Physiologic concentrations of β-lipotropin stimulate lipolysis in rabbit adipocytes

Physiologic

Concentrations of &Lipotropin Lipolysis in Rabbit Adipocytes W.O.

Stimulate

Richter and P. Schwandt

The proopiocorticomelanotropin (POMC) sequence #?-lipotropin stimulates glycerol release from incubated rabbit adipocytes at a minimal concentration of lOme mol/L. However, when lipolysis inhibiting substances (eg, fatty acids and adenosine) and contaminating peptide degrading activity are continuously removed by fat cell perifusion, the sensitivity is This higher sensitivity of the perifused adipocyte could also be increased to lo-l3 and partly to lo-l4 mol/L &lipotropin. demonstrated with a-MSH (from 5 x lo-” to 10-l’ mol/L). The restimulation of glycerol release was shown for both peptides. We conclude that POMC peptides might be involved in the regulation of lipolysis since the minimal effective concentrations are near to plasma concentrations. Q 1985 by Grune & Stratton, Inc.

I

N 1964, Li and co-workers’** purified B-lipotropin (P-LPH) from sheep pituitary glands. This lipolytic polypeptide contains the amino acid sequences of #I-melanocyte stimulating hormone (fi-MSH), metenkephalin, and P-endorphin. Moreover, it shares a common heptapeptide sequence (/3-LPH 47-53) with adrenocorticotropic hormone (ACTH 410). The lipolytic activity of fi-LPH in vitro was characterized by incubating either fat pads or isolated fat cells in flasks. Under those conditions in vitro, 8-LPH stimulated lipolysis in rabbit adipocytes’ down to a concentration of 10e9 mol/L.4 Since plasma concentrations of P-LPH are in the range from 21 to 66 pg/mL,’ this polypeptide was not regarded to be involved in the regulation of adipose tissue metabolism, although the secretion of P-LPH into the circulation is regulated, eg, by stress.5 In vivo, P-LPH increases free fatty acid and/or glycerol plasma concentrations when injected into fasting mice3 and rabbits.6*7 Although ,&LPH rapidly disappears from the circulation after intravenous (IV) injection into rats* or humans’ (half-life of about four minutes), even 1 pg (lo-” mol/kg) of /3-LPH IV stimulated lipolysis in the rabbit.’ The high metabolic clearance rate suggests that the local lipolytic concentrations of @-LPH at the fat cell level could be lower than calculated from the injected dose. Thus, the minimal effective concentration should be lower than 10e9 mol/L in incubated rabbit adipocytes. Therefore, we applied a fat cell perifusion system with continuous removal of extracellular metabolic products, which is also suitable for studying reversible processes.‘G’2 MATERIALS

AND

METHODS

Porcine @-lipotropin was prepared by high-performance liquid chromatography (HPLC) as described previously.” Synthetic (YMSH was purchased from Bacbem, Burgdorf, Switzerland. Bovine serum albumin (BSA) fraction V (Roth, Karlsruhe, West Germany) was defatted by charcoal treatment after the method of Chen.” Perirenal adipose tissue was obtained from female rabbits (white Neuseelander, 3.5 to 4.5 kg body weight) starved for 12 hours. Isolated fat cells were prepared according to Rodbell” using collagenase CLS I (Lot 495258, 166 U/mg from Worthington (Seromed, Munich, West Germany) in a concentration of 150 U/mL. A portion Metabolism, Vol 34, No 6 (June), 1985

of the fat cell solution was used for the determination of fat cell number in a Fuchs-Rosenthal-chamber (0.2 mm height, 0.0625 mm’ area, mean of 20 determinations). Fat cell size was estimated by measuring the diameter of 50 isolated adipocytes microscopically. The mean value of fat cell size was 63.2 + IO.9 pm. Perifusion experiments were performed with an automated system that allows continuous flow determination of released glycerol (n - 12),‘* or for obtaining log/dose-response curves with a sixchamber device (n = 6) where the perifusate (five-minute fractions) was analyzed for glycerol content as parameter of lipolysis. Glycerol was also determined enzymatically with the automated system.” About one million isolated fat cells were transferred into each perifusion chamber. Krebs-Ringer-bicarbonate buffer (pH 7.4, containing 3 mmol/L glucose and 1% defatted BSA fraction V, gassed with 95% 0,/5% CO*) was pumped with a flow rate of 1 mL/min through each perifusion chamber thermostated at 37 OC.The perifusion chamber consisted of a plastic tuberculine syringe with a volume of 1 mL. The stream in the cell was from the top to the bottom; the cell layer was 3 to 4 cm high. A preperifusion of 20 minutes was performed to eliminate substances that affect lipolysis left from the cell isolation procedure or released from the cells. &LPH and (u-MSH were tested in concentrations from lo-’ to lo-” mol/L. The conventional fat cell incubation method was applied with adipocytes from the same animal. Isolated fat cells (about 70,000 per tube) were incubated with ,!I-LPH and a-MSH (IO-’ to lo-” mol/L) in 1 mL Krebs-Ringer-bicarbonate buffer (pH 7.4, containing 3 mmol/L glucose and 4% BSA fraction V, gassed with 95% 0,/5% COr) under continuous shaking for one hour at 37 OC. The reaction was stopped by removing the fat cell layer after centrifugation at 12,000 g for four minutes. The infranatant was deep-frozen at -35 “C until glycerol analysis. Glycerol was determined enzymatitally as previously described.‘* Degradation experiments were performed by incubating 30 Hg porcine @-LPH (resulting in a concentration of 3 x 10-6mol/L) with 70,000 isolated rabbit fat cells in Krebs-Ringer-bicarbonate buffer pH 7.4 for 1,15, and 60 minutes at 37 OC.The reaction was stopped by removal of isolated fat cells after centrifugation through a semisynthetic oil (dinonylphtalate). The infranatant was either deep-frozen at -90 “C or lyophilized prior to

From the Department of Internal Medicine II, Klinikum Grosshadern. University of Munich. Marchioninistr 1 S. D-8000 Munich 70, West Germany. Supported by Deutsche Forschungsgemeinschaft (SFB 0207/LP 30). Address reprint requests to Dr P. Schwandt. Department of Internal Medicine II, Klinikum Grosshadern, University of Munich. Marchioninistr 15, D-8000 Munich 70. West Germany. o 1985 by Grune & Stratton, Inc. 0026-0495/85/3406~07$3.00/0 539

540

RICHTERAND SCHWANDT Table 1. Glycerol Release by Porcine &Lipotropin and n-MSH+ Peotide Concentrations

in P&fused

10-Q

lo-’

Isolated Rabbit Fat Cells ImollLI to-”

lo-”

14.9 + 6.4t

6.4 r 5.8

5.6 r 4.2

8.2 + 2.8t

4.7 + 3.6

5.2 + 3.6

@LPH

22.8

? 9.4t

17.5 + 8.6t

16.4 + 9.2t

a-MSH

20.6

? 12t

22.2

26.3

r 11.1t

+ lot

10

‘5

Basal Lipolysls

*Glycerol (nanomoles) released from 1 million adipocytes per minute determined in a five-minute perifusate (5 mL) Mean + SEM of six experiments. tP 5 0.05 (Wilcoxon matched pair, signed rank statistic) when compared

analysis on HPLC. Similar experiments were performed with washing buffer of the isolated fat cells and several dilutions of the collagenase solution (1 :l to 1: I million). For degradation experiments during perifusion, 1 million isolated fat cells were perifused with p-LPH in a concentration of 10m6 mol/L for five minutes (after 20 minutes preperifusion). This five-minute perifusate was collected and freeze-dried for analysis on HPLC. HPLC experiments were performed with a programmer model 200 and two model 410 pumps (Kontron, Eching, West Germany) connected with a variable UV wave length detector LCD 720. The column (Aquapore RP 300) 25 x 0.4 cm internal diameter was purchased from Kontron. Separation conditions were ambient temperature, flow rate at 1 mL/min, detection at 225 nm, methanol eluent, 0.1 mol/L sodium phosphate buffer A (pH 2.1), and linear gradient from 80% to 35% buffer A in 120 minutes. The column was calibrated with 15 partial amino acid sequences of &LPH.‘6

RESULTS

Porcine P-lipotropin stimulated glycerol release from perifused rabbit adipocytes in all 18 animals at a minimal effective concentration of lo-l3 mol/L. The results obtained with the six-chamber perifusion system are summarized in Tables 1 and 2. Figure 1 shows a typical experiment with restimulation at equimolar concentrations. In six of 12 assays done with the continuous flow determination system, glycerol release was increased also at concentrations of lo-l4 mol/L (Fig 2). When rabbit adipocytes were incubated with fl-LPH for one hour, the minimal effective concentration for glycerol release was 10m9mol/L (Tables 1 and 2). cu-MSH stimulated glycerol release under incubation conditions at a minimal effective concentration of 5 x lo-” mol/L but was ineffective at lo-” mol/L (Tables 1 and 2). During perifusion, the minimal effective concentrations was lo-l3 mol/L (Tables 1

with basal lipolysis.

and 2). A restimulation with equimolar concentrations (after a five-minute wash-out period) of (r-MSH (Fig 3) caused again a release of glycerol, though at a lower level. The incubation of fi-LPH with adipocytes prepared with collagenase CLS I caused a complete disappearance of the ,&LPH peak and the occurrence of several new peaks already after one minute (Fig 4). Identical degradation occurs when @-LPH is incubated with the washing buffer of the isolated fat cells or with collagenase solutions up to a dilution of 1: 1 million. None of these peaks had the retention time of pendorphin, P-MSH, met-enkephalin, or the common heptapeptide (/3-LPH 47-53). During perifusion of collagenase CLS I-treated adipocytes (after 20 minutes preperifusion) only a small decrease of the P-LPH peak occurred, and the new peaks did not have the retention time of any of the unspecific degradation products during incubation (Fig 5). DISCUSSION

Porcine P-lipotropin and (Y-MSH stimulate lipolysis down to concentrations of lo-l4 mol/L and lo-l3 mol/L, respectively, in perifused but not in incubated rabbit adipocytes. This change in sensitivity of the adipocytes toward @-LPH from pharmacologic to physiologic lipolytic concentrations was due to the test system: perifused fat cells are closer to conditions in vivo than incubated adipocytes since accumulating free fatty acids and increasing adenosine concentrations, both known to inhibit lipolysis,‘7-‘9 are continuously removed by the new perifusion medium. Furthermore, unspecific degradation of P-LPH by proteases from the adipocyte isolation procedure can be avoided

Table 2. Glycerol Release by Porcine @-Lipotropin and a-MSH* Pemide Concentrations lo-’

in Incubated Isolated Rabbit Fat Cells (mol/Ll

10-u

loms

lo-‘0

Basal LlPOlysiS

@-LPH

25.8

+ 2.8t

16.4 i 2.4t

13.6 ? 1.4t

10.0 + 1.4

9.9 + 1.8

CY-MSH

24.9

+ 3.2t

24.4

17.4 * 0.5t

9.9 + 1.7

9.9 + 1.8

i 2.2t

*Glycerol release (nanomoles) per 60 minutes into the incubation medium after one hour incubation of about 70.000 Krabs-Ringer-bicarbonate buffer pH 7.4. Mean t SEM of six determinations. tP 5 0.05 (Wilcoxon matched pair, signed rank statistic) when compared with basal lipolysis.

adipowtes

in 1 mL

541

PHYSlOLOGlCCONCENTRATIONSOF &LIPOTROPIN

GLYCEROL

jJtil/l

15

lo-

5-

I O'A

Fig 1.

15 $3 11 -log (c Imol 4041

OQ'

7

Stimulation (0) and restimulation (0) of glycerol release

by &lipotropin in perifused isolated rabbit fat cells. Glycerol concentrations in a five-minute perifusate of 1 million adipocytes. Results of a typical experiment.

by preperifusion. The increase in sensitivity of perifused adipocytes compared with incubated fat cells by 10’ to 1O4times for @-LPH, and a-MSH corresponds to the data with glucagon in rat adipocytes.” As shown in Fig 2, /3-LPH induced maximal glycerol release within two and a half minutes, followed by a declining glycerol release for about three minutes. The

TIME (mid

&I

15

c,

Fig 2. Continuous flow determination of released glycerol from 1 million perifused isolated rabbit fat cells stimulated by lo-” and lo-” mol/L @-lipotropin.

15

13 11 9 7 - log Ic Imd~lO-ll)

Stimulation (01 and restimulation WI of glycerol release Fig 3. by (r-MSH in perifused isolated rabbit adipocytes. Glycerol concentrations in a five-minute perifusate from 1 million fat cells. Results of a typical experiment.

bars in Fig 2 demonstrate the time for which any adipocytes (at least the cells at the bottom of the cell column) were exposed to P-LPH. The configuration of the glycerol peak reflects at least partly the lipolytic response to the decreasing concentrations of fl-LPH in the perifusion chamber. In addition, we cannot exclude the possibility that substances inhibiting lipolysis generated throughout an even short, but strong stimulation of lipolysis do occur. Similar observations are reported throughout longer (about 150 minutes) stimulation of perifused rat adipocytes with glucagon.” The restimulation at equimolar concentrations with /‘II-LPHas well as with a-MSH produces lower levels of glycerol release by a mechanism that is unclear. Longer wash-out periods between the stimulation with the peptide hormones did not result in a stronger restimulation, showing that this finding is not due to accumulating lipolysis inhibiting substances. Yet, a comparable stimulation at equimolar concentrations was obtained when the adipocytes were replaced by new isolated fat cells from the same animals (data not shown). Whether this phenomenon is due to receptor occupancy or degradation cannot be decided from our experiments. Though the P-LPH peak disappeared already after one-minute incubation with collagenase-treated fat

RICHTER AND SCHWANDT

542

0,Ol]A

IbLPH

‘11, 30

60

9’0

WLPH

RETENTIONTIME

d

(mid

HPLC analysis of the infranatant after the incubation of Fig 4. /3-LPH (10 pg injected) with 70,000 collagenase CLS I-treated fat ceils, showing the complete disappearance of the &LPH peak within one minute.

cells, glycerol release was significantly higher after one hour of incubation with 10m9 mol/L /3-LPH than without P-LPH. From our experiments we cannot conclude whether this was due to a small amount of undegraded /3-LPH or whether partial sequences of P-LPH stimulated glycerol release. The latter possibility seems to be less probable since none of the new peaks had the retention time of the lipolytic active partial sequences b-endorphin, /3-MSH, or /3-LPH 41-53.2’*22 B-LPH, /3-endorphin, ACTH, and (r-MSH stimulate

90

60 ‘30 RETENTIONklE (mid

HPLC analysis of j%LPH (10 pg injectedj after perifusion Fig 5. through 1 million isolated fat cells (prepared with collagenase CLS I) of the rabbit.

in different test systems. The lipolysis in vitro3*4*2’-24 minimal effective concentrations of fi-LPH and (YMSH in perifused rabbit adipocytes are in the range of human @-LPH plasma levels, suggesting a possible peripheral role of proopiocortin peptides in the regulation of adipose tissue metabolism. ACKNOWLEDGMENT The technical ciated.

assistance

of Elisabeth

Schuster

is greatly

appre-

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543

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