechanisms underlying regional differences in lipolysis in human adipose tissue. J Clin Invest84 :458 eC467,1989
Jansson P-AE, Smith U, Lnnroth P: Interstitial glycerol concentration measured by microdialysis in two subcutaneous regions in humans. Am J Physiol258 :E918 eCE922,1990
Jansson P-AE, Larsson A, Smith U, Lnnroth P: Glycerol production in subcutaneous adipose tissue in lean and obese humans. J Clin Invest89 :1610 eC1617,1992
Bolinder J, Kerckhoffs DAJM, Moberg E, Hagstrm-Toft E, Arner P: Rates of skeletal muscle and adipose tissue glycerol release in nonobese and obese subjects. Diabetes49 :797 eC892,2000
Qvisth V, Hagstrm-Toft E, Enoksson S, Sherwin RS, Sjberg S, Bolinder J: Combined hyperinsulinemia and hyperglycemia, but not hyperinsulinemia alone, suppresses human skeletal muscle lipolytic activity in vivo. J Clin Endocrinol Metab89 :4693 eC4700,2004
Intaglietta M, Johnson PC: Principles of capillary exchange. In Peripheral Circulation. Johnson PC, Ed. New York, Wiley,1978 , p.141 eC166
Lassen NA, Perl WA: Multiple indicators: capillary permeability. In Tracer Kinetic Methods in Mammalian Physiology. Lassen NA, Perl WA, Eds. New York, Raven Press,1979 , p.156 eC175
Gudbjrnsde畉tir S, Sjstrand M, Strindberg L, Wahren J, Lnnroth P: Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle. J Clin Endocrinol Metab88 :4559 eC4564,2003
Biolo G, Chinkes DL, Zhang X-J, Wolfe RR: A new model to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle. J Parent Ent Nutr16 :305 eC315,1992
Patterson BW, Horowitz JF, Wu G, Watford M, Coppack SW, Klein S: Regional muscle and adipose tissue amino acid metabolism in lean and obese humans. Am J Physiol Endocrinol Metab282 :E931 eCE936,2002
Enevoldsen LH, Simonsen L, Stallknecht B, Galbo H, Beow J: In vivo human lipolytic activity in preperitoneal and subdivisions of subcutaneous abdominal adipose tissue. Am J Physiol Endocrinol Metab281 :E1110 eCE1114,2001
Larsen OA, Lassen NA, Quaade F: Blood flow through human adipose tissue determined with radioactive xenon. Acta Physiol Scand66 :337 eC345,1966
Coppack SW, Evans RD, Fisher RM, Frayn KN, Gibbons GF, Humphreys SM, Kirk MJ, Potts JL, Hockaday TDR: Adipose tissue metabolism in obesity: lipase action in vivo before and after a mixed meal. Metabolism41 :264 eC272,1992
Rosenblatt J, Wolfe RR: Calculation of substrate flux using stable isotopes. Am J Physiol254 :E526 eCE531,1988
Wolfe RR: Radioactive and Stable Isotope Tracers in Biomedicine:Principles and Practice of Kinetic Analysis. Wiley-Liss, New York,1992
Mulla NA, Simonsen L, Beow J: Post-exercise adipose tissue and skeletal muscle lipid metabolism in humans: the effect of exercise intensity. J Physiol Lond254 :919 eC928,2000
Lassen NA: Capillary diffusion capacity of sodium studied by the clearances of Na-24 and Xe-133 from hyperemic skeletal muscle in man. Scand J Clin Lab Invest Suppl99 :24 eC26,1967
Linde B, Chisholm G, Rosell S: The influence of sympathetic activity and histamine on blood-tissue exchange of solutes in canine adipose tissue. Acta Physiol Scand92 :145 eC155,1974
Paaske WP: Absence of restricted diffusion in adipose tissue capillaries. Acta Physiol Scand100 :430 eC436,1977
Paaske WP, Sejrsen P: Permeability of continuous capillaries. Dan Med Bull36 :570 eC590,1989
Cinti S: Adipose tissues and obesity. Ital J Anat Embryol104 :37 eC51,1999
Cinti S: The adipose organ: morphological perspectives of adipose tissue. Proc Nutr Soc60 :319 eC328,2001
Fielding BA, Humphreys SM, Allman RF, Frayn KN: Mono-, di- and triacylglycerol concentrations in human plasma: effects of heparin injection and of a high-fat meal. Clin Chim Acta216 :167 eC173,1993
Lin ECC: Glycerol utilization and its regulation in mammals. Annu Rev Biochem46 :765 eC795,1977
Lee DP, Deonarine AS, Kienetz M, Zhu Q, Skrzypczak M, Chan M, Choy PC: A novel pathway for lipid biosynthesis: the direct acylation of glycerol. J Lipid Res42 :1979 eC1986,2001
Kurpad AV, Khan K, Calder G, Coppack SW, Macdonald IA, Elia M: Effect of noradrenaline on glycerol turnover and lipolysis in the whole body and subcutaneous adipose tissue in humans in vivo. Clin Sci86 :177 eC184,1994
Maggs DG, Jacob R, Rife F, Lange R, Leone P, During MJ, Tamborlane WV, Sherwin RS: Interstitial fluid concentration of glycerol, glucose and amino acids in human quadricep muscle and adipose tissue. J Clin Invest96 :370 eC377,1995
Jansson P-AE, Veneman T, Nurjhan N, Gerich JE: An improved method to calculate the adipose tissue interstitial substrate recovery for microdialysis studies. Life Sci54 :1621 eC1624,1994
Fisher RM, Miles JM, Kottke BA, Coppack SW: Very-low-density lipoprotein subfraction composition and metabolism by adipose tissue. Metabolism46 :605 eC610,1997
Eckel RH: Lipoprotein lipase: a multifunctional enzyme relevant to common metabolic diseases. N Engl J Med320 :1060 eC1068,1989
Verkman AS: Water permeability measurement in living cells and complex tissues. J Membr Biol15 :73 eC87,2000
Hoffstedt J, Arner P, Hellers G, Lonnqvist F: Variation in adrenergic regulation of lipolysis between omental and subcutaneous adipocytes from obese and non-obese men. J Lipid Res38 :795 eC804,1997
Lafontan M, Barbe P, Galitzky J, Tavernier G, Langin D, Carpene C, Bousquet-Melou A, Berlan M: Adrenergic regulation of adipocyte metabolism. Human Reprod1 (Suppl. 12) :6 eC20,1997
Tan GC, Goossens GH, Humphreys SM, Vidal H, Karpe F: Upper and lower body adipose tissue function:a direct comparison of fat mobilization in humans. Obes Res12 :114 eC118,2004
Havel RJ, Kane JP, Balasse EO, Segel N, Basso LV: Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest49 :2017 eC2035,1970
Metropolitan Life Assurance Company: Net weight standard for men and women. Stat Bull Metrol Life Found40 :1 eC4,1959(Simon W. Coppack, David L)
2 Shriners’s Hospital for Children, University of Texas Medical Branch, Galveston, Texas
3 Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota
4 Clinical Nutrition Research Unit, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
Lipolysis of adipose tissue triglycerides releases glycerol. Twenty-four volunteers, of whom 6 were obese and 13 were women, received a primed-constant infusion of 2H5-glycerol for 120 min during postabsorptive steady-state conditions. Arterial, abdominal venous, and interstitial (microdialysis) samples were taken, and a four-compartment model was applied to assess subcutaneous abdominal adipose tissue glycerol kinetics. Adipose tissue blood flow was measured using 133Xe washout. Venous glycerol concentrations (median 230 eol/l [interquartile range 210eC268]) were consistently greater than those of arterial blood (69.1 eol/l [56.5eC85.5]), while glycerol isotopic enrichments (tracer-to-tracee ratio) were greater in arterial blood (8.34% [7.44eC10.1]) than venous blood (2.34% [1.71eC2.69], P < 0.01). Microdialysate glycerol enrichment was 1.44% (1.11eC1.79), indicating incomplete permeability of glycerol between capillary blood and interstitium. Calculated interstitial glycerol concentrations were between 270 eol/l (256eC350) and 332 eol/l (281eC371) (examining different boundary conditions). The calculated capillary diffusion capacity (ps) was between 2.21 ml · 100 g tissueeC1 · mineC1 (1.31eC3.13) and 3.09 ml · 100 g tissueeC1 · mineC1 (1.52eC4.90) and correlated inversely with adiposity (Rs eC0.45, P < 0.05). Our results support previous estimates of interstitial glycerol concentration within adipose tissue and reveal capillary diffusion capacity is reduced in obesity.
Triglycerides in adipose tissue represent the body’s major source of endogenous fuel and are mobilized when energy requirements exceed exogenous energy supply, such as during exercise (1) and fasting (2). However, excessive body fat, particularly increased abdominal fat mass, is associated with increased rates of systemic lipolysis (3) and excessive release of free fatty acids into the circulation, contributing to insulin resistance, diabetes, and dyslipidemia (3,4). Therefore, dysregulation of lipolysis has important physiological and clinical implications.
The breakdown of endogenous fat involves the conversion of triglyceride to fatty acids and glycerol; complete hydrolysis of 1 mol triglyceride releases 3 mol fatty acids and 1 mol glycerol. Therefore, the rate of release of glycerol can be used to assess the lipolytic rate. Several methods have been used to measure glycerol kinetics in vivo in human subjects. Whole-body adipose tissue glycerol kinetics can be studied with intravenously infused isotopically labeled glycerol (5eC7). However, interpretation of results from this technique is complicated by the contribution of glycerol derived from lipolysis of circulating triglyceride-rich lipoproteins (such as chylomicrons) by lipoprotein lipase (LPL). Regional abdominal adipose tissue glycerol kinetics can be studied by two methods that rely on arteriovenous balance principles (8). One method involves placing a small catheter in a superficial abdominal vein to determine glycerol concentration in venous effluent from subcutaneous abdominal adipose tissue (7,9eC12). The other more widely used approach places thin microdialysis tubes into subcutaneous adipose tissue, allowing the estimation of glycerol concentration in interstitial adipose tissue fluid (10eC19). Correct interpretation of microdialysis results depends upon knowledge of the permeability of the capillary endothelium to glycerol diffusion, which is needed to convert interstitial glycerol concentrations to venous concentrations (15,18,19). Changes in capillary permeability regulate glycerol release, but this issue has previously been little considered, perhaps in part because there has been no viable method of measuring it in vivo. Capillary permeability is determined by capillary diffusion capacity, which is measured as the product of permeability and surface area (ps) (20eC22). Recently, Gudbjrnsde畉tir et al. (22) published the first in vivo data for the ps for glucose and insulin in human muscle capillaries, using a combination of arteriovenous difference microdialysis and mathematical modeling methods.
The combined use of glycerol tracers, abdominal vein catheterization, and microdialysis probes, in conjunction with a mathematical modeling approach (23), provides a novel potential approach for evaluating adipose tissue glycerol kinetics in vivo in human subjects and for estimating the ps for glycerol from in vivo data. This article reports the first application of this approach.
RESEARCH DESIGN AND METHODS
Twenty-four subjects participated in this study (Table 1). All subjects were weight stable for at least 2 months before the study and were considered to be in good health after completing a comprehensive medical evaluation, including history and physical examination, blood tests, and electrocardiogram. Six subjects were obese (BMI >30 kg/m2). In 15 subjects (12 female), body composition was measured by using dual-energy X-ray absorptiometry (Lunar Instruments, Madison, WI) within 2 weeks before the study. None of the subjects were taking regular medication, and premenstrual female subjects were studied during the follicular phase of their cycle. All obese subjects had normal glucose tolerance confirmed by a standard oral glucose tolerance test. The study was approved by the Human Studies Committee and the Clinical Research Center Scientific Advisory Board, and all subjects gave informed written consent. Other aspects of these studies have been previously reported (24).
Subjects were admitted to the Clinical Research Center in the afternoon before the study. At 1800, subjects ingested a meal containing 12 kcal/kg body wt for lean subjects and 12 kcal/kg adjusted body wt for obese subjects (adjusted body weight = ideal body weight [IBW] + [actual body weight eC IBW] x 0.25). At 2000, subjects ingested a defined liquid formula snack containing 250 kcal, 40 g carbohydrate, 6.1 g fat, and 8.8 g protein (Ensure; Ross Laboratories, Columbus, OH). After this snack, all subjects fasted until completion of the study the following day.
The following morning, 20-gauge catheters were inserted into a forearm vein for isotope infusion and into a radial artery for arterial blood sampling. A superficial abdominal vein was cannulated with a 10- to 20-cm, 22-gauge polyurethane catheter (Hydrocath; Viggo-Spectramed, Oxnard, CA) (7,9,12,24). Blood obtained from this site represents effluent from adipose tissue and overlying skin. All vascular catheters were kept patent by continuous saline infusion.
Three or four microdialysis probes (CMA, Acton, MA) were placed percutaneously without anesthesia into subcutaneous abdominal adipose tissue. Each probe consisted of dialysis tubing (10 x 0.5 mm, 20,000 MW cutoff) and was perfused overnight with Ringer’s lactate solution (0.3 e蘬/min) before insertion to ensure the elimination of any glycerol from the catheter itself. Perfusion of the probe permits the equilibration of adipose tissue interstitial glycerol with perfusate, which can be collected. All probes were placed within 10 cm of the midline and were at least 3 cm apart from each other. Each was continuously perfused with lactated Ringer’s solution at a rate of 0.1 e蘬/min by using a syringe infusion pump (Harvard Apparatus, South Natick, MA). Both venous and microdialysis catheters and the 133Xe depot were positioned so as to sample the more superficial subcutaneous tissue as described by Enevoldsen et al. (25).
Subjects remained supine throughout the study, and room temperature was kept constant at 23°C during the entire study. Baseline arterial and abdominal venous blood samples were obtained 60 min after catheters were placed. Adipose tissue interstitial fluid samples were collected 60 min after probe insertion. The first 60-min fraction of the dialysate effluent was discarded to eliminate the influence of the initial trauma caused by probe insertion on glycerol measurements. Previous studies (11,12) have shown a transient rise in interstitial fluid ATP, an index of tissue damage, during the first 15 min after probe insertion. After baseline, samples were collected and a primed (3.6 eol/kg)-continuous (0.24 eol · kgeC1 · mineC1) infusion of 1,1,2,3,3-[2H5]glycerol (Tracer Technologies, Somerville, MA) was started and maintained for 120 min using a syringe infusion pump (Harvard Apparatus, South Natick, MA). Arterial and abdominal venous blood samples were taken at 45, 60, 75, 90, 105, 110, 115, and 120 min. Dialysate from the microdialysis probes was collected for 60 min, between 60 and 120 min of isotope infusion, and the fluid from all probes were pooled to ensure recovery of adequate amounts of glycerol to permit accurate measurement of isotopic enrichment.
Abdominal subcutaneous adipose tissue blood flow (ATBF) was evaluated using the 133Xe washout technique (7,11,12,19,25). Immediately after starting the isotope infusion, 120eC150 mCi of 133Xe dissolved in 0.1 ml saline was injected over 60 s into the subcutaneous abdominal adipose tissue space. The decline in 133Xe activity was monitored continuously from 60 to 120 min after injection with a sodium iodide scintillation detector (Canberra Industries, Meriden, CT) placed 40 cm from the 133Xe depot and coupled with a multichannel analyzer (ND 600; Schaumburg, IL) set to measure the 81 keV 133Xe photopeak.
Analyses.
Arterial and venous plasma triglyceride concentrations were measured enzymatically (26). Arterial and venous blood concentrations of glycerol were determined enzymatically with an automated analyzer (Technicon, Tarrytown, NY). Isotopic enrichment (tracer-to-tracee ratio [TTR]) of glycerol in plasma and in microdialysate fluid was determined by gas chromatographyeCmass spectrometry by using an MSD 5971 system (Hewlett-Packard, Palo Alto, CA) with a 12 m x 0.2eCmm HP-1 fused silica capillary column (Hewlett-Packard). Plasma was deproteinized with barium hydroxide and zinc sulfate and then cetrifuged to isolate the protein-free supernatant. The microdialysate samples and the supernatant of the plasma samples were passed through stacked cation (Dowex AG-50W-X8) and anion (Dowex AG-1-X8) exchange columns. A trimethylsilyl derivative of glycerol was formed and injected into the gas chromatographyeCmass spectrometer. Ions, produced by electron impact ionization, were selectively monitored at mass-to-charge ratios 205.1, 206.1, and 208.1.
Calculations.
ATBF was calculated from 133Xe clearance as previously described (7,19,26), assuming an adipose tissueeCtoeCblood partition coefficient for xenon of 10 ml/100 g for all subjects (17eC19,27). Whole-body glycerol rate of appearance, Ra(systemic) (eol · kgeC1 · mineC1), in blood was calculated by using Steele’s equation for steady-state conditions modified for use with stable isotopes (28,29): Ra(systemic) = I/TTRart, where I is the isotope infusion rate in eol · kgeC1 · mineC1 and TTRart is the TTR of glycerol in arterial plasma at isotopic equilibrium.
Regional subcutaneous abdominal adipose tissue net glycerol release rate [Ra(local)], in ng · l00 g adipose tissueeC1 · mineC1, was calculated by using standard principles of arteriovenous balance and blood glycerol concentrations (29): Ra(local) = ATBF x (Glyvein eC Glyart), where ATBF is the rate of subcutaneous ATBF in ml · 100 g adipose tissueeC1 · mineC1 and Glyart and Glyvein are arterial and venous blood glycerol concentrations in eol/l.
Ra(local) (eol · 100 g tissueeC1 · mineC1) was also calculated by using standard arteriovenous balance methodology in conjunction with isotope tracer enrichment data (29): Ra(local) = ATBF x Glyart x ([TTRart/TTRvein] eC 1).
Model of adipose tissue glycerol kinetics.
We adapted the approach of Biolo et al. (23) to produce a four-compartment model for adipose tissue glycerol metabolism (Fig. 1) that contains several components. First, glycerol enters adipose tissue interstitial fluid after it is released by hormone-sensitive lipaseeCmediated lipolysis of adipocyte triglyceride. Interstitial glycerol can enter the venous blood compartment (FVI) by a process that depends on the permeability of capillary endothelium. We assumed that permeability for glycerol efflux is the same as that of glycerol influx in adipose tissue. Second, arterial delivery of glycerol to adipose tissue [F(in)] is determined by the product of Glyart and blood flow. Glycerol present in the arteriolar compartment can pass directly into the vein by functional shunting (FVA) or into adipose tissue interstitium (FIA). Third, glycerol released into adipose tissue venous blood by LPL action on circulating triglycerides (FLPL) was considered to originate on the intraluminal side of the endothelial barrier. This glycerol can enter the interstitial compartment (FIL) or be delivered to the systemic circulation by directly entering veins that drain adipose tissue (FVL).
Our model is not able to determine the exact proportion of LPL-derived glycerol directed to the vein or adipose tissue interstitium. However, we can examine two boundary conditions: 1) when all LPL-derived glycerol enters the interstitium and none enters the vein directly (boundary condition P) and 2) when all LPL-derived glycerol enters the vein directly and none enters the interstitium (boundary condition Q). For any individual subject, the true value of each model parameter estimate lies between the two boundary conditions.
Boundary condition P.
By definition, FVL = 0 and FIL = F(LPL). The relationship between arterial, abdominal venous, and interstitial glycerol is calculated as (20,21) F(out) = FVI + FVA;
The fluxes F(out), FVI, and FVA between compartments are indicated in Fig. 1. TTRart, TTRvein, and TTRinterstitial are glycerol TTR in arterial blood, venous blood, and interstitial fluid, respectively. This equation can be rearranged to (TTRart eC TTRinterstitial) x FVI = (TTRart eC TTRvein)F(out);
Boundary condition Q.
By definition, FVL = 0 and FIL = F(LPL). The relationships between arterial, abdominal venous, and interstitial glycerol are calculated as FVA + FVI + FLPL = F(out);
Therefore,
F(out) is calculated as the product of glycerol concentration in the vein, and ATBF and FLPL are the product of plasma flow and the arteriovenous difference in triglyceride concentration. Summers et al. (12) considered that glycerol released by LPL would not enter the interstitium in significant amounts, i.e., boundary condition Q is physiologically more plausible.
For both boundary conditions, we used the parameters from our model in conjunction with single-pass unidirectional extraction (E) principles (21) to estimate the capillary diffusion capacity (ps), which is a measure of the capillary surface area and the permeability of the capillaries to glycerol. The single-pass unidirectional extraction, which is the amount of glycerol arriving in the arterial blood that is transported into the tissue, is estimated by the model E = FIA/F(in), where FIA = F(in) eC FVA.
However, E is also fundamentally related to capillary diffusion capacity (21): 1 eC E = e(eCps/ATBF), where ps is the capillary diffusion capacity expressed in ml · 100 g tissueeC1 · mineC1. These equations can be combined to the following: ps = eCATBF 1ogn (l eC E).
Having calculated ps and E allows determination of interstitial glycerol concentration Glyinterstitial, because Glyinterstitial = (Glyvein eC Glyart x [1 eC E])/E;
Data from previous studies allowed us to expect additional constraints on the model. First, the data from previous studies (7,19) demonstrated no metabolism of labeled glycerol by adipose tissue. Therefore, the model does not need to include uptake of plasma-free glycerol into adipose tissue. Second, we assumed that the incorporation of labeled glycerol into triglyceride-rich lipoproteins was negligible during the short duration of the tracer infusion study. In similar studies, we found that isotopic enrichment of glycerol within triglyceride-rich lipoprotein was <3% plasma glycerol enrichment within this time of infusion (B.W.P., S.K., unpublished observations).
Statistics.
The glycerol concentration and enrichment data were normally distributed. To assess for steady-state conditions, we undertook an ANOVA of the plasma concentrations and enrichments, seeking an effect of time. Some model parameters were not normally distributed; therefore, nonparametric statistics were used when possible. Data are expressed as median (interquartile range), and Wilcoxon’s and Spearman’s (Rs) tests were used to analyze the data. A P value of 0.05 was considered to be statistically significant.
The model parameters for glycerol kinetics are expressed as the median of the parameters from the model run on the 24 subjects’ observational data (i.e., median of the models), which is shown in Tables 2 and 3. However, as an example of what individual data looked like, Figs. 2A and B show the parameter values obtained by entering the median observational data (concentrations and specific enrichments) from 24 subjects into the model (i.e., the model of the median data).
RESULTS
Glycerol concentrations and TTR.
The measured glycerol TTR in artery, abdominal vein, and abdominal interstitial fluid and concentration in artery and abdominal vein are shown in Fig. 3. The time course for circulating glycerol concentration and TTR demonstrate the presence of physiologic and isotopic steady-state conditions; ANOVA showed no significant changes with time. Arterial blood glycerol concentration (median [interquartile range] was 69.1 eol/l [56.5eC85.5], significantly lower than abdominal venous blood glycerol (230 eol/l [210eC268], P < 0.01). The model-derived values (using both boundary conditions) of adipose tissue interstitial glycerol concentration (Table 2) were greater than measured abdominal venous blood glycerol concentration (P < 0.001 for both).
Glycerol TTR was consistently greater in arterial plasma (8.34% [7.44eC10.1]) than abdominal venous plasma (2.34% [1.71eC2.69]) (P < 0.001), which was consistently greater than glycerol TTR in abdominal interstitial fluid (1.44% [1.11eC1.79]) (P < 0.001 for all comparisons), indicating flux of glycerol from adipocytes to interstitial fluid to the vascular compartment (Fig. 3). There was no significant uptake of glycerol tracer across adipose tissue; labeled blood glycerol content was 2.4% (eC3.7 to 5.0) lower in abdominal venous than arterial samples, not statistically significantly different from zero.
Triglyceride concentration.
Triglyceride concentrations were consistently lower in abdominal venous plasma (731 eol/l [647eC1,133]) than arterial plasma (784 eol/l [678eC1,188], P < 0.001).
ATBF.
Median abdominal subcutaneous ATBF was 3.07 ml · 100 g tissueeC1 · mineC1 (2.13eC4.06). ATBF correlated with ps (for both boundary conditions (Rs 0.55, P < 0.01) (Fig. 4), and blood flow was lower in obese than lean subjects (e.g., Rs with BMI = eC0.625, P < 0.001).
Glycerol release rates.
Systemic glycerol Ra was 2.11 eol · kgeC1 · mineC1 (1.71eC2.31). Local adipose tissue glycerol release rate calculated by arteriovenous balance was 483 nmol · 100 g tissueeC1 · mineC1 (339eC630). Local glycerol release calculated from isotope balance was not significantly different. Glycerol release by LPL action contributed 18.3% (15.0eC21.2) of the total local glycerol release. The proportion of local glycerol release that came from LPL action decreased with BMI and percentage IBW (Rs > 0.46, P < 0.05 for both).
Compartmental model parameters.
The results of the compartmental modeling of boundary conditions P and Q are shown in Table 2. Figs. 2A and B give an example of the parameter estimates from a single dataset. Table 3 shows parameters for lean (BMI <25 kg/m2) and obese (BMI >30 kg/m2) subjects.
Boundary condition P.
The proportion of arterial glycerol that entered the interstitial space, Fia/Fin, was 60.1% (47.8eC81.0) in boundary condition P. This proportion also represents the single-pass unidirectional extraction value. The boundary condition P values for capillary diffusion capacity (ps) and interstitial glycerol concentration are shown in Table 2. Neither ps nor interstitial glycerol concentration was significantly different between male and female subjects. Age was related to ps (Rs = 0.43, P < 0.05) but not to interstitial glycerol concentration. There was an inverse relationship between ps value and adiposity (correlation ps with BMI Rs = eC0.49, ps with percentage IBW Rs = eC0.45, ps with percent body fat Rs = eC0.54, P < 0.05 for all). There were positive correlations between interstitial glycerol concentration and adiposity (correlation with BMI Rs = 0.50, with percentage IBW Rs = 0.52, with percent body fat Rs = 0.52, P < 0.05 for all).
Boundary condition Q.
In boundary condition Q, by definition, all glycerol released by LPL action entered the venous blood without traversing the interstitial space. Each of the glycerol model parameters, FIA, FVA, and FVI, calculated for boundary condition Q were different from the parameters calculated for boundary condition P in all subjects (P < 0.001 for all model parameters).
The proportion of arterial glycerol that entered the interstitial space, Fia/Fin, was 52.8% (40.8eC65.3), which was consistently less than that seen in boundary condition P (P < 0.001). This proportion also represents the single-pass unidirectional extraction value.
The calculated values for capillary diffusion capacity (ps) and interstitial glycerol concentration in boundary condition Q are shown in Table 2. Neither ps nor interstitial glycerol concentration was significantly different between male and female subjects. Permeability surface area (ps) was related to age (Rs = 0.42, P < 0.05), but interstitial glycerol concentration was not. There was significance between ps value and adiposity (correlation with BMI Rs = eC0.58, with percentage IBW Rs = eC0.49, with percent body fat Rs = eC0.68, P < 0.05 for all). There were positive correlations between interstitial glycerol concentration and adiposity (correlation with BMI Rs = 0.41, with percentage IBW Rs = 0.41, with percent body fat Rs = 0.52, P < 0.05 for all).
DISCUSSION
In the present study, we adopted a novel approach to study postabsorptive regional abdominal subcutaneous adipose tissue glycerol kinetics in human subjects. Our data demonstrate that glycerol released from subcutaneous adipocytes travels along a concentration gradient from adipose tissue interstitial space to local veins before entering the systemic circulation. Of the glycerol appearing in the venous drainage from adipose tissue (median 732 nmol · 100 g tissueeC1 · mineC1), 52% (383 nmol · 100 g tissueeC1 · mineC1) was derived from lipolysis of adipocyte triglycerides and 14% (99 nmol · 100 g tissueeC1 · mineC1) from lipolysis of circulating plasma triglycerides. Between 8 and 11% (58eC82 nmol · 100 g tissueeC1 · mineC1, for boundary condition P and Q, respectively) of venous glycerol had been functionally shunted directly from arterial to venous blood without uptake into the interstitium. The remainder of glycerol that appeared in adipose tissue venous drainage was comprised of arterial glycerol that passed into the interstitium before reappearing in the local venous circulation.
Our approach also allowed calculation of the ps index of the capillary perfusion capacity for glycerol in subcutaneous abdominal human adipose tissue. Unfortunately, ps is difficult to measure, and other groups have suggested values ranging from 3.0 to 5.0 ml · 100 g tissueeC1 · mineC1 (11,17eC19,30), based in vitro methods (21,31eC34). Our calculated values for ps were based on measurements made in vivo and were generally lower than ps values assumed in studies (11,15,17eC19,30). In our study, ps values declined with increasing adiposity, consistent with the histological changes in adipose tissue observed in obese subjects (35,36). Our data suggest that studies of regional lipolytic activity that depend on interstitial glycerol concentration measurements should assume different ps values in lean and obese subjects. It is not known whether different adipose tissue beds have different capillary diffusion capacities, although data from a recent study (22) have shown that ps in human muscle changes in response to physiological stimuli. In our study, variation in ps was significantly related to obesity and age rather than sex, but further work would be necessary to examine whether other factors (e.g., insulin resistance or abnormal LPL activity) are more closely linked to variation in ps. In our study, ps was also related to ATBF, which itself is known to be reduced in obesity (17,18,27). It is likely that both ATBF and ps are reduced by structural alterations that occur in adipose tissue with increased adiposity (i.e., increased distance between capillary and adipocyte, and decreased capillary density); however, the association with blood flow does make it difficult to resolve the independent effects on ps of obesity and of low blood flow.
The ps for glycerol is probably related to the ps for other substances within the same tissue, because glycerol appears to diffuse in and out of tissues without any active transport processes. The methods we have used would allow future exploration of the factors that change the capillary diffusion capacity of human adipose tissue. The consequence of assuming an erroneous value for ps on local glycerol release rate is underscored in Table 3. Assuming a ps value of 5, rather than using the values calculated from our observations, caused an artifactual increase in local glycerol release rate but did not change the comparison between lean and obese subjects.
The validity of our calculations of ps depends upon the limitations and assumptions of our methods. These assumptions include the following. 1) There was no metabolism of glycerol by adipose tissue, 2) a true steady state was achieved in all sampling sites, 3) microdialysate samples were in equilibrium with interstitial glycerol with no isotope effect, 4) samples obtained by microdialysis were representative of adipose tissue interstitial fluid that was drained by the abdominal vein cannula, and 5) LPL-induced clearance of plasma triglycerides produces glycerol in equimolar amounts with little generation of mono- or diglycerides (37). We also assumed that there was no significant glycerol tracer recycling from adipocyte or plasma triglycerides. The possibility of tracer recycling is unlikely because the adipocyte triglyceride pool is very large, and any labeled glycerol that found its way there would be infinitely diluted. Moreover, there was little incorporation of labeled glycerol into plasma triglyceride during the course of this study.
In the present study, there was no significant glycerol uptake by adipose tissue, which is consistent with previous studies (7,19) involving labeled glycerol, and the absence of significant glycerol kinase activity in adipose tissue (38). In contrast, Lee et al. (39) reported acylation of glycerol in several tissues and Kurpad et al. (40) reported significant glycerol-isotope uptake by subcutaneous adipose tissue. However, the Kurpad et al. study may not have achieved steady-state conditions, glycerol balance being assessed after only 60 min of labeled glycerol infusion. In the present study, labeled glycerol was infused for 120 min, and the time-course data (Fig. 3 and ANOVA) for plasma glycerol concentration and TTR suggest that steady-state conditions were achieved. Although the other assumptions cannot be specifically tested, they have been accepted in previous studies.
Many results seen in this study were similar to previous values. Thus, whole-body glycerol Ra was similar to previous reports (5eC7), as were arterial and abdominal venous blood glycerol concentrations (7,11,12,27). The values of calculated interstitial glycerol concentration in the current study are similar to those reported previously and, as expected, show a direct correlation with increasing adiposity (17eC18). However, our calculated interstitial glycerol concentrations were higher than measured abdominal venous blood glycerol. In contrast, in a previous study (12) that measured interstitial glycerol concentration directly by microdialysis sampling, the value for adipose tissue interstitial glycerol concentration was lower than venous plasma glycerol concentration. The reason for this discrepancy between studies may be related to the technical difficulty in accurately measuring adipose tissue interstitial glycerol concentration directly, which have at times (e.g., Maggs et al. [41]) yielded results markedly discrepant with other studies (13) and the values we report. Early studies (14eC17) that measured adipose tissue interstitial glycerol concentration by using microdialysis involved a system that had poor glycerol recovery and required time-consuming calibrations. Our calculated values for adipose tissue interstitial glycerol concentration did not require probe calibration or an estimate of probe glycerol recovery. Current probes have >90% glycerol recovery when the perfusion rate is very slow (42).
The action of LPL on intravascular circulating triglycerides can release glycerol into the circulation. This study and previous studies (7,12,27,43) show significant clearance of circulating triglyceride by adipose tissue. In this study, the proportion of local glycerol release from LPL action declined significantly with obesity in accordance with previous comparisons of lean and obese subjects (27,44).
Some LPL-generated glycerol could diffuse into the interstitium before later release into venous plasma, while another fraction of LPL-generated glycerol would go to the venous blood without traversing the interstitium. Because we did not know the fraction of the LPL glycerol following these two paths, we considered the boundary conditions where 100% of the LPL-generated glycerol goes into the vein directly and none goes into the interstitium (boundary condition Q) or the converse (boundary condition P). The true situation lies between these two extremes, but one can draw inferences from the similarities of the two boundary conditions. LPL action on triglyceride-rich lipoproteins occurs within the capillary lumen (44) so it is plausible that most LPL-generated glycerol is "swept away" directly into the venous blood rather than accessing the interstitial fluid glycerol pool, as argued by Summers et al. (12). However, some LPL-generated glycerol might be taken up into the interstitial space without mixing with venous blood. Unstirred water layers exist at the boundary of most cell surfaces (45) and such local conditions could theoretically allow LPL-generated glycerol to cross the capillary barrier and enter the interstitial space.
The findings of the present study may not represent glycerol kinetics in other fat depots. In vitro studies of isolated adipocytes demonstrate regional differences in lipolytic activity and hormonal regulation of lipolysis. For example, abdominal adipose tissue is more lipolytic, more sensitive to -adrenergic stimulation, and more resistant to insulin-mediated antilipolysis than gluteal or femoral fat depots (46eC48). Such heterogeneity of adipose tissue emphasizes that results from a single depot such as the one studied here should be extrapolated to whole-body fat metabolism with caution.
Our results also confirm some limitations of using systemic glycerol Ra as a measure of whole-body adipose tissue lipolysis. We found that glycerol released during lipolysis of plasma triglycerides could have accounted for 15eC20% of total glycerol added to the bloodstream as it passes through abdominal adipose tissue. In addition, systemic glycerol Ra could miss 10eC20% of whole-body adipose tissue lipolysis because of the inability to detect lipolysis of intraperitoneal fat, when glycerol released into the portal vein is cleared by the liver (49). Therefore, measuring systemic glycerol Ra by infusing a labeled glycerol tracer probably provides a reasonable estimate of whole-body adipose tissue lipolytic activity under postabsorptive conditions that simultaneously overestimates (glycerol released by lipolysis of plasma triglycerides) and underestimates (glycerol released by lipolysis of intraperitoneal triglycerides) selected areas of regional adipose tissue glycerol release.
In summary, the data from this study show that glycerol released during lipolysis of adipose tissue triglycerides moves along a concentration gradient from interstitial space, to local veins, and into the systemic circulation. The calculated capillary diffusion capacity (ps) for glycerol increased with increasing adiposity and was lower than values previously proposed by assessments made in vitro.
ACKNOWLEDGMENTS
This study was supported by National Institutes of Health Grants DK37948, DK56341 (Clinical Nutrition Research Unit), RR00036 (General Clinical Research Center), and RR00594 (Biomedical Mass Spectrometry Resource), The Special Trustees of St. Bartholomew’s and The London School of Medicine, and The Wellcome Trust.
We thank the staff of the Clinical Research Centre for help with the studies and the volunteer subjects for participation.
ATBF, adipose tissue blood flow; IBW, ideal body weight; LPL, lipoprotein lipase; TTR, tracer-to-tracee ratio
REFERENCES
Klein S, Coyle EF, Wolfe RR: Fat metabolism during low-intensity exercise in endurance-trained and untrained men. Am J Physiol267 :E934 eCE940,1994
Klein S, Sakurai Y, Romijn JA, Carroll RM: Progressive alterations in lipid and glucose metabolism during short-term fasting in young adult men. Am J Physiol265 :E801 eCE806,1993
Jensen MD: Regulation of forearm lipolysis in different types of obesity: in vivo evidence for adipocyte heterogeneity. J Clin Invest87 :187 eC193,1991
Krotkiewski M, Bjrntorp P, Sjstrm L, Smith U: Impact of obesity on metabolism in men and women. J Clin Invest72 :1150 eC1162,1983
Klein S, Young VR, Blackburn GL, Bistrian BR, Wolfe RR: Palmitate and glycerol kinetics during brief starvation in normal weight young adult and elderly subjects. J Clin Invest78 :928 eC933,1986
Judd RL, Nelson R, Klein S, Jensen MD, Miles JM: Measurement of plasma glycerol specific activity by high performance liquid chromatography to determine glycerol flux. J Lipid Res39 :1106 eC1110,1988
Coppack SW, Persson M, Judd RL, Miles JM: Glycerol and non-esterified fatty acid metabolism in human muscle and adipose tissue in vivo. Am J Physiol276 :E233 eCE240,1999
Zierler KL: Theory of the use of arteriovenous concentration differences for measuring metabolism in steady and non-steady states. J Clin Invest40 :2111 eC2115,1961
Frayn KN, Coppack SW, Humphreys SM, Whyte PL: Metabolic characteristics of human adipose tissue in vivo. Clin Sci76 :509 eC516,1989
Arner P, Beow J: Assessment of adipose tissue metabolism in man: comparison of Fick and microdialysis techniques. Clin Sci85 :247 eC256,1993
Simonsen L, Beow J, Madsen J: Adipose tissue metabolism in humans determined by vein catheterization and microdialysis techniques. Am J Physiol266 :E357 eCE365,1994
Summers LKM, Arner P, Ilic V, Clark ML, Humphreys SM, Frayn KN: Adipose tissue metabolism in the postprandial period: microdialysis and arteriovenous techniques compared. Am J Physiol274 :E651 eCE655,1998
Samra JS, Ravell CL, Giles SL, Arner P, Frayn KN: Interstitial glycerol concentration in human skeletal muscle and adipose tissue is close to the concentration in blood. Clin Sci90 :453 eC456,1996
Arner P, Bolinder J, Eliasson A, Lundin A, Ungerstedt U: Microdialysis of adipose tissue and blood for in vivo lipolysis studies. Am J Physiol255 :E737 eCE742,1988
Wahrenberg H, Lonnqvist F, Arner P: Mechanisms underlying regional differences in lipolysis in human adipose tissue. J Clin Invest84 :458 eC467,1989
Jansson P-AE, Smith U, Lnnroth P: Interstitial glycerol concentration measured by microdialysis in two subcutaneous regions in humans. Am J Physiol258 :E918 eCE922,1990
Jansson P-AE, Larsson A, Smith U, Lnnroth P: Glycerol production in subcutaneous adipose tissue in lean and obese humans. J Clin Invest89 :1610 eC1617,1992
Bolinder J, Kerckhoffs DAJM, Moberg E, Hagstrm-Toft E, Arner P: Rates of skeletal muscle and adipose tissue glycerol release in nonobese and obese subjects. Diabetes49 :797 eC892,2000
Qvisth V, Hagstrm-Toft E, Enoksson S, Sherwin RS, Sjberg S, Bolinder J: Combined hyperinsulinemia and hyperglycemia, but not hyperinsulinemia alone, suppresses human skeletal muscle lipolytic activity in vivo. J Clin Endocrinol Metab89 :4693 eC4700,2004
Intaglietta M, Johnson PC: Principles of capillary exchange. In Peripheral Circulation. Johnson PC, Ed. New York, Wiley,1978 , p.141 eC166
Lassen NA, Perl WA: Multiple indicators: capillary permeability. In Tracer Kinetic Methods in Mammalian Physiology. Lassen NA, Perl WA, Eds. New York, Raven Press,1979 , p.156 eC175
Gudbjrnsde畉tir S, Sjstrand M, Strindberg L, Wahren J, Lnnroth P: Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle. J Clin Endocrinol Metab88 :4559 eC4564,2003
Biolo G, Chinkes DL, Zhang X-J, Wolfe RR: A new model to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle. J Parent Ent Nutr16 :305 eC315,1992
Patterson BW, Horowitz JF, Wu G, Watford M, Coppack SW, Klein S: Regional muscle and adipose tissue amino acid metabolism in lean and obese humans. Am J Physiol Endocrinol Metab282 :E931 eCE936,2002
Enevoldsen LH, Simonsen L, Stallknecht B, Galbo H, Beow J: In vivo human lipolytic activity in preperitoneal and subdivisions of subcutaneous abdominal adipose tissue. Am J Physiol Endocrinol Metab281 :E1110 eCE1114,2001
Larsen OA, Lassen NA, Quaade F: Blood flow through human adipose tissue determined with radioactive xenon. Acta Physiol Scand66 :337 eC345,1966
Coppack SW, Evans RD, Fisher RM, Frayn KN, Gibbons GF, Humphreys SM, Kirk MJ, Potts JL, Hockaday TDR: Adipose tissue metabolism in obesity: lipase action in vivo before and after a mixed meal. Metabolism41 :264 eC272,1992
Rosenblatt J, Wolfe RR: Calculation of substrate flux using stable isotopes. Am J Physiol254 :E526 eCE531,1988
Wolfe RR: Radioactive and Stable Isotope Tracers in Biomedicine:Principles and Practice of Kinetic Analysis. Wiley-Liss, New York,1992
Mulla NA, Simonsen L, Beow J: Post-exercise adipose tissue and skeletal muscle lipid metabolism in humans: the effect of exercise intensity. J Physiol Lond254 :919 eC928,2000
Lassen NA: Capillary diffusion capacity of sodium studied by the clearances of Na-24 and Xe-133 from hyperemic skeletal muscle in man. Scand J Clin Lab Invest Suppl99 :24 eC26,1967
Linde B, Chisholm G, Rosell S: The influence of sympathetic activity and histamine on blood-tissue exchange of solutes in canine adipose tissue. Acta Physiol Scand92 :145 eC155,1974
Paaske WP: Absence of restricted diffusion in adipose tissue capillaries. Acta Physiol Scand100 :430 eC436,1977
Paaske WP, Sejrsen P: Permeability of continuous capillaries. Dan Med Bull36 :570 eC590,1989
Cinti S: Adipose tissues and obesity. Ital J Anat Embryol104 :37 eC51,1999
Cinti S: The adipose organ: morphological perspectives of adipose tissue. Proc Nutr Soc60 :319 eC328,2001
Fielding BA, Humphreys SM, Allman RF, Frayn KN: Mono-, di- and triacylglycerol concentrations in human plasma: effects of heparin injection and of a high-fat meal. Clin Chim Acta216 :167 eC173,1993
Lin ECC: Glycerol utilization and its regulation in mammals. Annu Rev Biochem46 :765 eC795,1977
Lee DP, Deonarine AS, Kienetz M, Zhu Q, Skrzypczak M, Chan M, Choy PC: A novel pathway for lipid biosynthesis: the direct acylation of glycerol. J Lipid Res42 :1979 eC1986,2001
Kurpad AV, Khan K, Calder G, Coppack SW, Macdonald IA, Elia M: Effect of noradrenaline on glycerol turnover and lipolysis in the whole body and subcutaneous adipose tissue in humans in vivo. Clin Sci86 :177 eC184,1994
Maggs DG, Jacob R, Rife F, Lange R, Leone P, During MJ, Tamborlane WV, Sherwin RS: Interstitial fluid concentration of glycerol, glucose and amino acids in human quadricep muscle and adipose tissue. J Clin Invest96 :370 eC377,1995
Jansson P-AE, Veneman T, Nurjhan N, Gerich JE: An improved method to calculate the adipose tissue interstitial substrate recovery for microdialysis studies. Life Sci54 :1621 eC1624,1994
Fisher RM, Miles JM, Kottke BA, Coppack SW: Very-low-density lipoprotein subfraction composition and metabolism by adipose tissue. Metabolism46 :605 eC610,1997
Eckel RH: Lipoprotein lipase: a multifunctional enzyme relevant to common metabolic diseases. N Engl J Med320 :1060 eC1068,1989
Verkman AS: Water permeability measurement in living cells and complex tissues. J Membr Biol15 :73 eC87,2000
Hoffstedt J, Arner P, Hellers G, Lonnqvist F: Variation in adrenergic regulation of lipolysis between omental and subcutaneous adipocytes from obese and non-obese men. J Lipid Res38 :795 eC804,1997
Lafontan M, Barbe P, Galitzky J, Tavernier G, Langin D, Carpene C, Bousquet-Melou A, Berlan M: Adrenergic regulation of adipocyte metabolism. Human Reprod1 (Suppl. 12) :6 eC20,1997
Tan GC, Goossens GH, Humphreys SM, Vidal H, Karpe F: Upper and lower body adipose tissue function:a direct comparison of fat mobilization in humans. Obes Res12 :114 eC118,2004
Havel RJ, Kane JP, Balasse EO, Segel N, Basso LV: Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest49 :2017 eC2035,1970
Metropolitan Life Assurance Company: Net weight standard for men and women. Stat Bull Metrol Life Found40 :1 eC4,1959(Simon W. Coppack, David L)