Introduction

The balance among the synthesis, oxidation, and hydrolysis of triacylglycerols (TGs)¹ in adipocytes determines the net quantity of cellular fat content. Over time, small shifts in this delicate balance accumulate and may produce profound alterations in fat mass (1, 2, 3, 4, 5, 6). Although it has been shown that adipocytes have active mitochondrial machinery for free fatty acid (FFA) oxidation (7), very little information is available in the literature regarding these oxidative regulatory mechanisms. For a long time, many believed that adipose tissue served only for fat storage, not for fat burning. This concept derived, in part, from early studies on FFA oxidation in isolated fat cells, which generally showed very low values of oxidation compared with liver or muscle cells (8, 9, 10, 11, 12). However, in many of these studies, cells were incubated with isotope-labeled FFAs in the exogenous incubation medium, and the amount of label appearing in the oxidative products was measured. We consider that this method has at least two flaws. First, on entry into the cells, labeled FFAs are mixed and, hence, diluted, with unlabeled FFAs released from the endogenous TG pool. Therefore, the specific activity (SA) calculated using the concentration in the exogenous pool is inaccurate. Second, exogenous FFA is commonly added in complex with bovine serum albumin (BSA). With its large buffering capacity for FFAs (one BSA molecule binds one to eight FFA molecules depending on FFA availability), the true FFA concentration in the exogenous pool may differ substantially from the initial calculated concentration. This is further complicated by the fact that FFA transport into the cells may depend on the exogenous concentration of monomeric FFAs (not bound to BSA). Therefore, the appearance of label in the oxidative products indicates only that a certain amount of the labeled FFAs is oxidized, but a calculation of the actual rate of FFA oxidation using the initial exogenous concentration is inaccurate and provides no information about the actual oxidative consumption of FFAs.

With the ever-increasing prevalence of overweight and obesity in our time, understanding how adipocyte FFA metabolism can be regulated is not only scientifically interesting but also clinically important. Considering that white adipose tissue may constitute a significant percentage of the body mass, especially in the obese state, even a moderate up-regulation of FFA oxidation in this organ could contribute significantly to a long-term modulation of the total body energy balance. Toward this goal, we have developed a new strategy to measure the partitioning of endogenous FFAs among the three possible metabolic pathways: oxidation, re-esterification, or release into the exogenous medium. Briefly, the TG pool is prelabeled with a radioactive isotope. The products of subsequent lipolysis (FFAs and glycerol), oxidation (¹⁴CO₂ or ³H₂O), and re-esterification of released FFAs (measured by the incorporation of [U-¹⁴C]glucose into TG-glycerol moiety) were measured independently. Using this strategy, we measured a small consistent flow of endogenously released FFAs into the oxidative pathway. This flux was regulated by changes in nutritional state (fed or fasted) or changes in extracellular insulin concentration. To our knowledge, this is the first report that accurately measures the metabolic fate of endogenously produced FFAs in primary rat adipocytes. In addition, this study presents an important tool to determine FFA oxidation in adipocytes and moves us one step closer to our goal of controlling energy disposition in adipose tissue as a means of weight control.

Research Methods and Procedures

Isolation of Adipocytes from Adipose Tissue

White adipocytes were isolated from rat fat pads by collagenase digestion as described previously (13, 14). Briefly, fat pads from the perirenal and epididymal depots were removed and transferred into Krebs-Ringer phosphate HEPES buffer (KRH) (130 mM NaCl, 4.7 mM KCl, 1.24 mM MgSO₄, 2.5 mM CaCl₂, 1 mM HEPES, 2.5 mM NaH₂PO₄, 5 mM D-glucose, 2% BSA, and 200 nM adenosine, pH 7.4) at 37 °C. Adipose tissue pieces were minced and digested with collagenase B (2 mg/mL) in KRH buffer for 35 minutes at 37 °C in a shaking water bath. Thus, the fat cell suspension obtained was filtered through a 250-m nylon mesh and centrifuged for 15 seconds at 1000 rpm. The adipocytes collected from the top phase were washed with KRH buffer three times. The cells were resuspended in 3 volumes of KRH buffer, allowed to equilibrate for 15 minutes at 37 °C, and then used directly for the subsequent experiments.

FFA Oxidation Measured by ³H₂O Release

Oxidized [9,10-³H]palmitate was measured on the basis of ³H₂O released into the media from cells pre-incubated with 2 Ci/mL [9,10-³H]palmitate using a modified protocol from the literature (15). Briefly, adipocytes were prelabeled with 2.0 Ci/mL [9,10-³H]palmitate in 2% FFA-free BSA-KRH buffer for 30 minutes at 37 °C with gentle shaking. Cells were washed with 2% FFA-free BSA-KRH buffer three times to remove unincorporated [9,10-³H]palmitate from the medium. Four milliliters of adipocyte suspension containing 1 mL of adipocytes were incubated for 2 hours at 37 °C. Palmitate oxidation was assessed by measuring the amount of ³H₂O released into the medium. Briefly, 1.0 mL of medium was transferred into a 1.5-mL center well. The center well was placed in a scintillation vial (20 mL) containing 1 mL of unlabeled water. The vial sealed with parafilm was incubated at 50 °C for 18 hours. This allowed the equilibration of ³H₂O in the center well with the water phase outside the center well while retaining the labeled FFAs inside the well. After carefully removing the center wells, the scintillation vials were added with 4 mL of scintillation fluid and used for -counting. Blank was treated the same as above except that cells were lysed at time 0. To determine the equilibrium coefficient between the water phase inside and outside the center well, 1 mL of ³H₂O was added to a 1.5-mL center well in parallel with the experimental samples. The recovery of ³H₂O from known amounts of ³H₂O was 46% to 48% under these experimental conditions, indicating nearly complete equilibration between the two water pools. The total amount of labeled palmitate incorporated into the adipocyte lipid pool during the 30-min pre-incubation period was 6.5 10⁶ counts per minute (CPM)/mL of cell volume. Each milliliter of cells contained 5 mol FFAs (enzymatic colorimetry) and 7 mg of protein (Bradford). The amount of FFA oxidized was calculated as the following:

FFA Oxidation Measured by CO₂ Release

The method for measurement of ¹⁴CO₂ released was adapted from a published protocol (16). Briefly, adipocytes were prelabeled with 1Ci/mL [U-¹⁴C]palmitate in 2% FFA-free BSA-KRH buffer for 30 minutes at 37 °C with gentle shaking. Cells were washed with 2% FFA-free BSA-KRH buffer three times to remove unincorporated [U-¹⁴C]palmitate from the medium. Four milliliters of adipocyte suspension (1 mL of cell volume) labeled with [U-¹⁴C]palmitate were incubated in a T 25 culture flask for 2 hours at 37 °C. The flasks were sealed with a rubber stopper with a center well suspended. A folded filter paper (1 cm²) was placed inside the center well. At the end of the incubation period, the ¹⁴CO₂ produced by the adipocytes was released by injection of 0.5 mL of 10 N H₂SO₄ into the flask. -Phenethylamine (0.3 mL) was injected into the center well to absorb ¹⁴CO₂. After overnight equilibration at room temperature, each center well with filter paper containing the absorbed ¹⁴CO₂ was quickly transferred into a 20-mL scintillation vial with 5 mL of scintillation fluid for -counting. Blank was treated the same as above except the cells were lysed at time 0. The total amount of labeled palmitate incorporated into the adipocyte lipid pool during the 30-minute pre-incubation period was 4.5 10⁶ CPM/mL of cell volume. Each milliliter of cells contained 5 mol of FFAs and 7 mg of protein. The amount of FFA oxidized was calculated as the following:

FFA Re-esterification

Adipocytes were prepared in parallel with those used for oxidation studies described above except without radioactive labeling. After a 30-min pre-incubation, cells were washed and then incubated with 2.5 Ci/mL [U-¹⁴C]glucose in 2% FFA-free BSA-KRH buffer for 2 hours at 37 °C with gentle shaking. At the end of the incubation, medium was removed and 5 mL of Dole's reagent (heptane:1 N H₂SO₄:isopropanol 10:1:40, v/v) was added to 1 mL of adipocytes to stop the reaction. The lipids in the adipocytes were extracted with 3 mL of heptane. One milliliter of the organic phase (extract) was dried and then methylated with 0.5 mL of BF₃-methanol (10%, w/w) and 0.5 mL of water-free heptane at 90 °C for 1 hour. At the end of the incubation, 1 mL of water was added to stop the reaction. The aqueous phase containing the glycerol moiety was washed with 5 mL of heptane three times to remove the FFA moiety (in the form of fatty acyl methyl esters). After the wash, >99% of the fatty acyl methyl esters were removed, and the aqueous phase was used for scintillation counting. The SA of labeled glucose in initial buffer (5 mM glucose) was 202 CPM/nmol glucose. Each molecule of glucose produces two molecules of glycerol, which, in turn, form the backbone of two molecules of TG, each containing three molecules of FFA. The amount of FFA re-esterification was calculated from the amount of labeled glucose converted to the TG-glycerol moiety.

Lipolysis

Lipolysis was assessed by measuring the glycerol (GPO-Trinder triglycerol kit, Sigma, St. Louis, MO) and FFA (NEFAC kit, Wako Chemicals, Richmond, VA) released from adipocytes into the medium during the 2-h incubation.

Protein and TG Content

Adipocyte protein was measured by the Bradford method using BSA as a standard. TG content of adipocytes was determined using the GPO-Trinder TG kit.

Animals

Male Sprague-Dawley rats (220 to 250 g; Charles River Breeding Laboratories, Wilmington, MA) were maintained at 21 °C with a 12-h-light/12-h-dark cycle (light on from 6 AM to 6 PM). They were fed standard Purina chow ad libitum. For animals studied under fasting conditions, food was removed 24 hours before the experiment. The protocol of using animals for this study has been approved by the Boston University School of Medicine.

Materials

Collagenase B was purchased from Boehringer Mannheim (Mannheim, Germany). [9,10-³H]palmitate and [U-¹⁴C]palmitate were from Amersham Life Science Co. (Buckinghamshire, UK). D-[U-¹⁴C]glucose was from NEN Life Science Co. (Boston, MA). TG and glycerol test kits and protein assay reagent (Bradford) were from Sigma. The FFA test kit was from Wako Chemicals.

Statistic Analysis

Data were expressed as mean SE. For groups of three, comparisons were performed using ANOVA. For groups of two, comparisons were performed using Student's t test. Significance was set at p < 0.05.

Results

Linearity of FFA Oxidation

Isolated adipocytes from rat adipose tissue are fragile with relatively short-term viability in suspension. To determine their functional viability and to choose an optimal duration for experiments, FFA oxidation and glycerol release were measured at different time-points up to 6 hours in adipocytes from fed and fasted rats after cells were prelabeled with [9,10-³H]palmitate. The effects of insulin were also studied using adipocytes from fasted rats. As shown in Figure 1 (top), the amount of FFA oxidation, determined as TG-derived ³H₂O, increased linearly with time for up to 6 hours. In parallel, glycerol release in adipocytes from normal and fasted rats also increased linearly up to 6 hours and increased linearly up to 4 hours in adipocytes (treated with 10 nM insulin) from fasted rats (Figure 1, bottom). Based on these findings, the following studies were conducted using 2 hours of incubation. Although label incorporation increased with pre-incubation time (p < 0.05) (Figure 2, top), there were no differences in FFA oxidation (Figure 2, middle) or glycerol release (Figure 2, bottom) during the 2-h incubation. The effects of pre-incubation duration were measured by prelabeling adipocytes with [9,10-³H]palmitate for 30, 60, and 90 minutes. We chose 30 minutes as the prelabeling time for the following experiments.

Figure 1:.

The time course of FFA oxidation and lipolysis in isolated rat adipocytes. Adipocytes were labeled with [9,10-³H]palmitate as tracer for 30 minutes. After the removal of exogenous labeled FFA (zero time-point), the production of ³H₂O from endogenous FFA oxidation (left) and the glycerol released (right) from adipocytes was measured at 1, 2, 3, 4, 5, and 6 hours. Values are mean SE of (n = 4). Top, adipocytes from normal fed rats. Middle, adipocytes from fasted rats (starved for 24 hours). Bottom, Insulin-treated (1 nM) adipocytes from fasted rats (starved for 24 hours). Results of two experiments performed in duplicate are expressed as fold increase relative to a value of 1 at 1 hour.

Full figure and legend (55K)

Figure 2:.

FFA oxidation and lipolysis during a 2-h incubation after different periods of prelabeling. Adipocytes from normal fed rats were labeled with [9,10-³H]palmitate as tracer for 30, 60, and 90 minutes, and the exogenous FFAs were removed after each time interval. There was an increase in the incorporated label with increasing pre-incubation time (top). The prelabeled adipocytes were then incubated for 2 hours in the absence of exogenous FFA, and the production of ³H₂O from endogenous FFA oxidation (middle) and the glycerol release (bottom) from adipocytes were measured. Values are mean SE of two experiments performed in duplicate.

Full figure and legend (36K)

SA of FFA

SA of endogenous FFAs (in the TG pool) was calculated at zero time and at different time-points up to 6 hours after exogenous FFAs were removed. There were no significance differences in SA at different time-points (p > 0.05) (Figure 3). Variation in SA was negligible within the experimental time frame, suggesting that the newly labeled TGs were not being preferentially hydrolyzed. The SA of the following studies was calculated at time zero.

Figure 3:.

SA of FFA at different times after exogenous FFA was removed. Adipocytes from normal fed rats were labeled with [9,10-³H]palmitate as tracer for 30 minutes, after which the exogenous FFAs were removed. The SA of FFA was calculated at zero time-point (immediately after the exogenous FFA was removed), and 1, 2, 3, 4, 5, and 6 hours after the removal of exogenous FFA. The calculation of SA was as described in "Research Methods and Procedures." Values are mean SE of two experiments performed in duplicate.

Full figure and legend (60K)

Comparison of FFA Oxidation Measured by ³H₂O Release and ¹⁴CO₂ Release

Beta-oxidation of FFAs produces H₂O and acetyl coenzyme A (CoA), whereas complete oxidation of acetyl CoA through the Kreb's cycle produces CO₂. In the literature, FFA oxidation is commonly measured by detecting isotopically labeled ³H₂O or ¹⁴CO₂. However, the two methods seldom have been compared in the same reaction system. While establishing conditions for prelabeling the endogenous TG pool in adipocytes, we also compared results measured by each technique. As shown in Table 1, FFA oxidation measured as the release of ³H₂O did not differ significantly from that measured as the release of ¹⁴CO₂ (p > 0.05), indicating that most of the acetyl CoA produced from -oxidation was completely oxidized through the Kreb's cycle.

Table 1 - Oxidation of FFA and lipolysis in adipocytes from fed and fasted rats.

Full table

FFA Partitioning among the Major Metabolic Fates

As shown above, FFAs produced from the lipolysis of the endogenous TG pool in adipocytes could be readily oxidized, but this represented only a small proportion of the released FFAs (0.2%). Major metabolic fates included re-esterification into TG and FFA efflux of the cells, an important energy source for other cell types in vivo. The partitioning of FFA among these three metabolic pathways can be assessed by independent measurement of the oxidative products (³H₂O or ¹⁴CO₂), the esterification products ([U-¹⁴C]glucose incorporation into TG-glycerol), and the released products (glycerol and FFA in the medium). As shown in Table 1, in adipocytes from normal fed animals, lipolytically released FFA distributed nearly evenly between the re-esterification (49.7%) and release of cells (50.1%), whereas the amount of FFA oxidation accounted for 0.2% of total released endogenous FFA. Fasting caused a significant increase in lipolysis (1.4-fold increase, p < 0.05), accompanied by a 2-fold increase in FFA oxidation and a substantial increase in FFA release (1.6-fold increase, p < 0.05) into the medium. There was no specific difference in the absolute amount of FFA re-esterification, but fasting decreased the ratio of FFA partitioning into the pathway (Table 1). Exposure of cells from fasted animals to insulin suppressed FFA oxidation to a level less than that found in cells from fed animals (35% reduction, p < 0.05). This was accompanied by a substantial increase in FFA re-esterification (98% increase, p < 0.05).

Discussion

Extensive studies have been conducted on the regulation of lipolysis and TG synthesis in adipocytes (17, 18, 19, 20, 21). There is also a limited number of reports on FFA oxidation in whole cells (11, 20, 22, 23, 24, 25, 26) and isolated mitochondria (8, 23) that have measured the oxidative products from labeled exogenous FFA. The capacity for adipocytes to oxidize endogenous FFA has remained largely unexplored, despite its obvious importance considering the large abundance of endogenous FFA stored in adipocytes. In the present study, we demonstrated that endogenous FFA could be oxidized and that the partitioning of endogenous FFA among the metabolic pathways was regulated hormonally or physiologically. Although the net percentage of FFA disposed of through the oxidative pathway might seem to be small (0.2%) (Table 1), this figure is significant considering that the measurement was conducted over a 2-h period. Over time, it is possible that a slight up-regulation in this pathway might produce substantial effects in body energy balance. For instance, if the results in Table 1 were extrapolated to humans, the increase in oxidation accompanying a fast would decrease body fat mass by more than 1 kg per 50 kg of fat mass per year. This hypothesis needs to be tested.

Although it has been well recognized that metabolism in white adipose tissue contributes to the control of body fat content (1, 3, 6), prior studies in this area have focused on lipolysis and re-esterification. A number of studies also explored the feasibility of increasing the expression of uncoupling protein-1 in white adipocytes using genetic (27) or pharmacological manipulations (28). There has been generally very little interest in FFA oxidation in native white adipocytes. However, a thorough understanding of FFA metabolism in white adipocytes represents an important step toward the regulation of this process to increase FFA disposal and reduce storage. In this study, we showed that although the percentage of endogenous FFA oxidized (0.2%) might be small compared with those partitioned in other pathways (49.7% of endogenous FFA re-esterified and 50.1% released) (Table 1), the net amount of FFA disposed through the oxidative pathway could be significant and increased with fasting. The mechanism through which fasting up-regulated FFA oxidation is not well understood. A number of possibilities might contribute to some extent. First, total TG hydrolysis (measured by glycerol release) was increased, which might increase the intracellular acyl CoA concentration. Because fasting generally down-regulates the enzymes of TG synthesis (29), more acyl CoA might become available to the mitochondria. Secondly, fasting down-regulates the expression and activity of acetyl CoA carboxylase (30, 31, 32); hence, the production of malonyl CoA, the allosteric inhibitor of carnitine palmitoyl transferase-1, might be reduced. The latter has been known as the gatekeeper for acyl CoA to enter the -oxidation pathway (33, 34). Hence, reduced malonyl CoA production during fasting likely contributed to increased carnitine palmitoyl transferase-1 flux to promote FFA oxidation. Additionally, although our study was performed at identical glucose concentrations (5 mM) for adipocytes from fed and fasted animals, glucose uptake in the latter might be diminished (35, 36). Hence, cells shifted to increased FFA oxidation for energy production. This argument is supported by the findings that insulin, which stimulates glucose uptake, completely abolished this fasting-mediated increase in FFA oxidation (61% reduction) (Table 1). Because fasting causes profound alternations in the physiological environment, additional mechanisms responsible for the regulation of FFA oxidation are likely to be involved. Understanding these mechanisms will help to identify the control points in the regulation of FFA metabolism in adipocytes and contribute to the development of new means to control these processes and optimize energy balance.

Notes

¹ Nonstandard abbreviations: TG, triacylglycerol; FFA, free fatty acid; SA, specific activity; BSA, bovine serum albumin; KRH, Krebs-Ringer phosphate HEPES buffer; CoA, coenzyme A; CPM, counts per minute.

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Acknowledgments

This work was supported by NIH Grants DK 56690, 46200 (to B. E. C.), and 59261 (to W. G.)