104 Proc. roy. Soc. Med. Volume 69 1976 Supplement 2

Properties of Sterol Biosynthesis in Human Leukocytes: Effects of Gemfibrozil Dr D J Betteridge, Dr M J P Higgins and Dr D J Galton (Lipid Research Laboratory, St Bartholomew's Hospital, London, UK)

Errors in metabolic regulation are due to defects in the regulatory properties of enzymes, receptors or carrier proteins (Galton et al. 1975). If there is loss of negative feedback inhibition of an enzyme it can give rise to enhanced activity of metabolic pathways and lead to accumulation of terminal metabolites. Regulatory defects differ from the classical inborn errors of metabolism, where there is a defect in the catalytic activity of enzymes with a consequent accumulation of intermediary metabolites that are not normally found at high concentrations in the body. Therapy directed at catalytic defects is difficult and involves attempts to replace mutant enzymes. However, therapy of regulatory defects of enzymes should, in principle, be simpler, especially when there is loss of negative feedback inhibition of- the enzyme. In such cases the use of enzyme inhibitors may restore the activity of the unregulated enzyme (and therefore the metabolic pathway) towards normal.

Dr D J Galton

it has recently been demonstrated that familial hypercholesterolmmia is associated with a defect in the regulation of an important ratedetermining enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme-A reductase (E.C.I.I.I.34; HMG-CoA reductase). In cultured fibroblasts from patients homozygous for familial hypercholesterolaemia, pre-incubated in lipid-free serum, there is almost complete lack of suppression of HMG-CoA reductase by added low density lipoprotein (Goldstein & Brown 1973). The failure to suppress HMG-CoA

--Mwww-m.

NUCLEUS

!-I,." i... 11-I'l'-l.l'l'-'l'l,;,-l'l

I

I

of MG CoAJ rLKuctase -

Inducers

;-

,mRNA

I

I

Feedbacko repression

.---- _-Feedback inhibition-- -.1MG - CoA reductase (microsomal)

Cholesterol_----__ Mevalonate

K '14

Steroid

-

-

HMG - CoA

Acetoacetyl CoA

! )~ ~ ~ ~ ~_.0 I

I

Fig 1 A hypothetical scheme for the regulation of HMG-CoA reductase in leukocytes by low-density lipoproteins (LDL)

105

Gemfibro'zil

thesis of enzyme (Higgins & Rudney 1973). Enzyme repression possibly involves a nuclear or cytoplasmic aporepressor (Fig 1) for control of transcription of mRNA and results in a decrease in de novo synthesis of HMG-CoA reductase. In familial hypercholesterolEmia it has been variously postulated that there is: (1) A defective cell receptor for combination with LDL (Brown & Goldstein 1974). (2) A decreased intracellular sterol pool for repression of nuclear transcription (Fogelman et al. 1975). This could be due to a greater rate of egress of intracellular cholesterol or a steroid derivative. (3) A defect in LDL which prevents combination with cell receptors (Higgins etal. 1975). The observable results of all three cases is that the activity of HMG-CoA reductase is not suppressed in cells incubated with LDL.

80 70

/

062

V

60

/

-

2

4

6

8

12

10

Time (hours)

Fig 2 The

effiects offetal calf serum (ECS) on the

activity of HMG-CoA reductase in leukocytes of patients with familial

hypercholesterokeFmia.

Leukocytes were preparedfrom whole blood and after washing were incubated in Minimal Essential Medium (1

ml) with 20% FCS or lipid-free FCSfor periods

up to 12 h. At the end of incubation the activity of

HMG-CoA reductase was measured in leukocytes as described by Brown & Goldstein (1974). Points are means ofduplicate assays in cells heterozygous for

hypercholesterolwrmia incubated in 20 % ECS (-l *); duplicate assays in cells heterozygous forfamilial hypercholesterotaemia familial

lipid-free

incubated in 20 % FCS (A-A); in control cells

ECS (0

incubated in 20% lipid-free

0); and in

control cells incubated with 20% ECS

(A\

- - - -

A\)

reductase by low density lipoprotein (LDL) can also

be demonstrated

heterozygous

for

in leukocytes

familial

of patients

hypercholesterolbemia

(Betteridgeetal. 1975).

Methods Leukocytes were prepared by mixing blood with two parts by volume of 3% gelatin in isotonic saline containing disodium edetate (2 mmol/l). The mixture was allowed to separate at 37°C for 40 min, after which the supernatant was carefully withdrawn using a siliconized wide-bore pipette. After isolation, the leukocytes were suspended in a tissue culture medium (Minimal Essential Medium, Gibco) and incubated for periods up to 12 h in the presence of lipid-free serum. Transfer of leukocytes to fresh incubation medium containing whole serum was used to study enzyme suppression. Leukocytes prepared in this way actively E E

100

90 E M

go E~~~

70

-

Although it is not yet understood whether the regulatory defect of HMG-CoA reductase is of etiological significance for

hypercholesterolxmia,

X50

a possible line of therapy for such disorders is to inhibit the unregulated enzyme. We have therefore

(2,5

examined

xylyloxy)-valeric

prenoid for

effects

the

or

2,2

in

leukocytes

repressors

in

of

on a

iso-

search

HMG-CoA

reductase. A

scheme

for

the

regulation

of HMG-CoA

reductase by LDL in leukocytes is presented in Fig 1. Following the combination of LDL with specific

high-affinity

cell

receptors,

there

are

probably two mechanisms for regulation of HMG-CoA reductase. Feedback inhibition involves a direct, but not allosteric modulation of

the

enzyme,

which

may

be

S 30

dimethyl-5

acid (gemfibrozil)

biosynthesis

inhibitors

of

mediated

by

the

endoplasmic reticulum or microtubular system, and this is followed by changes in de novo syn-

o 10

r

A

2

4

6 8 Time (hours)

10

Fig 3 The effect of low density lipoprotein (LDL) on HMG-CoA reductase in leukocytes from patients with familial hypercholesterolamia. Leukocytes were preparedfrom whole blood and preincubated with lipid-free serum (20 %.) for 12 h. They were then transferred to fresh buffer containingLDL (100 j.g proteinlml) and incubatedfor periods up to 8 h. HMG-CoA reductase was measured at the end of incubation as described by Brown & Goldstein (1974). Points are means ofduplicate assays in cells heterozygousforfamilial hypercholesterolamia (U- *); and assays in control cells (- 0)

106 Proc. roy. Soc. Med. Volume 69 1976 Supplement 2

6s

n=6

Very little increase in enzyme activity is observed if cells are incubated in untreated 20% fetal calf serum (FCS). When leukocytes are now transferred to an incubation medium containing LDL (100 ,ug protein/ml) there is marked suppression of enzyme activity over about 8 h (Fig 3). Patients heterozygous for familial hypercholesterolmmia show incomplete suppression of HMGCoA reductase when their leukocytes are incubated with LDL (Fig 3). The effects of gemfibrozil on the derepression phase of the enzyme in leukocytes is shown in Fig 4. The ethanol blank produces slight inhibition of acetate incorporation into isoprenoids, and this inhibition is more marked in the presence of gemfibrozil at 2 and 3 mM. However at high concentrations of the drug, aggregation of leukocytes occurs, and this may be partly responsible for the reduced incorporation of acetate into isoprenoids.

n=5 n=3

4.

2.

Conclusions Isoprenoid biosynthesis in human leukocytes is regulated by lipoproteins, and this is probably mediated by changes in the activity of a rateDrug concentration determining enzyme HMG-CoA reductase. DeFig 4 The effect ofgemfibrozil on the incorporation (Y%) fects in the regulation of this enzyme can be of14C-acetate into isoprenoids of isolated human observed in leukocytes from patients with familial leukocytes. Leukocytes were preparedfrom whole hypercholesterolkmia. One defect is enhanced blood and incubated in lipid-free serum (20 %.) for 8 h activity of the enzyme when leukocytes are inin the presence ofincreasing concentrations of gemfibrozil (dissolved in ethanol). During the last cubated in a lipid-free medium; another is a 90 min of incubation the rate of "4C-acetate incorporated failure of suppression of HMG-CoA reductase into isoprenoids was measured. Histograms represent when leukocytes are incubated in a lipoproteinmean values as a percent ofactivities in lipid-free rich medium. Agents which can prevent 'derepresmedium with s.e. mean between bars; n=number ofobservations sion' of the enzyme, or alternatively, agents which can enhance suppression of the enzyme, may be of incorporate 14C-acetate or 3H20 into isoprenoid, use in the treatment of familial hypercholesconfirming the results of Fogelman et al. (1975). terolhmia. Use of gemfibrozil has been studied, HMG-CoA reductase activity was also measured and at concentrations of 2 to 3 mM it reduces in leukocytes following the methods previously 14C-acetate incorporation into isoprenoids as well described for fibroblasts (Brown et al. 1973). The as producing leukocyte aggregation. drug was added in 10 ,lI of ethanol to 2 ml of incubation medium. Ethanol was added to REFERENCES control incubations. M J P & Galton D J ethanol blank

1mM

2mM

3mM

Results The effects of incubating leukocytes for periods up to 12 h in the presence of lipid-free fetal calf serum (20% FCS) is shown in Fig 2. Under these conditions the activity of HMG-CoA reductase is 'derepressed' and isoprenoid biosynthesis is enhanced. Leukocytes from patients heterozygous for familial hypercholesterolhmia show a greater degree of 'depression' than control leukocytes, confirming the results of Fogelman et al. (1975).

Betteridge D J, Higgins (1975) British Medical Journaliv, 500

Brown M S, Dana S E & Goldstein J L (1973) Proceedings of the National Academy of Sciences (USA) 70, 2162 Brown M S & Goldstein J L (1974) Proceedings of the National Academy of Sciences (USA) 71, 788 Fogelman A M, Edmond J, Seager J & Popjak G (1975) Journal ofBiological Chemistry 250, 2045 Galton D J, Higgins M J P & Reckless J P D (1975) Lancet i, 1224 Goldstein J L & Brown M S (1973) Proceedings of the National Academy of Sciences (USA) 70, 2804 Higgins M J P & Rodney H (1973) Nature (New Biology) 246,60 Higgins M J P, Lecamwasam D S & Galton D J (1975) Lancet ii, 737

Properties of sterol biosynthesis in human leukocytes: effects of gemfibrozil.

104 Proc. roy. Soc. Med. Volume 69 1976 Supplement 2 Properties of Sterol Biosynthesis in Human Leukocytes: Effects of Gemfibrozil Dr D J Betteridge,...
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