Studies on the function of hepatic lipase in the cat after immunological blockade of the enzyme in vivo

Atherosclerosis, 69 (1988) 173-183 Elsetier Scientific Publishers Ireland,

173 Ltd.

ATH 04067

Studies on the function of hepatic lipase in the cat after immunological blockade of the enzyme in vivo Pierre N.M. Demacker, Anneke G.M. Hijmans, Anton F.H. Stalenhoef and Albert van ‘t Laar Department

of Medicine, Division of General Internal Medicine,

St. Radboud Hospital,

University of Nijmegen

(The Netherlands)

(Received 29 April, 1987) (Revised, received 21 August, 1987) (Accepted 24 August, 1987)

In order to investigate the in vivo function of hepatic lipase, cats were injected with anti-cat hepatic lipase antibodies which produced a complete and specific inhibition of heparin-releasable hepatic lipase. The cat was chosen as an animal model because it displays, like man, a relative deficiency of lipoprotein lipase compared to hepatic lipase and because the possession of two subfractions of high density lipoproteins, HDL, and HDL,. In fasted cats no changes were observed in plasma triglycerides or phospholipids. In fed animals triglycerides increased considerably, indicating that hepatic lipase may have a function in the postprandial phase. In fat-loaded cats (6 g of fat/kg) triglycerides in the d < 1.019 g/ml fraction increased from 4 h after the blockade due to accumulation of lipoproteins with pre&mobility containing the apoproteins, apo B-100, apo E and apo A-I. Apo B-48 did not accumulate consistently. Phospholipids in the HDL,-fraction and those in the HDL,-fraction of the fat-loaded cats tended to increase and decrease from 6 and 9 h after the blockade, respectively. The absolute change in HDL, phospholipids approximated that of HDL,phospholipids. Overall, the density of HDL particles decreased, apparently secondary to the accumulation of apo A-I in the d < 1.019 g/ml fraction. Our findings suggest that hepatic lipase is involved in the hydrolysis of a special class of apo A-I containing triglyceride-rich lipoproteins synthesised in the postprandial phase.

Key words: Hepatic triglyceride lipase; Animal model; Chylomicrons; Apoproteins

This study was presented at the Washington Spring Symposium, 20-23 May, 1986, and has been published in part in Arteriosclerosis, 4 (1984) 516a. Correspondence to: P.N.M. Demacker, Department of Medicine, Division of General Internal Medicine, St. Radboud Hospital, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Abbreviations: LPL, lipoprotein hpase; HTGL, hepatic triglyceride lipase or hepatic lipase; VLDL, very low density 0021-9150/88/$03.50

0 1988 Elsevier Scientific

Publishers

Ireland,

Lipoproteins;

Postprandial

phase;

lipoproteins (d < 1.006 g/ml); IDL, intermediate density lipoproteins (1.006 < d < 1.019 g/ml); LDL, low density lipoproteins (1.019 < d < 1.063 g/ml); HDL, and HDL,, subfractions of high density lipoproteins with densities 1.063 -C d < 1.100 g/ml and 1.100 -C d < 1.185 g/ml, respectively; apo-, apolipoprotein; EDTA, ethylenediamine tetraacetate; TG, triglycerides.

Ltd.

174 Introduction

Intravenous injection of heparin releases two lipolytic enzymes into the circulation, lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) [l]. The former enzyme has a function in the hydrolysis of triglycerides both of chylomicrons and VLDL [2,3]. The role of HTGL in lipoprotein metabolism is still not clear. Based on in vitro and in vivo studies a function has been suggested in the conversion of VLDL-remnants to LDL [4-61, in the metabolism of apo B-48 containing lipoproteins [7] and in the conversion of HDL, to HDL, [8-131. To elucidate the specific function of HTGL, several studies have been performed in rats [4, 6-91 and one in cynomolgus monkeys [5], in which HTGL was blocked by specific antibodies in vivo. Because these animals have a relative deficiency of HTGL compared to LPL [14,15], primary and secondary effects caused by a rise of LPL are not readily to distinguish. Even in rats different results have been reported in hooded and albino rats ascribed to the differences in the remaining LPL activity [7]. To minimise the eventual secondary effects, animal models should be used with a relative deficiency of LPL compared to HTGL. In this respect the cat is a suitable model [16]. Besides, in this animal the removal of chylomicrons is as rapidly as in man and apo B-48 is synthesised exclusively in the intestine. Furthermore, like man, this animal model possesses two distinct HDL subfractions [16]. For the present studies highly purified cat HTGL was used for immunisation. The antiserum produced was analysed by Ouchterlony’s immunodiffusion test. Precipitation arcs were made visible by protein staining which considerably enhanced the sensitivity of this method. The antiserum was found to be monospecific and showed no cross-reactivity with cat lipoprotein lipase, apoproteins and, after purification, also not with other cat serum proteins. This purified antiserum was used to completely inhibit HTGL in vivo for up to 24 h. Because there is evidence that the enzyme may have a function in the post-prandial phase of lipoprotein metabolism [4,17,18], experiments were performed in fasted and fed animals. A clear response was only observed in the latter group. Therefore, the

effect of HTGL fat-loaded cats.

blockade

was also studied

in

Materials and methods

Animals Adult male cats (Felix domesticus), 3.5-5 kg, were maintained as colonies of 4-6 animals on a commercial diet (LF-32, cat diet, Hope Farms, Woerden, The Netherlands). Most studies were done in pairs, one cat receiving anti-HTGL-IgG and the other non-immune IgG. The body weights of both were matched within 5%. Isolation of hepatic Iipase From 3 cats previously used in a neurological experiment, livers were perfused at 37°C with Krebs-Ringer bicarbonate buffer (pH 7.4) to flush out the blood 1191.Perfusion with the same buffer containing 40 IU of heparin/ml resulted in a rapid release of a triglyceride lipase, not inhibitable by 1 M NaCl. To the pooled samples NaCl (0.2 M) and 60 ml heparin-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) pre-equilibrated with 0.01 M Tris-HCI buffer (pH 7.4) containing 0.2 M NaCl was added. Binding was performed by end-over-end rotation at 4” C for 2 h (40 cycles/mm). The gel was then washed on a Buchner funnel with 5 x 200 ml of the Tris-HCl buffer containing 0.2 M NaCl and 10% (v/v) glycerol, followed by 2 washings with the same buffer, containing 0.1% (w/v) Triton X-100. Coupling efficiency was 70%. The gel was then packed into a column, diameter 2.5 cm, and the washing with the Triton X-100 containing buffer was continued for 4 h at a flow rate of 1 ml/mm until the absorbance at 280 nm did not decrease any further. Only one peak of protein and enzyme activity (not inhibitable by 1.0 m NaCl in the substrate) was released at 0.8 M NaCl, when a linear gradient of 2 x 200 ml, 0.3-2.0 M NaCl in the buffer containing 10% glycerol was used for elution. The fractions with the highest enzymatic activity were pooled and concentrated by dialyzing against 3.6 M (NH,),SO, overnight at 4°C [20]. The precipitated protein was then recovered by centrifugation for 30 n-tin at 3500 X g at 4” C, dissolved in 1% glycine/glycerol (8 : 2, v/v) and purified further by preparative flat bed isoelectric

175 focusing. (LKB application note 198): 100 ml of 4% Ultrodex (LKB) slurry containing 5% glycerol and 5% ampholine, pH 4-6, was spread evenly into a tray with dimensions of 24.5 X 11 cm. By use of a small fan 27% evaporation of the fluid was obtained (checked by weighing). The anode strip was soaked in 1 M phosphoric acid and the cathode strip in M NaOH. The sample (5.2 mg) dissolved in 3 ml glycine/glycerol solution (8 : 2, v/v) and 0.15 ml of the ampholine was applied as a narrow zone using the sample applicator inserted at l-3 cm from the anode. Electrofocusing was performed for 20 h at 4” C, at a power limit of 8 W and an initial voltage and current of 500 V and 13 mA, respectively. An almost linear pH gradient from pH 4.25 to 6.25 was obtained. The gel was fractionated into 30 equal segments by the fractionating grid and these sections were transferred to elution columns and eluted with 2 x 1 ml Tris-HCl buffer containing 0.8 M NaCl and 10% glycerol. The enzyme activity was completely transferred to the fractions with pH 4.95-5.45. The enzyme protein (recovery 30% activity 3994 pmol FFA/h/mg enzyme protein) was used for raising antibodies after concentrating and dissolution as described above. Preparation and characterisation of the antibodies against hepatic lipase 150 pg of the purified enzyme was mixed with

1.1 ml Freund’s complete adjuvant and injected subcutaneously into a sheep. Boosters mixed with incomplete Freund’s adjuvant, were given every 6 weeks. Blood was collected after the third and following boosters into EDTA, 1 mg/ml, and the IgG fraction was isolated with sodium sulphate precipitation. The IgG fraction was purified further by affinity chromatography to remove unspecific antibodies against a protein present in the d > 1.21 g/ml fraction as follows: the d > 1.21 g/ml fraction of preheparin cat serum not containing LPL or HTGL was eluted over a heparin-Sepharose column; 5 mg of the protein released between 0.3 and 0.8 M NaCl was coupled to 40 ml of activated Bio-Gel A-5m (Affigel 10, BioRad) according to the instructions of the manufacturer. This Bio-Gel column was used for purification of the IgG samples injected. Fractions were only used which reacted negatively with whole

cat serum as examined by a sensitive double immunodiffusion test; 10 ~1 (approximately 150 pg of protein) of the purified IgG preparation was sufficient to completely (> 95%) inhibit the 1.0 M NaCl lipolytic activity from 50 ~1 of cat postheparin plasma. The anti-HTGL-IgG did not inactivate LPL from cat adipose tissue isolated as described [21]. Lipolytic activity, released from the liver by heparin and resistant to 1.0 M NaCl, was inactivated completely by the antibodies. Before injection, the IgG preparations were assayed for activity in vitro against HTGL, centrifuged for 1 h at 120000 X g to remove aggregates and sterilized by passage through a 0.45~pm filter (Millipore). Three protocols were followed. Protocol I: anesthesia (intramuscular injection of 10 mg vetalar followed by iv. injection of pentobarbital (30 mg/kg with a maximum of 90 mg) was started in 7 cats, including 2 controls, at 8.30 a.m. after 16 h of fasting. To prevent stress, the whole colony was kept fasting instead of isolating individual animals. Protocols II: anesthesia was started at 8.30 a.m. in 4 cats, including 2 controls, having continuous access to food. Protocol III: cream (6.0 g of fat/kg bodyweight) was administered orally at 8.30 a.m. without anesthesia to 12 cats including 6 controls, which had been fasting for 16 h. These cats were left alone for approximately 90 min, followed by anesthesia. Blood was sampled and the IgG was administered through a polyethylene catheter inserted into the jugular vein. The animals were ventilated with N,O and 0, (2 : 1, v/v) through a trachea tube with a LoosCo Amsterdam Infant ventilator at 20 cycles/mm, volume 2 litre, pressure 10 cm H,O, under ECG and temperature monitoring (Datascope 850). Additional pentobarbital (6 mg/kg) was administered when required (every 2-4 h, up to 8 h after the first administration of the IgG, similar amounts were injected into non-immune IgG and anti-HTGL-IgG-treated cats). Ventilation was discontinued at t = 13 h. Further blood samples up to 24 h after the first IgG administration could be drawn without anesthesia. Body temperature was maintained by a thermostated mattress. All cats recovered completely within 24 h after drawing the last blood samples. At t = 0, 180 mg of anti-HTGL-IgG was in-

176 jetted which was at least twice the amount sufficient to completely inhibit the HTGL activity in cat postheparin plasma. Half this amount was given on t = 1.5 and 3 h (protocol I and II) and on t = 6 and 12 h (protocol III). Controls received similar amounts of nonimmune IgG. Blood samples were collected into EDTA (1 mg/ml of blood) and stored in ice until separation of the plasma. At the end of the experiments heparin (100 IU/kg Thromboliquine, Organon, Oss, The Netherlands) was injected for determination of postheparin LPL and HTGL. Postheparin plasma was obtained 15 min after injection. Liver biopsies

Inhibition of HTGL in situ by i.v. injection of the antibodies was tested in liver biopsy samples. During complete anesthesia, a 5-cm abdominal incision was made. At the indicated times, a wedge-shaped piece of tissue (So-100 mg) was obtained. The site of biopsy was sutured. The liver biopsy was washed at 4°C in saline, blotted on gauze and divided in 2-3 segments which were weighed and transferred to 150 ~1 of .saline containing 10 IU of heparin/ml. After homogenisation by repeated passage through a 19-gauge needle [5], the homogenate was frozen at - 20” C until assay; 20 ~1 of the homogenate was assayed in the same way as postheparin plasma or perfused rat liver fractions. Enzyme activity was expressed/mg of homogenate protein. Complete linearity of enzyme activity with biopsy mass was obtained in the range of 20-120 mg (r = 0.99, n = 6). Analytical methods

LPL and HTGL were selectively measured with a routine immunological technique [16,22], in which HTGL was precipitated with anti-HTGL antibodies during incubation at 4 o C for 1 h followed by centrifugation to remove the immune complex. In parallel experiments it was found that no preincubation or centrifugation of the putative immune complex was required for complete inhibition, suggesting that inactivation of hepatic lipase occurs as a result of binding at or near the active site. LPL was determined at low salt concentration of 0.1 M NaCl in the presence of 10% (v/v) human serum as source for apoprotein C-II. In the assay of HTGL, the activity of LPL was

inhibited by 1.0 M NaCl and omitting the serum activator. Incubations were performed as described [16]. Lipoproteins were isolated by density gradient ultracentrifugation [16,23] total cholesterol, unesterified cholesterol, phospholipids and triglycerides were determined by enzymatic kits [16], and protein by the method of Lowry et al. [24]. Electrophoresis of lipoproteins was performed in 1% (w/v) agarose at 10 V/cm, lipoprotein bands were stained with Sudan Black B [25]. SDS gel electrophoresis of apoproteins was performed in 0.75% discontinuous gels [16]. Student’s and Wilcoxon’s paired and unpaired t-tests were used to test differences for significance. Results Effectiveness of the blockade of hepatic lipase in vivo

The intravenous injection of anti-HTGL-IgG at t = 0, 1.5 and 3 h in the protocols I and II rapidly and steadily inactivated the HTGL-IgG activity in situ, as appeared from HTGL measurements in liver biopsies taken at these time intervals. The enzyme activity in biopsies from the controls decreased only slightly (Fig. 1). The remaining HTGL-activity in situ 15 min after i.v. heparin injection was considerable lower in the controls, but did not decrease any further in those which had received the anti-HTGL-IgG (Fig. 1). This indicates, that the remaining 30% of enzyme activity which is not inhibitable by the antibodies and not releasable by heparin, (Fig. 1) must be located intracellularly. The frequent intravenous injections of the anti-HTGL-IgG inactivated HTGL activity in postheparin plasma by more than 85% (Table 1). When compared to values found in a reference group after sedation by ketamine [16], LPL at the end of the experiments in the present study was lower, probably due to the continuous fasting. This indicates that during the experiment LPL activity decreases gradually. However, the decrease was independent of whether anti-HTGLIgG or nonimmune IgG had been injected. In any case, our animal model displays a relative deficiency of LPL compared to HTGL, and sec-

177 %Acttvity

remaining

ondary effects due to changes of LPL activity can be expected to be minimal.

HeDarln lOblU/kg

Changes in the plasma and lipoprotein lipids during blockade of hepatic lipase

cr

h

-2

IG 1

0

I

1

2

3

L

5 Time(h)

I

PH

J

Fig. 1. In vivo test on the inhibition of hepatic lipase in liver biopsies by the anti-HTGL-IgG. On t = 0, 1.5 and 3 h, 6 animals received anti-HTGL-IgG (O0) and 4 animals received nonimmune IgG (0 - - - 0). Liver biopsies were taken at regular time intervals during 5 h. At the end of the experiment, heparin (100 IU/kg) was injected intravenously; 15 min later the last biopsy postheparin (pH) was taken. The numbers 5-8 represent nonfasting cats participating in protocol II, the others were fasted overnight (protocol I). The absolute values of HTGL/mg of liver tissues at t = 0 were taken as 100%.

The concentrations of plasma triglycerides in the fasted animals treated with anti-HTGL-IgG did not change (Fig. 1). In two fasted cats, otherwise not included in the protocol, the experiment was prolonged from 5 to 24 h. Continuous blockade of HTGL was maintained by additional injection of anti-HTGL-IgG at t = 6 and 12 h. However, the highest concentration of plasma triglycerides (0.39 mmol/l) had already been reached within 5 h of the blockade, indicating that the absence of any accumulation of plasma triglycerides in the fasting cats was not due to insufficient duration of the blockade (data not shown). In the fed and fat-loaded animals, the blockade of HTGL resulted in average in a gradual increase of the plasma and VLDL + IDL-triglycerides which was significant from 4 h after the start of the blockade (Figs. 1 and 2). In the nonimmune-IgGtreated cats, triglycerides in plasma and in the d < 1.019 g/ml fraction did not increase. These results indicate that HTGL has a function in the hydrolysis of VLDL + IDL-triglycerides. Plasma cholesterol did not change during HTGL blockade in the fasted nor in the fed animals. In the fat-loaded HTGL-blocked cats, plasma cholesterol increased concomitantly with the triglycerides (Fig. 1). In the fat-loaded cats, blockade of HTGL resulted in an increase of HDL,-phospholipids, significantly different from

TABLE 1 EFFECT OF ANTI-HTGL-IgG

ADMINISTRATION

ON THE POSTHEPARIN

PLASMA ACTIVITY OF LPL AND HTGL

LPL and HTGL (mean+SD, activities pmol FFA/h/ml) were determined in postheparin plasma at the end of the experiments performed according to protocols I, II, III. The controls received similar amounts of nonimmune sheep IgG. The animals were anesthetised with pentobarbital and ventilated with N20: Oz. Anesthetising in the fat-loaded animals was discontinued 13 h after the first IgG injection. LPL

Fasted Fed Fat-loaded

HTGL

Anti-HTGL-IgG

Nonimmune IgG

Anti-HTGL-IgG

Nonimmune IgG

3.0 f OS(5) a 5.7-7.1 2.4 f 0.9(6)

3.2-5.5 5.2-5.4 1.85 f LO(6)

0,4 f 0.2(5) 1.6-4.4 0.2 & 0.3(6)

28.0-32.5 18.3-22.7 21.0+ 3.8(6)

a Number of animals studied.

178 Cholesterol

(mmol/l

h-

1

,

I

Triglycerides

I

1

(mmol/i

I I

II

III

I

) I

0

2

4

6

0

2

4

6

0

2

4

6

8

10

12

19 Time

24 (h)

Fig. 2. Plasma cholesterol and triglyceride concentrations in the cats treated according to the various protocols. Overnight fasted cats (I), fed cats (II) and fat-loaded cats (III) received anti-HTGL-IgG (O?? ) or control IgG (0 - - - 0) on t = 0. Continuous blockade was maintained for up to 24 h by repeated injection of the IgG. In protocol III the fat load was given approximately 90 min before the first injection of the IgG. Values were expressed as mean + SEM. In protocol I, 5 immune-treated cats and 2 controls were studied. In protocol II the individual data of 2 controls and 2 immune-treated cats are shown. In protocol III the average results are given obtained in 6 immune-treated cats and 6 controls. * P < 0.05 (paired Student’s t-test); * * P < 0.01.

the values in the nonimmune serum-treated cats at t = 19 and 24 h (Fig. 3). No significant differences were obtained in HDL,-phospholipids in both groups of fat-loaded cats. Nevertheless, within the group of fat-loaded cats treated with anti-HTGLIgG a trend to increasing HDL,- and decreasing HDL,-phospholipids, respectively, could be seen. These changes occurred largely between 6 and 9 h for HDL,- and between 9 and 19 h for HDL,phospholipids. The absolute change in HDL,phospholipids approximated that of HDL,-phospolipids (Fig. 3). However, the changes in the latter fraction occurred later than those in the former and these in turn amply followed the changes in triglycerides which, as shown before, were on average higher from 4 h after the start of the blockade. The absence of any consistent change

in HDL,- and HDL,-phospholipids in the fasted or fed cats during blockade of HTGL (Fig. 3) also indicates that HTGL acts primarily on VLDL + IDL-triglycerides. Characterization of the accumulated lipoproteins During blockade of HTGL in the fat-loaded cats, a pre-&lipoprotein band arose (Fig. 4). The mobility of the accumulated lipoproteins appeared to be concentration-dependent. When a concentrated d < 1.019 g/ml fraction of the antiHTGL-IgG-treated cats was analysed a decreased mobility was observed (Fig. 4). During the blockade of hepatic lipase in fat-loaded cats no consistent changes were seen in the a-fraction. Chylomicrons were sometimes visible in the first 3 h, up to 4.5 h af.ter the fat load, suggesting that the

179 VLDL.

32

IDL-Trlglycerldes

1

(mmol/l

absorption of fat is not greatly delayed by the anesthesia compared to cats sedated with ketamine [16]. On SDS-gel electrophoresis the accumulated lipoproteins contained 3 major bands co-migrating with human apo B-100, apo E and apo A-I, respectively (Fig. 5). The latter protein also comigrated with the main protein band in cat HDL, (data not shown).

1

2.8

J

24

-

20.

1.6

-

12

-

Fig. 3. VLDL+IDL-triglycerides after oral administration of cream and injection of anti-HTGL-IgG. The oral fat load (6 g of fat/kg body weight) was given 90 min prior to anesthesia and the administration of the anti-HTGL-IgG (e#) or control IgG (0 - - - 0) in 6 animals each. Values are expressed as mean f SEM. * P < 0.05 (Student’s paired r-test vs basal values); * *P < 0.01.

08-

0.41 ~~~__I___n_~F-q---_p____u____* I

6 HDL,

2

4

6

10

l/-r-f/--T-

12

19

24

ume

( h )

1

-phosphollprds

1.0 -

HDL3 -phospholipids

(mmo//l)

??

1 I I I I I

\.

2.0 -

18-

’ ‘\ \ L-0

,o----0

I,

I

2

f,

60

I

2

L

60

2I

L

6I

8

10 I

’

12

‘55% ( Time

h

Fig. 4. Changes in the concentrations of HDL,- and HDLs-phospholipids in the cats treated according to the various protocols. Ovemigth fasted cats (I), fed cats (II) and fat-loaded cats (III) received anti-HTGL-IgG (O0) or control IgG (0- -0) on t = 0. Continuous blockade was maintained for up to 24 h by repeated injections of the IgG. In protocol III, the fat load was given 90 min prior to the first injection of the IgG. Values are expressed as mean + SEM. In protocol I and II the individual data of the two controls are compared to the average results of 5 immune-treated cats and the individual data of 2 immune-treated cats, respectively. In protocol III 6 cats received immune IgG and 6 cats control IgG. * P c 0.05 (Student’s unpaired f-test vs controls); * * P < 0.01.

Fig. 5. Agarose gel electrophoresis of plasma lipoproteins in fat-loaded cats treated with nonimmune IgG or anti-HTGL-IgG. From left to right are shown: plasma samples obtained at t = 0, 6 and 19 h from a cat which received nonimmune IgG; plasma samples obtained at t = 0 to 19 h from an anti-HTGL-IgG-treated cat; concentrated d < 1.019 (t) and t > 1.019 (b) fractions of the plasma obtained at t =ll h from the cat treated with anti HTGL-IgG. a, pre-8 and /3 represent the electrophoretic migration of human serum, HDL, VLDL and LDL, respectively.

Fig. 6. Changes in the apoprotein pattern of fat-loaded cats treated 0.75% SDS gel apoprotem,pattem of the d c 1.019 g/ml, fraction at same samples from a cat treated with anti-HTGL-IgG. Apoproteins human serum (all apoproteins), VLDL fasting cat serum (apo B-100), right were from an animal which showed a moderate response to the apo E and apo A-I show a gradual increase.

with nonimmune IgG or anti-HTGL-IgG. From left to right t = 0 to 24 h from a cat treated with nonimmune IgG and the were identified by comparison with the colored band from cat lymph (apo B-48) and cat HDL, (apo A-I). The samples at HTGL-blockade; no apo B-48 band is visible, but apo B-100,

181 Apo B-48 was sometimes present in the first 2-3 h after the fat load, independent of the treatment. No consistent accumulation of apo B-48 was observed in the cats treated with anti-HTGLIgG. In some anti-HTGL-IgG-treated cats which showed an extreme response in the triglycerides, apo B-48 was present in variable amounts up to 19 h after the start of the blockade. At that time LPL activity is decreased to a minimum (Table 1) and the removal of the remainder of chylomicrons may be extra delayed by the competition with the abundant amounts of apo B-100 containing lipoproteins for hydrolysis by HTGL. During the experiment, the intensity of the apo B-100, apo E and apo A-I bands increased clearly in the HTGL-IgG-treated cats (Fig. 5). The changes in apo A-I corresponded to those in apo B-100 and apo E, but not to those in apo B-48. Discussion

Reports in the literature suggest that LPL and HTGL have overlapping specificities in the hydrolysis of the triglyceride-rich lipoproteins [5]. However, our data indicate that chylomicron clearance proceeds normally when HTGL is inhibited, because there was no accumulation of triglycerides at early times and little or no apo B-48 accumulated. Apparently, the relatively low LPL activity is still capable of hydrolysing the chylomicrons to their remnants. Thus, our data do not support a role of HTGL in the metabolism of chylomicrons. Rather our results indicate that HTGL is involved in the hydrolysis of VLDL + IDL-triglycerides probably of endogenous origin, because apo B-100 accumulated. The effect of blockade was especially evident in fed or fat-loaded animals, apparently because the synthesis of these triglyceride-rich lipoproteins is enhanced in the postprandial phase. As postulated earlier, if LPL and HTGL have overlapping specificities, inhibition of HTGL would lead to increased degradation of VLDL by LPL and the transfer of a larger mass of lipids and apoproteins to HDL [5]. To minimize these secondary effects on the HDL composition an animal should be used with a relative deficiency of LPL compared to HTGL. In this respect the cat is more appropriate than rats or the cynomolgus

monkey [14,15]. Indeed, by using the cat as animal model, changes in the HDL phospholipids were found to be secondary to those in the VLDL + IDL-triglycerides. The increase of HDL,-phospholipids was associated with a tendency to lower HDL,-phospholipid values, secondary to the accumulation of apo A-I in the d < 1.019 g/ml fraction. Apparently, at a normal lipolysis by HTGL, the apo A-I does not accumulate in the d < 1.019 g/ml fraction and is transferred to HDL,. The density of HDL, returns to normal and these lipoproteins are then probably more readily converted to HDL,. This hypothesis explains that during blockade of HTGL, changes in the lipoproteins occur both in the VLDL and in HDL, but those in the HDL are clearly secondary. These results are not in line with the reports which suggest a role for HTGL in the metabolism of HDL, especially HDL,-phospholipids [8-131. Indeed, HTGL has both phospholipase as well as triglyceride lipase activity. However, a primary function of HTGL in the metabolism of HDL,phospholipids can only be taken seriously when the data are supported by in vivo experiments, especially because the hydrolysis of HDL,-phospholipids in vitro by HTGL is not very specific and can completely be taken over by snake venom phospholipase A, [13]. However, the only data on the preferential enrichment of HDL,-phospholipids after blockade of HTGL in vivo [9] are difficult to interpret. Although the HDL,-phospholipid concentration 1 h after blockade was 22% higher in the antiserum-treated group compared to that in the controls, the relative phospholipid contents in the HDL, fractions were similar. The difference of 54% in the HDL,-phospholipid concentrations in both groups at 4 h after blockade was primarily due to a decrease in the control group; the concentration in the antiserum-treated group remained unchanged [9]. Furthermore, the relative phospholipid content of the HDL, at 4 h after blockade was similar to that in the control group (mean k SD: 41.9 f 4.6% vs 35.2 f lO.l%, respectively, P = 0.08, n = 10). Because the magnitude of the remaining LPL activity may influence the density and size of that lipoprotein which is the specific substrate for HTGL, but which can also be hydrolyzed by LPL, density criteria are unreliable for characterizing

182

this special class of lipoproteins. Therefore, its origin can best be studied by quantitative determination of the apoprotein concentrations in the various lipoprotein fractions. Unfortunately, in most previous studies the changes in apoprotein composition have seldom been determined. An enrichment of apo B-48 in the LDL density class was found in one study in rats, suggesting a role of HTGL in the catabolism of chylomicrons [7]. However, the apo B-48 band in fat-loaded rats was most intense at 6 h after blockade and accumulated more in fed than in fasted rats. Unlike man, rabbit and cat, rats synthesise apo B-48 both in the intestine and liver [26]. It has been shown that the synthesis of apo B-48-VLDL is enhanced after feeding and decreased after fasting [27]. Therefore, both the study of Daggy and Bensadoun [7] as well as our study point to a specific function of HTGL in the hydrolysis of postprandial synthesised VLDL. In another study, in which only the low-molecular weight apoproteins were followed, an increase of apo E was found in the VLDL fraction [28], in agreement with our results. The presence of apo A-I in the d < 1.019 g/ml fraction at low HTGL activity is not a unique finding. For example, in similar experiments in rats [28] an abnormal apoprotein accumulated in the VLDL fraction which could not be identified. However, on the basis of the isoelectric point, between apo E and apo Cm, this apoprotein may be apo A-I. This is stressed by the fact that the protein focused as two isoproteins [28]. Furthermore, in cholesterol-fed hypothyroid rats having a deficiency of HTGL, apo A-I was present in the d-c 1.019 g/ml fraction as well [17]. However, whether this apoprotein is from exogenous or endogenous origin remained unanswered. Unfortunately, Daggy and Bensadoun [7] did not follow the fate of the low-molecular weight apoproteins which could have revealed more on the relationship of apo A-I to apo B-100 and apo B-48, and thus on the origin of apo A-I. In the present study blockade of HTGL in all fat-loaded cats resulted in a consistent increase of apo B-100, apo E and apo A-I. The changes in the intensity of the apo B-48 band were not consistent and were not related to those of the apo A-l band. Furthermore, apo A-I was frequently present in samples in which no chylomicron band could be

detected on agarose gel electrophoresis and no apo B-48 band could be seen on SDS gel electrophoresis. These findings suggest that apo A-I in the d < 1.019 g/ml fraction is an integrated part of the B-100 containing particle and is from hepatic rather than from intestinal origin. Acknowledgements

We thank Pietemel Van Heijst and Heidi HakLemmers for their excellent technical assistance, Mr. Th. Arts, Mr. A. Peters and their coworkers of the Central Animal Laboratory of the Medical Faculty for their expert assistance in performing the animal experiments and Prof. J.H. Veerkamp for his comments on the manuscript. The secretarial help of Mrs Ans Ruesen-Maandag and Mrs Emy Meij-Van Kesteren is gratefully acknowledged. References 1 La Rosa, J.C., Levy, RI., Windmueller, H.G. and Fredrick-

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