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The role of free fatty acids in the regulation of lipolysis by human adipose tissue cells
The Role of Free Fatty Acids in the Regulation Lipolysis by Human Adipose Tissue Cells Thomas The
effect
of added
and cyclic pose of
AMP
tissue
sodium
response
ceils
was
Inhibition quantity
to 1V7
was
and linoleic
of added acids
were
acid in suppressing Suppression
adi-
tion was detectable the
addition
addition
tion, suppressed
the
lipolytic
could
M
isoprote-
fresh
with
cumulated
added,
0.2
the mM,
not
buffer
with
increas-
stimulated
fatty
acid.
Palmitic
findings
as oleic
fatty
isoproterenol-stimulated of cyclic
AMP
fatty
change, by are
acids
regulators
AMP
acids
acids,
addition
after
were
after forma-
fatty
by the
However,
the
removed
cyclic
AMP
fresh
isoproterenol.
consistent are
one minute
Cyclic
by accumulated
be stimulated
isoproterenol.
greater
as effective
within
of oleate.
The
detectable
of oleate
Boyd E. Terry, and G. Alan Robinson
on lipolysis
of human
studied.
progressively
ing quantities
acid
decreased
of adipocytes
lowest
lipolysis.
fatty
concentration
oleate
renol.
and was
W. Burns, Paul E. Langley.
of
formation
with
physiologically
the
of acby was
These view
that
significant
of lipolysis.
forma-
I
T IS WELL KNOWN that the accumulation of free fatty acids (FFA) inhibits the rate of lipolysis by adipose tissue. ‘s2 This effect is diminished in the presence of albumin because albumin binds FFA, reducing their inhibitory effect until the albumin binding sites are saturated. Studies have been done in our laboratory that indicate that at least one aspect of the action of FFA involves the inhibition of cyclic AMP formation.:’ Preliminary observations suggested that the inhibitory effect of FFA commences at concentrations of FFA less than that required to saturate albumin binding sites. The present studies were undertaken to obtain additional information on this and other points that bear on the question of whether or not FFA inhibition of lipolysis might be physiologically significant. MATERIALS
AND METHODS
Isolated adipocytes were prepared from subcutaneous tissue samples obtained from patients undergoing abdominal surgery. The method of preparing cells, incorporating modifications of Rodbell’s procedure,4 has been described.5 Cells were suspended in Krebs bicarbonate buffer containing 2.5 mM glucose and 4% bovine serum albumin. Flasks containing aliquots of cell suspensions were incubated with gentle shaking in an atmosphere of 95% 0, and 5% CO,. For the determination of cyclic AMP, test substances were incubated in IO ml of cell suspension. At the end of incubation, the contents of each incubation flask were quick-frozen in liquid nitrogen. Extracts were prepared and purified essentially as described by Murad et al.6 and cyclic AMP content determined by radioimmunoassay.’ Cyclic AMP content was expressed as pmole/g of triglyceride. To evaluate their effects on lipolysis, test substances were incubated with 1 ml of cell suspension in each of 3 to 6 flasks. Following incubation, cell-free filtrates were prepared and analyzed for glycerol and FFA using the methods of Garland and RandIe and Dole,g respectively. Concentrations of glycerol and FFA were expressed as gmolefg of triglyceride. Since it has been shown that human adipocytes have both cy- and @-adrenergic receptor sites,l”,ll rsoproterenol, a pure fi agonist, was chosen to stimulate cyclic AMP forma-
From the Division of Endocrinology and Metabolism, Department of Medicine and the Department of Surgery, Universit_v of Missouri School of Medicine, Columbia, MO.; and The Department of Pharmacology, University of Texas MedicalSchool. Houston. Tex. Receivedforpublication January 18. 1978. Supported b.v USPHS Grants Am I1265 and HL 16552. Address reprint requests to Thomas W. Burns. M.D.; Department oJMedicine. Division of Endocrinology and Metabolism, University of Missouri, School of Medicine, Columbia, MO. 65212. (B 1978 by Grune & Stratton, Inc. 0026~0495/78/2712-OOO$Ol.OO/O Metabolism, Vol. 27, No. 12 IDecemberJ. 1978
1755
BURNS
1756
0
isoproterenol-stimulated lipolysis. Human cells were incubated for 4 hr in Krebs buffer containing 4% albumin and either 10-xM, lo-‘M, or 10mhM isoproterenol. The concentration of FFA was increased by adding sodium oleate at the beginning of incubation. Each point represents the mean fatty acid concentration of three flasks. The smallest quantity of added FFA, 1.5 mM. reduced lipolysis stimulated by each concentration of isoproterenol.
3
2 1 ADDED OLEATE, mM
tion and lipolysis. The use of the physiologic cathecholamines, lead to problems in interpretation For experiments Krebs-Ringer
requiring
bicarbonate
epinephrine
and norepinephrine,
could
since these substances have both CYand 6 agonist properties.
added
FFA,
sodium oleate,
palmitate
or linoleate
was emulsified
in
solution and then put in solution by the addition of powdered albumin. The
albumin buffer with added FFA
was dialyzed
buffer was 7.4. The quantity of FFA
twice against bicarbonate
buffer. The final pH of the
released by cells during incubation was calculated
the initial FFA content of the cell suspension (added FFA) FFA).
ET AL
FFA values were obtained by titration
by subtracting
from the tinal content (added and released
before and after incubation. RESULTS
The Efect
of Varying FFA Concentrations
on FFA Inhibition
~~/‘Lipol~~.si.s
To gain insight into the quantity of FFA required to affect lipolysis, a series of experiments were done in which the concentrations of both isoproterenol and added oleate were varied. The quantity of FFA present in the media at 4 hr was determined and corrected for the quantity of FFA added. The results of a representative experiment are shown in Fig. I. The smallest quantity of added oleate, 1.5 mM, consistently reduced lipolysis stimulated by IO-‘. IO-‘, and IO-” M isoproterenol. Subsequent experiments were done employing only one concentration of isoproterenol, IO-’ M and smaller increments of added oleate, from 0.2 to 1.4 mM. The results of four experiments are shown in Fig. 2. The lowest concentra-
40. Fig. 2. The effect of low concentrations of added FFA on lipolysis stimulated by 10-‘M isoproterenol. Human cells were incubated for 4 hr in Krebs buffer 4% albumin contained and isoproterenol. The concentration of FFA wes increesed by adding sodium oleate at the beginning of incubation. The results are expressed as the percent inhibition of lipolysis achieved by added FFA. Each point represents the mean value of four experiments. The lines above and below each point represent the standard error of the mean.
30PER CENT INHIBITION OF FFA RELEASE
20_
lo-
O0
0.5 ADDED
1.0 OLEATE,
1.5 mM
FFA IN THE REGULATION
OF LIPOLYSIS
1757
50 Fig. 3. Comparison of the effect of added sodium paimitate. linolate and oleate on FFA release stimulated by lo-‘M isoproterenol. Human cells were incubated for 4 hr in Krebs buffer containing 4% albumin and isoproterenol. The concentration of FFA was increased by adding FFA at the beginning of incubation. The results are expressed as a percent inhibition of FFA release achieved by added FFA. Each point represents the mean fatty acid concentration of three flasks.
40 PER CENT 30 INHIBITION OF FFA go RELEASE 10 0 0
0.5 ADDED FREE FATTY
1.0
1.5
ACID, mM
tion of added oleate, 0.2 mM, appeared to suppress lipolysis; this effect was progressive with increasing quantities of added FFA and was greatest with the highest concentration of oleate employed, I .5 mM. The E#ect of Palmitic and Linoleic Acids on Lipolysis
All previous experiments concerned with the effect of added FFA on lipolysis had been done with oleic acid, the most abundant fatty acid in human plasma. To determine the effectiveness of other fatty acids in inhibiting lipolysis, palmitic and linoleic acids were incubated with adipocytes in the presence of isoproterenol, lo-’ M; FFA released into the medium was estimated at the end of 4 hr. The results of typical experiments are shown in Fig. 3. Both palmitic and linoleic acids were essentially equal to oleic acid in suppressing lipolysis. The Duration of Exposure to FFA Required to Aflect Cyclic AMP Formation
To assess the exposure time to FFA required to inhibit cyclic AMP formation, it was first necessary to define how quickly human adipocytes respond to catecholamines such as isoproterenol. In a majority of other previous studies, cyclic AMP had been measured after a 30 min exposure to test substances; glycerol and FFA released into the media were estimated at 4 hr. To gain more precise information on the rapidity of the human adipocyte response, intracellular cyclic AMP was measured at various times between 0 and 10 min after the addition of
c-AMP Pmols/g
1,500 1,000
a 0
2
4 6 TIME, min
B
10
Fig. 4. Alterations in cyclic AMP concentration following the addition of isoproterenol. 1O+M. Human cells were incubated for varying times from. 0 to 10 min in Krebs buffer containing 4% albumin and isoproterenol. The results of four experiments are plotted. An increase in cyclic AMP was evident by 1 min.
BURNS
1758
GLYCEROL wols/g
ET AL
Fig. 5. Alterations in glycerol release following the addition of isoproterenol 1 O-‘hl. Human cells
0.6-
0.3-
2
0
6 4 TIME, min
6
isoproterenol. The results of four are plotted. An experiment5 increase in buffer glycerol was evident by 1 min.
10
isoproterenol, IO-“M. Representative results are given in Fig. 4. By I min, there was a substantial rise in cyclic AMP; this response increased over the ensuing 10 min. In other experiments, glycerol release was measured at frequent intervals from 0 to 10 min after the addition of isoproterenol. A definite increase in glycerol in the buffer was evident as early as I min (Fig. 5). To more closely define the time exposure required for FFA to influence cyclic AMP formation, aliquots of cells were exposed to added oleate for 5, 3, I, and 0 min, respectively. All flasks contained isoproterenol, lo-“M. The results of two such experiments are given in Table 1. At 1 min, exposure to added oleate reduced cyclic AMP accumulation stimulated by isoproterenol by approximately 10%. A 5-min exposure resulted in a reduction in cyclic AMP of 60%. Response of‘Suppressed
Cyclic AMP to Fresh Isoproterenol
Conceivably, the low level of cyclic AMP noted after 3 ‘2-4 hr of incubation noted in earlier experiments” could be due to loss of activity of isoproterenol rather than to an impaired responsiveness of the cyclic AMP generating system. To evaluate this possibility, aliquots of cells were incubated for 4 hr with isoproterenol, 10-5M; to one set of flasks, fresh isoproterenol was added at 312 hr. Cyclic AMP concentration in the two groups was essentially the same: 600 pmole/g in the group to which no fresh isoproterenol had been added and 580 pmole/g in the group to which additional catecholamine had been added (Fig. 6). Table 1. Exposure Time Required for FFA Inhibition of Isoproterenol-Stimulated Cyclic AMP Formation Exposure Time IminI ISOprOterenOl (lo-”
Cyclic AMP. pm&/g Ole.3t.Z
Ml
Experiment
(2 5 mM)
third
oleate
Experwnent 2
0
5
0
12726
12111
5
1
10909
11104
5
3
6698
7972
5
5
4032
3616
49
Human cells were incubated for 5 min in Krebs buffer Sodium
1
0
was
added
to one set of flasks
set at 4 min. The results
of 2 experiments
containing
at the beginning are given.
4%
a7
albumin
of incubation,
and
to another
isoproterenol.
lo-’
M.
set at 2 mm and to a
FFA IN THE REGULATION
OF LIPOLYSIS
1759
Fig. 6. The effect of fresh isoproterenol and buffer change on cyclic AMP concentration (left panel) and on glycerol accumulation (right panel). Human ceils were incubated in Krebs buffer containing 4% albumin and. except for control flasks, isoproterenol 10-5M. The height of each bar represents the mean (*SEMI of four experiments. (A) control before incubation; (6) half-hour incubation; (Cl 4 hr incubation; and (D) 4-hr incubation with fresh isoproterenol, 10m5M added at 3’ :! hr; (E) 4-hr incubation, with buffer change and fraseh isoproterenol added at 3’9 hr.
Reversibility
ABCDE
of Cyclic AMPSuppression
Associated
A
B
C
D
E
with FFA Accumulation
To test the reversibility of the suppressed cyclic AMP generation that occurred with FFA build-up during prolonged incubation, cells were incubated with lo-“M isoproterenol for 3!+ hr; they were then transferred to fresh buffer and isoproterenol and incubated for an additional half hour. Other aliquots of cells were incubated for half hour and 4 hr in the usual manner without buffer change. Cells and buffer from all flasks were frozen for subsequent cyclic AMP assay. The mean and standard error of the mean of 3 experiments are shown in Fig. 6. As noted before, the cyclic AMP concentrations in flasks undergoing 4-hr incubation was substantially less than that noted in flasks incubated for a half hour, 600 pmole/g and 8,800 pmole/g, respectively. The flasks incubated for 4 hr but with fresh buffer provided at 31s hr had a mean cyclic AMP concentration of 3300 p mole/g. DISCUSSION In studies previously reported,” dose response experiments were done in which the effects of oleate in concentrations varying between 0.6 and 3.0 mM on cyclic AMP formation were observed. Only a slight inhibitory effect was found until a concentration of 2.5 mM oleate was reached. The concentration of isoproterenol in those experiments was 10m5M, a quantity that stimulates lipolysis maximally in our assay system. In the studies described in this report, we found that cells stimulated by a lower concentration of isoproterenol, lo-‘M, could be suppressed with correspondingly lower concentrations of oleate. In fact, under these conditions, the addition of 0.2 mM oleate (a FFA/albumin ratio of 0.3) had a detectable effect on cyclic AMP formation. * Because the albumin is thought to have 2 high affinity binding sites and 5 sites with lower affinity for FFA, it has been suggested that there is no inhibitory effect of FFA until FFA/albumin molar ratios exceed two.2 The present findings argue against this view and suggest that the inhibitory
*These calculations do not take into account albumin preparation used in these experiments 0.35 mM FFA.
the endogenous FFA present in buffer albumin. The (Sigma Co; lot #16C-0028) contained approximately
1760
BURNS
ET AL
effect of FFA is a continuum. Thus, even when the FFA/albumin ratio is less than two (i.e., high affinity sites not completely saturated), a small fraction of FFA apparently remains unbound. The finding that small increments in FFA concentration can rapidly influence cyclic AMP formation lends weight to the hypothesis that FFA are physiologically important in the direct regulation of iipoiysis. It is not surprising that paimitic and iinoieic acids were comparable to oieic in suppressing cyclic AMP generation. In general, their physiochemical properties are not very different from oieic acid, and they are present in large quantities in human plasma. The concentrations of paimitic, iinoieic and oieic acids are 29%, that some 13%, and 40%, respectively. “L Some years ago, Rodbeii speculated specificity may exist among fatty acids relative to their inhibitory potential.” He suggested that long-chain saturated fatty acids may be more potent inhibitors because of their slower rates of diffusion out of ceils compared with unsaturated, polar fatty acids. Recently, Fain and Shepherd’” reported the effects of oieate on iipoiysis, cyclic AMP accumulation and the activities of adenyiyi cyclase, protein kinase and triglyceride iipase. These studies were done on either intact ceils or ceil “ghosts” prepared from rat fat pads. Oieate inhibited ail five processes but the concentration of oieate required to influence iipase and protein kinase activities was much greater than that needed to suppress adenyiyi cyclase, cyclic AMP accumulation and iipoiysis. The shorter chain fatty acid, octanoate, was effective in inhibiting cyciase activity, cyclic AMP accumulation and iipoiysis but had no effect on iipase activity.” The inhibition of cyclic AMP formation by added FFA appears to be rapid in onset. A definite decrease in cyclic AMP concentration was observed one minute after the addition of sodium oieate to isoproterenoi-stimulated ceils. This finding is consistent with those reported by Fain and Shepherd,13 who found a near maximal inhibition of cyclic AMP accumulation within 30 set after the addition of oieate to rat fat ceils. Fain and his coworkers have extensively evaluated the question of inhibitors of iipoiysis and have concluded that the principal inhibitors released by rat fat ceils during stimulation with iipoiytic hormones are FFA and that it is unlikely that adenosine is an important feed-back regulator of adenyiyl cyciase activity.‘” The findings of Maigieri et al. Ifi relative to chicken fat ceils are of interest inasmuch as they point to a potentially significant species difference in this regard. When these ceils are stimulated with giucagon, a large rise in cyclic AMP occurs and is maintained over an incubation period of one hour, suggesting that chicken fat ceils do not exhibit FFA feed-back regulation of either cyclic AMP accumulation or of iipoiysis. The addition of sufficient oieate to raise the FFA/aibumin ratio to 8 did not effect the level of cyclic AMP; FFA/aibumin ratios as high as 12 did not inhibit the adenyiyi cyciase activity of chicken fat ceil ghosts. If anything, fatty acids at a concentration of 1 mM tended to stimulate the isoproterenoi-sensitive adenyiyi cyciase activity of turkey erythrocyte membranes.” Although Rodbell demonstrated that iipoiysis by isolated rat fat ceils in the presence of hormones virtually ceased when the ratio of FFA/aibumin exceeded 3, only recently has the possible role of FFA as physiologic regulators of iipolysis been appreciated. The following observations support the view that FFA are physiologically important in the control of iipoiysis in human adipose tissue: (1) FFA are effective in concentrations of 1.0 mM or less; (2) Their onset of action is
FFA IN THE REGULATION
OF LIPOLYSIS
1761
rapid, inhibition being demonstrable at 1 min; and (3) FFA inhibition of cyclic AMP accumulation is reversible, i.e., inhibited cells resuspended in fresh buffer containing isoproterenol will respond with a brisk increase in cyclic AMP. The crucial question remains, however, as to whether the inhibitory effect of FFA on lipolysis is operative in intact man. Renold18 had earlier suggested that the rate of delivery of albumin through the sinusoidal intravascular spaces of adipose tissue might be an important determinant of the rate of lipolysis. The availability of ample albumin should prevent the buildup of intracellular FFA and favor fat mobilization. On the contrary, in the face of reduced albumin, FFA concentration would increase and lipolysis would be inhibited. Indirect evidence supporting such a view is contained in a study done by Bogdonoff and colleagues some years ago.lg In this study, 1 I patients with low plasma albumin concentrations (0.8 to 2.2 g/dl) were infused with norepinephrine, and plasma FFA determined every 10 min for the ensuing hour. On a different day, the experiment was repeated after the infusion of 50 g of albumin. With Dr. Bogdonoff’s permission, we have recalculated some of these data. In response to the first norepinephrine infusion, plasma FFA rose from a mean control value of 432 f 44 pEq/l to 556 & 42 pEq/ 1, an increment of 124 pEq/l or 29% (increase by albumin). After the albumin infusion, the response to norepinephrine was from a mean control value of 431 =t 63 pEq/l to 841 * 116 pEq/l, an increase of 410 pEq/l or 95%. Thus, hypoalbuminemic patients appeared to have an impaired lipolytic response to norepinephrine that was at least partially corrected by albumin infusion. Although it is possible, or even probable, that released FFA are the most important feedback regulators of human fat cells, it should be noted that these cells may also be subject to other types of regulation. For example, it can be seen from Fig. 6 that the cyclic AMP response to the second application of isoproterenol was less than the response to the first, even after the accumulated FFA had been removed. This could reflect the agonist-specific type of tachyphylaxis seen in many other types of cells.““~“’ ACKNOWLEDGMENT
We are grateful
for the excellent technical assistance provided by Barbara Couture-Murillo. REFERENCES
I. Steinberg
D, Vaughan
M: Release
of free.
5.
Burns
TW,
Hales
fatty acids from adipose tissue in vitro in relation
lipolysis in isolated
to rate of triglyceride
Lancet 1:796-798,
in Renold Physiology, American
A,
Cahill
synthesis and degradation, G (eds):
Section
5,
Handbook
Washington
Physiological
Society,
of
D.C., 1965,
pp
3355347 2. Rodbell
M: Modulation
pose tissue by fatty
cells. Ann NY Acad Sci I3 3. Burns TW, of free-fatty-acid
of lipolysis in adi-
acid concentration
Langley
1:302-3
in fat
14. 1965
24: 265276,
4. Rodbell M: Metabolism
1975
1. Effects of hormones on glucose metabolism 1964
sensitive
guanosine
and
Regulation
V, Vaughan
protein-binding
3’:5’
- Monophosphate. Acad Sci 68:736-739, 1971 7. Steiner AL,
Wehmann,
al: Radioimmunoassay 2:51-61,
nucleotides.
of
adipose tissue cells.
1966
F, Manganiello
M: A
assay Proc
RE, Parker
CW,
for the measurement
Adv Cyclic
for Natl
Nucleotide
et of
Res
1972
8. Garland
PB, Randle
assay for glycerol.
of isolated fat cells
lipolysis. J Biol Chem 239:375-380,
simple,
cyclic
PE, Robison GA: Site
inhibition of lipolysis by human
adipocytes. Metabolism
6. Murad
CN:
human
Nature
PJ: A rapid enzymatic 196:987-988,
1962
9. Dole VP: A relation between non-esterified fatty acids in plasma and the metabolism cose. J Clin Invest 35: l50- 154, 1956
of glu-
1762
IO. Burns TW, Langley PE: Lipolysis by human adipose tissue: The role of cyclic 3’,5’adenosine monophosphate and adrenergic receptor sites. J Lab Clin Med 75:983-997, 1970 11. Robison GA. Langley PE, Burns TW: Adrenergic receptors in human adipocytes: Divergent effects on adenosine-3’,5’-monophosphate and lipolysis. Biochem Pharmacol 2 I :589-592, 1972 12. Burns TW, Gehrke CW. Anigian MJ, et al: Effect of insulin on plasma free fatty acids of normal subjects. J Lab Clin Med 62:646-656, I963 13. Fain JN, Shepherd RE: Free fatty acid as feedback regulators of adenylate cyclase and cyclic 3’:5’-AMP accumulation in rat fat cells. .I Biol Chem 250:6586-6592, 1975 14. Fain JN, Shepherd RE: Inhibition of adenosine 3’:5’-monophosphate accumulation in white fat cells by short chain fatty acids, Lactate, and 8-hydroxybutyrate. J Lipid Res 17:377-385, 1976 15. Fain JN, Shepherd RE: Hormonal regulation of lipolysis: Role of cyclic nucleotides. adenosine and free fatty acids, in Klachko DM, Anderson RR, Heimberg M (eds): Hormones
BURNS
ET AL.
and Energy Metabolism. New York, Plenum (in press) 16. Malgieri JA, Shepherd RE, Fain JN: Lack of feedback regulation of cyclic 3’:5’-AMP accumulation by free fatty acids in chicken fat cells. J Biol Chem 250:6593-6598. 1975 17. Orly J, Schramm M: Fatty acids as modulators of membrane functions: Catecholamineactivated adenylate cyclase of the turkey erythrocyte. Proc Nat Acad Sci 723433 3437, 1975 18. Renold AE: A brief and fragmentary introduction to some aspects of adipose tissue metabolism with emphasis on glucose uptake. Ann NY AcadSci 131:7 12, 1965 19. Bogdonoff MD, Linhart JW. Estes EH: Effect of serum albumin infusion on lipid metabolism in man. J Appl Physiol 17:974 978, 1962 20. Conolly ME. Greenacre JK: The lymphocyte fi-adrenoceptor in normal subjects and patients with bronchial asthma. J Clin Invest 58:1307 1316. 1976 21. Morishima I, Robison GA, Thompson WJ, et al: Mechanism of catecholaminedesensitization of cultured libroblasts. Pharmacologist l&186, 1976
effect
of added
and cyclic pose of
AMP
tissue
sodium
response
ceils
was
Inhibition quantity
to 1V7
was
and linoleic
of added acids
were
acid in suppressing Suppression
adi-
tion was detectable the
addition
addition
tion, suppressed
the
lipolytic
could
M
isoprote-
fresh
with
cumulated
added,
0.2
the mM,
not
buffer
with
increas-
stimulated
fatty
acid.
Palmitic
findings
as oleic
fatty
isoproterenol-stimulated of cyclic
AMP
fatty
change, by are
acids
regulators
AMP
acids
acids,
addition
after
were
after forma-
fatty
by the
However,
the
removed
cyclic
AMP
fresh
isoproterenol.
consistent are
one minute
Cyclic
by accumulated
be stimulated
isoproterenol.
greater
as effective
within
of oleate.
The
detectable
of oleate
Boyd E. Terry, and G. Alan Robinson
on lipolysis
of human
studied.
progressively
ing quantities
acid
decreased
of adipocytes
lowest
lipolysis.
fatty
concentration
oleate
renol.
and was
W. Burns, Paul E. Langley.
of
formation
with
physiologically
the
of acby was
These view
that
significant
of lipolysis.
forma-
I
T IS WELL KNOWN that the accumulation of free fatty acids (FFA) inhibits the rate of lipolysis by adipose tissue. ‘s2 This effect is diminished in the presence of albumin because albumin binds FFA, reducing their inhibitory effect until the albumin binding sites are saturated. Studies have been done in our laboratory that indicate that at least one aspect of the action of FFA involves the inhibition of cyclic AMP formation.:’ Preliminary observations suggested that the inhibitory effect of FFA commences at concentrations of FFA less than that required to saturate albumin binding sites. The present studies were undertaken to obtain additional information on this and other points that bear on the question of whether or not FFA inhibition of lipolysis might be physiologically significant. MATERIALS
AND METHODS
Isolated adipocytes were prepared from subcutaneous tissue samples obtained from patients undergoing abdominal surgery. The method of preparing cells, incorporating modifications of Rodbell’s procedure,4 has been described.5 Cells were suspended in Krebs bicarbonate buffer containing 2.5 mM glucose and 4% bovine serum albumin. Flasks containing aliquots of cell suspensions were incubated with gentle shaking in an atmosphere of 95% 0, and 5% CO,. For the determination of cyclic AMP, test substances were incubated in IO ml of cell suspension. At the end of incubation, the contents of each incubation flask were quick-frozen in liquid nitrogen. Extracts were prepared and purified essentially as described by Murad et al.6 and cyclic AMP content determined by radioimmunoassay.’ Cyclic AMP content was expressed as pmole/g of triglyceride. To evaluate their effects on lipolysis, test substances were incubated with 1 ml of cell suspension in each of 3 to 6 flasks. Following incubation, cell-free filtrates were prepared and analyzed for glycerol and FFA using the methods of Garland and RandIe and Dole,g respectively. Concentrations of glycerol and FFA were expressed as gmolefg of triglyceride. Since it has been shown that human adipocytes have both cy- and @-adrenergic receptor sites,l”,ll rsoproterenol, a pure fi agonist, was chosen to stimulate cyclic AMP forma-
From the Division of Endocrinology and Metabolism, Department of Medicine and the Department of Surgery, Universit_v of Missouri School of Medicine, Columbia, MO.; and The Department of Pharmacology, University of Texas MedicalSchool. Houston. Tex. Receivedforpublication January 18. 1978. Supported b.v USPHS Grants Am I1265 and HL 16552. Address reprint requests to Thomas W. Burns. M.D.; Department oJMedicine. Division of Endocrinology and Metabolism, University of Missouri, School of Medicine, Columbia, MO. 65212. (B 1978 by Grune & Stratton, Inc. 0026~0495/78/2712-OOO$Ol.OO/O Metabolism, Vol. 27, No. 12 IDecemberJ. 1978
1755
BURNS
1756
0
isoproterenol-stimulated lipolysis. Human cells were incubated for 4 hr in Krebs buffer containing 4% albumin and either 10-xM, lo-‘M, or 10mhM isoproterenol. The concentration of FFA was increased by adding sodium oleate at the beginning of incubation. Each point represents the mean fatty acid concentration of three flasks. The smallest quantity of added FFA, 1.5 mM. reduced lipolysis stimulated by each concentration of isoproterenol.
3
2 1 ADDED OLEATE, mM
tion and lipolysis. The use of the physiologic cathecholamines, lead to problems in interpretation For experiments Krebs-Ringer
requiring
bicarbonate
epinephrine
and norepinephrine,
could
since these substances have both CYand 6 agonist properties.
added
FFA,
sodium oleate,
palmitate
or linoleate
was emulsified
in
solution and then put in solution by the addition of powdered albumin. The
albumin buffer with added FFA
was dialyzed
buffer was 7.4. The quantity of FFA
twice against bicarbonate
buffer. The final pH of the
released by cells during incubation was calculated
the initial FFA content of the cell suspension (added FFA) FFA).
ET AL
FFA values were obtained by titration
by subtracting
from the tinal content (added and released
before and after incubation. RESULTS
The Efect
of Varying FFA Concentrations
on FFA Inhibition
~~/‘Lipol~~.si.s
To gain insight into the quantity of FFA required to affect lipolysis, a series of experiments were done in which the concentrations of both isoproterenol and added oleate were varied. The quantity of FFA present in the media at 4 hr was determined and corrected for the quantity of FFA added. The results of a representative experiment are shown in Fig. I. The smallest quantity of added oleate, 1.5 mM, consistently reduced lipolysis stimulated by IO-‘. IO-‘, and IO-” M isoproterenol. Subsequent experiments were done employing only one concentration of isoproterenol, IO-’ M and smaller increments of added oleate, from 0.2 to 1.4 mM. The results of four experiments are shown in Fig. 2. The lowest concentra-
40. Fig. 2. The effect of low concentrations of added FFA on lipolysis stimulated by 10-‘M isoproterenol. Human cells were incubated for 4 hr in Krebs buffer 4% albumin contained and isoproterenol. The concentration of FFA wes increesed by adding sodium oleate at the beginning of incubation. The results are expressed as the percent inhibition of lipolysis achieved by added FFA. Each point represents the mean value of four experiments. The lines above and below each point represent the standard error of the mean.
30PER CENT INHIBITION OF FFA RELEASE
20_
lo-
O0
0.5 ADDED
1.0 OLEATE,
1.5 mM
FFA IN THE REGULATION
OF LIPOLYSIS
1757
50 Fig. 3. Comparison of the effect of added sodium paimitate. linolate and oleate on FFA release stimulated by lo-‘M isoproterenol. Human cells were incubated for 4 hr in Krebs buffer containing 4% albumin and isoproterenol. The concentration of FFA was increased by adding FFA at the beginning of incubation. The results are expressed as a percent inhibition of FFA release achieved by added FFA. Each point represents the mean fatty acid concentration of three flasks.
40 PER CENT 30 INHIBITION OF FFA go RELEASE 10 0 0
0.5 ADDED FREE FATTY
1.0
1.5
ACID, mM
tion of added oleate, 0.2 mM, appeared to suppress lipolysis; this effect was progressive with increasing quantities of added FFA and was greatest with the highest concentration of oleate employed, I .5 mM. The E#ect of Palmitic and Linoleic Acids on Lipolysis
All previous experiments concerned with the effect of added FFA on lipolysis had been done with oleic acid, the most abundant fatty acid in human plasma. To determine the effectiveness of other fatty acids in inhibiting lipolysis, palmitic and linoleic acids were incubated with adipocytes in the presence of isoproterenol, lo-’ M; FFA released into the medium was estimated at the end of 4 hr. The results of typical experiments are shown in Fig. 3. Both palmitic and linoleic acids were essentially equal to oleic acid in suppressing lipolysis. The Duration of Exposure to FFA Required to Aflect Cyclic AMP Formation
To assess the exposure time to FFA required to inhibit cyclic AMP formation, it was first necessary to define how quickly human adipocytes respond to catecholamines such as isoproterenol. In a majority of other previous studies, cyclic AMP had been measured after a 30 min exposure to test substances; glycerol and FFA released into the media were estimated at 4 hr. To gain more precise information on the rapidity of the human adipocyte response, intracellular cyclic AMP was measured at various times between 0 and 10 min after the addition of
c-AMP Pmols/g
1,500 1,000
a 0
2
4 6 TIME, min
B
10
Fig. 4. Alterations in cyclic AMP concentration following the addition of isoproterenol. 1O+M. Human cells were incubated for varying times from. 0 to 10 min in Krebs buffer containing 4% albumin and isoproterenol. The results of four experiments are plotted. An increase in cyclic AMP was evident by 1 min.
BURNS
1758
GLYCEROL wols/g
ET AL
Fig. 5. Alterations in glycerol release following the addition of isoproterenol 1 O-‘hl. Human cells
0.6-
0.3-
2
0
6 4 TIME, min
6
isoproterenol. The results of four are plotted. An experiment5 increase in buffer glycerol was evident by 1 min.
10
isoproterenol, IO-“M. Representative results are given in Fig. 4. By I min, there was a substantial rise in cyclic AMP; this response increased over the ensuing 10 min. In other experiments, glycerol release was measured at frequent intervals from 0 to 10 min after the addition of isoproterenol. A definite increase in glycerol in the buffer was evident as early as I min (Fig. 5). To more closely define the time exposure required for FFA to influence cyclic AMP formation, aliquots of cells were exposed to added oleate for 5, 3, I, and 0 min, respectively. All flasks contained isoproterenol, lo-“M. The results of two such experiments are given in Table 1. At 1 min, exposure to added oleate reduced cyclic AMP accumulation stimulated by isoproterenol by approximately 10%. A 5-min exposure resulted in a reduction in cyclic AMP of 60%. Response of‘Suppressed
Cyclic AMP to Fresh Isoproterenol
Conceivably, the low level of cyclic AMP noted after 3 ‘2-4 hr of incubation noted in earlier experiments” could be due to loss of activity of isoproterenol rather than to an impaired responsiveness of the cyclic AMP generating system. To evaluate this possibility, aliquots of cells were incubated for 4 hr with isoproterenol, 10-5M; to one set of flasks, fresh isoproterenol was added at 312 hr. Cyclic AMP concentration in the two groups was essentially the same: 600 pmole/g in the group to which no fresh isoproterenol had been added and 580 pmole/g in the group to which additional catecholamine had been added (Fig. 6). Table 1. Exposure Time Required for FFA Inhibition of Isoproterenol-Stimulated Cyclic AMP Formation Exposure Time IminI ISOprOterenOl (lo-”
Cyclic AMP. pm&/g Ole.3t.Z
Ml
Experiment
(2 5 mM)
third
oleate
Experwnent 2
0
5
0
12726
12111
5
1
10909
11104
5
3
6698
7972
5
5
4032
3616
49
Human cells were incubated for 5 min in Krebs buffer Sodium
1
0
was
added
to one set of flasks
set at 4 min. The results
of 2 experiments
containing
at the beginning are given.
4%
a7
albumin
of incubation,
and
to another
isoproterenol.
lo-’
M.
set at 2 mm and to a
FFA IN THE REGULATION
OF LIPOLYSIS
1759
Fig. 6. The effect of fresh isoproterenol and buffer change on cyclic AMP concentration (left panel) and on glycerol accumulation (right panel). Human ceils were incubated in Krebs buffer containing 4% albumin and. except for control flasks, isoproterenol 10-5M. The height of each bar represents the mean (*SEMI of four experiments. (A) control before incubation; (6) half-hour incubation; (Cl 4 hr incubation; and (D) 4-hr incubation with fresh isoproterenol, 10m5M added at 3’ :! hr; (E) 4-hr incubation, with buffer change and fraseh isoproterenol added at 3’9 hr.
Reversibility
ABCDE
of Cyclic AMPSuppression
Associated
A
B
C
D
E
with FFA Accumulation
To test the reversibility of the suppressed cyclic AMP generation that occurred with FFA build-up during prolonged incubation, cells were incubated with lo-“M isoproterenol for 3!+ hr; they were then transferred to fresh buffer and isoproterenol and incubated for an additional half hour. Other aliquots of cells were incubated for half hour and 4 hr in the usual manner without buffer change. Cells and buffer from all flasks were frozen for subsequent cyclic AMP assay. The mean and standard error of the mean of 3 experiments are shown in Fig. 6. As noted before, the cyclic AMP concentrations in flasks undergoing 4-hr incubation was substantially less than that noted in flasks incubated for a half hour, 600 pmole/g and 8,800 pmole/g, respectively. The flasks incubated for 4 hr but with fresh buffer provided at 31s hr had a mean cyclic AMP concentration of 3300 p mole/g. DISCUSSION In studies previously reported,” dose response experiments were done in which the effects of oleate in concentrations varying between 0.6 and 3.0 mM on cyclic AMP formation were observed. Only a slight inhibitory effect was found until a concentration of 2.5 mM oleate was reached. The concentration of isoproterenol in those experiments was 10m5M, a quantity that stimulates lipolysis maximally in our assay system. In the studies described in this report, we found that cells stimulated by a lower concentration of isoproterenol, lo-‘M, could be suppressed with correspondingly lower concentrations of oleate. In fact, under these conditions, the addition of 0.2 mM oleate (a FFA/albumin ratio of 0.3) had a detectable effect on cyclic AMP formation. * Because the albumin is thought to have 2 high affinity binding sites and 5 sites with lower affinity for FFA, it has been suggested that there is no inhibitory effect of FFA until FFA/albumin molar ratios exceed two.2 The present findings argue against this view and suggest that the inhibitory
*These calculations do not take into account albumin preparation used in these experiments 0.35 mM FFA.
the endogenous FFA present in buffer albumin. The (Sigma Co; lot #16C-0028) contained approximately
1760
BURNS
ET AL
effect of FFA is a continuum. Thus, even when the FFA/albumin ratio is less than two (i.e., high affinity sites not completely saturated), a small fraction of FFA apparently remains unbound. The finding that small increments in FFA concentration can rapidly influence cyclic AMP formation lends weight to the hypothesis that FFA are physiologically important in the direct regulation of iipoiysis. It is not surprising that paimitic and iinoieic acids were comparable to oieic in suppressing cyclic AMP generation. In general, their physiochemical properties are not very different from oieic acid, and they are present in large quantities in human plasma. The concentrations of paimitic, iinoieic and oieic acids are 29%, that some 13%, and 40%, respectively. “L Some years ago, Rodbeii speculated specificity may exist among fatty acids relative to their inhibitory potential.” He suggested that long-chain saturated fatty acids may be more potent inhibitors because of their slower rates of diffusion out of ceils compared with unsaturated, polar fatty acids. Recently, Fain and Shepherd’” reported the effects of oieate on iipoiysis, cyclic AMP accumulation and the activities of adenyiyi cyclase, protein kinase and triglyceride iipase. These studies were done on either intact ceils or ceil “ghosts” prepared from rat fat pads. Oieate inhibited ail five processes but the concentration of oieate required to influence iipase and protein kinase activities was much greater than that needed to suppress adenyiyi cyclase, cyclic AMP accumulation and iipoiysis. The shorter chain fatty acid, octanoate, was effective in inhibiting cyciase activity, cyclic AMP accumulation and iipoiysis but had no effect on iipase activity.” The inhibition of cyclic AMP formation by added FFA appears to be rapid in onset. A definite decrease in cyclic AMP concentration was observed one minute after the addition of sodium oieate to isoproterenoi-stimulated ceils. This finding is consistent with those reported by Fain and Shepherd,13 who found a near maximal inhibition of cyclic AMP accumulation within 30 set after the addition of oieate to rat fat ceils. Fain and his coworkers have extensively evaluated the question of inhibitors of iipoiysis and have concluded that the principal inhibitors released by rat fat ceils during stimulation with iipoiytic hormones are FFA and that it is unlikely that adenosine is an important feed-back regulator of adenyiyl cyciase activity.‘” The findings of Maigieri et al. Ifi relative to chicken fat ceils are of interest inasmuch as they point to a potentially significant species difference in this regard. When these ceils are stimulated with giucagon, a large rise in cyclic AMP occurs and is maintained over an incubation period of one hour, suggesting that chicken fat ceils do not exhibit FFA feed-back regulation of either cyclic AMP accumulation or of iipoiysis. The addition of sufficient oieate to raise the FFA/aibumin ratio to 8 did not effect the level of cyclic AMP; FFA/aibumin ratios as high as 12 did not inhibit the adenyiyi cyciase activity of chicken fat ceil ghosts. If anything, fatty acids at a concentration of 1 mM tended to stimulate the isoproterenoi-sensitive adenyiyi cyciase activity of turkey erythrocyte membranes.” Although Rodbell demonstrated that iipoiysis by isolated rat fat ceils in the presence of hormones virtually ceased when the ratio of FFA/aibumin exceeded 3, only recently has the possible role of FFA as physiologic regulators of iipolysis been appreciated. The following observations support the view that FFA are physiologically important in the control of iipoiysis in human adipose tissue: (1) FFA are effective in concentrations of 1.0 mM or less; (2) Their onset of action is
FFA IN THE REGULATION
OF LIPOLYSIS
1761
rapid, inhibition being demonstrable at 1 min; and (3) FFA inhibition of cyclic AMP accumulation is reversible, i.e., inhibited cells resuspended in fresh buffer containing isoproterenol will respond with a brisk increase in cyclic AMP. The crucial question remains, however, as to whether the inhibitory effect of FFA on lipolysis is operative in intact man. Renold18 had earlier suggested that the rate of delivery of albumin through the sinusoidal intravascular spaces of adipose tissue might be an important determinant of the rate of lipolysis. The availability of ample albumin should prevent the buildup of intracellular FFA and favor fat mobilization. On the contrary, in the face of reduced albumin, FFA concentration would increase and lipolysis would be inhibited. Indirect evidence supporting such a view is contained in a study done by Bogdonoff and colleagues some years ago.lg In this study, 1 I patients with low plasma albumin concentrations (0.8 to 2.2 g/dl) were infused with norepinephrine, and plasma FFA determined every 10 min for the ensuing hour. On a different day, the experiment was repeated after the infusion of 50 g of albumin. With Dr. Bogdonoff’s permission, we have recalculated some of these data. In response to the first norepinephrine infusion, plasma FFA rose from a mean control value of 432 f 44 pEq/l to 556 & 42 pEq/ 1, an increment of 124 pEq/l or 29% (increase by albumin). After the albumin infusion, the response to norepinephrine was from a mean control value of 431 =t 63 pEq/l to 841 * 116 pEq/l, an increase of 410 pEq/l or 95%. Thus, hypoalbuminemic patients appeared to have an impaired lipolytic response to norepinephrine that was at least partially corrected by albumin infusion. Although it is possible, or even probable, that released FFA are the most important feedback regulators of human fat cells, it should be noted that these cells may also be subject to other types of regulation. For example, it can be seen from Fig. 6 that the cyclic AMP response to the second application of isoproterenol was less than the response to the first, even after the accumulated FFA had been removed. This could reflect the agonist-specific type of tachyphylaxis seen in many other types of cells.““~“’ ACKNOWLEDGMENT
We are grateful
for the excellent technical assistance provided by Barbara Couture-Murillo. REFERENCES
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D, Vaughan
M: Release
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and Energy Metabolism. New York, Plenum (in press) 16. Malgieri JA, Shepherd RE, Fain JN: Lack of feedback regulation of cyclic 3’:5’-AMP accumulation by free fatty acids in chicken fat cells. J Biol Chem 250:6593-6598. 1975 17. Orly J, Schramm M: Fatty acids as modulators of membrane functions: Catecholamineactivated adenylate cyclase of the turkey erythrocyte. Proc Nat Acad Sci 723433 3437, 1975 18. Renold AE: A brief and fragmentary introduction to some aspects of adipose tissue metabolism with emphasis on glucose uptake. Ann NY AcadSci 131:7 12, 1965 19. Bogdonoff MD, Linhart JW. Estes EH: Effect of serum albumin infusion on lipid metabolism in man. J Appl Physiol 17:974 978, 1962 20. Conolly ME. Greenacre JK: The lymphocyte fi-adrenoceptor in normal subjects and patients with bronchial asthma. J Clin Invest 58:1307 1316. 1976 21. Morishima I, Robison GA, Thompson WJ, et al: Mechanism of catecholaminedesensitization of cultured libroblasts. Pharmacologist l&186, 1976