Effects of dietary cholesterol on adipose tissue lipoprotein lipase in the baboon

44

Biochimicu et Bioph~pwu Actu 879 (1986) 44-50 Elsevier

BBA 52403

Effects of dietary cholesterol on adipose tissue lipoprotein lipase in the baboon Douglas S. Lewis

(Received 4 March 1986) (Revised manuscript received 30 June 1986)

Key words:

Lipoprotein

hpase; Adipose

tissue: Cholesterol;

Infant diet: (Baboon)

The effects of infant diet (breast milk or formula containing 2,30 or 60 mg/dl cholesterol) and subsequent dietary cholesterol (0.02, 1.0 or 1.7 mg/kcal) and fat (saturated or unsaturated) on heparin-releasable lipolytic activity from omental adipose tissue was estimated from 99 baboons of 5-8 years of age. This lipase activity was characterized as li~protein lipase based on salt inhibition and a~li~protein C-II activation. Lipoprotein Iipase activity released from adipose tissue by heparin was significantly (P < 0.002) lower in high cholesterol-fed baboons than in those fed low cholesterol. Most of this difference was due to impaired long-term heparin release of lipoprotein lipase. Adipose tissue lipoprotein lipase increased with increasing fat cell size regardless of diet, but there was no effect of diet on adipocyte size. There were no significant effects of infant cholesterol intake nor adult saturated or unsaturated fat on lipoprotein lipase activity. Adult baboons breast fed as infants had lower adipose tissue li~protein lipase activity (P < 0.07) than adults fed formula as infants.

Introduction In many expe~mental animals increased cholesterol intake leads to marked elevations of cholesterol-containing plasma lipoproteins and to the accumulation of cholesterol into tissues. Because lipoprotein lipase is a major enzyme involved with the catabolism of plasma triacylglycerol-rich lipoproteins and may also be involved in the uptake of cholesterol into adipose tissue [1,2] and cultured cells [3] the effect of cholesterol feeding on lipoprotein lipase is of interest. Although triacylglycerol-rich substrates, high in cholesterol content, inhibit lipoprotein lipase activity in vitro [4,5], there have been conflicting reports as to whether diets rich in cholesterol Correspondence address: Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, P.O. Box 2814’7, San Antonio. TX 78284, U.S.A.

0005-2760/86/$03,50

0 1986 Elsevier Science Publishers

influence lipoprotein lipase activity in vivo 16-91. This report describes the effects of high cholesterol diets on adipose tissue lipoprotein lipase in adult baboons fed various levels of cholesterol since infancy. Methods

Animais and diets The baboons in this study were fed commercial infant formulas [lo] containing one of three levels of cholesterol (approximately 2, 30, or 60 mg/dl) or were breast fed from birth to 16 weeks of age [ll]. At weaning (16 weeks of age) these baboons (Pa&o sp.) were randomly assigned to one of the following diets: low cholesterol (0.02 mg/ kcat)saturated fat, low cholesterol-unsaturated fat, high cholesterol (1.0 mg/kcal)-saturated fat and high cholesterol-unsaturated fat. The four semi purified diets were prepared with Special Monkey Chow 25

B.V. (Biomedical

Division)

45

(no. 5045 : 6; Ralston-Purina, St. Louis, MO) containing 1.0 or 0.02 mg cholesterol per kcal with either saturated or unsaturated fat. All diets contained approximately 40% of calories as fat, 38% of calories as carbohydrate (nitrogen-free extract), and 22% of calories as protein (high protein gluten meal). These diets are described in greater detail elsewhere [11,12]. Another group of baboons, different from above, were weaned onto a standard atherogenic diet [12] containing 40% of calories as lard and 1.7 mg cholesterol per kcal, denoted very high cholesterol-saturated fat diet. Animals were housed in a gang cage environment and fed the described diets for 5-8 years. Baboons fed the very high cholesterol-saturated fat diet were sampled at 5 years of age and those baboons fed low and high cholesterol diets were sampled at 8 years of age. Collection of adipose tissue The fat depot chosen for study was the omental fat depot which comprises approximately 26% of the total fat depot mass and about 10% of the total carcass fat mass of young adult baboons (Lewis, unpublished data). The omental fat depot was obtained from fasted (18 h) baboons at necropsy. Adipose tissue samples (l-5 g) were placed in saline at 37°C carefully dissected free of non-fat tissues, washed free of blood, weighed, and minced. The minced adipose tissue was incubated with 10 ml per g tissue of Krebs-Ringer bicarbonate buffer (pH 7.4), containing 1% albumin (Pentex, Miles), 10 mM glucose and 10 units/ml heparin derived from porcine intestine (Elkins-Sinn, Inc., Cherry Hill, NJ) per ml. Samples for lipoprotein lipase assays were removed at the indicated times and assayed immediately. Adipocyte number and volume were determined as follows. Adipose tissue was incubated with collagenase for 1 h at 37°C and the freed adipocytes were isolated, microscopically sized and the mean cell volume was calculated using the technique of Stiles et al. [13]. Triacylglycerols of a known weight of fat tissue were extracted with CHCl,/methanol (2 : 1) and the triacylglycerol weight determined [13]. Adipocyte number is estimated by dividing the ml triacylglycerol extracted (weight x specific gravity) by the mean cell volume.

Assay for lipoprotein lipase Heparin-released lipoprotein lipase was assayed by a method similar to that described by NilssonEhle and Schotz [14]. Preliminary studies indicated that extraction of lipoprotein lipase from acetone powder was not feasible because of relatively low yields of enzyme activity. Thus, only heparin-released lipoprotein lipase activity is reported. A synthetic triolein substrate was prepared by emulsifying on ice (Brinkmann Polytron, setting 7 for 5 min) 2.5 mCi of purified glycerol tri[9,10-3H]oleate (Amersham-Searle), 600 mg of nonlabeled triolein (Supelco), 36 mg egg phosphatidylcholine (Sigma), and 10 of spectrophotometric grade glycerol. This concentrated substrate was stored under N, at room temperature. Lipoprotein lipase activity was measured by incubating the 0.1 ml of diluted enzyme preparation (0.02 ml of extract with 0.08 ml of 0.05 M Tris (pH 8.0)) with 0.1 ml of diluted substrate containing 0.008 ml of heat-inactivated baboon serum (previously tested to provide maximal activation of lipoprotein lipase activity), 0.017 ml of the glycerol tri(9,10-3H)oleate stock and 0.075 ml of 0.2 M Tris (pH 8.0) containing 3% bovine serum albumin (Miles, Pentex). In some experiments, purified apolipoprotein C-II from baboons was kindly provided by Dr. Rampratap S. Kushwaha. After 30 min at 37 “C, 3H-labeled fatty acids were extracted and measured as described by Nilsson-Ehle and Schotz [14]. Enzyme activity was expressed as pmol free fatty acid released/h per lo6 adipocytes. Because these animals were necropsied over 2.5 years, the following precautions were taken to minimize the effects of different substrates and serum activators on lipoprotein lipase activity. Serum that was used in lipoprotein lipase assays was derived from a single baboon and was stored in small aliquots at -20°C. These storage conditions had no significant effect on the serum activation of lipoprotein lipase. Potential differences in the emulsified triacylglycerol substrate and effects on lipoprotein lipase activity were normalized by comparison of post-heparinized plasma lipolytic activity, from a control animal, measured with each stock substrate.

46

Serum lipid analysis The serum cholesterol concentration was measured by an enzymatic procedure [15,16]. Serum triacylglycerol concentration was measured by an enzymatic procedure which corrected for free glycerol [17]. These procedures met the criteria of the Center for Disease Control Lipid Standardization Program. Statistical analysis The data were analyzed by analysis of variance. The linear model included terms for the overall mean and effects of infant diet, juvenile dietary cholesterol, juvenile dietary fat, sex, and sire, and the two-factor interactions: infant diet by cholesterol, infant diet by fat, cholesterol by fat, cholesterol by sex, fat by sex. All effects were assumed to be fixed. The measurements were logarithmically transformed before statistical analysis to better satisfy the assumptions of the statistical model. Results Body weight and plasma lipid levels of baboons fed different cholesterol diets There were no significant effects of infant diet, either cholesterol or breast vs. formula fed, on body weight, serum cholesterol or serum triacylglycerol. The effect of adult diet on body weight and serum lipids is reported in Table I. Each diet group had equal male and females with the exceptions of the low-cholesterol-saturated fat (nine males and eight females) and the high cholesterol-saturated fat (eight females and four males) diet groups. The larger percentage of females in the high cholesterol-saturated fat diet group accounts for the lower body weight observed. There were no significant differences in omental fat depot mass due to diet. Dietary cholesterol increased (P < 0.01) serum cholesterol levels, but did not affect serum triacylglycerols. There was no significant difference in serum triacylglycerol levels between baboons fed saturated fat and those fed unsaturated fat. Another group of baboons (n = 38) 5 years old, fed a very high cholesterol diet (1.7 mg/ kcal) with 40% of kcal as lard had even higher serum cholesterol levels (188 f 16 mg/dl) than those baboons reported in Ta-

TABLE

I

EFFECT OF DIET ON BODY MASS, AND SERUM LIPIDS

WEIGHT,

FAT

DEPOT

Results are expressed as mean f SE. LC-UF, low cholesterolunsaturated fat; LC-SF, low cholesterol-saturated fat; HC-UF, high cholesterol-unsaturated fat: HC-SF, low cholesterolsaturated fat. Diet

n

LC-UF LC-SF HC-UF HC-SF

17 18 14 12

Serum

Body weight

Fat depot

(kg)

(9)

22.3k1.6 22.3*1.4 20.8*1.5 17.9k1.4

12Ok26.2 117i17.4 109k27.5 116+26.3

Cholesterol

Triacylglycerol

(mg/dl)

(mg/dl)

11Ok 4.6 124+ 6.1 142k10.7 152k12.1

42k3.9 47k4.5 36*3.7 54k8.9

ble I. The very high cholesterol-saturated fat diet baboons had similar serum triacylglycerols (45 f 11 mg/dl) as other baboons fed saturated fat. Characterization of adipose tissue lipoprotein lipase The enzymatic characteristics of lipoprotein lipase extracted with heparin from adipose tissue from low cholesterol, unsaturated fat fed baboons are summarized in Table II. Adipose tissue lipoprotein lipase was strongly inhibited by 1.0 M NaCl and by protamine sulfate. In addition, serum or apolipoprotein C-II was required for optimal TABLE

II

CHARACTERIZATION TIVITY

OF

LIPOPROTEIN

LIPASE

AC-

Heparin-releasable activity was from adipose tissue from baboons (n = 4) fed a low cholesterol-unsaturated fat diet. Minced adipose tissue was incubated with (1 ml/g tissue) Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 units heparin per ml for 60 min at 37°C. Control assays contain heat-inactivated serum as activator. For the apolipoprotein C-II assay (apo CII) 2 pg protein were preincubated with the triacylglycerol substrate in place of heat-inactivated serum. Assay conditions

% activity adipose

Control Apo C-II No serum +l.OMNaCl + Protamine

sulfate

tissue

?kSEM 100 100 6k1.5 12% 1.0 lo* 1.5

47

TABLE

III

EXTRACTION OF LIPOPROTEIN FROM BABOON ADIPOSE TISSUE

LIPASE

ACTIVITY

Results are representative data from ten experiments. Extraction conditions: minced fat tissue was extracted with heparin (lo/ml) for 1 h. Activity is expressed as units per g tissues. One unit is equal to 1 pmol fatty acid released per h. Activity

Extraction Conditions No 10 10 10

heparin, units/ml units/ml units/ml

37°C heparin, heparin, heparin,

37°C 4OC cycloheximide

a Tissue was preincubated

a. 37OC

0.01 2.55 0.50 1.12

for 1 h with 1 mM cycloheximide.

activity. Both salt sensitivity and activation of adipose tissue lipoprotein lipase by serum were similar in other diet groups (data not shown). The heparin release of adipose tissue lipoprotein lipase is illustrated in Fig. 1. This release of lipoprotein lipase occurs in two stages: first an immediate release of lipoprotein lipase (first 15-30 min), and a second long-term (30-110 min) release of lipoprotein lipase. The long-term release was abolished at 4°C and inhibited in the presence of cycloheximide (Table III).

enzyme

Effects of adult diet and infant diet on lipoprotein lipase activity in adult baboons Table IV summarizes the lipoprotein lipase activity in baboon adipose tissue by adult dietary cholesterol and type of fat. The high cholesterol diets significantly (P < 0.002) decreased adipose tissue lipoprotein lipase compared to baboons fed low cholesterol. The baboons fed a very high cholesterol-saturated fat diet (1.7 mg cholesterolkcal) had adipose tissue lipoprotein lipase levels similar to those of animals fed 1.0 mg cholesterol per kcal. Saturated fat lowered mean adipose tissue lipoprotein lipase activities but this effect was not statistically significant. The effects of infant diet on adipose tissue lipoprotein lipase were examined as breast fed vs. formula fed and as formula cholesterol content (2, 30, 60 mg/kcal and breast fed). Adult baboons that were breast fed as infants had lower, but not statistically significant, adipose tissue lipoprotein lipase (P < 0.07) than baboons fed formula (data not shown). Infant dietary cholesterol had no significant effect on adipose tissue lipoprotein lipase in adult baboons. The effect of adult dietary cholesterol on the heparin release of adipose tissue lipoprotein lipase in 11 baboons fed 1.7 mg cholesterol per kcal and in 11 baboons fed 0.02 mg cholesterol per kcal is reported in Fig. 2. In baboons maintained on a

TABLE

I \ r

> I= ::

EFFECT OF DIETARY CHOLESTEROL, SATURATED AND UNSATURATED FAT ON HEPARIN-RELEASABLE LIPOPROTEIN LIPASE ACTIVITY FROM ADIPOSE TISSUE OF ADULT BABOONS

0.6 t

Adipose tissue was incubated with 10 units heparin per ml for 60 min at 37OC. Results are expressed as mean * SE. The fat content of the very high cholesterol diet was primarily lard. 0.2 -

Cholesterol content

aJ -I 0

I 20

I 40

TIME Fig. from units with

IV

I 60

I 80

1 100

(min)

1. Lipoprotein Iipase (LPL) activity released by heparin omental fat tissue. Tissue pieces were incubated with 10 heparin per ml Krebs-Ringer bicarbonate buffer (pH 7.4) 1.0% albumin/l0 mM glucose at 37°C (0) or at 4OC (0).

Type of fat

(pmol fatty acid released/h per lo6 cells)

(w/k4

unsaturated

saturated

Low (0.02)

1.70*0.31 (n =17)

1.04+0.31 (n =17)

High

0.60*0.38 (n =12)

0.44 * 0.52 (n=8)

(1.0) Very high (1.7)

0.40 i 0.26 (n=36)

MEAN

0

I

2

FAT

CELL

SIZE

nl

Fig. 3. Relationship of lipoprotein lipase (LPL) activity to mean fat cell size. Lipoprotein lipase activity per fat depot is expressed as units (1 unit -1 pmol of fatty acid released per h). 0, baboons fed the low cholesterol diet; 0, baboons fed the high cholesterol diet.

TIME (hrs) Fig. 2. Heparin release of lipoprotein hpase (LPL) activity from adipose tissue in baboons fed high fat diets with either high or low cholesterol. Fat tissue slices were incubated at 37°C with 10 units heparin per ml Krebs-Ringer bicarbonate buffer (pH 7.4) with 1.0% albumin/l0 mM glucose. Aliquots of medium were removed at the indicated times and immediately assayed for lipoprotein lipase activity. Data are presented as a percentage of the total lipoprotein lipase activity released during the 2 h incubation. 0, baboons fed high cholesterol; 0, baboons fed low cholesterol.

high cholesterol diet 80% of the heparin-releasable lipoprotein lipase was released in the first 30 min incubation with heparin. In contrast, in the low cholesterol-fed baboons, only 42% of the heparinreleasable lipoprotein lipase was released in the first 30 min incubation with heparin. Most of the lipoprotein lipase activity was released over 2 h incubation. The differences in adipose tissue lipoprotein lipase among diet groups was not due to any difference in mean adipocyte volumes. The high cholesterol diet group had a mean volume of 0.38 f 0.35 nl (mean _rt S.D.) and the low cholesterol diet group had a mean volume of 0.44 k 0.35 nl. However, Fig. 3 illustrates that the total depot lipoprotein lipase activity increases with fat cell volume regardless of diet.

Discussion The present study demonstrates that the heparin release of lipoprotein lipase from adipose tissue is significantly depressed by approximately 63% in baboons made hypercholesterole~c by feeding a high fat diet, high in cholesterol, compared to more normal cholesterolemic baboons fed a high fat diet, low in cholesterol. Since lipoprotein lipase activity was studied only at necropsy it is not known whether the decreased level of adipose tissue lipoprotein lipase in the high cholesterol-fed baboons was due to either the long term feeding (5 or 8 years) or due to the cholesterol content of the diet being fed at the sampling time. In a similar experiment Kotze and Menne [9] showed that baboons fed a high fat diet (including cholesterol) for 2 years had lower adipose tissue lipoprotein lipase than baboons fed a low fat, low cholesterol diet. However, it is difficult to determine whether the decreased adipose tissue lipoprotein lipase activity described by Kotze and Menne was due to the fat, cholesterol, and/or carbohydrate content of their experimental and control diets. Both fat 118,191 and carbohydrate diets 120,211 can influence adipose tissue lipoprotein lipase. In the current study only the cholesterol content and the degree of saturation of dietary fat were altered. In contrast to its effects in baboons, dietary

49

cholesterol had no effect on adipose tissue lipoprotein lipase in the guinea pig [8]. These conflicting results may be due to species differences, the duration of feeding the diets, and methodological differences. In this experiment, all baboons were fed a high fat diet for 5 or 8 years, and lipoprotein lipase activity was expressed on a cellular basis. The latter may be of particular importance because of the metabolic differences, including lipoprotein lipase activity, between fat cells of various sizes [22-261. The other diet variable in this experiment was the infant diet (breast vs. formula feeding) of the adult baboons studied. While infant cholesterol intake did not influence lipoprotein lipase in adipose tissue in 8-year-old adults, baboons breast fed as infants have less adipose tissue lipoprotein lipase activity (P < 0.07) than baboons fed formula as infants. The level of significance for the effect of breast feeding on lipoprotein lipase activity at 8 years of age is not at the classical P -c0.05. However, because baboons were either breast fed or formula fed 8 years earlier and other factors could modulate infant diet effects, these results should not be totally ignored. Indeed, the importance of these infant diet effects may be related to the observed deferred effects of breast feeding on cholesterol metabolism in the same baboons [11,27], but at an earlier age (3.5 years). Dietary cholesterol appears to influence the ability of adipose tissue to respond to a heparin stimulus. Heparin releases lipoprotein lipase from baboon adipose tissue in two phases similar to that described in rat preadipocytes [29] and mouse Ob17 fat cells [30]. The initial release of lipoprotein lipase activity is independent of temperature and protein synthesis and presumably represents a pool of preexisting enzyme [30]. Increased dietary cholesterol appears to suppress the second phase release of lipoprotein lipase, which is dependent on temperature and is inhibited by cycloheximide. These results suggest that the capacity of adipose tissue to generate new lipoprotein lipase activity by either protein synthesis or activation of inactive enzyme may be altered by dietary cholesterol. Anti-lipoprotein lipase antibodies to measure lipoprotein lipase mass [31] could be used to test this hypothesis. The physiological significance of the effect of

dietary cholesterol on adipose tissue lipoprotein lipase in the baboon is not clear because changes in lipoprotein lipase activity did not correlate with fasting serum triacylglycerol levels. It is possible that the omentum is not representative of the total fat depot mass. However, it is more likely that the amount of lipoprotein lipase located on the endothelial cell surface in adipose tissue is low in the fasting state [28], and small changes in this pool of lipoprotein lipase could have negligible effect on fasting plasma triacylglycerol. By contrast, significant depression of the capacity of fat cells to produce lipoprotein lipase on demand (measured in this study using heparin) could result in decreased lipolysis of serum triacylglycerol-rich lipoproteins in adipose tissue during the postprandial state. Acknowledgements The author thanks Amy Allison for technical help, Dr. Glen Mott for serum lipid data, and Dr. C.A. McMahan for help with statistical analysis. This work was supported in part by the National Heart, Lung and Blood Institute grants HL-26890 and HL-28972. References 1 Angel, A. and Farkas, J. (1974) J. Lipid Res. 15, 491-499 2 Raymond, T.L.. Lofland, H.B. and Clarkson, T.B. (1976) Exp. Mol. Pathol. 25, 344-354 3 Stein, 0.. Friedman, G., Chajek-Shaul, T., Halperin. G.. Olivecrona, T. and Stein, Y. (1983) Biochim. Biophys. Acta 750, 306-316 4 Rossner, S. and Vessby, B. (1977) Nutr. Metab. 21, 349-357 5 Fielding, C.J. (1970) Biochim. Biophys. Acta 218. 221-226 6 Van Zutphen, L.F.M., Den Bieman, M.G.C.W., Hulsmann, W.C. and Fox, R.R. (1981) Lab. Anim. 15. 61-67 7 Sasinowski, F. and Oswald, R. (1981) Lipids 16, 380-383 8 Heller, F.R. (1983) Biochim. Biophys. Acta 752, 357-360 9 Kotze, J.P. and Menne, I.V. (1977) S. Afr. J. Sci. 73, 91-92 10 Mott, G.E., McMahan, CA. and McGill, H.C., Jr. (1978) Circ. Res. 43, 364-371 11 Mott, G.E., McMahan, C.A., Kelley, J.L., Farley, C.M. and McGill, H.C., Jr. (1982) Atherosclerosis 45, 191-202 12 McGill, H.C., Jr., McMahan, CA., Kruski, A.W., and Mott, G.E. (1981) Arteriosclerosis 1, 3-12 13 Stiles. J.W., Francendese, A.A. and Masoro, E.J. (1975) Am. J. Physiol. 229, 1561-1568 14 Nilsson-Ehle, P. and Schotz, M.C. (1976) J. Lipid. Res. 17, 536-541 15 Allain, CC., Poon. L.S., Chan, C.S.G., Richmond, W. and Fu, P.C. (1974) Clin. Chem. 20: 470-475

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16 Witte, D.L., Barrett, II, D.A. and Wycoff, D.A. (1974) Clin. Chem. 20, 1282-1286 17 Mott, G.E. and Rogers, M.L. (1978) Clin. Chem. 24, 354-357 18 Taskinen, M.R., Nikkila, E.A. and Hilden, H. (1983) Diabetes, Obes. Hyperlipidemias 2 (Proc. Eur. Meet. Metabol.), 3rd 1981, pp. 105-108 19 Sadur, C.N., Yost, T.J. and Eckel, R.H. (1984) Metab. Clin. Exp. 33, 1043-1047 20 Granneman, J.G. and Wade, G.N. (1983) Metab. Clin. Exp. 32,202-207 21 Pedersen, M.E. and Schotz, M.C. (1980) J. Nutr. 110, 481-487 22 Zinder, 0. and Shapiro, B. (1971) J. Lipid Res. 12, 91-95 23 Puppione, D.L., Sardet, C., Yamanaka, W., Oswald, R. and Nichols, A.V. (1971) Biochim. Biophys. Acta 231, 295-301

24 Chapman, M.J., Mills, G.L. and Taylaur, C.E. (1973) Biothem. J. 131, 177-185 25 Bjorntorp, P. and Karlsson, M. (1970) Eur. J. Clin. Invest. 1, 112-117 26 Guy-Grand, B. and Bigorie, B. (1975) Horm. Metab. Res. 7.471-475 27 Mott, G.E., Jackson, E.M., McMahan, C.A., Farley, C.M. and McGill, H.C., Jr. (1985) Arteriosclerosis 5, 347-354 28 Nilsson-Ehle, P., Garfinkel, A.S. and Schotz, M.C. (1980) Annu. Rev. B&hem. 49, 667-693 29 Click, J.E. and Rothblat, G. (1980) Biochim. Biophys. Acta 618, 163-172 30 Vannier, C. and Ailhaud, G. (1986) B&him. Biophys. Acta 875, 324-333 31 Olivecrona, T. and Bengtsson, G. (1982) Biochim. Biophys. Acta 752, 38-45