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Regulation of lipid synthesis by low density lipoproteins in cultured skin fibroblasts in homozygous familial hypercholesterolemia
445
Biochimica el Biophysics Acta, 487 (1977) 445-457 @ Elsevier~North-Holland Biomedical Press
BBA 57005
REGWLATfUN OF LIPID SYNTHESIS BY LOW DENSITY LIPOPROTEINS 1N CULTURED SKIN F~BROBLASTS IN HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA
CHIEN H. FUNG, AVEDIS K. KHAGHADURIAN IBRAHIM F. DURR Departments and Dentislry
uf Medicine
*, CHING-HUA WANG and
and Biochemistry, Rutgers Medicat School, of New Jersey, Piscataway, N.J. 08854 U.S.A.)
Cattege
of Medicirae
(Received December 14th, 1976)
Summary The regulation of cholesterol and fatty acid syntheses by low density lipoproteins (LDL) was studied in cultured skin fibroblasts from a normal subject and a subject with homozygous familial hypercholesterolemia the fibroblasts of which had no LDL binding to specific cell surface receptors (receptor negative) and no cholesterol esterifying activity. Assays were done in cells preincubated in media containing lipoprotein-deficient serum (serum A) or serum A delipidated with ether-ethanol (serum B). In homozygous familial hypercholesterolemia cells preincubated in serum B a second preincubation with saturating levels of LDL caused a 34% suppression in acetate incorporation into cholesterol and a 32% reduction of the activity of 3-hydr~~y-3-methylgl~t~yl coenzyme A (HMG-Co A) reductase compared to 9X and 93% reduction, respectively, in the normal cell line. In homozygous familial hypercholesterolemia cells grown in 10% fetal calf serum there was an increased incorporation of acetate into total fatty acids as well as into phospholipids and triacylglycerols, indicating a lack of suppression of fatty acid synthesis by medium lipids in the mutant cells. In homozygous familial hyperchoIesterolemia cells preincubated in serum B a second preincubation with LDL caused a 51% suppression of acetate incorporation into phospholipids compared to ‘i6% in normal cells. Our data indicate that in homozygous familial hypercholesterolemia there is a derangement in the regulation of fatty acid synthesis. It is, however, less marked than the derangement of sterol synthesis. The inhibitory action of LDL both on sterol and fatty acid syntheses in cells that do not show any LDL binding activity by the presently available tech-
* Ta
whom correspondence should be addressed. Abbreviations: serum A, lipoprotein-deficient serum; serum b, lipoprotein-deficient serum delipidated with ether/ethanol; HMG-CoA. 3-hydroxy-3-methylglutaryl coenzyme A; antibiotic mixture, penicillin, streptomycin and neomycin; VLDL. very low density lipoproteins (d G 1.019 g/ml); LDL, low density lipoproteins (d = 1.019-1.063 g/ml); HDL. high density lipoproteins (d = 1.063-l 210 glml).
446
nique is in contrast to the findings of Goldstein et al. ((1975) Proc. Natl. Acad. Sci. U.S. 72, 1092-1096) which indicate that LDL has no inhibitory effect in receptor negative cell lines. -.Introduction Cultured fibroblasts have been used extensively to study the regulation of sterol and fatty acid syntheses. In established cell lines Bailey [l] and Rothblat [2] demonstrated inhibition of sterol synthesis by the sterols contained in the growth medium. Similarly, Avigan et al. [3] and Williams and Avigan [4] have shown that overnight preincubation of human skin fibroblasts in medium containing lipid-free serum results in a marked increase in cholesterol synthesis. This stimulation was reversed by whole serum, low- or high-density lipoproteins or cholesterol suspension. The remarkable increase in cholesterol synthesis was found to be associated with a parallel increase in the activity of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase [5]. Jacobs et al. [6] and Jacobs and Majerus [7] have shown that in primary cultures of human skin fibroblasts the rate of acetate incorporation into fatty acids is markedly inhibited by fatty acids and serum lipids. Volpe and Marasa [8] have made similar observations in cultured glial cells. Cultured skin fibroblasts have been useful in the delineation of the metabolic defect in a group of human errors of metabolism commonly known as the familial hypercholesterolemias [g-17]. In patients with the homozygous form of this disorder Khachadurian and Kawahara [9] and Khachadurian et al. [lo] reported a decrease in the feedback inhibition of cholesterol synthesis by medium lipids. Similar results were reported by Avigan et al. [15]. On the other hand the extensive studies of Brown and Goldstein [11,14] indicate a complete lack of feedback inhibition of HMG-CoA reductase activity, resulting from an absence of binding of LDL to specific cell surface receptors. These differences could result from the inherent difficulties in the experimental procedures or from genetic heterogeneity [ 10,16,18]. The regulation of fatty acid synthesis in familial hypercholesterolemia has attracted little attention so far. Khachadurian and Kawahara [9] noted a decrease in the inhibition of acetate incorporation into the total saponifiable fraction in homozygous familial hypercholesterolemia cells grown in medium containing 10% fetal calf serum. These findings suggest a defective regulation of fatty acid synthesis as well. The present studies were carried out to determine whether in severe homozygous familial hypercholesterolemia the feedback inhibition of cholesterol synthesis was totally lacking or only decreased, and to study the regulation of fatty acid synthesis by measuring the incorporation of acetate into the major lipid classes. Materials and Methods Materials. [2-14C]Acetic acid, sodium salt (56-58 Ci/mol) and PCS * solubilizer were purchased from Amersham/Searle Corporation. DL-[3-‘4C]HMG-CoA * Phase-combining
system.
(26 Ci/mol) and [l-‘4C]oleic acid (50 Ci/mol) were obtained from New England Nuclear. Lipid standards containing cholesterol (20.2%), cholesterol oleate (20.0%), methyl oleate (20.0%), oleic acid (19.9%) and trioleoylglycerol (19.9%) were obtained from Sigma Chemical Co. Mevalonic acid lactone was purchased from K and K Co. Minimum essential medium Earle’s powder (Cat. No. F-11), nutrient mixture with L-glutamine (Cat. No. 155), heat-inactivated fetal calf serum, trypsin-EDTA solution (1 X) and antibiotic mixture (penicillin, streptomycin and neomycin) (100 X) were purchased from Grand Island Biological Co. Pre-coated thin-layer chromatography sheets of silica gel were obtained from Brinkman Instruments, Inc. All other reagents and organic solvents were reagent grade or of the highest purity commercially available. Cell lines. The normal cell line GM 240 was obtained from Dr. A.E. Green, Institute for Medical Research, Camden, N.J. Normal cells 1516 and 32 were from two healthy individuals of age 5 and 37 years who had plasma cholesterol of 140-190 mg per 100 ml [lo ]. Homozygote MH 25 fibroblasts were derived from a patient homozygous for familial hypercholesterolemia as described previously by Khachadurian [ 191 and Khachadurian and Uthman 1201. Growth of cells. Cells were grown in a 75 cm’ Falcon flask at 37°C in 95% sir/5% CO, humidified environment in 20 ml nutrient mixture F-10 or Eagle’s minimum essential medium containing 0.2 ml of antibiotic mixture (100 X) supplemented with 10% heat-inactivated fetal calf serum. The cells were subcultured at 7-day intervals by trypsinization and seeded into three 75 cm2 Falcon flasks, each containing one million cells. In some experiments the cells were seeded into 25 cm’ Falcon flasks, each containing 0.3 million cells. The medium was changed every 3 days. Cells in the 6th to 16th passages were used in all experiments. Incubation with radioactive acetate was done under two different conditions: (a), resting cells: confluent cells were washed three times with 5 ml Krebs-Ringer’s phosphate buffer, pH 7.4, and incubated with [2-“Clacetate in 5 ml of the buffer for 2 h at 37°C on a Thomas Rotating Apparatus with a speed of 100 oscillations per min; (b) growing cells: [ 2-14C]acetate was added to the medium on day 1 of growth. On day 3 the medium was changed. Cells were washed with Krebs-Ringer’s phosphate buffer. Fresh growth medium with radioactive acetate was added, and cells were allowed to reach conflu-
ency . Preparation of low-density lipidated lipoprotein-deficient
lipoproteins, lipoprotein-deficient serum. Low-density lipoproteins
serum and de-
(LDL) were isolated from human plasma by ultracentrifugal flotation according to the method of Have1 et al. [21]. Lipoprotein-deficient serum (serum A) was obtained after removal of LDL, very low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by ultracentrifugal flotation. Because of the possibility that serum A so prepared could contain trace amounts of cholesterol, it was further extracted once with ten volumes of ether/ethanol (1 : 1, v/v) solution at-30°C and stirred vigorously for 2 h. The precipitated protein was collected by centrifugation at 4°C. The protein was then washed first with 300 ml of cold 95% ethanol and subsequently with 300 ml of cold ether. The protein precipitate was again collected by centrifugation. The residual ether was removed by vacuum evaporation at 25°C. The dry protein was then dissolved in a minimal volume of Dulbecco phosphate-buffered saline and dialyzed once against ten
448
volumes of the same solution at 4°C for 24 h. The resulting preparation of delipidated lipoprotein-deficient serum (serum B) and the solution of LDL were sterilized by passing through a Millipore filter with a pore size of 0.22 ym. Assays. The content of total cholesterol in LDL, serum A and serum B was determined by the calorimetric method of Abel1 et al. [22] and by gas-liquid chromatography according to the procedure reported by Ishikawa et al. [ 231. A Hewlett-Packard model 421 gas chromatograph equipped with flame ionization detectors and an Autolab Minigrator was used. The cell protein was measured as described by Lowry et al. [ 241. [ 1-‘4C]Oleate . albumin complex was prepared as described by Van Harken et al. (251. Incorporation of [ I-‘“Cloleate into lipids was measured by the method reported by Goldstein et, al. 1261. On day 6, after cells had been incubated 24 h in medium coK~taining 2.5 mg protein per ml of serum B, LDL was added to give the final concentration of 200 pg LDL-cholesterol per ml. After a further 17 h incubation at 37”C, albumin-bound [‘4C]oleate (0.1 mM, 4250 cpm/nmol) was added, and the cells were harvested 2 h later. The cellular content of [‘4C]cholesterol esters was determined. Cell extracts for measurement of HMG-CoA reductase activity were prepared by the method of Goldstein and Brown [ 111. Cells from two 75 cm2 Falcon flasks were pooled to prepare each extract. HMG-CoR reductase activity was measured by the method described by Shapiro et al. ]27]. Ahquots of the cell extract (50-80 pg of protein) were used for assaying enzyme activities. Aliquots (50 ~1) of the reaction mixture after centrifu~ation were applied to silica gel thin-layer chromato~aphy sheets. After drying, 10 mg of mevalonic acid lactone in 10 i_tl of water was applied to each spot as a carrier. The plates were developed in a benzene/acetone solution (1 : 1, v/v), and then air dried. Mevalonic acid lactone, which locates in the region of Rr = 0.72 + 0.02, may be detected as a quenching band when the chromatogram is viewed under an ultraviolet lamp. The area with RF = 0.72 was removed and added to 10 ml of PCS solubilizer and counted for radioactivity which was used to calculate HMG-CoA reductase activity (Method 1). The area with RF = 0.60 was also removed and counted for radioactivity. The sum of the counts in the two spots was also used to calculate HMG-CoA reductase activity (Method 2) 1271. extraction and fr~ctio~ut~o~ of lipids. Two independent methods were used: (a) Cells were saponified and extracted as previously described [9]. The incubation mixture together with the cells was mixed with ethanolic NaOH, saponified and extracted with ligroin to recover the unsaponifiable lipids. Subsequently, the aqueous layer was acidified and extracted with ligroin to recover the fatty acids. Where indicated, cholesterol was isolated from the unsaponifiable fraction by thin-layer chromatography on aluminum plates coated with silica gel with gypsum as described by Gloster and Fletcher [28]. In addition, the saponifiable fraction was dried under vacuum, then mixed with 5 ml of methanolic HzS04 and placed in a boiling water bath for 30 min. After cooling, the mixture was extracted three times, each with 10 ml ligroin to recover the methyl esters of fatty acids and was chromato~aphed on silica gel plates using methyl palmitate as the standard ester. (b) Lipids were alternatively estracted by the procedure of Folch et al. 1291 as follows: The incubation mixture was decanted, and the cells were washed twice with 5 ml of phosphate-buffere?
449
saline buffer, pH 7.4. The cells were scraped and suspended in 5 ml of KrebsRinger’s phosphate buffer. An aliquot (0.1-0.2 ml) of the cell suspension was removed for protein determination. The remaining cell suspension was centrifuged. The cell pellet was resuspended into 1 ml of Krebs-Ringer’s phosphate buffer and extracted vigorously once with a chloroform/methanol (2 : 1, v/v) solution on a Vortex-Genie mixer for 2 min. The chloroform layer was recovered and then evaporated to dryness at 40°C in an Evapo-Mix evaporator connected to a water aspirator. The lipids were dissolved in 0.2 ml chloroform and subjected to thin-layer chromatography as described above. Spots corresponding to phospholipids, cholesterol, free fatty acids, triacylglycerols and cholesterol esters were identified against lipid standards. The spots were cut off and dissolved in a PCS scintillation solution. All radioactivities were determined in an Intertechnique SL30 Liquid Scintillation Spectrometer with an efficiency of 90% for 14C. Since the specific radioactivity of [2-‘4C]acetate used in the experiments varied, the values espressed as counts per min per mg protein (cmp/mg protein) in tables and figures have been normalized using the specific radioactivity of [2-14C]acetate = 8.50 Ci/mol as a factor. Results Effect of 10% fetal calf serum on the incorporation of acetate into cholesterol and fatty acid esters In confluent cells grown in the nutrient mixture F-10 medium containing 10% fetal calf serum, the rate of the incorporation of acetate into cholesterol in homozygous cells (6.9 + 0.2 . 10” cpm/mg cell protein) is about ten times greater than in normal cells (0.7 + 0.2 * lo3 cpm/mg cell protein) while the rate of acetate incorporation into fatty acid esters (6.2 i 0.6 . 10” cpm/mg cell protein) is approximately three times greater than normal (2.4 2 0.4 . 10’ cpm/ mg cell protein). These observations are consistent with the previous results obtained from other cell lines homozygous for familial hypercholesterolemia
[91. Table I shows results of experiments in which radioactive acetate was added to resting confluent cells or growing cells. The cells were washed and their lipids were separated by thin-layer chromatography. Results indicate increased incorporation of acetate into all lipid classes by resting as well as growing homozygous cells. In growing homozygous cells, 45% of the radioactivity is recovered in the cholesterol fraction, 51% in the phospholipid fraction and 1.4% in triacylglycerols. The release of the newly synthesized lipids into the medium by these cells was assessed by dividing the radioactivity in each lipid class recovered in the medium (extracellular) by the sum of the intracellular and extracellular counts. In normal cells 70% of the cholesterol, 38% of phospholipid and 97% of the triacylglycerol counts were recovered in the medium. The corresponding values for the homozygous cells were 63, 33 and 94%. Therefore, the low radioactivity in the triacylglycerols compared to phospholipids is partly explained by the difference in the loss of these two lipids into the medium. The limited uptake of medium triacylglycerols noted in other types of cells [30], as well as the shift of fatty acids to phospholipids [31,32] could also con-
tribute to the low level of radioactivity in intracellular triacylglycerols. ‘I’hc smallest amount of radioactivity is found in the cholesterol esters. Goldst,ein et al. [ 26 J show that in normal cells, incorporation of acetate into the cholesterol ester is about 40%’ of that found in the phospholipid fraction but is negliUnder our experimental conditions, such a gible in cells from homozygotes. difference is not evident. In other types of cells the poor incorporation of acetate into the fatty acid moiety of cholesterol esters was also noted 1331. ‘I’hG> possibility that the low radioactivity in cholesterol ester is due to a loss to the medium is precluded by our analysis of the medium (data not shown). In resting cells the distribution of the label in various lipid classes is similar to that in growing cells except for a slightly higher incorporation of acetate into cholesterol than into phospholipids.
Comparison of acetate incorporation by cells prcincubated deficient serum (serum A) and delipidated lipoprotein-deficient B)
in lipoproteinserum (serum
Cholesterol analysis of serum A obtained from ultracentrifugal flotation of normal human plasma by calorimetric method and gas-liquid chromatography indicated that serum A contained approx. 3 ~-(g cholesterol per mg protein (range 2-4 i-1~ per mg protein); and that, after extraction of serum .I with ethanol/ether (1 : 1, v/v) solution, the resulting serum R contained no appreciable cholesterol (~0.1 pg cholesterol per mg protein ). Results shown in ‘I’ahle II indicate that normal cells preincubated in serum B showed greater incorporation of acetate into all lipid classes as compared to cells prcincubated in serum A. The increased incorporation ranged from 1.5- to 4fold, being highest for phospholipids. These differences were much less marked in homozygous cells. Homozygous cells prcincubated in serum A or serum B incorporate more acetate into various lipid fractions l.han normal cells under similar conditions. Such a diffrrencsr hctwren homozygous and normal cell lines is not a constant
451 TABLE
II
INHIBITORY NORMAL
EFFECTS AND
On day 6 nearly
OF HUMAN
HOMOZYGOTE conftucnt
LDL
ON SYNTHESIS
OF LlPlDS
FROM
[2-‘4ClACETATE
IN
FIBROBLASTS
cultures
were
fed with
fresh minimum
essential medium
containing
either
2.2 me/ml of serum A or serum B. The cultures were further incubated for 44 h at 37’C (first incubation). Half of the cultures were then supplemented with 200 ug/ml of LDL-cholesterol. and the other half served as controls (without LDL). All cultures were again incubated at 37’C for 22 h (second incubation). At the end of the second incubation. cells were washed with Dulbecco phosphate-buffered saline solution. 5 ml of Krebs-Ringer’s phosphate buffer containing 0.22 mM of [2-14Clacetate (4.6 Ci/molf were added. Thr cells were incubated at 37OC for 2 h and then washed and assayed for the radioactivities of ail classes of Iipids as described in Materials and Mrthods. The values arc means + S.D. Homowgotc -...
NOlTtlal cpm
x lo-J/mg
lipid A A + LDL B B + LDL
25.0 6.4 65.4 6.5
+ 2.1 z+0.7 +_4.0 ? 1.3
(B) Phospholipid Serum A Serum A + LDL Serum B Srrum B + LDL
4.4 2.5 17.9 2.3
? i + +
0.8 0.2 1.1 0.3
(C) Cholrsterol Serum A Serum A + LDL Serum B Serum B + LDL
13.4 0.3 19.8 0.3
+ t ? f
1.4 0.03 2.1 0.06
(II) Free fatty acid Serum A Serum A + LDL Serum B Swum B + LDL
0.8 0.4 1.1 0.5
It 0.2 .+ 0.1 f 0.1 ? 0.09
(E) Triacylglycerol Serum A Serum A + LDL Serum B Serum B + LDL
0.8 0.6 1.6 0.5
i 0.1 f 0.1 i 0.2 _t 0.1
(F) Chalrstarol Esters Serum A Serum A + LDL Serum B Serum B + LDL
0.3 1.0 0.2 0.8
+_0.1 + 0.3 * 0.1 + 0.1
(A) Total Serum Serum Serum Serum
Suppression
74 88
43 87
98 98
50
55
25 66 -
-
(%)
cpm x 10-3 /me
71.0 42.3 83.4 39.4
i- 8.1 + 2.9 % 9.0 ? 3.4
14.2? 7.0 ! 25.7 + 9.4 +
0.9 0.4 2.6 1.2
39.6 t 1.6 23.5 +_2.0 42.2? 5.1 19.2 f 2.9 1.3 0.7 2.3 1.0
?- 0.3 -f 0.1 + 0.3 ? 0.2
1.3 0.9 2.8 1.4
2 + * f
0.3 0.2 0.3 0.3
k 0.1 + 0.1 ?- 0.1 t 0.1
0.1 0.2 0.3 0.1
Suppression (%
40 53
51 64
41 54
46 57
31 48 ._~ ~~ _._
* Washed and replaced.
finding and was not noted in our previously reported experiments [9] or in subsequent experiments in which normal and other homozygous cell lines were preincubated in lipid extracted fetal calf serum. Effect
of addition of LDL to cells preincubated
in serum A or serum R media
Addition of LDL to cells previously preincuhated with serum A or serum B caused a suppression of acetate incorporation into all lipid classes except cho-
452
lesterol esters in normal cells, the suppression being most marked (98%) for cholesterol. The percent inhibition by LDL was greater in cells preincubated in serum B as expected (Table II). A suppression of acetate incorporation by LDL is also noted in the same lipid classes in homozygous cells, the suppression being slightly higher in cells preincubated in serum B. Unlike normal cells, suppression of cholesterol synthesis by LDL is not significantly greater than suppression of other lipid classes. It is noteworthy that following addition of LDL the rates of incorporation are similar in cells preincubated in serum A or serum B. This observation holds both for normal and homozygous cells. In normal cells LDL caused an increase in the incorporation of acetate into cholesterol esters. This increase can be attributed to an activation of cholesterol ester synthesis by LDL and is consistent with results reported by Goldstein et al. [ 261. In homozygous cells LDL did not have an appreciable effect on incorporation of acetate into the cholesterol ester fraction. Fig. 1A and 1B show the effect of medium LDL concentrations on the rate of acetate incorporation into the various lipid classes in cells preincubated with serum B. In normal cells (Fig. 1A) 50% inhibition (k’i) and maximum inhibition
6
HOMOZYGOTE .
Fw Fatty Aads
m
Trlotylglyc*rol,
100 A
NORMAL
80.
i,
0
0
60 120 LDL-Cholesterol
40
40
80 I20 160 20 LDL- Chok8terol (pglml
1
160 200 (pqlmll
20~~~z&~s ; I ,
0
40
80
120
LDL-
160
200
Cholesterol
0
(pq/ml1
40
, , /
80
I20 I60 200 L~~-C~leste~oi
Lag/ml)
Fig. 1. Effect of LDL-cholesterol on incorporation rrf 114C1acetateinto total iipid, ch<)lesterol, phusphw lipids. free fatty acids and triacylglycerals in normal (A) and homoaygote (B) cells. On day 6 nearly confluent cell m~nolayrrs were rrplacrd with fresh minimum essential medium containing wrum B (2.2 mg! ml). ‘I’hv cultures were then incuhatrd for 44 h at 37OC (first incubati
453
of acetate incorporation into cholesterol occur at LDL-cholesterol concentrations of lo-20 and 80 pg/ml, respectively. In homozygous cells (Fig. 1B) the k’i value for LDL inhibitio~l of cholesterol synthesis was similar to t.hat obtained in normal cells, and maximum inhibition by LDL was approx. 30%. Results with other lipid classes show a similar pattern of inhibition. The figure of 30% inhibition for cholesterol synthesis obtained in this experiment has to be compared to 54% inhibition shown on Table II. In a total of four experiments in the same homozygous cell lines, the range of inhibition has varied between 18 and 54% wit.h a mean of 34 + 8% (S.E.) (compared to 97 + 1% in the normal cell line). The range in the suppression of phospholipid synthesis was less variable, ranging between 34 and 65% with a mean of 51 + 7% (S.E.) compared to 76 Z!Z 4% in the normal). I~cor~o~atio~ of ~14C~oleate into cholesterol esters In normal cells preincubated in serum B addition of LDL caused an increase in cholesterol ester formation from 14 rt 3 to 1230 I 30 S.D. pmol/h per mg protein. In homozygous cell lines the corresponding values were 11 rt 2 and 13 -t_ 5 indicating a lack of activation of cholesterol ester synthesis by medium LDL. These results are consistent with similar measurement done by Brown and Goldstein on the same cell lines (personal communication). HMG-CoA reductase activity Table III shows the effect of LDL on the activity of the enzyme HMG-CoA reductase in normal and homozygous cells preincubated in serum B. The results are calculated by two different methods. In method 1 the spot on the thinlayer plate corresponding to the carrier mevalonate (R, = 0.72) is counted. In method 2 a larger segment of the chromatogram (RF = 0.6-0.72) is counted as recommended by Shapiro et al. [27]. At the moment we do not know the nature of the radioactive compound having the RF of 0.6. Results in Table III indicate that there is a suppression of HMG-CoA reductase activity by LDL in the homozygous cells. The suppression is 32% by method 1 and 15% by method 2 compared to 93 and 95%, respectively, in the normal cells. TABLE
111
EFFECT OF LDL ON THE GOTE F‘IBROBLASTS
Addition CUlt.UW
to
Serum B Serum B + LDL
ACTIVITIES
Normal
fpmoJ/mg
Method
1
OF HMG-CoA
2
Method
30.3 + 2.8 2.1 i 0.6
45.3 i 1.3 2.1 r 0.6
22.2 15.1
(93)
(95)
(32)
* Washed and rcplaccd.
IN NORMAL
Homozyeote
per minf Mvthod
REDUCTASE
1
+ 2.4 f 1.7
(pmoljmg
AND HOMOZY-
per min)
Method 31.9 27.2 (15)
2
” 1.6 ? 1.1
-
Discussion The familial hypercholesterolemias are inborn errors of metabolism characterized by elevation of plasma cholesterol and LDL, deposition of cholesterol and its esters in various tissue compartments and accelerated atherosclerosis. Clinical, genetic and laboratory studies suggest that several genotypes are grouped under this designation. In one subgroup of these patients with severe clinical manifestations and marked hypercholesterolemia the condition appears to represent the homozygous state for a single mutation at an autosomal gene [20,34]. These patients have been referred to as homozygous familial hypercholesterolemia in this and previous studies. The metabolic defect in this group of patients is under active study. Khachadurian [ 191 and Khachadurian and Kawahara [9] reported a decrease in the feedback inhibition of cholesterol synthesis by medium lipids in cultured skin fibroblasts. Simultaneously, Goldstein and Brown [ 11,121 reported on their elegant and extensive studies on fibroblasts from patients who had similar clinical and laboratory manifestations. Their studies indicated that the primary defect is at a high affinity cell surface receptor for LDL. Patients with homozygous familial hypercholesterolemia had an absence of specific binding hence no inhibition of HMG-CoA reductase. Other cellular events that are secondary to the binding e.g. degradation of the protein component of LDL; transfer of LDL-cholesterol into the cell; stimulation of the cellular mechanism for the esterification OF cholesterol were also absent in these patients. In experiments reported by Brown and Goldstein medium LDL has no inhibitory effect on HMG-CoA reductase activity or on the incorporation of acetate into cholesterol in homozygous cell lines [ 111. In contrast in our experiments in cells from this patient as well as from our other homozygous patients ]9,10] there is partial suppression of these two parameters by medium LDL. Because Goldstein et al. [lS] described a second group of patients with homozygous familial hypercholesterolemia in whom the high affinity receptor for LDL was deficient (receptor defective) rather than absent (receptor negative), we carried out the experiments described in this paper on a cell line that was found to be receptor negative by Brown and Goldstein (personal communication). Moreover, the incorporation of oleate into cholesterol esters by these cells was not stimulated by added LDL in our experiments, consistent with the findings of Brown and Goldstein in “receptor negative” cells. The possibility that the differences between our results and those of Goldstein and Brown are due to the method of preparation of lipoprotein-deficient serum has to be considered. Since these authors prepared the lipoprotein-deficient serum by ultracentrifugation, it is possible that traces of lipids left in the serum were producing a baseline inhibition which was not further increased by adding LDL to homozygous cells. Therefore, we ran parallel experiments using serum A as well as serum B. Our results indicate that acetate incorporation is higher in cells preincubated in serum B. However, inhibition by LDL is present even in cells preincubated in serum A. Therefore, the observed differences cannot be explained entirely on the basis of different methods of preparation of lipoprotein-deficient serum. A similar inhibition of acetate incorporation and of the HMG-CoA reductase activity was also demonstrated by Avigan et al. ]l[i] in
455
all cell lines derived from patients with homozygous familial hypercholesterolemia. A second explanation for the discrepancies observed could be the inherent difficulties in defining and quantifying LDL binding to specific cell surface receptors which would preclude a clear distinction between “receptor negative” and “receptor defective” cell lines by the available assays. Such difficulties may explain why Breslow et al. [16] found a specific binding of 12% of normal in their patient with severe homozygous familial hypercholesterolemia, and why Stein et al. 1171 observed near normal binding in their homozygous cell line. If it is assumed that our cell line lacks entirely specific receptor sites for LDL, then another mechanism for interaction of LDL with the cell must be operative. Such an interaction of LDL with the homozygous cells is also needed to explain the effect of LDL on fatty acid synthesis observed by us. The regulation of fatty acid synthesis in homozygous familial hypercholesterolemia has received relatively little attention. In most of our earlier experiments [9] we had noted an increased incorporation of acetate into the total saponifiable fraction by homozygous familial hypercholesterolemia cells grown in a medium containing 10% fetal calf serum. Avigan et al. [15] showed that homozygous familial hypercholesterolemia cells preincubated in whole serum had a 23% greater incorporation of acetate into fatty acids compared to normal cells; following addition of whole serum to cells preincubated in delipidated serum, there was a 29% suppression of incorporation in homozygous familial hypercholesterolemia cells compared to 44% in normals. Our more extensive studies, reported in this paper, clearly demonstrate that in homozygous familial hypercholesterolemia there is a derangement in fatty acid synthesis, resulting in an increased incorporation of acetate into this fraction by resting as well as growing cells, and a decreased suppression of acetate incorporation into fatty acids by medium LDL in cells preincubated in serum B. In cells preincubated in serum A, suppression by LDL was slightly higher in homozygous cells. It is possible that the small amounts of lipids contained in the serum A had already suppressed the normal cell line. This may also explain the observation of Goldstein and Brown [ll] who found no suppression of acetate incorporation into fatty acids, triacylglycerols or phospholipids by LDL added to normal cells preincubated in serum A. Our findings of increased incorporation of acetate into fatty acids in homozygous familial hypercholesterolemia is consistent with the hypothesis that the primary defect in familial hypercholesterolemia is in the binding or internalization of LDL. However, as with acetate incorporation into sterols and suppression of HMG-CoA reductase, the LDL effect is only defective rather than absent. A coordinate depression of acetate incorporation by L cells into both cholesterol and fatty acids by serum lipids has been reported by Howard et al. [ 351. Similar results were obtained in other cell lines by Alberts et al. [ 361 and Volpe and Marasa [8]. The sites of regulation of fatty acid synthesis by medium lipids has been studied by several investigators. Thus in the permanent cell lines L-M and NCTC 1469 (mouse liver) Alberts et al. [36] showed that transfer of cells from serum-containing medium to serum-free medium resulted in increased activities of both acetyl-CoA carboxylase and fatty acid synthetase. Similar results were obtained by Volpe and Marasa [8] in glial cell cultures. In human skin fibroblasts Jacobs et al. [6] showed a 50%’ increase in acetyl-CoA
456
carboxylase upon incubation of cells in delipidated medium with a “slow” increase in fatty acid synthetase. Howard et al. [35] also noted an approx. 4-fold stimulation of acetyl-CoA synthetase in L cells preincubated in delipidated serum. If the abnormal regulation of fatty acid synthesis in homozygous familial hypercholesterolemia is secondary to a defect in binding of LDL, then one would expect a lack of regulation at the various enzymes normally regulated by medium LDL. At the present, the only information available on this point is the report of Avigan et al. [ 151 indicating that the inhibition of acetyl-CoA carboxylase by medium lipids was similar in normal and homozygous familial hypercholesterolemia cells and amounted to 20-40%. Since these authors did not observe a definite defect in the regulation of incorporation of acetate into fatty acids under their experimental conditions, it becomes difficult to interpret their results on the activity of acetyl-CoA carboxylase. A detailed study of the enzymes of the fatty acid synthetic pathway would be valuable in delineating the metabolic defect in homozygous familial hypercholesterolemia as well as other forms of hereditary hyperlipidemias. Acknowledgements We are grateful to Y.S. Huang for his cooperation in the early phase of this study and thank B. Olsen and G. Avigad for their critical review of this manuscript. This work was supported by United States Public Health Service Grant No. HL 18878-01, National Foundation-March of Dimes Grant No. l-352, and CMDNJ-Rutgers Medical School Grants: USPHS-PE15 and GRS-RR 5576. References 1
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Biochimica el Biophysics Acta, 487 (1977) 445-457 @ Elsevier~North-Holland Biomedical Press
BBA 57005
REGWLATfUN OF LIPID SYNTHESIS BY LOW DENSITY LIPOPROTEINS 1N CULTURED SKIN F~BROBLASTS IN HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA
CHIEN H. FUNG, AVEDIS K. KHAGHADURIAN IBRAHIM F. DURR Departments and Dentislry
uf Medicine
*, CHING-HUA WANG and
and Biochemistry, Rutgers Medicat School, of New Jersey, Piscataway, N.J. 08854 U.S.A.)
Cattege
of Medicirae
(Received December 14th, 1976)
Summary The regulation of cholesterol and fatty acid syntheses by low density lipoproteins (LDL) was studied in cultured skin fibroblasts from a normal subject and a subject with homozygous familial hypercholesterolemia the fibroblasts of which had no LDL binding to specific cell surface receptors (receptor negative) and no cholesterol esterifying activity. Assays were done in cells preincubated in media containing lipoprotein-deficient serum (serum A) or serum A delipidated with ether-ethanol (serum B). In homozygous familial hypercholesterolemia cells preincubated in serum B a second preincubation with saturating levels of LDL caused a 34% suppression in acetate incorporation into cholesterol and a 32% reduction of the activity of 3-hydr~~y-3-methylgl~t~yl coenzyme A (HMG-Co A) reductase compared to 9X and 93% reduction, respectively, in the normal cell line. In homozygous familial hypercholesterolemia cells grown in 10% fetal calf serum there was an increased incorporation of acetate into total fatty acids as well as into phospholipids and triacylglycerols, indicating a lack of suppression of fatty acid synthesis by medium lipids in the mutant cells. In homozygous familial hyperchoIesterolemia cells preincubated in serum B a second preincubation with LDL caused a 51% suppression of acetate incorporation into phospholipids compared to ‘i6% in normal cells. Our data indicate that in homozygous familial hypercholesterolemia there is a derangement in the regulation of fatty acid synthesis. It is, however, less marked than the derangement of sterol synthesis. The inhibitory action of LDL both on sterol and fatty acid syntheses in cells that do not show any LDL binding activity by the presently available tech-
* Ta
whom correspondence should be addressed. Abbreviations: serum A, lipoprotein-deficient serum; serum b, lipoprotein-deficient serum delipidated with ether/ethanol; HMG-CoA. 3-hydroxy-3-methylglutaryl coenzyme A; antibiotic mixture, penicillin, streptomycin and neomycin; VLDL. very low density lipoproteins (d G 1.019 g/ml); LDL, low density lipoproteins (d = 1.019-1.063 g/ml); HDL. high density lipoproteins (d = 1.063-l 210 glml).
446
nique is in contrast to the findings of Goldstein et al. ((1975) Proc. Natl. Acad. Sci. U.S. 72, 1092-1096) which indicate that LDL has no inhibitory effect in receptor negative cell lines. -.Introduction Cultured fibroblasts have been used extensively to study the regulation of sterol and fatty acid syntheses. In established cell lines Bailey [l] and Rothblat [2] demonstrated inhibition of sterol synthesis by the sterols contained in the growth medium. Similarly, Avigan et al. [3] and Williams and Avigan [4] have shown that overnight preincubation of human skin fibroblasts in medium containing lipid-free serum results in a marked increase in cholesterol synthesis. This stimulation was reversed by whole serum, low- or high-density lipoproteins or cholesterol suspension. The remarkable increase in cholesterol synthesis was found to be associated with a parallel increase in the activity of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase [5]. Jacobs et al. [6] and Jacobs and Majerus [7] have shown that in primary cultures of human skin fibroblasts the rate of acetate incorporation into fatty acids is markedly inhibited by fatty acids and serum lipids. Volpe and Marasa [8] have made similar observations in cultured glial cells. Cultured skin fibroblasts have been useful in the delineation of the metabolic defect in a group of human errors of metabolism commonly known as the familial hypercholesterolemias [g-17]. In patients with the homozygous form of this disorder Khachadurian and Kawahara [9] and Khachadurian et al. [lo] reported a decrease in the feedback inhibition of cholesterol synthesis by medium lipids. Similar results were reported by Avigan et al. [15]. On the other hand the extensive studies of Brown and Goldstein [11,14] indicate a complete lack of feedback inhibition of HMG-CoA reductase activity, resulting from an absence of binding of LDL to specific cell surface receptors. These differences could result from the inherent difficulties in the experimental procedures or from genetic heterogeneity [ 10,16,18]. The regulation of fatty acid synthesis in familial hypercholesterolemia has attracted little attention so far. Khachadurian and Kawahara [9] noted a decrease in the inhibition of acetate incorporation into the total saponifiable fraction in homozygous familial hypercholesterolemia cells grown in medium containing 10% fetal calf serum. These findings suggest a defective regulation of fatty acid synthesis as well. The present studies were carried out to determine whether in severe homozygous familial hypercholesterolemia the feedback inhibition of cholesterol synthesis was totally lacking or only decreased, and to study the regulation of fatty acid synthesis by measuring the incorporation of acetate into the major lipid classes. Materials and Methods Materials. [2-14C]Acetic acid, sodium salt (56-58 Ci/mol) and PCS * solubilizer were purchased from Amersham/Searle Corporation. DL-[3-‘4C]HMG-CoA * Phase-combining
system.
(26 Ci/mol) and [l-‘4C]oleic acid (50 Ci/mol) were obtained from New England Nuclear. Lipid standards containing cholesterol (20.2%), cholesterol oleate (20.0%), methyl oleate (20.0%), oleic acid (19.9%) and trioleoylglycerol (19.9%) were obtained from Sigma Chemical Co. Mevalonic acid lactone was purchased from K and K Co. Minimum essential medium Earle’s powder (Cat. No. F-11), nutrient mixture with L-glutamine (Cat. No. 155), heat-inactivated fetal calf serum, trypsin-EDTA solution (1 X) and antibiotic mixture (penicillin, streptomycin and neomycin) (100 X) were purchased from Grand Island Biological Co. Pre-coated thin-layer chromatography sheets of silica gel were obtained from Brinkman Instruments, Inc. All other reagents and organic solvents were reagent grade or of the highest purity commercially available. Cell lines. The normal cell line GM 240 was obtained from Dr. A.E. Green, Institute for Medical Research, Camden, N.J. Normal cells 1516 and 32 were from two healthy individuals of age 5 and 37 years who had plasma cholesterol of 140-190 mg per 100 ml [lo ]. Homozygote MH 25 fibroblasts were derived from a patient homozygous for familial hypercholesterolemia as described previously by Khachadurian [ 191 and Khachadurian and Uthman 1201. Growth of cells. Cells were grown in a 75 cm’ Falcon flask at 37°C in 95% sir/5% CO, humidified environment in 20 ml nutrient mixture F-10 or Eagle’s minimum essential medium containing 0.2 ml of antibiotic mixture (100 X) supplemented with 10% heat-inactivated fetal calf serum. The cells were subcultured at 7-day intervals by trypsinization and seeded into three 75 cm2 Falcon flasks, each containing one million cells. In some experiments the cells were seeded into 25 cm’ Falcon flasks, each containing 0.3 million cells. The medium was changed every 3 days. Cells in the 6th to 16th passages were used in all experiments. Incubation with radioactive acetate was done under two different conditions: (a), resting cells: confluent cells were washed three times with 5 ml Krebs-Ringer’s phosphate buffer, pH 7.4, and incubated with [2-“Clacetate in 5 ml of the buffer for 2 h at 37°C on a Thomas Rotating Apparatus with a speed of 100 oscillations per min; (b) growing cells: [ 2-14C]acetate was added to the medium on day 1 of growth. On day 3 the medium was changed. Cells were washed with Krebs-Ringer’s phosphate buffer. Fresh growth medium with radioactive acetate was added, and cells were allowed to reach conflu-
ency . Preparation of low-density lipidated lipoprotein-deficient
lipoproteins, lipoprotein-deficient serum. Low-density lipoproteins
serum and de-
(LDL) were isolated from human plasma by ultracentrifugal flotation according to the method of Have1 et al. [21]. Lipoprotein-deficient serum (serum A) was obtained after removal of LDL, very low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by ultracentrifugal flotation. Because of the possibility that serum A so prepared could contain trace amounts of cholesterol, it was further extracted once with ten volumes of ether/ethanol (1 : 1, v/v) solution at-30°C and stirred vigorously for 2 h. The precipitated protein was collected by centrifugation at 4°C. The protein was then washed first with 300 ml of cold 95% ethanol and subsequently with 300 ml of cold ether. The protein precipitate was again collected by centrifugation. The residual ether was removed by vacuum evaporation at 25°C. The dry protein was then dissolved in a minimal volume of Dulbecco phosphate-buffered saline and dialyzed once against ten
448
volumes of the same solution at 4°C for 24 h. The resulting preparation of delipidated lipoprotein-deficient serum (serum B) and the solution of LDL were sterilized by passing through a Millipore filter with a pore size of 0.22 ym. Assays. The content of total cholesterol in LDL, serum A and serum B was determined by the calorimetric method of Abel1 et al. [22] and by gas-liquid chromatography according to the procedure reported by Ishikawa et al. [ 231. A Hewlett-Packard model 421 gas chromatograph equipped with flame ionization detectors and an Autolab Minigrator was used. The cell protein was measured as described by Lowry et al. [ 241. [ 1-‘4C]Oleate . albumin complex was prepared as described by Van Harken et al. (251. Incorporation of [ I-‘“Cloleate into lipids was measured by the method reported by Goldstein et, al. 1261. On day 6, after cells had been incubated 24 h in medium coK~taining 2.5 mg protein per ml of serum B, LDL was added to give the final concentration of 200 pg LDL-cholesterol per ml. After a further 17 h incubation at 37”C, albumin-bound [‘4C]oleate (0.1 mM, 4250 cpm/nmol) was added, and the cells were harvested 2 h later. The cellular content of [‘4C]cholesterol esters was determined. Cell extracts for measurement of HMG-CoA reductase activity were prepared by the method of Goldstein and Brown [ 111. Cells from two 75 cm2 Falcon flasks were pooled to prepare each extract. HMG-CoR reductase activity was measured by the method described by Shapiro et al. ]27]. Ahquots of the cell extract (50-80 pg of protein) were used for assaying enzyme activities. Aliquots (50 ~1) of the reaction mixture after centrifu~ation were applied to silica gel thin-layer chromato~aphy sheets. After drying, 10 mg of mevalonic acid lactone in 10 i_tl of water was applied to each spot as a carrier. The plates were developed in a benzene/acetone solution (1 : 1, v/v), and then air dried. Mevalonic acid lactone, which locates in the region of Rr = 0.72 + 0.02, may be detected as a quenching band when the chromatogram is viewed under an ultraviolet lamp. The area with RF = 0.72 was removed and added to 10 ml of PCS solubilizer and counted for radioactivity which was used to calculate HMG-CoA reductase activity (Method 1). The area with RF = 0.60 was also removed and counted for radioactivity. The sum of the counts in the two spots was also used to calculate HMG-CoA reductase activity (Method 2) 1271. extraction and fr~ctio~ut~o~ of lipids. Two independent methods were used: (a) Cells were saponified and extracted as previously described [9]. The incubation mixture together with the cells was mixed with ethanolic NaOH, saponified and extracted with ligroin to recover the unsaponifiable lipids. Subsequently, the aqueous layer was acidified and extracted with ligroin to recover the fatty acids. Where indicated, cholesterol was isolated from the unsaponifiable fraction by thin-layer chromatography on aluminum plates coated with silica gel with gypsum as described by Gloster and Fletcher [28]. In addition, the saponifiable fraction was dried under vacuum, then mixed with 5 ml of methanolic HzS04 and placed in a boiling water bath for 30 min. After cooling, the mixture was extracted three times, each with 10 ml ligroin to recover the methyl esters of fatty acids and was chromato~aphed on silica gel plates using methyl palmitate as the standard ester. (b) Lipids were alternatively estracted by the procedure of Folch et al. 1291 as follows: The incubation mixture was decanted, and the cells were washed twice with 5 ml of phosphate-buffere?
449
saline buffer, pH 7.4. The cells were scraped and suspended in 5 ml of KrebsRinger’s phosphate buffer. An aliquot (0.1-0.2 ml) of the cell suspension was removed for protein determination. The remaining cell suspension was centrifuged. The cell pellet was resuspended into 1 ml of Krebs-Ringer’s phosphate buffer and extracted vigorously once with a chloroform/methanol (2 : 1, v/v) solution on a Vortex-Genie mixer for 2 min. The chloroform layer was recovered and then evaporated to dryness at 40°C in an Evapo-Mix evaporator connected to a water aspirator. The lipids were dissolved in 0.2 ml chloroform and subjected to thin-layer chromatography as described above. Spots corresponding to phospholipids, cholesterol, free fatty acids, triacylglycerols and cholesterol esters were identified against lipid standards. The spots were cut off and dissolved in a PCS scintillation solution. All radioactivities were determined in an Intertechnique SL30 Liquid Scintillation Spectrometer with an efficiency of 90% for 14C. Since the specific radioactivity of [2-‘4C]acetate used in the experiments varied, the values espressed as counts per min per mg protein (cmp/mg protein) in tables and figures have been normalized using the specific radioactivity of [2-14C]acetate = 8.50 Ci/mol as a factor. Results Effect of 10% fetal calf serum on the incorporation of acetate into cholesterol and fatty acid esters In confluent cells grown in the nutrient mixture F-10 medium containing 10% fetal calf serum, the rate of the incorporation of acetate into cholesterol in homozygous cells (6.9 + 0.2 . 10” cpm/mg cell protein) is about ten times greater than in normal cells (0.7 + 0.2 * lo3 cpm/mg cell protein) while the rate of acetate incorporation into fatty acid esters (6.2 i 0.6 . 10” cpm/mg cell protein) is approximately three times greater than normal (2.4 2 0.4 . 10’ cpm/ mg cell protein). These observations are consistent with the previous results obtained from other cell lines homozygous for familial hypercholesterolemia
[91. Table I shows results of experiments in which radioactive acetate was added to resting confluent cells or growing cells. The cells were washed and their lipids were separated by thin-layer chromatography. Results indicate increased incorporation of acetate into all lipid classes by resting as well as growing homozygous cells. In growing homozygous cells, 45% of the radioactivity is recovered in the cholesterol fraction, 51% in the phospholipid fraction and 1.4% in triacylglycerols. The release of the newly synthesized lipids into the medium by these cells was assessed by dividing the radioactivity in each lipid class recovered in the medium (extracellular) by the sum of the intracellular and extracellular counts. In normal cells 70% of the cholesterol, 38% of phospholipid and 97% of the triacylglycerol counts were recovered in the medium. The corresponding values for the homozygous cells were 63, 33 and 94%. Therefore, the low radioactivity in the triacylglycerols compared to phospholipids is partly explained by the difference in the loss of these two lipids into the medium. The limited uptake of medium triacylglycerols noted in other types of cells [30], as well as the shift of fatty acids to phospholipids [31,32] could also con-
tribute to the low level of radioactivity in intracellular triacylglycerols. ‘I’hc smallest amount of radioactivity is found in the cholesterol esters. Goldst,ein et al. [ 26 J show that in normal cells, incorporation of acetate into the cholesterol ester is about 40%’ of that found in the phospholipid fraction but is negliUnder our experimental conditions, such a gible in cells from homozygotes. difference is not evident. In other types of cells the poor incorporation of acetate into the fatty acid moiety of cholesterol esters was also noted 1331. ‘I’hG> possibility that the low radioactivity in cholesterol ester is due to a loss to the medium is precluded by our analysis of the medium (data not shown). In resting cells the distribution of the label in various lipid classes is similar to that in growing cells except for a slightly higher incorporation of acetate into cholesterol than into phospholipids.
Comparison of acetate incorporation by cells prcincubated deficient serum (serum A) and delipidated lipoprotein-deficient B)
in lipoproteinserum (serum
Cholesterol analysis of serum A obtained from ultracentrifugal flotation of normal human plasma by calorimetric method and gas-liquid chromatography indicated that serum A contained approx. 3 ~-(g cholesterol per mg protein (range 2-4 i-1~ per mg protein); and that, after extraction of serum .I with ethanol/ether (1 : 1, v/v) solution, the resulting serum R contained no appreciable cholesterol (~0.1 pg cholesterol per mg protein ). Results shown in ‘I’ahle II indicate that normal cells preincubated in serum B showed greater incorporation of acetate into all lipid classes as compared to cells prcincubated in serum A. The increased incorporation ranged from 1.5- to 4fold, being highest for phospholipids. These differences were much less marked in homozygous cells. Homozygous cells prcincubated in serum A or serum B incorporate more acetate into various lipid fractions l.han normal cells under similar conditions. Such a diffrrencsr hctwren homozygous and normal cell lines is not a constant
451 TABLE
II
INHIBITORY NORMAL
EFFECTS AND
On day 6 nearly
OF HUMAN
HOMOZYGOTE conftucnt
LDL
ON SYNTHESIS
OF LlPlDS
FROM
[2-‘4ClACETATE
IN
FIBROBLASTS
cultures
were
fed with
fresh minimum
essential medium
containing
either
2.2 me/ml of serum A or serum B. The cultures were further incubated for 44 h at 37’C (first incubation). Half of the cultures were then supplemented with 200 ug/ml of LDL-cholesterol. and the other half served as controls (without LDL). All cultures were again incubated at 37’C for 22 h (second incubation). At the end of the second incubation. cells were washed with Dulbecco phosphate-buffered saline solution. 5 ml of Krebs-Ringer’s phosphate buffer containing 0.22 mM of [2-14Clacetate (4.6 Ci/molf were added. Thr cells were incubated at 37OC for 2 h and then washed and assayed for the radioactivities of ail classes of Iipids as described in Materials and Mrthods. The values arc means + S.D. Homowgotc -...
NOlTtlal cpm
x lo-J/mg
lipid A A + LDL B B + LDL
25.0 6.4 65.4 6.5
+ 2.1 z+0.7 +_4.0 ? 1.3
(B) Phospholipid Serum A Serum A + LDL Serum B Srrum B + LDL
4.4 2.5 17.9 2.3
? i + +
0.8 0.2 1.1 0.3
(C) Cholrsterol Serum A Serum A + LDL Serum B Serum B + LDL
13.4 0.3 19.8 0.3
+ t ? f
1.4 0.03 2.1 0.06
(II) Free fatty acid Serum A Serum A + LDL Serum B Swum B + LDL
0.8 0.4 1.1 0.5
It 0.2 .+ 0.1 f 0.1 ? 0.09
(E) Triacylglycerol Serum A Serum A + LDL Serum B Serum B + LDL
0.8 0.6 1.6 0.5
i 0.1 f 0.1 i 0.2 _t 0.1
(F) Chalrstarol Esters Serum A Serum A + LDL Serum B Serum B + LDL
0.3 1.0 0.2 0.8
+_0.1 + 0.3 * 0.1 + 0.1
(A) Total Serum Serum Serum Serum
Suppression
74 88
43 87
98 98
50
55
25 66 -
-
(%)
cpm x 10-3 /me
71.0 42.3 83.4 39.4
i- 8.1 + 2.9 % 9.0 ? 3.4
14.2? 7.0 ! 25.7 + 9.4 +
0.9 0.4 2.6 1.2
39.6 t 1.6 23.5 +_2.0 42.2? 5.1 19.2 f 2.9 1.3 0.7 2.3 1.0
?- 0.3 -f 0.1 + 0.3 ? 0.2
1.3 0.9 2.8 1.4
2 + * f
0.3 0.2 0.3 0.3
k 0.1 + 0.1 ?- 0.1 t 0.1
0.1 0.2 0.3 0.1
Suppression (%
40 53
51 64
41 54
46 57
31 48 ._~ ~~ _._
* Washed and replaced.
finding and was not noted in our previously reported experiments [9] or in subsequent experiments in which normal and other homozygous cell lines were preincubated in lipid extracted fetal calf serum. Effect
of addition of LDL to cells preincubated
in serum A or serum R media
Addition of LDL to cells previously preincuhated with serum A or serum B caused a suppression of acetate incorporation into all lipid classes except cho-
452
lesterol esters in normal cells, the suppression being most marked (98%) for cholesterol. The percent inhibition by LDL was greater in cells preincubated in serum B as expected (Table II). A suppression of acetate incorporation by LDL is also noted in the same lipid classes in homozygous cells, the suppression being slightly higher in cells preincubated in serum B. Unlike normal cells, suppression of cholesterol synthesis by LDL is not significantly greater than suppression of other lipid classes. It is noteworthy that following addition of LDL the rates of incorporation are similar in cells preincubated in serum A or serum B. This observation holds both for normal and homozygous cells. In normal cells LDL caused an increase in the incorporation of acetate into cholesterol esters. This increase can be attributed to an activation of cholesterol ester synthesis by LDL and is consistent with results reported by Goldstein et al. [ 261. In homozygous cells LDL did not have an appreciable effect on incorporation of acetate into the cholesterol ester fraction. Fig. 1A and 1B show the effect of medium LDL concentrations on the rate of acetate incorporation into the various lipid classes in cells preincubated with serum B. In normal cells (Fig. 1A) 50% inhibition (k’i) and maximum inhibition
6
HOMOZYGOTE .
Fw Fatty Aads
m
Trlotylglyc*rol,
100 A
NORMAL
80.
i,
0
0
60 120 LDL-Cholesterol
40
40
80 I20 160 20 LDL- Chok8terol (pglml
1
160 200 (pqlmll
20~~~z&~s ; I ,
0
40
80
120
LDL-
160
200
Cholesterol
0
(pq/ml1
40
, , /
80
I20 I60 200 L~~-C~leste~oi
Lag/ml)
Fig. 1. Effect of LDL-cholesterol on incorporation rrf 114C1acetateinto total iipid, ch<)lesterol, phusphw lipids. free fatty acids and triacylglycerals in normal (A) and homoaygote (B) cells. On day 6 nearly confluent cell m~nolayrrs were rrplacrd with fresh minimum essential medium containing wrum B (2.2 mg! ml). ‘I’hv cultures were then incuhatrd for 44 h at 37OC (first incubati
453
of acetate incorporation into cholesterol occur at LDL-cholesterol concentrations of lo-20 and 80 pg/ml, respectively. In homozygous cells (Fig. 1B) the k’i value for LDL inhibitio~l of cholesterol synthesis was similar to t.hat obtained in normal cells, and maximum inhibition by LDL was approx. 30%. Results with other lipid classes show a similar pattern of inhibition. The figure of 30% inhibition for cholesterol synthesis obtained in this experiment has to be compared to 54% inhibition shown on Table II. In a total of four experiments in the same homozygous cell lines, the range of inhibition has varied between 18 and 54% wit.h a mean of 34 + 8% (S.E.) (compared to 97 + 1% in the normal cell line). The range in the suppression of phospholipid synthesis was less variable, ranging between 34 and 65% with a mean of 51 + 7% (S.E.) compared to 76 Z!Z 4% in the normal). I~cor~o~atio~ of ~14C~oleate into cholesterol esters In normal cells preincubated in serum B addition of LDL caused an increase in cholesterol ester formation from 14 rt 3 to 1230 I 30 S.D. pmol/h per mg protein. In homozygous cell lines the corresponding values were 11 rt 2 and 13 -t_ 5 indicating a lack of activation of cholesterol ester synthesis by medium LDL. These results are consistent with similar measurement done by Brown and Goldstein on the same cell lines (personal communication). HMG-CoA reductase activity Table III shows the effect of LDL on the activity of the enzyme HMG-CoA reductase in normal and homozygous cells preincubated in serum B. The results are calculated by two different methods. In method 1 the spot on the thinlayer plate corresponding to the carrier mevalonate (R, = 0.72) is counted. In method 2 a larger segment of the chromatogram (RF = 0.6-0.72) is counted as recommended by Shapiro et al. [27]. At the moment we do not know the nature of the radioactive compound having the RF of 0.6. Results in Table III indicate that there is a suppression of HMG-CoA reductase activity by LDL in the homozygous cells. The suppression is 32% by method 1 and 15% by method 2 compared to 93 and 95%, respectively, in the normal cells. TABLE
111
EFFECT OF LDL ON THE GOTE F‘IBROBLASTS
Addition CUlt.UW
to
Serum B Serum B + LDL
ACTIVITIES
Normal
fpmoJ/mg
Method
1
OF HMG-CoA
2
Method
30.3 + 2.8 2.1 i 0.6
45.3 i 1.3 2.1 r 0.6
22.2 15.1
(93)
(95)
(32)
* Washed and rcplaccd.
IN NORMAL
Homozyeote
per minf Mvthod
REDUCTASE
1
+ 2.4 f 1.7
(pmoljmg
AND HOMOZY-
per min)
Method 31.9 27.2 (15)
2
” 1.6 ? 1.1
-
Discussion The familial hypercholesterolemias are inborn errors of metabolism characterized by elevation of plasma cholesterol and LDL, deposition of cholesterol and its esters in various tissue compartments and accelerated atherosclerosis. Clinical, genetic and laboratory studies suggest that several genotypes are grouped under this designation. In one subgroup of these patients with severe clinical manifestations and marked hypercholesterolemia the condition appears to represent the homozygous state for a single mutation at an autosomal gene [20,34]. These patients have been referred to as homozygous familial hypercholesterolemia in this and previous studies. The metabolic defect in this group of patients is under active study. Khachadurian [ 191 and Khachadurian and Kawahara [9] reported a decrease in the feedback inhibition of cholesterol synthesis by medium lipids in cultured skin fibroblasts. Simultaneously, Goldstein and Brown [ 11,121 reported on their elegant and extensive studies on fibroblasts from patients who had similar clinical and laboratory manifestations. Their studies indicated that the primary defect is at a high affinity cell surface receptor for LDL. Patients with homozygous familial hypercholesterolemia had an absence of specific binding hence no inhibition of HMG-CoA reductase. Other cellular events that are secondary to the binding e.g. degradation of the protein component of LDL; transfer of LDL-cholesterol into the cell; stimulation of the cellular mechanism for the esterification OF cholesterol were also absent in these patients. In experiments reported by Brown and Goldstein medium LDL has no inhibitory effect on HMG-CoA reductase activity or on the incorporation of acetate into cholesterol in homozygous cell lines [ 111. In contrast in our experiments in cells from this patient as well as from our other homozygous patients ]9,10] there is partial suppression of these two parameters by medium LDL. Because Goldstein et al. [lS] described a second group of patients with homozygous familial hypercholesterolemia in whom the high affinity receptor for LDL was deficient (receptor defective) rather than absent (receptor negative), we carried out the experiments described in this paper on a cell line that was found to be receptor negative by Brown and Goldstein (personal communication). Moreover, the incorporation of oleate into cholesterol esters by these cells was not stimulated by added LDL in our experiments, consistent with the findings of Brown and Goldstein in “receptor negative” cells. The possibility that the differences between our results and those of Goldstein and Brown are due to the method of preparation of lipoprotein-deficient serum has to be considered. Since these authors prepared the lipoprotein-deficient serum by ultracentrifugation, it is possible that traces of lipids left in the serum were producing a baseline inhibition which was not further increased by adding LDL to homozygous cells. Therefore, we ran parallel experiments using serum A as well as serum B. Our results indicate that acetate incorporation is higher in cells preincubated in serum B. However, inhibition by LDL is present even in cells preincubated in serum A. Therefore, the observed differences cannot be explained entirely on the basis of different methods of preparation of lipoprotein-deficient serum. A similar inhibition of acetate incorporation and of the HMG-CoA reductase activity was also demonstrated by Avigan et al. ]l[i] in
455
all cell lines derived from patients with homozygous familial hypercholesterolemia. A second explanation for the discrepancies observed could be the inherent difficulties in defining and quantifying LDL binding to specific cell surface receptors which would preclude a clear distinction between “receptor negative” and “receptor defective” cell lines by the available assays. Such difficulties may explain why Breslow et al. [16] found a specific binding of 12% of normal in their patient with severe homozygous familial hypercholesterolemia, and why Stein et al. 1171 observed near normal binding in their homozygous cell line. If it is assumed that our cell line lacks entirely specific receptor sites for LDL, then another mechanism for interaction of LDL with the cell must be operative. Such an interaction of LDL with the homozygous cells is also needed to explain the effect of LDL on fatty acid synthesis observed by us. The regulation of fatty acid synthesis in homozygous familial hypercholesterolemia has received relatively little attention. In most of our earlier experiments [9] we had noted an increased incorporation of acetate into the total saponifiable fraction by homozygous familial hypercholesterolemia cells grown in a medium containing 10% fetal calf serum. Avigan et al. [15] showed that homozygous familial hypercholesterolemia cells preincubated in whole serum had a 23% greater incorporation of acetate into fatty acids compared to normal cells; following addition of whole serum to cells preincubated in delipidated serum, there was a 29% suppression of incorporation in homozygous familial hypercholesterolemia cells compared to 44% in normals. Our more extensive studies, reported in this paper, clearly demonstrate that in homozygous familial hypercholesterolemia there is a derangement in fatty acid synthesis, resulting in an increased incorporation of acetate into this fraction by resting as well as growing cells, and a decreased suppression of acetate incorporation into fatty acids by medium LDL in cells preincubated in serum B. In cells preincubated in serum A, suppression by LDL was slightly higher in homozygous cells. It is possible that the small amounts of lipids contained in the serum A had already suppressed the normal cell line. This may also explain the observation of Goldstein and Brown [ll] who found no suppression of acetate incorporation into fatty acids, triacylglycerols or phospholipids by LDL added to normal cells preincubated in serum A. Our findings of increased incorporation of acetate into fatty acids in homozygous familial hypercholesterolemia is consistent with the hypothesis that the primary defect in familial hypercholesterolemia is in the binding or internalization of LDL. However, as with acetate incorporation into sterols and suppression of HMG-CoA reductase, the LDL effect is only defective rather than absent. A coordinate depression of acetate incorporation by L cells into both cholesterol and fatty acids by serum lipids has been reported by Howard et al. [ 351. Similar results were obtained in other cell lines by Alberts et al. [ 361 and Volpe and Marasa [8]. The sites of regulation of fatty acid synthesis by medium lipids has been studied by several investigators. Thus in the permanent cell lines L-M and NCTC 1469 (mouse liver) Alberts et al. [36] showed that transfer of cells from serum-containing medium to serum-free medium resulted in increased activities of both acetyl-CoA carboxylase and fatty acid synthetase. Similar results were obtained by Volpe and Marasa [8] in glial cell cultures. In human skin fibroblasts Jacobs et al. [6] showed a 50%’ increase in acetyl-CoA
456
carboxylase upon incubation of cells in delipidated medium with a “slow” increase in fatty acid synthetase. Howard et al. [35] also noted an approx. 4-fold stimulation of acetyl-CoA synthetase in L cells preincubated in delipidated serum. If the abnormal regulation of fatty acid synthesis in homozygous familial hypercholesterolemia is secondary to a defect in binding of LDL, then one would expect a lack of regulation at the various enzymes normally regulated by medium LDL. At the present, the only information available on this point is the report of Avigan et al. [ 151 indicating that the inhibition of acetyl-CoA carboxylase by medium lipids was similar in normal and homozygous familial hypercholesterolemia cells and amounted to 20-40%. Since these authors did not observe a definite defect in the regulation of incorporation of acetate into fatty acids under their experimental conditions, it becomes difficult to interpret their results on the activity of acetyl-CoA carboxylase. A detailed study of the enzymes of the fatty acid synthetic pathway would be valuable in delineating the metabolic defect in homozygous familial hypercholesterolemia as well as other forms of hereditary hyperlipidemias. Acknowledgements We are grateful to Y.S. Huang for his cooperation in the early phase of this study and thank B. Olsen and G. Avigad for their critical review of this manuscript. This work was supported by United States Public Health Service Grant No. HL 18878-01, National Foundation-March of Dimes Grant No. l-352, and CMDNJ-Rutgers Medical School Grants: USPHS-PE15 and GRS-RR 5576. References 1
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