Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Getty-Kaushik, L.
Right arrow Articles by Corkey, B. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Getty-Kaushik, L.
Right arrow Articles by Corkey, B. E.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Diabetes 54:629-637, 2005
© 2005 by the American Diabetes Association, Inc.

Free Fatty Acid Regulation of Glucose-Dependent Intrinsic Oscillatory Lipolysis in Perifused Isolated Rat Adipocytes

Lisa Getty-Kaushik, Ann-Marie T. Richard, and Barbara E. Corkey

From the Obesity Research Center, Department of Molecular Medicine, Boston Medical Center, Boston, Massachusetts


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Free fatty acids (FFAs) and glycerol oscillate in plasma. This study examined intrinsic lipolytic oscillations within adipocytes. Rat adipocytes were perifused with Krebs-Ringer bicarbonate buffer: 1) ± 2 mmol/l glucose; 2) +1 µmol/l isoproterenol ± 2 mmol/l glucose; 3) + increasing oleate; and 4) + increasing percent BSA. At 2 mmol/l glucose, there were 9 ± 1 glycerol, FFAs, and lactate pulses per hour with a pulse duration of 5 ± 1 min. Lipolytic stimulation caused a 50–80% increase in the amplitude of lipolytic oscillations. Removal of glucose caused a 40–70% decrease in the amplitude of lipolytic oscillations and disturbed the pulsatility. Exogenous FFAs suppressed lipolysis and oscillatory amplitude, possibly because of increased cytosolic long-chain coenzyme A (LC-CoA). Increasing percent BSA increased stimulated lipolysis and oscillatory amplitude, possibly because of decreased intracellular LC-CoA. These data show, for the first time, intrinsic lipolytic oscillations, which are glucose dependent and modulated by FFAs. We hypothesize that lipolytic oscillations are driven by oscillatory glucose metabolism, which leads to oscillatory relief of LC-CoA inhibition of triglyceride lipase(s). The results contribute to the understanding of physiological and biochemical regulators of lipolysis, such as glucose and FFAs. Lipolytic oscillations may be beneficial in the delivery of FFAs to liver, pancreas, and other tissues.

Abnormal fat metabolism plays an important role in the pathogenesis of obesity-related type 2 diabetes (1–4), and elevated plasma free fatty acid (FFA) concentrations are associated with peripheral and hepatic insulin resistance (5–7). Adipose tissue is a dynamic organ that is vital to the regulation of glucose homeostasis, whole-body energy fuel regulation, feeding behavior, and body composition. It has previously been shown by Getty et al. (8), in dogs, that in the basal fasted state, FFA and glycerol oscillate in plasma with an average of nine pulses per hour and an average pulse duration of 5 min. It has also been shown that there is oscillatory lipolysis from the omentum with an average of 10 pulses per hour and an average pulse length of 6 min.

Because lipolysis is primarily regulated by adrenergic modulation and insulin concentration, it is possible that either could be driving the plasma FFA oscillation. With the plasma insulin oscillation removed by the insulin clamp, FFAs still showed an oscillation in plasma, suggesting that insulin does not drive the FFA oscillation (8). The study also looked at the effect of ß-adrenergic blockade. In three of the nine dogs studied, propranolol infusion seemed to suppress the FFA oscillation. In dogs where the FFA oscillation remained, propranolol infusion significantly disrupted the regularity of the plasma FFA oscillation (9,10). Further investigation of the role of the central nervous system in the regulation of in vivo lipolytic oscillations by Hucking et al. (11) suggested that lipolysis in the fasting state consisted of an oscillatory component dependent upon sympathetic innervation and a nonoscillatory component. In both above-mentioned studies, it is possible that lipolysis was still oscillating on the level of individual fat pads or even individual adipocytes and that these oscillations became unsynchronized and thus were lost, dampened, or below detection when sympathetic input was blocked.

Thus, the present study determined whether the plasma FFA oscillation originates within the adipocyte from an internal pacemaker similar to that seen in the ß-cell of the pancreas (12–14). The basal profiles of FFA, glycerol, and lactate release from isolated perifused adipocytes were determined in the basal state (± glucose), during stimulation of lipolysis with 1 µmol/l isoproterenol (± glucose), during perifusion with increasing concentrations of oleate, and during perifusion with increasing percent BSA (+ isoproterenol). The results demonstrated that there were intrinsic lipolytic oscillations in adipocytes that were dependent on glucose and regulated by FFAs. The study also showed that glucose metabolism oscillated and that there appeared to be a link between glycolytic and lipolytic oscillations. The data support a model where lipolytic oscillations are driven by oscillatory glucose metabolism, which leads to oscillatory relief of inhibition of triglyceride lipase(s) (TGL) by long-chain coenzyme A (LC-CoA).


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sprague-Dawley male rats weighing 150–250 g were used. The animals were housed in the Laboratory Animal Science Center at Boston University Medical Center. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Boston University Medical Center. The animals were fed normal rat chow and water ad libitum until time of death by anesthesia and cervical dislocation.

The perifusing buffer consisted of Krebs-Ringer bicarbonate buffer (KRBB) composed of the following: 120 mmol/l NaCl, 25 mmol/l NaHCO3, 5 mmol/l KCl, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 2.5 mmol/l CaCl2, and 20 mmol/l MOPS. The final pH of the perifusing buffer was 7.4 at 37°C and contained either 0.05% or 0.1% BSA as indicated. Dulbecco’s PBS, isoproterenol, and oleic acid were purchased from Sigma (St. Louis, MO). BSA, fraction V, was purchased from U.S. Biochemical (Cleveland, OH). The BSA was purified by charcoal treatment to remove lipids (15) and then dialyzed against KRBB (16). Collegenase, type 1, was purchased from Worthington Biochemical (Lakewood, NJ).

Adipocyte isolation.
Adipocytes were isolated from rat epididymal fat pads as described by Rodbell (16) using collagenase digestion as modified by Turpin et al. (17). The adipose tissue was collected and minced in room temperature PBS and then digested in KRBB containing 0.1% BSA, 2 mmol/l glucose, and 1 mg/ml collagenase at 37°C with shaking. The cells were then filtered through polyester mesh and washed three times with KRBB containing 0.1% BSA and 2 mmol/l glucose.

Adipocyte perifusion.
Adipocytes were perifused in a column as described by Turpin et al. (17). After washing, 400-µl packed cells (2–4 million cells) were loaded onto a column 0.7 cm in diameter and 4 cm high. The column was contained in a temperature-controlled environment maintained at 37°C. The cells were perifused (KRBB + 0.05% BSA + 2 mmol/l glucose) for 30 min to allow for equilibration. Perifused solutions were pumped through the column in the direction of gravity using an analog tubing pump (Ismatech REGLO pump, type ISM 827, model 78016-30; Cole-Parmer Instruments, Chicago, IL) at a flow rate of 0.7 ml/min. This flow rate allowed for gentle mixing of the cells due to the balance between the buoyancy of the adipocytes and the downward flow of the buffer, thus providing equal distribution of buffer to all cells. After the equilibration period, samples were taken to determine basal lipolysis for 1 h. The cells were then perifused with either KRBB (± glucose), isoproterenol (± glucose), increasing concentrations of oleate, or increasing concentrations of BSA (+ isoproterenol) for 2–3 h.

In all experiments, samples of the effluent were collected every minute and measured for FFA, glycerol, and lactate. Samples were stored at –20°C until processing. Because of the low levels of FFA, glycerol, and lactate in the collected effluent, the samples were concentrated. Samples were dried down in a high-performance vacuum pump (Speedvac system, model SS11; Savant Instruments, Holbrook, NY), reconstituted in distilled water, and then assayed.

Assays.
FFAs were measured using a colorimetric kit from Wako (NEFA C; Wako Pure Chemical Industries, Richmond, VA) that uses acylation of coenzyme A. Glycerol was measured using a colorimetric kit from Sigma (Infinity Triglyceride Reagent) that uses glycerol kinase and glycerol phosphate oxidase. Lactate was measured using a colorimetric kit from Sigma (Lactate Reagent) that uses lactate oxidase. The intra-assay coefficient of variations (CVs) were as follows (determined from historical data and from experiments in this study that were assayed in duplicate): FFA = 3% (8), glycerol = 5% (8,18), and lactate = 3% (19). Because the samples were concentrated, they contained a relatively high salt concentration. However, it is reported that slight to moderate turbidity causes no significant interference with any of the assays. Interference was tested for and found to be insignificant.

Calculations.
Many hormones are secreted in an episodic manner (9,20,21) and appear as a sequence of irregular pulses or "bursts"; thus, there has been a proliferation of methods developed to quantify pulsatility (10,22,23). In the present study, pulse analyses of the temporal profiles of FFA, glycerol, and lactate were performed using ULTRA (a pulse detection algorithm obtained from E. Van Cauter, Department of Medicine, University of Chicago, Chicago, IL) (23). This algorithm was chosen to compare the in vitro results obtained in this study with the published in vivo results (8,11). ULTRA eliminates all peaks of plasma concentration for which either the increment or the decrement does not exceed a certain threshold. The threshold is determined by parameters set by the user as the intra-assay CV and the number of CVs to be used as threshold significance. In this study, three times the CV of each assay was used as a threshold (23). ULTRA is largely insensitive to unstable baseline hormone concentrations and is not adversely affected by varying pulse amplitudes, widths, or configurations within the series.

To determine that oscillations were due to oscillations in lipolysis and not due to an artifact from sample collection or handling, effluent from basally treated adipocytes was collected, pooled, realiquoted, concentrated, assayed, and analyzed with ULTRA. No significant pulses were detected (data not shown).

The FFA-to-glycerol ratio was determined at each time point and then averaged. The FFA-to-glycerol ratio was not calculated from the average FFA and glycerol concentrations.

Statistical analysis.
Values are reported as means ± SE. The data were analyzed using ANOVA. Comparisons were made using the Student’s t test.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lipolytic oscillations in isolated adipocytes.
The time courses of FFA, glycerol, and lactate release from perifused adipocytes were examined in the basal and stimulated state and can be seen in Fig. 1 (summary in Table 1). Figure 1A shows basal lipolysis and lactate release (100 min of the 120-min basal experiment), Fig. 1B shows the effect of isoproterenol (last 100 min of the stimulated experiment; the entire response to isoproterenol can be seen in Fig. 2B), and Fig. 1C shows basal and stimulated FFA and glycerol oscillatory profiles together to demonstrate the correlation of the two profiles (data in Fig. 1A, B, and C are from separate experiments). Adipocytes were perifused with KRBB containing 0.05% BSA and 2 mmol/l glucose for 1 h to determine basal lipolysis and lactate release. After the basal period, the adipocytes were either continued in the basal state for 1 h or were stimulated with 1 µmol/l isoproterenol for 2 h. The open circles show raw data, whereas the solid lines show the significant pulse profile as determined by ULTRA (Fig. 1C shows only the ULTRA profiles). There were an average of 9 ± 1 pulses per hour (pulse frequency) of FFA, glycerol, and lactate, with each pulse ("burst" of release) lasting an average of 5 ± 1 min (pulse duration).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1. Basal (A) and stimulated (B) lipolytic and lactate oscillations. A: Samples were collected every minute in the basal state (KRBB + 0.05% BSA + 2 mmol/l glucose) for 2 h (only 100 min are shown). Profiles of glycerol (top panel), FFA (middle panel), and lactate (bottom panel) are shown from one representative experiment (n = 6). B: Samples were collected every minute in the basal state (KRBB + 0.05% BSA + 2 mmol/l glucose) for 1 h, and then samples were collected every minute during stimulation with 1 µmol/l isoproterenol for 2 h (only the last 100 min of stimulation are shown). Profiles of glycerol (top panel), FFA (middle panel), and lactate (bottom panel) are shown from one representative experiment (n = 6). C: FFA and glycerol ULTRA profiles for basal and stimulated experiments are shown on the same graph to demonstrate the correlation of the two profiles (different experiments from A and B). In all panels, the raw data are shown in the open circles, whereas the thick line shows the significant pulses as determined by ULTRA.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1 Effect of glucose removal on basal and stimulated adipocyte metabolism and oscillations

 


View larger version (56K):
[in this window]
[in a new window]
  		FIG. 2. Effect of glucose removal on basal (A) and stimulated (B) lipolytic and lactate oscillations. A: Samples were collected every minute in the basal state (KRBB + 0.05% BSA) in the presence ({circ}) or absence ( {zdb0030537640005}) of 2 mmol/l glucose for 2 h. Profiles of glycerol (top panel), FFA (middle panel), and lactate (bottom panel) are shown from one representative experiment (n = 6). B: Samples were collected every minute in the basal state (KRBB + 0.05% BSA + 2 mmol/l glucose) for 1 h, and then samples were collected every minute during stimu-lation with 1 µmol/l isoproterenol in the presence ({circ}) or absence ( {zdb0030537640005}) of 2 mmol/l glucose for 2 h. Profiles of glycerol (top panel), FFA (middle panel), and lactate (bottom panel) are shown from one representative experiment (n = 6).

 
Isoproterenol caused the expected significant increase in FFA and glycerol release with an increase in the FFA-to-glycerol ratio. Isoproterenol caused a significant decrease in lactate release, suggesting that glucose was being used to re-esterify FFA rather than being metabolized to lactate. Interestingly, as can be seen in Fig. 1B, isoproterenol caused a significant increase in the amplitude of FFA and glycerol oscillations and a significant decrease in the amplitude of lactate oscillations. Stimulation with isoproterenol did not change FFA, glycerol, or lactate pulse frequency or pulse duration when compared with basal. For the duration of most experiments, the FFA and glycerol oscillations remained in phase and appeared to be correlated (Fig. 1C).

Glucose dependence of oscillatory lipolysis.
Isolated adipocytes were perifused, as described above, in the presence or absence of 2 mmol/l glucose. The effects of glucose removal on the basal profiles of FFA, glycerol, and lactate release are illustrated in Fig. 2A (summary in Table 1). In the basal state with no glucose, there was a tendency for FFA and glycerol release to be suppressed, which also caused a nonsignificant decrease in the FFA-to-glycerol ratio. Lactate release, however, was significantly decreased when glucose was absent from the perifusing buffer. More importantly, the absence of glucose in the basal state disrupted the lipolytic oscillations in that no significant pulses were detected of the duration expected, and thus there were fewer FFA and glycerol pulses per hour. The amplitude of the FFA and glycerol pulses that were detected, as well as the lactate pulses, was decreased in the absence of glucose.

The effects of glucose removal on the profiles of FFA, glycerol, and lactate release during isoproterenol stimulation are illustrated in Fig. 2B (Table 1). The stimulation of lipolysis and the increase in the amplitude of lipolytic oscillation by isoproterenol seen in the presence of glucose was not seen when glucose was removed from the perifusing buffer. However, stimulation did prevent the fall in lipolytic oscillatory amplitude. With stimulation in the absence of glucose, lactate release was suppressed by >80% and even transiently dropped below the detection level of the assay.

Fatty acid regulation of oscillatory lipolysis.
Isolated adipocytes were perifused with KRBB + increasing concentrations of oleate during the basal state for 2 h or increasing percent BSA during the stimulated state (1 µmol/l isoproterenol) for 2 h. As can be seen in Fig. 3A (Table 2), exogenous FFA significantly suppressed both lipolysis and lactate release in a concentration-dependent manner. Exogenous FFA also significantly suppressed the amplitude of both lipolytic and lactate oscillations in a concentration-dependent manner. There was no change in the pulse frequency or pulse duration of either lipolytic or lactate oscillations. As seen in Fig. 3B (Table 3), increasing the percent BSA of the perifusion buffer significantly increased stimulated lipolysis in a concentration-dependent manner. (The suppressive effect of isoproterenol on lactate release was unchanged.) Increasing the percent BSA of the perifusion buffer also increased the amplitude of stimulated lipolytic oscillations in a concentration-dependent manner. (The suppressive effect of isoproterenol on lactate oscillations was unchanged.) Increasing percent BSA did not change the pulse duration or pulse frequency of lipolytic or lactate oscillations.



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 3. Effect of exogenous FFA concentration on lipolytic and lactate oscillations. A: Samples were collected every minute in the basal state (KRBB + 0.05% BSA + 2 mmol/l glucose) in the presence of increasing concentrations of oleate (magenta = basal, blue = 0.02 mmol/l, purple = 0.04 mmol/l, green = 0.08 mmol/l, black = 0.12 mmol/l, red = 0.30 mmol/l). B: Samples were collected every minute in the stimulated state (KRBB + 2 mmol/l glucose + 1 µmol/l isoproterenol) in the presence of increasing percent BSA (black = 0%, red = 0.1%, purple = 0.5%, blue = 1.0%, magenta = 1.5%). Profiles of glycerol (top panel), FFA (middle panel), and lactate (bottom panel) are shown from one representative experiment (n = 3 for each concentration).

 

View this table:
[in this window]
[in a new window]
  		TABLE 2 Effect of increasing exogenous FFA concentration on basal and stimulated adipocyte metabolism and oscillations

 

View this table:
[in this window]
[in a new window]
  		TABLE 3 Effect of increasing percent exogenous BSA on basal and stimulated adipocyte metabolism and oscillations

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides the first documentation of intrinsic lipolytic oscillations in isolated adipocytes. The oscillations had a pulse duration and pulse frequency similar to that seen in plasma in vivo. When lipolysis was stimulated with isoproterenol, there was a significant increase in FFA and glycerol release, with a significant increase in the pulse amplitude with no change in the pulse duration or number of pulses per hour. The discovery of an intrinsic lipolytic oscillation is important, since it contributes to the understanding of the normal physiological and biochemical processes that control whether FFAs are re-esterified to be stored as fat or released through the process of lipolysis to be used as fuel. Recognition of the regulators of lipolytic oscillations, such as glucose and FFAs, may help in determining the pathophysiology of dyslipidemia, which occurs in obesity and obesity-related type 2 diabetes.

The data suggested that there was oscillatory glucose metabolism within adipocytes, since release of lactate was oscillatory. This result is consistent with previous work done by Lipkin et al. (24), who examined glucose oxidation to CO2 in isolated perifused rat adipocytes and showed that initiation of perifusion with insulin induced oscillatory glucose oxidation. Also, preliminary data from our laboratory have shown oscillations in oxygen consumption in the minute time range from single 3T3-L1 adipocytes (R.F. Corkey, B.E. Corkey, unpublished data). It is well known that oscillations in glycolysis exist in many cell types (25–29). Like oscillatory insulin secretion from ß-cells, we suggest that oscillatory glycolysis may be the pacemaker for the intrinsic lipolytic oscillation in adipocytes. The mechanism of glycolytic oscillations in skeletal muscle extracts involves autocatalytic AMP-dependent activation of phosphofructokinase by its product fructose 1,6-bisphosphate, thus leading to bursts in phosphofructokinase activity (26). It is possible that glycolytic oscillations would cause secondary oscillations in most of cellular metabolism, including lipolysis (29,30).

Because the similarity in frequency between glycolytic and lipolytic oscillations suggested a relationship between the two pathways, we examined the requirement for glucose in lipolytic oscillations. In the basal state, glucose removal from the perifusion buffer tended to decrease the amount of FFA and glycerol released, while markedly decreasing lactate release. Interestingly, the absence of glucose disrupted the pulsatility of lipolytic oscillations in that no significant pulses were detected in the range of pulse duration expected. Furthermore, the absence of glucose caused a significant decrease in the amplitude of the FFA and glycerol pulses that were detected. These results show that lipolytic oscillations depended on the presence of glucose, suggesting that oscillatory glucose metabolism may play a role in generating the lipolytic oscillation. The results also showed that the lipolytic response to isoproterenol depended on glucose. Without glucose, there was no increase in lipolysis or in the amplitude of lipolytic oscillations upon stimulation with isoproterenol. Although lipolytic oscillations depended on glucose to maintain their amplitude, removal of glucose did not completely abolish either lipolytic or lactate oscillations or significantly suppress lipolysis, suggesting that 1) basal lipolysis does not depend on external glucose and 2) there must be an internal source of glucose metabolism within the adipocyte. It is probable that the adipocyte was able to provide glucose from the breakdown of glycogen, and this source was enough to maintain basal lipolysis. McMahon and Frost (31) showed that in glucose-deprived 3T3-L1 cells, glycogen is depleted in a time-dependent manner with a half-time of 6 h.

In interpreting the results of this study, one must keep in mind the role that glucose, BSA, and ATP play in lipolysis. Both Fassina et al. (32) and Giudicelli et al. (33) have shown that omission of glucose from the incubation buffer reduced the basal intracellular ATP level in adipocytes, as well as decreased the amount of cAMP formation. However, removal of glucose did not affect dibutyryl cyclic AMP-induced lipolysis, suggesting that ATP issued from glucose oxidation is not essential for stimulation of hormone-sensitive lipase (HSL). Thus, Giudicelli et al. (33) concluded that, in conditions of reduced glucose availability, activation of lipolysis by HSL is still possible, with the ATP required being supplied in sufficient amounts by mitochondrial oxidation of noncarbohydrate substrates such as fatty acids. This may also play a role in the results from this study, where basal lipolysis continued even in the absence of glucose. It is also important to note that all the above-mentioned experiments were carried out at a very low BSA concentration (0.05%). It has been shown that the accumulation of intracellular FFA in adipocytes inhibits the rate of lipolysis and that this effect can be diminished by the presence of albumin (33–36). Burns et al. (35) showed that when human adipocytes were incubated in albumin-free buffer, isoproterenol failed to stimulate lipolysis or increase cAMP and that this was due to the increase of intracellular FFA. This is similar to the results of this study, where lipolytic stimulation was abolished in the presence of low BSA and no glucose.

Model of lipolytic oscillations in adipocytes.
We hypothesize a model where lipolytic oscillations are driven by osillatory glucose metabolism, which leads to oscillatory inhibition of TGL by LC-CoA (Fig. 4). LC-CoA is formed from FFA and removed from the cytosol by esterification with glucose-derived {alpha}-glycerophosphate ({alpha}-GP) to form triglycerides. It has been shown by Jepson and Yeaman (37) that LC-CoA directly inhibits HSL, thus inhibiting triglyceride breakdown and the release of FFA and glycerol. This effect has not been shown for intracellular FFA per se. It has long been believed that HSL is the main triglyceride lipase active in adipocytes (and other tissues); however, several recent studies suggest the presence of other triglyceride lipases that may play a role equally as important as HSL. Both Jenkins et al. (38) and Soni et al. (39) have recently described novel triglyceride lipases present in adipocytes. Okazaki et al. (40) have reported the presence of a non-HSL triglyceride lipase in adipocytes whose hormonal regulation is similar to that of HSL. Also, work with HSL knockout mice has shown that HSL maintains very little basal lipolysis but accounts for a significant portion of catecholamine-induced lipolysis (41).



View larger version (77K):
[in this window]
[in a new window]
  		FIG. 4. Model of the proposed mechanism of intrinsic lipolytic oscillations. 1) LC-CoA esters inhibit TGL, thus inhibiting lipolysis. 2) LC-CoA is removed by glycolytically generated {alpha}-GP through conversion to triglyceride, thus removing the inhibition of TGL. 3) Glucose metabolism within the cell is oscillatory. 4) This would lead to oscillatory formation of {alpha}-GP and oscillatory removal of LC-CoA, which causes oscillatory removal of the inhibition of TGL, thus leading to oscillatory release of FFA and glycerol. DHAP, dihydroxyacetone phosphate; F16BP, fructose 1,6-bisphosphate; GAP, glyceraldehyde-3 phosphate; TG, triglycerides.

 
In our model, oscillatory glycolysis would result in oscillatory formation of {alpha}-GP, leading to oscillatory removal of LC-CoA to form triglycerides, thus causing oscillatory levels of LC-CoA. These oscillatory levels of LC-CoA within the cytosol would cause oscillatory inhibition of TGL and thus lead to oscillatory release of FFA and glycerol. LC-CoA could inhibit only HSL, all triglyceride lipases present in the adipocyte, or perhaps a specific non-HSL lipase. The model is consistent with our results showing that glucose is necessary to maintain lipolytic oscillations and the response to isoproterenol. Without sufficient glucose, there would be inadequate production of {alpha}-GP, causing LC-CoA levels to rise. This would inhibit TGL and thus suppress lipolysis and dampen lipolytic oscillations. However, as mentioned earlier, basal lipolysis, while decreased, was not significantly suppressed in the absence of glucose. This could be due to the above-mentioned contribution of glycogen to {alpha}-GP formation or to the above-mentioned presence of other triglyceride lipases besides HSL that may not be sensitive to LC-CoA inhibition and may maintain basal lipolysis.

One way to test this model is to examine the effect of increased intracellular LC-CoA on lipolytic oscillations. We hypothesized that an increase in FFA within the adipocyte would decrease lipolysis and lipolytic oscillations, whereas a decrease in FFA would increase lipolysis and lipolytic oscillations. Increasing concentrations of exogenous FFA significantly suppressed both lipolysis and lactate release in a concentration-dependent manner, consistent with suppression due to increased cytosolic LC-CoA. And more importantly, exogenous FFA suppressed the amplitude of both lipolytic and lactate oscillations.

In a further test of the model, we examined the effect of increasing the percent BSA of the perifusion buffer on stimulated lipolysis and lipolytic oscillations. Increasing the percent BSA significantly stimulated lipolysis and the amplitude of lipolytic oscillations in a concentration-dependent manner. These results suggested that in conditions of low BSA, increased intracellular FFA, due to lipolytic stimulation, inhibited lipolysis and lipolytic oscillations due to increased LC-CoA. Increasing BSA concentrations outside the cell allowed for increased outward movement of FFA and thus diminished the LC-CoA inhibition of lipolysis. These results support our hypothesis and suggest that in situations of high plasma FFA, LC-CoA inhibition of TGL may play a role in preventing further increases in plasma FFA.

Although all the above-mentioned experiments support our hypothesis of glycolytically driven oscillatory LC-CoA regulation of triglyceride lipase(s) within the adipocyte, there are other mechanisms for oscillatory lipolysis that should be considered. It is possible that the intracellular levels of {alpha}-GP do not oscillate but remain constant. {alpha}-GP can be formed from glyceroneogenesis (mostly during fasting) (42) as well as glycolysis, and it is not known whether glyceroneogenesis oscillates. If this is the case, then it is possible that the process of fatty acid recycling within the adipocyte oscillates per se. Or it is possible that perhaps both the glycolytic and lipolytic oscillations are entrained by another common oscillator within the cell (for example, oscillatory mitochondrial metabolism).

One question that arises is how the isolated adipocytes are synchronized to release FFA, glycerol, and lactate in an oscillatory manner. One possibility is that the released FFA act in a paracrine manner to synchronize neighboring cells. Another possibility is that the proximity of the cells to each other allows for cell-to-cell communication, as has been suggested in perifusion studies measuring insulin secretion (43). Also, a recent study by Deeney et al. (29) showed synchronous oscillations in insulin release among clonal ß-cells grown in separate wells. These cells were physically separated from each other and yet were stimulated by glucose to act in unison. Similar results were shown by Lipkin et al. (24), who showed that the oscillations in glycolysis in isolated adipocytes were not apparent until a step-jump in insulin or hydrogen peroxide was imposed.

Although the role of lipolytic and glycolytic oscillations in adipose tissue is unknown, we anticipate that they may be important in maintaining normal fuel homeostasis and insulin sensitivity in various tissues. FFA oscillations may prevent insulin resistance and promote insulin secretion. It is not yet known whether FFA oscillations are lost or diminished in type 2 diabetes. Lipkin et al. (24) suggest that the oscillations of glycolysis in fat cells may allow metabolic efficiency to be optimized. This may also be true for lipolytic oscillations, in that delivery of FFA in an oscillatory pattern to tissues such as the liver and the ß-cell may be beneficial, providing the optimum balance between FFA and glucose uptake and metabolism, thus contributing to the maintenance of glucose homeostasis.

In conclusion, there are basal intrinsic lipolytic oscillations present within adipocytes with a pulse duration of 5 min. The lipolytic oscillations depend on glucose and are regulated by FFA. It is hypothesized that lipolytic oscillations are driven by oscillatory glucose metabolism, which leads to oscillatory removal of LC-CoA inhibition of TGL. The results of this study contribute to the understanding of physiological and biochemical regulators of lipolysis, such as glucose and FFA. It is anticipated that lipolytic oscillations in adipose tissue may play an important role in the delivery of FFA to liver, ß-cells, muscle, and other tissues.


   ACKNOWLEDGMENTS
 
This work was supported by the National Institutes of Health (DK56690 and 1F32DK10110–01).

The authors extend extreme gratitude to their colleagues.

Address correspondence and reprint requests to Barbara E. Corkey, PhD, Obesity Research Center, Boston Medical Center, EBRC 840, 650 Albany St., Boston, MA 02118. E-mail: bcorkey{at}bu.edu

Received for publication April 5, 2004 and accepted in revised form December 7, 2004

Abbreviations: {alpha}-GP, {alpha}-glycerophosphate; FFA, free fatty acid; HSL, hormone-sensitive lipase; KRBB, Krebs-Ringer bicarbonate buffer; LC-CoA, long-chain coenzyme A; TGL, triglyceride lipase(s)


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Milburn JL, Hirose H, Lee YH, Nagasawa Y, Ogawa A, Ohneda M, BeltrandelRio H, Newgard CB, Johnson JH, Unger RH: Pancreatic ß-cells in obesity: evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. J Biol Chem270 :1295 –1299,1995[Abstract/Free Full Text]
  2. McGarry JD, Dobbins RL: Fatty acids, lipotoxicity, and insulin secretion. Diabetologia42 :128 –138,1999[Medline]
  3. McGarry JD: Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes51 :7 –18,2002[Free Full Text]
  4. Prentki M, Corkey BE: Are the ß-cell signaling molecules malonyl-CoA and cytosolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes45 :273 –283,1996[Abstract]
  5. Boden G, Chen X: Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes. J Clin Invest96 :1261 –1268,1995
  6. Kim JK, Wi JK, Youn JH: Plasma free fatty acids decrease insulin-stimulated skeletal muscle glucose uptake by suppressing glycolysis in conscious rats. Diabetes45 :466 –453,1996
  7. Boden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes45 :3 –10,1996
  8. Getty L, Panteleon AE, Mittelman SD, Dea MK, Bergman RN: Rapid oscillations in omental lipolysis are independent of changing insulin levels in vivo. J Clin Invest106 :421 –430,2000[Medline]
  9. Pincus SM, Keefe DL: Quantification of hormone pulsatility via an approximate entropy algorithm. Am J Physiol262 :E741 –E754,1992
  10. Pincus SM, Goldberger AL: Physiological time-series analysis: what does regularity quantify? Am J Physiol266 :H1643 –H1656,1994
  11. Hucking K, Hamilton-Wessler M, Ellmerer M, Bergman RN: Burst-like control of lipolysis by the sympathetic nervous system in vivo. J Clin Invest111 :257 –264,2003[Medline]
  12. Marchetti P, Scharp DW, Mclear M, Gingerich R, Finke E, Olack B, Swanson C, Giannarelli R, Navalesi R, Lacy PE: Pulsatile insulin secretion from isolated human pancreatic islets. Diabetes43 :827 –830,1994[Abstract]
  13. O’Meara NM, Sturis J, Blackman JD, Byrne MM, Jaspan JB, Roland DC, Thistlethwaite JR, Polonsky KS: Oscillatory insulin secretion after pancreas transplant. Diabetes42 :855 –861,1993[Abstract]
  14. Longo EA, Tornheim K, Deeney JT, Varnum BA, Tillotson D, Prentki M, Corkey BE: Oscillations in cytosolic free Ca++, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets. J Biol Chem266 :9314 –9319,1991[Abstract/Free Full Text]
  15. Chen I, Golay A, Swislocki LM, Reaven GM: Resistance to insulin suppression of plasma free fatty acid concentrations and insulin stimulation of glucose uptake in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab64 :17 –21,1987[Abstract]
  16. Rodbell M: Metabolism of isolated fat cells. J Lipid Res22 :375 –380,1964
  17. Turpin BP, Duckworth WC, Solomon SS: Perifusion of isolated rat adipose cells. J Clin Invest60 :442 –448,1977
  18. Hansen BC, Jen KC, Pek SB, Wolfe RA: Rapid oscillations in plasma insulin, glucagon, and glucose in obese and normal weight humans. J Clin Endocrinol Metab54 :785 –792,1982[Abstract]
  19. Barhan D, Trinder P: An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst97 :142 –145,1972[Medline]
  20. Lang DA, Matthews DR, Burnett M, Ward GM, Turner RC: Pulsatile, synchronous basal insulin and glucagon secretion in man. Diabetes31 :22 –26,1982[Abstract]
  21. Goodner CJ, Walike BC, Koerker DJ, Ensinck JW, Brown AC, Chideckel EW, Palmer J, Kalnasy L: Insulin, glucagon, and glucose exhibit synchronous, sustained oscillations in fasting monkeys. Science195 :177 –179,1977[Abstract/Free Full Text]
  22. Veldhuis JD, Johnson ML: Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection. Am J Physiol250 :E486 –E493,1986
  23. Van Cauter E: Estimating false-positive and false-negative errors in analyses of hormonal pulsatility. Am J Physiol254 :E786 –E794,1988
  24. Lipkin EW, Teller DC, Haen C: Dynamic aspects of insulin action: synchronization of oscillatory glycolysis in isolated perifused rat fat cells by insulin and hydrogen peroxide. Biochemistry22 :792 –799,1983[Medline]
  25. Andres V, Schultz V, Tornheim K: Oscillatory synthesis of glucose 1,6-bisphosphate and frequency modulation of glycolytic oscillations in skeletal muscle extracts. J Biol Chem265 :21441 –21447,1990[Abstract/Free Full Text]
  26. Tornheim K: Are metabolic oscillations responsible for normal oscillatory insulin secretion? Diabetes46 :1375 –1380,1997[Abstract]
  27. Werrlein RJ, Glinos AD: Oxygen microenvironment and respiratory oscillations in cultured mammalian cells. Nature251 :317 –319,1974[Medline]
  28. Corkey BE, Tornheim K, Deeney JT, Glennon C, Parker JC, Matschinsky FM, Ruderman NB, Prentki M: Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract. J Biol Chem263 :4254 –4258,1988[Abstract/Free Full Text]
  29. Deeney JT, Kohler M, Kubik K, Brown G, Schultz V, Tornheim K, Corkey BE, Berggren PO: Glucose-induced metabolic oscillations parallel those of Ca2+ and insulin release in clonal insulin-secreting cells. J Biol Chem276 :36946 –36950,2001[Abstract/Free Full Text]
  30. Nillson T, Schultz V, Berggren PO, Corkey BE, Tornheim K: Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate and cytoplasmic free calcium in glucose-stimulated pancreatic ß-cells. Biochem J314 :91 –94,1996
  31. McMahon RJ, Frost SC: Glycogen: a carbohydrate source for GLUT-1 glycosylation during glucose deprivation of 3T3–L1 adipocytes. Am J Physiol270 :E640 –E645,1996
  32. Fassina G, Dorigo P, Perini G, Toth E: Effect of glycolysis inhibitors on cyclic AMP synthesis in rat adipose tissue. Biochem Pharma21 :2295 –2301,1972[Medline]
  33. Giudicelli Y, Pecquery R, Provin D, Agli B, Nordmann R: Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells. Biochim Biophys Acta486 :385 –398,1976
  34. Angel A, Desai K, Halperin ML: Free fatty acid and ATP levels in adipocytes during lipolysis. Metabolism20 :87 –99,1971[Medline]
  35. Burns TW, Langley PE, Robison GA: Site of free-fatty-acid inhibition of lipolysis by human adipocytes. Metabolism24 :265 –276,1975[Medline]
  36. Fain JN, Shepherd RE: Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells. J Biol Chem250 :6586 –6592,1974
  37. Jepson CA, Yeaman SJ: Inhibition of hormone-sensitive lipase by intermediary lipid metabolites. FEBS Lett310 :197 –200,1992[Medline]
  38. Jenkins CM, Mancuso DJ, Yan W, Sims HF, Gibson B, Gross RW: Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J Biol Chem279 :38079 –39186,2004[Free Full Text]
  39. Soni GK, Lehner R, Metalnikov P, O’Donnell P, Semache M, Gao W, Ashman K, Pshezhetsky AV, Mitchell GA: Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase. J Biol Chem279 :40683 –40689,2004[Abstract/Free Full Text]
  40. Okazaki H, Osuga J, Tamura Y, Yahagi N, Tomita S, Futoshi S, Iizuka Y, Ohashi K, Harada K, Kimura S, Gotoda T, Shimano H, Yamada N, Ishibashi S: Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases. Diabetes51 :3368 –3375,2002[Abstract/Free Full Text]
  41. Harada K, Shen W-J, Patel S, Natu V, Wang J, Osuga J-I, Ishibashi S, Kraemer FB: Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice. Am J Physiol285 :E1182 –E1195,2000
  42. Forest C, Tordjman J, Glorian M, Duplus E, Chauvet G, Quette J, Beale EG, Antoine B: Fatty acid recycling in adipocytes: a role for glyceroneogenesis and phosphoenolpyrubate carboxykinase. Biochem Society Transactions31 :1125 –1129,2003
  43. Cunningham BA, Deeney JT, Bliss CR, Corkey BE, Tornheim K: Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal B-cells (HIT). Am J Physiol271 :E702 –E710,1996

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
L. Hu, J. T. Deeney, C. J. Nolan, M.-L. Peyot, A. Ao, A. M. Richard, E. Luc, N. J. Faergeman, J. Knudsen, W. Guo, et al.
Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes
Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E1085 - E1092.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Getty-Kaushik, L.
Right arrow Articles by Corkey, B. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Getty-Kaushik, L.
Right arrow Articles by Corkey, B. E.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum