Perifused Fat Cells

of changes in in the rate of lipolysis of alterations in the rates of glycerol the initiation of lipolysis. the in glycerol

The perifused fat cell system utilized in this investigation offers a simple, reproducible method by which minute-to-minute changes in the rate of lipolysis of isolated fat cells may be monitored. In this technique, isolated fat cells are placed in a plastic column and perifused with albumin-containing buffer in the absence and presence of various lipolytic agents.
Using this technique, we have been able to observe alterations in the rates of glycerol release during the initiation of hormone-induced lipolysis. In addition, we have been able to observe the decline in glycerol release following the removal of the lipolytic hormone.
The combined administration of theophylline and epinephrine, both at submaximal concentrations, resulted in rates of glycerol release which were significantly greater than the sum of the rates observed during individual administration.
Considerable emphasis in the last decade has been placed on the elucidation of the mechanisms involved in the hormonal * This invcstjgation was supported by National In vitro experiments have been conducted by incubating intact adipose tissue or isolated adipocytes in the presence of various stimulatory agents and measuring the release of fatty acids, or glycerol, or bot,h into the incubation medium.
Although utilization of the incubation technique has made it, possible to observe hormonally induced changes in lipolysis, it, has been difficult to observe the rates of lipolysis during the activation process on a minute-tominute basis. In addition, it has been impossible to follow the changes in lipolytic rates occurring after the removal or cessation of hormonal stimulus.
These limitations have recentl) been overcome with the development of a perifused fat cell technique (1). Using glycerol release as an index of lipolysis, it is possible to measure the rates of lipolysis during both the activation and inactivation phases of hormone-induced lipolysis. This technique offers the advantage of being able to follow sequentially the release of glycerol by isolated fat cells at time intervals as short as 5 s.
The purpose of the present investigation was to determine the ability of various agents to stimulate lipolysis in the perifusrtl fat cell system. III addition, this study was undertaken to examine the rates of change in glycerol production occurring during the initiation and cessation of lipolysis caused by various stimulatory agents. Perifusion of Fat Cells-Isolated fat cells were perifused as previously described (I). Briefly, 2 ml of packed cells were placed in a water-jacketed plastic column maintained at 37" and Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1.0% (w/v) albumin maint'ained at 37" under an atmosphere of 95% 0~~5% COZ was pumped into the top of the column at a flow rate of 10 ml per min. The fat cells were perifused for a IO-min equilibration period after which several samples were taken over the nest 10 min for t'he determination of basal rates of lipolysis. The lipolytic agent to be tested was then infused into the cell chamber by means of an injection port located at the top of the column.
The perfusate was collected at the bottom of the column over I-min intervals.
Anal&al Procedures-The concentration of glycerol in the perfusate was determined by the fiuorometric method of Chernick (4). Protein content of the isolated fat cells was determined by the method described by Lowry et al. (5).

EJect of illbunlin
on Lipolysis-The results obtained when isolated fat cells were perifused with buffer containing different concentrations of albumin in the presence of epinephrine (final concentration 1~1%) are presented in Fig. 1. In order to establish the bnsal rate of lipolysis, the cells were first perifused with buffer containing 0.5% (W/V) albumin for 10 min before the start of epincphrine infusion. Following the start of infusion of epinephrine, there occurred an increased rate of glycerol release which n-ached a ljlateau value in approximately 20 min. When the perifnsing buffer was changed to one containing 1.0% (w/v) albumin, there occurred a substantial increase in the lipolytic rate which reached a plateau value in approximately 10 min. Increasing the albumin concentration to 2.0% (K/V) resulted in a small additional increase in glycerol release. In other csperiments with epincphrinc-stimulated cells, when the perfusing medium was &tched from an albumin-containing to an albumin-free buffer, the rate of glycerol release decreased pre- cipitously to near basal levels. The experimental results in the following sections were obtained with buffer cont'aining l.OyO (w/v) albumin.
Epinephrine-stimulated Lipolysis-Rates of glycerol release obtained during the initiation and termination of cpinephrincstimulated lipolysis are recorded in Fig. 2. After a IO-min period of perifusion with drug-free buffer, epinephrine was infused into the cell chamber such that the final concentration was 10 PM. This concentration of epinephrine produced the maximal rate of lipolysis.
Following the start of infusion of epincphrine there was a sharp increase in the rate of lipolysis.
The lipolytic rate continued to increase until a peak response was attained al)proximately 20 min after the start of epinephrine administration. Upon cessation of the cpinephrine infusion, the rate of lipolysis returned to basal rates within 20 to 25 min.
Theophylline-stimulated Lipolysis-Rates of glycerol release obtained during the initiation and termination of theophyllineinduced lipolysis are recorded in Fig. 3. After establishing the basal rate of liyolysis, the fat cells were perifuscd with buffer containing theophylline at a final concentration of 1 mM. The masimal rate of lipolysis att'ainablc with theophylline was achieved with 1 mM concentration.
Following the start of thcophyllinc perifusion, there occurred a sharp increase in the rate of lipolysis. The peak lipolytic response was attained in approximately 20 min after the initiation of theophylline administration. Upon switching to theophylline-free buffer, the rate of glycerol release returned to basal levels within a IO-min period.
With this concentration of theophylline (1 mM), the inactivation of lipolysis appeared to proceed at a faster rate than the activation of lipolysis.
Dose Response Curves Obtained with Various Lipolytic Agents-In order to determine whether the lipolytic response T\-as doscdependent in the perifusion system, studies wcrc undertaken to determine dose-related responses with various known lipolytic agents, the results of which arc depicted in Figs. 4 and 5. Fig.  4 represents the alterations in the rate of lipolysis caused by sequential increases in the concentration of theophyllinc. After pose of this portion of the study was to demonstrate the ability of these agents to stimulate lipolysis in the perifused fat cell system. Like the response seen with the other tested lipolytic agents, there was a sharp increase in the rate of lipolysis after the administration of these polypeptide hormones.
The most outstanding observation with these agents was the ability of GH to function as an effective lipolytic stimulant in the perifused fat cell system. At the concentration employed (20 pg per ml), lipolysis was rapidly stimulated and the maximal rate of lipolysis was attained in 20 min after the initiation of GH administration.
Also included in Table II are the results obtained when isolated fat cells were perifused with serotonin (final concentration 1 mM). At this concentrat'ion, serotonin did not stimulate lipolysis.
Those cells which were refractory to serotonin administration were stimulated by epinephrine, thereby indicating that serotonin, at the concentration employed, does not exert lipolytic action in the perifused fat cell system. 8 Concentrations-Glycerol release by isolated cells when epinephrine and theophyllinc, both at submaximal concentrations, were infused individually and simultaneously have been recorded in Fig. 7. The infusion of cpinephrine (final concentration 0.05 PM) for 20 min resulted in ;I difference (peak value -basal value) of I6 nmoles of glycerol per min per mg of protein.
The infusion of theophyllinc (final concentration 10 PM) for a 20-min 1:eriod resulted in a change of 16 nmolcs of glycerol per min per mg protein.
When epincphrine and theophylline were infused together, the peak response was not obtained until approximately 30 min after the start of infusion.
However, there was a difference of 47 nmoles of glycerol per min per mg of protein after 20 min of infusion.
This value was considerably larger than the sum of the differences obtained individually wit'h epincphrine and theophylline over 20.min infusion periods.
Not,e that the difference obtained at any time after the start of the combined infusion was much greater than the sum of the differences of the indi\-idual iufusions at the same -I -I i 1 . doses of epinephrine and theophylline on glycerol release in the perifused fat cell. Two milliliters of packed isolated fat cells were perifused as described under "Experimental Procedure." P:pinephrine (final concentration 0.05 by) was infused for 20 min after which the rates of glycerol production were allowed to return to basal levels. Theophylline (final concentration 10 PM) was then infused for a 20.min period.
Upon the return to basal levels. both euineDhrine and theouhvlline were simultaneouslg inf&d for 'a 36.min period. G&erol is expressed as nanomoles of glycerol released per min per mg of protein content of the cells.

time.
Also the rate of activation of lipolysis, as determined by the slope of the linear portion of the activation curves, in the combined infusion was substantially greater than the sum of the rates of activation seen with the individual administrations of epinephrine and thcophylline. The result,s of this experiment were readily reproducible with different populations of isolated cells. When the same cspcriment was carried out with a submaximal concentration of epinephrine and a maximal concentration of theophylline, a greater than additive effect was not observed. In this experiment, 0.05 PM epinephrine produced a difference of 7 nmoles of glycerol per min per mg of protein, and 1 rnM theophylline produced a difference of 61 nmoles of glycerol per min per mg of protein.
When both drugs were administered simultaneously, the difference in glycerol production was 60 nmoles per min per mg of prot'ein. UISCUSSION The pcrifused fat cell system, in addition to enabling one to observe sequentially lipolytic rates in adipocytes during the activation and inactivation of hormone-induced lipolysis, has for the most part generated results comparable to those obtained in the incubated tissue met'hod.
Catecholamines, ACTH, glucagon (6), and TSH (7) have been shown to be effective lipolytic agents in incubated rat epididymal fat pads. The results of the present investigation indicate t,hat these substances also activat'e lipolysis in the perifused fat cell system. In contradistinction, the results obtained with GH in t'he perifused fat cell system are at variance with results obtained in the incubation method. It has been reported (8) that GH had little effect on the release of fatty acids from isolated fat cells. It was also reported that in the combined presence of GH and glucocorticoid, there was a 2-hour lag period before any significant lipolytic response was observed.
In the perifused fat cell, GH in the absence of glucocorticoid rapidly increased the lipolytic rate and maximal activation was obt'ained within 20 min after the administration of the hormone. To rule out the possibility that the preparation of GH used in this study may have been contaminated with ACTH, TSH, or other lipolytic peptides which were responsible for the noted lipolytic effect, the preparation was tcstcd for lipolytic activity using the flask incubation method.
After incubating isolated fat cells in the presence of GII at a final concentration of 20 pg per ml for 1 hour, there was no change in glycerol release from the control values. Therefore, it would appear that the lipolytic response observed in the perifused fat cell system with this particular GH preparation cannot be observed bp conventional incubation methods.
It is possible that an inhibitor substance accumulates in the incubated tissue method and this may be washed out in the perifused fat cell system thereby rnabling GH to exert its lipolytic action.
The incubation of adipose tissue with cyclic AMP, which has been implicated as an intracellular mediator of hormone-stimulated lipolysis, has generally resulted in a lack of lipolytic response. In epididymal fat pads, exogenous cyclic AMP has been reported to inhibit lipolysis (9) and counteract the lipolytic effect of cpinephrine (10). However, lipolysis in fat cells and fat pad fragments can be stimulated by cyclic AMP when incubated in a simple phosphate-0.9c/d XaCl medium (11). In the perifused fat cell system, cyclic ~0IF' was found to hare no effect on the basal rate of lipolysis.
In contrast, dibutyryl cyclic AMP, an effective lipolytic agent in incubated fat pads and isolated fat cells (la), substantially stimulated lipolysis in the perifuscd fat cell system. The greater cfflcacy of dibut'yryl cyclic AMP has been attributed to its resistance to degradation by phosphodiesterase and it's greater cell permeability (13). Referring to the results contained in Fig. 6, there n-as a 6-min lag period between the administration of dibutyryl cyclic AMP and the initial increase in the rate of lipolgsis.
It may be speculated that during this period, dibutyryl cyclic AMP was permeating the cell membrane and thereby increasing its int,racellular concent,ration to effective stimulatory levels. Thcophylline has been reported lo act synergistically with catecholamines in the in vitro stirnulation of lipolysis (14-16) and elevation of cyclic AMP levels (17) in adipose tissue. In the present study, the rate of glycerol release observed in the presence of theophylline and epinephrine at submaximal concentrations was greater than the sum of the rates of glycerol release observed during the administration of theophylline or cpincphrine alone. This greater than additive effect was also observed when the initial rates of act'ivation of lipolysis were compared. Similar to results obtained in the incubation method (14,15), the rate of lipolysis obtained with a maximal concentration of theophylline in the pcrifused system could not be further increased by epinephrine.
Thus, again, results obtained in the perifused fat cell system parallel results obtained in the tissue incubation technique.
The unparalleled feature of the perifused fat cell systern resides in the ability to measure sequentially rates of lipolysis over short intervals of time. An important, point to be made at this time is that the lipolytic rates observed during the activation and inactivation phases of hormone-induced lipolysis in this system appear not to be dependent upon drug equilibration and washout from the cell chamber, as evidenced by previous observations with labeled epinephrine (1).
Conflicting reports exist in the literature concerning the rate at which catecholamines activate the lipolytic process. Angel et al. (18) and Kuo and De Renzo (19) hare published data indicating a 5-to IO-min delay before norepinephrine stimulates lipolysis in isolated fat cells while >Ianganiello et al. (20) have indicated that maximum rates of lipolysis are observed within 1 min of addition of epinephrine.
Our data with the perifused fat cell system indicate that epinephrine produces an immediate stimulation of lipolysis but 10 to 15 min are required before maximum rates are obtained. This is in agreement with the work of Scow (21) using perfused rat adipose tissue which showed that fatty acid release was greater in the second lo-min period than in the first 10 min after addition of epinephrine.
With the perifused fat cell system it is possible not only to determine the maximal rate of lipolysis attainable with various lipolytic agents, but also to examine the rate of activation of lipolysis during the initiation process. More importantly, prior to the development of this system, methods were not available for observing the decline in rates of lipolysis during the inactivation process. Thus, the development of the perifused fat cell system should prove to be useful in elucidating the biochemical events occurring during the initiation, continuance, and cessation of hormone-induced lipolysis.