29
Clinica Chimica Acta, 63 (1975) 29-35 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7177
EFFECTS OF APOLIPOPROTEINS OF HUMAN ADIPOSE TISSUE
R. EKMAN*
ON LIPOPROTEIN
LIPASE ACTIVITY
and P. NILSSON-EHLE
Department of Clinical Chemistry, University Hospital, Lund, and Department Physiological Chemistry 4, University of Lund, Lund (Sweden) (Received
of
March 3,1975)
Summary The effect of apolipoproteins isolated from HDL and VLDL on the activity of lipoprotein lipase (LPL) of adipose tissue was studied. The CII apoprotein was found to activate LPL. This activation was strongly inhibited by CI, AI (apo-Lp-Gln I), and the arginine-rich apoprotein, whereas AI1 and CIII exhibited a considerably lower inhibitory effect.
Introduction Lipoprotein lipases (LPL) catalyze the hydrolysis of emulsified long chain triacylglycerols at maximal rate in the presence of serum apolipoproteins [l] . It is generally agreed that the most potent activator of lipoprotein lipase from various sources is a polypeptide of the C-family, CII (apo-Lp-Glu) [2-6]. The other C-peptides, CI (apo-Lp-Ser) and CIII (apo-Lp-Ala), have been reported to both activate and inhibit LPL activity. These diverging results may be explained partly by differences in experimental conditions [4,5] or by contaminants in the apolipoprotein preparations used, but it is evident that species differences and the varying enzyme sources employed could also account for such discrepancies [2-71. We have therefore investigated this problem further using human adipose tissue as enzyme source. Employing apolipoproteins purified from human HDL and VLDL, we found CII to be the main activator of LPL. CI, AI (apo-Lp-Gln I), and the arginine-rich peptide [8] strongly inhibited activation of LPL by CII.
* Correspondence
to:
Dr
S-221 85 Lund, Sweden.
Rolf
Ekman. Department
of Clinical Chemistry.
University
Hospital.
30
Materials and methods Enzyme sources Human subcutaneous adipose tissue was obtained by surgical biopsy. Acetone-ether powders were made as described earlier [9] except that 4% crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, U.S.A. stated purity >99%) was substituted for serum as bulk-increasing agent. The dried acetone-ether powders were extracted with ammonia buffer as described [9] . The suspensions were centrifuged and the clear supernatants pooled and used as enzyme source. As a second enzyme source, heparin eluates were prepared by incubating biopsy specimens for 30 min at 37°C in a physiological buffer medium (pH 7.7) with heparin, glucose, and insulin [lo]. The enzymatic activity (see below) of the enzyme sources varied from preparation to preparation, corresponding to 3-15 mU of enzymatic activity per g of adipose tissue. To facilitate comparison of separate experiments, all enzymatic activities are therefore expressed as percent of controls. Preparation of apolipoproteins Human VLDL and HDL were prepared from freshly collected serum from fasting adults with Dextran sulfate 2000 (Pharmacia Fine Chemicals) and CaCl, according to Burstein et al. [ 111 . The lipoprotein preparations were thoroughly dialyzed against 0.02 M Tris HCl, 0.01 M EDTA, and 0.15 M NaCl, pH 7.7, at +8”C for 24 hours. The purity of the lipoprotein preparation was checked by agarose gel electrophoresis [ 121 and crossed immunoelectrophoresis [ 131. Lipoprotein electrophoresis revealed no contamination, but in the sensitive crossed immunoelectrophoresis with an antiserum against human serum protein (Daco AS, Copenhagen, Denmark) VLDL and HDL showed trace amounts of plasma proteins. The lipoproteins were delipidated with 1,1,3,3-tetramethylurea (Sigma Chemical Co., U.S.A.) as described [14]. With optimal concentration (4.3 mol/l) of 1,1,3,3tetramethylurea, the technique permits delipidation which gives an almost complete recovery of proteins essentially devoid of lipids. The apoprotein mixture was dialyzed against 0.01 M Tris!HCl, 0.001 M EDTA, 6 M urea, pH 8.0, before chromatographic separation. The apoproteins of VLDL (CI, CII, CIII, arginine-rich) were separated by ion exchange chromatography as described [S] with some modifications in columns (1.5 cm X 30 cm) packed with Whatman DE-52 anion-exchange cellulose and equilibrated at +S”C with 50 ml of 10 mM Tris/HCl, 6 M urea, pH 8.0, containing 1 mM EDTA at a flow rate of 30 ml per hour. lo-20 mg of apoprotein was applied to the column. Elution was performed with a linear gradient from 0 to 0.15 M of NaCl. The effluent was collected in 5 ml fractions. Pooled fractions were immediately dialyzed extensively against 10 mM Tris/ HCl, 1 mM EDTA, pH 8.0, and concentrated by membrane ultrafiltration with UM-2 membrane (Amicon). AI and AI1 apoproteins from HDL were isolated by gel filtration as described [ 151 . The homogeneity of the preparations was established by polyacrylamide gel electrophoresis at pH 8.5 in 7.5% gels containing 8 M urea [16] (Fig. 1). Amino acid composition was determined on a Jeol-6-AH amino acid analyzer 1171 to assess purity of isolated apoproteins and agreed with published results
31
Fig. 1. Polyacrylamide gel electrophoresisin used in this investigation.
[8,25].
Protein
concentrations
7.5%acrylamide.
were determined
8 M urea. pH 8.5 of the isolated
by the method
apoproteins
of Lowry et al.
[IsI. As control proteins, two nonlipoprotein weight in the same range as the apoliproteins, colipase [20] , were used.
polypeptides with i.e. fl,-microglobulin
molecular [19] and
Incubations LPL activity was assayed using a triacylglycerol emulsion consisting of tri[3 H] oleoyl glycerol stabilized by lysolecithin in a calcium-containing Tris/ HCl buffer (pH 8.0) essentially as described before [21] except for the substitution of fasting serum by 4% crystalline bovine serum albumin. To test the effects of apolipoproteins on enzymatic activity, 50 1.11enzyme source and 50 ~1 apolipoprotein solution were preincubated for 10 min at 37°C and the incubation started by addition of 100 ~1 substrate solution. In the studies of the effects of various apolipoproteins on the CII activation of LPL, the enzyme was first mixed with CII and immediately thereafter the apolipoprotein to be tested was added. In these latter incubations, the final concentration of CII was generally 5 pg/ml, which was the lowest concentration which gave essentially maximal activation. All assays were performed in duplicate. Precision of the assay, tested on a series of 28 duplicates, was 3.7% (variation coefficient) in the range of 0.008+.15 mU of enzymatic activity.
32
Results Effects
of apolipoproteins
on LPL actiuity
With both enzyme sources, activity (Fig. 2). Maximal (about was obtained at low apolipoprotein
CII was the most potent activator of LPL a three-fold) increase in enzymatic activity concentrations (5-10 pg/ml) and essentially
2cu
arg-rich Cl
0.f 0 io
ary-rich
io Apolipoprotein
0
10
.
20 Apolipoprotein
W cont.
40
50
0
b
io
20 Apolipoprotein
ipgiml)
30 cont.
40
So
iugiml)
0
.
30 cont.
40 tug/ml)
gn
0
10
20 Apolipoprotein
30
40 cont.
M
bglml)
Fig. 2. Effect of apolipoproteins and two nonlipoprotein polypeptides (@: = &-microglobulin) on the lipolytic activity of two lipoprotein lipase preparations of human adipose tissue. A, extracts of aeetoneether powders; B, heparin eluates. Enzymatic activity is expressed as percent of the activity in the absence of apolipoprotein. Final concentrations were: trioleoyl glycerol, 1.25 ~mol/ml: lysolecithin, 0.05 mg/ml; crystalline bovine serum albumin, 0.17 % (w/v); CaC12, 10 mM: and apolipoproteins as indicated in 0.1 M Tris HCl (pH 8.0).
33
equalled that provided by saturating concentrations of whole serum, indicating that destruction of the biological activity of the peptide during preparation was small. Addition of CIII also enhanced LPL activity slightly (lo-20%) (Fig. 2). The other apolipoproteins as well as the nonlipoprotein polypeptides, f12microglobulin and colipase (which is known to modify pancreatic lipase activity [20] ), showed varying degrees of inhibition especially at high concentrations (Fig. 2). It is interesting to note, however, that at 20 pg/ml, only CI, AI, and the arginine-rich peptide significantly inhibited enzymatic activity in the heparin eluates (cf. below) (Fig. 2B). Effects
of apolipoproteins
In these
on LPL activation
experiments,
maximal
by CII
activation
of LPL was provided
by the
Cl arg-rich
Apolipoprotein
cont.
fpglml)
arg-rich rl
Apolipoprotein
cont.
(vglml)
0
Apolipoprotein
cont.
(pglml)
Apolipoprotein
cont.
(uglml)
Fig. 3. Effect of apolipoproteins and two nonlipoprotein polypeptides (& = &-microglobulin) on CIIstimulated lipolytic activity of two lipoprotein lipase preparations of human adipose tissue. A, extracts of acetone-ether powders: B, heparin eluates. Enzymatic activity is expressed as percent of activity in the presence of CII. In all assays. CII was present at a concentration of 5 &ml: other components of the incubation mixture are given under Fig. 2.
34
addition of CII (5 &g/ml) and the effects of other apolipoproteins were tested over the range O-LOO pg/ml (Fig. 3). With the heparin eluate as enzyme source CI, AI, and the arginine-rich peptide strongly inhibited the stimulation of LPL activity by Cl1 (7O-95% inhibition at 40 pg/ml) (Fig, 3B). CIII and A11 inhibited enzymatic activity to a much lesser extent (lo-1570 inhibition at 30 and 40 pg/ml, respectively). The effect of these apolipoproteins did not differ significantly from those observed with colipase and & -microglobulin. The results obtained with the extracts of acetone-ether powders of adipose tissue were qualitatively similar, but the effects of CI, AI, and the arginine-rich peptide were generally somewhat less pronounced with this enzyme source (Fig. 3A). Inhibition by CI, AI, and the argini~le-rich peptide seemed to depend on the absolute amount of these apoliproteins, since additional CII (to 50 pg/ml) did not relieve the inhibition. Discussion In agreement with earlier studies using other enzyme sources [ 2-61, we found that only CII could substitute for serum as an activator of human adipose tissue LPL. The slight stimulation of LPL activity by CIII is consistent with the findings of LaRosa et al. [ 2] and Have1 et al. [ 51 and does not seem to be due to contamination by CII (Fig. 1). The inhibitory effect of CI on CII activation of LPL was also found by Have1 et al. [5], Bensadoun et al. [6], and Twu et al. [22], using LPL from cow’s milk, porcine adipose tissue, and rat heart, respectively. In the two former studies [5,6], however, CIII was reported to be almost as potent an inhibitor of LPL activity as CI. This discrepancy with the present results may be due to the different enzyme preparations used. Human adipose tissue LPL also seems to differ from LPL from cow’s milk with regard to the effect of AI, which has been reported not to modify the enzymatic activity from the latter source [23,24]. An inhibitory effect of AI on LPL activity has been reported by Krauss et al. [43, using rat adipose tissue as enzyme source. In contrast to the present findings, however, this inhibition was seen only when substrate concentration was reduced below the saturation level. Another new finding in the present study was the strong inhibitory effect on LPL activity by the argininerich peptide. Although the inhibitor effect of CI, AI, and the ~ginine-rich peptide was found using both enzyme sources, the LPL activity eluted with heparin was more strongly inhibited than the enzymatic activity of the acetoneether preparation. The reason for this is unknown. Possible physiological implications of the inhibition of LPL activity by AI, CI, and the arginine-rich peptide are speculative, but at least the two latter apolipoproteins may conceivably modify the hydrolysis of triglyceride by LPL under physiological or pathophysiological conditions. Have1 et al. [ 251 reported high concentrations of the arginine-rich peptide in the P-migrating VLDL fraction of patients with primary dysbetalipoproteinemia (type III). It is possible that inhibition of lipolytic processes by the high concentrations of this apolipoprotein may be a cont~buto~ factor in the development of this lipoproteinemia. Similarly high concentrations of CI which we have found in the abnormal LDL fraction containing Lp-X from patients with obstructive jaundice (unpublished
35
observations) patients.
may
relate
to
the
hypertriglyceridemia
developing
in these
Acknowledgements Mrs Birgitta Rapp and Mrs Berit Johansson gave skilful technical assistance. This work was supported by grants from the Swedish Medical Research Council (03X-3362 and 03X-4147), the Swedish Nutrition Foundation, and the Medical Faculty, University of Lund. References 1
Kern.
2
LaRosa,
E.D.
(1955)
J.C.,
Commun. Brown,
W.V.
4
Krauss,
R.M..
5
Havel,
and
R.J..
chemistry Bensadoun,
7
Ganesan,
8
Shore,
A., D.,
11
Burstein. Johansson, Ganrot,
15 16
l-14
P., Lux.
S.E.
and
Fredrickson.
(1972)
A.,
Toth.
Davies,
B.J.
Berg&d,
Biophys.
Fredrickson, Shore,
T.,
D.S.
Res.
D.S.
V.G..
(1970)
Commun.
(1973)
Biochem. 46.
Circ.
Fielding,
J.O.
Biophys.
P.E.
Res.
Res.
and
3’75-382 33.
403-411
Egelrud,
T.
(1973)
20
Borgstrijm, Nilsson-Ehle, J.-S.,
Beam,
B. and
I.M.
22
Twu.
23
Havel,
R.J..
Garfinkel, Shore,
AS.
24
Miller.
A.L.
and Smith,
25
Havel.
R.J.
V.G.,
and Kane,
Chim.
L.C. J.P.
(1973)
J. Lipid Suppl.
Suppl.
Chem.
FEBS
Lett.
249, 15,
2220-2227 205-208
Acta
42,
383-390
730-741
Res.
11.
124.
124.
583-595
7-19
39-41
and
E. (1969)
Randall,
Chem.
Biochim.
Acta
54.
M.C.
J. Biol. Proc.
Stiller.
Natl.
Biochemistry
8. 3309-3316
404-427
J. Chromatogr.
A.L.
B. and Bier,
(1973)
(1970)
121,
J. Biol.
and Schotz, Shore,
J. Biol.
(1973)
Chim. 15,
Invest.
S. and
(1972) Farr,
C. (1971)
Clin.
Clin.
Metabolism
Invest.
(1974) W.J.
350-364 Sci.
(1968)
Erlanson,
P. (1974)
Lab. 53.
Acad.
N.J., A.G.
R.
W.V.
502-507
P. (1972)
Lab.
C., Koga,
N.Y.
and Karlsson.
I. and
21
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J., Edelstein.
12,
B. (1966)
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Biochem.
Rosebrough,
McConathy,
and Morfin.
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Ann.
Brown.
P. and
Belfrage,
Hood,
&and.
Anal.
(1964)
O.H.,
H.R.
D. and
Biochemistry
H. and
P. and
(1972)
Scan%
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and
Alaupovic,
B. (1973)
Sholnick,
B.G. (1973)
Lowry,
Biochem.
R.I.
C.. Steinberg, R.D..
P.. Tornqvist.
P.O.
Jeppsson.
Olivecrona,
Ehnholm,
J.P.
17
(1972)
C.J.,
Bjijmtorp,
M.,
18
M.L. Levy.
and Shore,
B.,
12
Kane,
215.
P.N..
Bradford,
Nilsson-Ehle, Persson,
13
Baginsky,
Herbert, Fielding,
V.G.
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93-103
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4095-4103 Acta
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Bio-
Clinica Chimica Acta, 63 (1975) 29-35 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7177
EFFECTS OF APOLIPOPROTEINS OF HUMAN ADIPOSE TISSUE
R. EKMAN*
ON LIPOPROTEIN
LIPASE ACTIVITY
and P. NILSSON-EHLE
Department of Clinical Chemistry, University Hospital, Lund, and Department Physiological Chemistry 4, University of Lund, Lund (Sweden) (Received
of
March 3,1975)
Summary The effect of apolipoproteins isolated from HDL and VLDL on the activity of lipoprotein lipase (LPL) of adipose tissue was studied. The CII apoprotein was found to activate LPL. This activation was strongly inhibited by CI, AI (apo-Lp-Gln I), and the arginine-rich apoprotein, whereas AI1 and CIII exhibited a considerably lower inhibitory effect.
Introduction Lipoprotein lipases (LPL) catalyze the hydrolysis of emulsified long chain triacylglycerols at maximal rate in the presence of serum apolipoproteins [l] . It is generally agreed that the most potent activator of lipoprotein lipase from various sources is a polypeptide of the C-family, CII (apo-Lp-Glu) [2-6]. The other C-peptides, CI (apo-Lp-Ser) and CIII (apo-Lp-Ala), have been reported to both activate and inhibit LPL activity. These diverging results may be explained partly by differences in experimental conditions [4,5] or by contaminants in the apolipoprotein preparations used, but it is evident that species differences and the varying enzyme sources employed could also account for such discrepancies [2-71. We have therefore investigated this problem further using human adipose tissue as enzyme source. Employing apolipoproteins purified from human HDL and VLDL, we found CII to be the main activator of LPL. CI, AI (apo-Lp-Gln I), and the arginine-rich peptide [8] strongly inhibited activation of LPL by CII.
* Correspondence
to:
Dr
S-221 85 Lund, Sweden.
Rolf
Ekman. Department
of Clinical Chemistry.
University
Hospital.
30
Materials and methods Enzyme sources Human subcutaneous adipose tissue was obtained by surgical biopsy. Acetone-ether powders were made as described earlier [9] except that 4% crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, U.S.A. stated purity >99%) was substituted for serum as bulk-increasing agent. The dried acetone-ether powders were extracted with ammonia buffer as described [9] . The suspensions were centrifuged and the clear supernatants pooled and used as enzyme source. As a second enzyme source, heparin eluates were prepared by incubating biopsy specimens for 30 min at 37°C in a physiological buffer medium (pH 7.7) with heparin, glucose, and insulin [lo]. The enzymatic activity (see below) of the enzyme sources varied from preparation to preparation, corresponding to 3-15 mU of enzymatic activity per g of adipose tissue. To facilitate comparison of separate experiments, all enzymatic activities are therefore expressed as percent of controls. Preparation of apolipoproteins Human VLDL and HDL were prepared from freshly collected serum from fasting adults with Dextran sulfate 2000 (Pharmacia Fine Chemicals) and CaCl, according to Burstein et al. [ 111 . The lipoprotein preparations were thoroughly dialyzed against 0.02 M Tris HCl, 0.01 M EDTA, and 0.15 M NaCl, pH 7.7, at +8”C for 24 hours. The purity of the lipoprotein preparation was checked by agarose gel electrophoresis [ 121 and crossed immunoelectrophoresis [ 131. Lipoprotein electrophoresis revealed no contamination, but in the sensitive crossed immunoelectrophoresis with an antiserum against human serum protein (Daco AS, Copenhagen, Denmark) VLDL and HDL showed trace amounts of plasma proteins. The lipoproteins were delipidated with 1,1,3,3-tetramethylurea (Sigma Chemical Co., U.S.A.) as described [14]. With optimal concentration (4.3 mol/l) of 1,1,3,3tetramethylurea, the technique permits delipidation which gives an almost complete recovery of proteins essentially devoid of lipids. The apoprotein mixture was dialyzed against 0.01 M Tris!HCl, 0.001 M EDTA, 6 M urea, pH 8.0, before chromatographic separation. The apoproteins of VLDL (CI, CII, CIII, arginine-rich) were separated by ion exchange chromatography as described [S] with some modifications in columns (1.5 cm X 30 cm) packed with Whatman DE-52 anion-exchange cellulose and equilibrated at +S”C with 50 ml of 10 mM Tris/HCl, 6 M urea, pH 8.0, containing 1 mM EDTA at a flow rate of 30 ml per hour. lo-20 mg of apoprotein was applied to the column. Elution was performed with a linear gradient from 0 to 0.15 M of NaCl. The effluent was collected in 5 ml fractions. Pooled fractions were immediately dialyzed extensively against 10 mM Tris/ HCl, 1 mM EDTA, pH 8.0, and concentrated by membrane ultrafiltration with UM-2 membrane (Amicon). AI and AI1 apoproteins from HDL were isolated by gel filtration as described [ 151 . The homogeneity of the preparations was established by polyacrylamide gel electrophoresis at pH 8.5 in 7.5% gels containing 8 M urea [16] (Fig. 1). Amino acid composition was determined on a Jeol-6-AH amino acid analyzer 1171 to assess purity of isolated apoproteins and agreed with published results
31
Fig. 1. Polyacrylamide gel electrophoresisin used in this investigation.
[8,25].
Protein
concentrations
7.5%acrylamide.
were determined
8 M urea. pH 8.5 of the isolated
by the method
apoproteins
of Lowry et al.
[IsI. As control proteins, two nonlipoprotein weight in the same range as the apoliproteins, colipase [20] , were used.
polypeptides with i.e. fl,-microglobulin
molecular [19] and
Incubations LPL activity was assayed using a triacylglycerol emulsion consisting of tri[3 H] oleoyl glycerol stabilized by lysolecithin in a calcium-containing Tris/ HCl buffer (pH 8.0) essentially as described before [21] except for the substitution of fasting serum by 4% crystalline bovine serum albumin. To test the effects of apolipoproteins on enzymatic activity, 50 1.11enzyme source and 50 ~1 apolipoprotein solution were preincubated for 10 min at 37°C and the incubation started by addition of 100 ~1 substrate solution. In the studies of the effects of various apolipoproteins on the CII activation of LPL, the enzyme was first mixed with CII and immediately thereafter the apolipoprotein to be tested was added. In these latter incubations, the final concentration of CII was generally 5 pg/ml, which was the lowest concentration which gave essentially maximal activation. All assays were performed in duplicate. Precision of the assay, tested on a series of 28 duplicates, was 3.7% (variation coefficient) in the range of 0.008+.15 mU of enzymatic activity.
32
Results Effects
of apolipoproteins
on LPL actiuity
With both enzyme sources, activity (Fig. 2). Maximal (about was obtained at low apolipoprotein
CII was the most potent activator of LPL a three-fold) increase in enzymatic activity concentrations (5-10 pg/ml) and essentially
2cu
arg-rich Cl
0.f 0 io
ary-rich
io Apolipoprotein
0
10
.
20 Apolipoprotein
W cont.
40
50
0
b
io
20 Apolipoprotein
ipgiml)
30 cont.
40
So
iugiml)
0
.
30 cont.
40 tug/ml)
gn
0
10
20 Apolipoprotein
30
40 cont.
M
bglml)
Fig. 2. Effect of apolipoproteins and two nonlipoprotein polypeptides (@: = &-microglobulin) on the lipolytic activity of two lipoprotein lipase preparations of human adipose tissue. A, extracts of aeetoneether powders; B, heparin eluates. Enzymatic activity is expressed as percent of the activity in the absence of apolipoprotein. Final concentrations were: trioleoyl glycerol, 1.25 ~mol/ml: lysolecithin, 0.05 mg/ml; crystalline bovine serum albumin, 0.17 % (w/v); CaC12, 10 mM: and apolipoproteins as indicated in 0.1 M Tris HCl (pH 8.0).
33
equalled that provided by saturating concentrations of whole serum, indicating that destruction of the biological activity of the peptide during preparation was small. Addition of CIII also enhanced LPL activity slightly (lo-20%) (Fig. 2). The other apolipoproteins as well as the nonlipoprotein polypeptides, f12microglobulin and colipase (which is known to modify pancreatic lipase activity [20] ), showed varying degrees of inhibition especially at high concentrations (Fig. 2). It is interesting to note, however, that at 20 pg/ml, only CI, AI, and the arginine-rich peptide significantly inhibited enzymatic activity in the heparin eluates (cf. below) (Fig. 2B). Effects
of apolipoproteins
In these
on LPL activation
experiments,
maximal
by CII
activation
of LPL was provided
by the
Cl arg-rich
Apolipoprotein
cont.
fpglml)
arg-rich rl
Apolipoprotein
cont.
(vglml)
0
Apolipoprotein
cont.
(pglml)
Apolipoprotein
cont.
(uglml)
Fig. 3. Effect of apolipoproteins and two nonlipoprotein polypeptides (& = &-microglobulin) on CIIstimulated lipolytic activity of two lipoprotein lipase preparations of human adipose tissue. A, extracts of acetone-ether powders: B, heparin eluates. Enzymatic activity is expressed as percent of activity in the presence of CII. In all assays. CII was present at a concentration of 5 &ml: other components of the incubation mixture are given under Fig. 2.
34
addition of CII (5 &g/ml) and the effects of other apolipoproteins were tested over the range O-LOO pg/ml (Fig. 3). With the heparin eluate as enzyme source CI, AI, and the arginine-rich peptide strongly inhibited the stimulation of LPL activity by Cl1 (7O-95% inhibition at 40 pg/ml) (Fig, 3B). CIII and A11 inhibited enzymatic activity to a much lesser extent (lo-1570 inhibition at 30 and 40 pg/ml, respectively). The effect of these apolipoproteins did not differ significantly from those observed with colipase and & -microglobulin. The results obtained with the extracts of acetone-ether powders of adipose tissue were qualitatively similar, but the effects of CI, AI, and the arginine-rich peptide were generally somewhat less pronounced with this enzyme source (Fig. 3A). Inhibition by CI, AI, and the argini~le-rich peptide seemed to depend on the absolute amount of these apoliproteins, since additional CII (to 50 pg/ml) did not relieve the inhibition. Discussion In agreement with earlier studies using other enzyme sources [ 2-61, we found that only CII could substitute for serum as an activator of human adipose tissue LPL. The slight stimulation of LPL activity by CIII is consistent with the findings of LaRosa et al. [ 2] and Have1 et al. [ 51 and does not seem to be due to contamination by CII (Fig. 1). The inhibitory effect of CI on CII activation of LPL was also found by Have1 et al. [5], Bensadoun et al. [6], and Twu et al. [22], using LPL from cow’s milk, porcine adipose tissue, and rat heart, respectively. In the two former studies [5,6], however, CIII was reported to be almost as potent an inhibitor of LPL activity as CI. This discrepancy with the present results may be due to the different enzyme preparations used. Human adipose tissue LPL also seems to differ from LPL from cow’s milk with regard to the effect of AI, which has been reported not to modify the enzymatic activity from the latter source [23,24]. An inhibitory effect of AI on LPL activity has been reported by Krauss et al. [43, using rat adipose tissue as enzyme source. In contrast to the present findings, however, this inhibition was seen only when substrate concentration was reduced below the saturation level. Another new finding in the present study was the strong inhibitory effect on LPL activity by the argininerich peptide. Although the inhibitor effect of CI, AI, and the ~ginine-rich peptide was found using both enzyme sources, the LPL activity eluted with heparin was more strongly inhibited than the enzymatic activity of the acetoneether preparation. The reason for this is unknown. Possible physiological implications of the inhibition of LPL activity by AI, CI, and the arginine-rich peptide are speculative, but at least the two latter apolipoproteins may conceivably modify the hydrolysis of triglyceride by LPL under physiological or pathophysiological conditions. Have1 et al. [ 251 reported high concentrations of the arginine-rich peptide in the P-migrating VLDL fraction of patients with primary dysbetalipoproteinemia (type III). It is possible that inhibition of lipolytic processes by the high concentrations of this apolipoprotein may be a cont~buto~ factor in the development of this lipoproteinemia. Similarly high concentrations of CI which we have found in the abnormal LDL fraction containing Lp-X from patients with obstructive jaundice (unpublished
35
observations) patients.
may
relate
to
the
hypertriglyceridemia
developing
in these
Acknowledgements Mrs Birgitta Rapp and Mrs Berit Johansson gave skilful technical assistance. This work was supported by grants from the Swedish Medical Research Council (03X-3362 and 03X-4147), the Swedish Nutrition Foundation, and the Medical Faculty, University of Lund. References 1
Kern.
2
LaRosa,
E.D.
(1955)
J.C.,
Commun. Brown,
W.V.
4
Krauss,
R.M..
5
Havel,
and
R.J..
chemistry Bensadoun,
7
Ganesan,
8
Shore,
A., D.,
11
Burstein. Johansson, Ganrot,
15 16
l-14
P., Lux.
S.E.
and
Fredrickson.
(1972)
A.,
Toth.
Davies,
B.J.
Berg&d,
Biophys.
Fredrickson, Shore,
T.,
D.S.
Res.
D.S.
V.G..
(1970)
Commun.
(1973)
Biochem. 46.
Circ.
Fielding,
J.O.
Biophys.
P.E.
Res.
Res.
and
3’75-382 33.
403-411
Egelrud,
T.
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