Apparent non-specific effect of bile salts on the terminal reactions of cholesterol biosynthesis by rat liver

SHORT COMMUNICATIONS

4oo BBA 53121

Apparent non-specific effect of bile salts on the terminal cholesterol biosynthesis by rat liver Cholesterol

is biosynthesized

is formed is further

converted

by rat-liver

into bile salts.

reactions of

enzymes I. Much of the cholesterol Thus, bile salts may be considered

that an

end-product of hepatic cholesterol biosynthesis. Recently, FIMOGNARI AND RODWELL (refs. 2, 3) demonstrated that certain bile salts inhibit the conversion of P-hydroxy,%methylglutaryl-CoA

into mevalonic

acid. These workers

suggested

that bile salts

thus serve as end-product inhibitors of this early reaction of cholesterol biosynthesis. In steroid-secreting tissues, steroid hormones are the end products of cholesterol metabolism. End-product steroids inhibit the oxidative demethylation of lanosterol which is one of the terminal reactions of cholesterol biosynthesis that are catalyzed by microsomal enzymes 4. Thus, the effect of bile salts on the lanosterol demethylation reactions of cholesterol biosynthesis was investigated to determine whether or not bile salts inhibit these terminal reactions of cholesterol biosynthesis by liver. Crude homogenate of rat liver was prepared by centrifuging Bucher homogenates of rat liver at IOOOOxg for 20 min (ref. 5). Microsomes were prepared by centrifugation of crude homogenate for I h at 105000~g; the pellet was suspended in 0.1 M potassium phosphate buffer (pH 7.4, 30 mM nicotinamide). The volume of microsomal suspension was adjusted to 25% of the volume of crude homogenate. The rate of oxidative demethylation of [14C]lanosterol (225 to 400 disint./min per mpmole) into [W]cholesterol and other C,,-sterols was determined by the collection

I OO

IO

20

30

First incubation (min) Fig. I. Time course of inactivation by bile salts. Microsomes (160-320 mg protein) and 12 ,umoles NAD were first incubated at 37O alone (O---C?), with 2.5 mM taurodeoxycholate (A---&, or with 0.3 mM glycolithocholate (m---m). After incubation times of o, 5, IO, 15, 20, and 30 min. r-ml samples (20-40 mg protein) of the first incubation mixture were added to 40 m/Lmoles of [‘%]lanosterol, and the mixtures were incubated a second time at 37’ for 5 min. The final volume for the second incubation was 1.2 ml. Fig. 2. Effect of glycolithocholate on the rate of demethylation. Microsomes (160-320 mg protein) and 12 pmoles NAD were first incubated for 12 min at 37’ either alone (o---c) or with 0.5 (A---A) or 0.3 (m--m) mM glycolithocholate. Microsomes (20-40 mg protein) from the first incubation were added to 16, 24, 40, and IOO m,umoles of [‘%Jlanosterol substrate; a second incubation was continued at 37’ for IO min. The final volume for the second incubation was I.5 ml.

Biochim.

Biophys.

Acta,

137 (1967) 400-402

SHORT

401

COMMUNICATIONS

of r4C0, as described previously5. [14C]Lanosterol was prepared from [z-14C]mevalonate5. When conditions of initial reaction rates were investigated (Figs. I and z), microsomes were incubated less than IO min (ref. 5) ; other samples were incubated for 15 or 30 min as described. Solutions of bile salts in 0.1 M potassium phosphate buffer (final pH 7.4) were added to the incubation flasks. Turbid samples were discarded. Incubation of crude homogenate with 1.3 mM deoxycholate, taurodeoxycholate, taurochenodeoxycholate, glycodeoxycholate, glycolithocholate, or glycochenodeoxycholate yielded inhibition of 25% or more (Table I). Many of the other compounds listed in Table I and taurine, biliverdin, and bilirubin were either less active or completely inactive at concentrations of 1.3 mM. Glycolithocholate inhibited the microTABLE APPARENT

I IKHIBITION

OF

LANOSTEROL

--f

CHOLESTEROL

CONVERSION

BY

BILE

SALTS

1.5 pmoles NAD, 40 m,umoles of [i4C]lanosterol and the indicated amounts of bile salt were incubated either with 4 ml of crude homogenate (IOO-200) mg protein) or with I ml of the suspension of microsomes (20-40 mg protein). Final volumes were 4.3 ml for incubation with crude homogenates and 1.3 ml for microsomes. Incubations were for 15 or 30 min for crude homogenates and microsomes, respectively. All incubations were at 37”. Inhibition is expressed as an average of the percentage of the respective control rate. Values in parentheses represent the number of separate determinations,

Inhibitor*

Deoxycholate Cholate Dehydrocholate Chenodeoxycholate Lithocholate Taurine conjugates Taurodeoxycholate Taurochenodeoxycholate Taurocholate Taurolithocholate Taurodehydrocholate Glycine conjugates Glycodeoxycholate Glycolithocholate Glycochenodeoxycholate Glycocholate Glycodehydrocholate

Homogenate “/6I+zhibition at 1.3 mM 25 (3) + 2.0 19** (2)

1.30

k 6.0

-

13 (2) + 8.5 3 !I) 0 (I) ~1~2.0 52 27 14 4 0

5 (6)

-

(2) (2) (3) (2)

* 17.0 F 17.0 i 2.0 i 2.0

1.30 1.30 -

(2)

_c

I.0

88 (r) 6g (2)

&

11.0

0.20

44 (2) * ‘7 (I) 6 (2) t

3.0

1.30

5:::

1;;

-

I.02

5o*** 5o*** 33 (I)

(4) (4)

-

0.0

* Deoxycholic acid (mp. r74-‘76”; [a]nzS 55 & 0.3’, c = 1.0 in ethanol) was purchased from Sigma Chemical Co. Cholic acid (m.p. log-200.5’; [cz]D’~ 38.4 + 0.3’. c = 0.6 in ethanol) was purchased from Eastman Organic Chemicals. Dehydrocholic acid (m.p. 237-239”; [alDzs 24.8 & 0,3’, c = 1.4 inethanol) andlithocholicacid (m.p. 182-186’; [a]nz3 34 + 0.3’, c = r.5 in ethanol) were purchased from Calbiochem. All other bile acids were purchased as the sodium salt from Calbiochem. The compounds were supplied with accompanying data describing elemental analyses, rotations, homogeneous (thinand melting points, and each compound was labeled “chromatographically layer chromatography)“. ** For ease of comparison, the inhibition observed for 1.3 mM concentrations was calculated from results of experiments conducted with various concentrations: cholate, 2 trials (0.28, 1.4 mM); taurodeoxycholate and taurochenodeoxycholate, 4 trials (0.65, 1.3, 2.6, 5.2 mM). *** The concentration required for 50% inhibition was calculated from results of trials conducted with various concentrations: glycodeoxycholate, 6 trials (o.r-0.9 mM) ; glycolithocholate, 4 trials (0.65, 1.3, 2.6, 5.2 mM).

B&him.

Biophys.

Acta, 137 (1967) 400-402

SHORTCOMMUNICATIONS

402

somal demethylation, and a higher concentration of glycodeoxycholate was inhibitory. In both cases, however, the concentrations required for approx. 50% inhibition were 2 to 5 times the effective concentrations reported by FIMOGNARI AND RODWELL~~~. In addition,

these

most

active

compounds

were glycine

conjugates

of bile acids

(Table I). Bile salts normally formed by rat liver are conjugated to taurine rather than to glycine6. Microsomes were incubated first with either 2.5 mM taurodeoxycholate (weak inhibitor) or 0.3 mM glycolithocholate (strong inhibitor) for o to 30 min. Samples were removed, substrate was added, and the rate of demethylation of lanosterol was measured by a second incubation. Some demethylase activity was lost during the first incubation of microsomes in the absence of bile salts (Fig. I). Treatment of microsomes with glycolithocholate during the first incubation rate of inactivation obtained for the control sample. affect the rate. Microsomes

were incubated

yielded approx. 3 times the Taurodeoxycholate did not

with 0.3 and 0.5 mM glycolithocholate

for 12 min

at 37O. Various amounts of lanosterol were added and the mixtures were incubated for an additional IO min. Graphical analysis of the data suggests that the apparent effect respect

of glycolithocholate

is neither

fully

competitive

nor non-competitive

to the concentration of lanosterol (Fig. 2). The results demonstrate clearly that rather high concentrations

with

of the bile salts

must be added to the incubation medium to obtain significant extents of inhibition of lanosterol demethylation by microsomal enzymes. Further evidence suggests that the inhibition is not specific. Removal of the inert soluble protein from microsomes resulted in the loss of inhibitory activity of some of the compounds (Table I). Bile salts normally biosynthesized in rat liver were inactive. In addition, because the enzymatic

activity

decreased

during prolonged

exposure

of the microsomes

to glyco-

lithocholate, the effect may be ascribed to an enhanced rate of irreversible, timedependent denaturation of the microsomal enzymes. Further, the nature of the inhibition was neither clearly competitive nor non-competitive with respect to lanosterol concentration. The effect of these high concentrations of bile salts may be similar to the less specific interaction of bile salts with lipid-rich microsomal membranes described by HESS AND LAGG'. Thus, the feedback inhibition of bile salts on the hepatic formation of mevalonic acid is the only specific end-product action of bile salts on cholesterol biosynthesis This work was supported of Arthritis

and Metabolic

that has been reported. by Grant AM-04505-06 from the National

Diseases

of the U.S. Public

Health

Graduate School of Nutrition and the Section of Biochenzistvy ami Molecular Biology, Cornell Uni?jersity, Ithaca, N.Y. (U.S.A.) I 2 3 4

LV. L. MILLER J. I,. GAYLOR

Ii.HLoCH, .‘kimce, 150 (1965) 19. (;.M. r;IMOGSARI .\ND v. \v. r
5 J. L. G~YLOR, ,J. Bid. Chem., 239 (1964) 756. 6 G. A. D. HASLEWOOD, Physiol. Rev., 35 (1955) 178. 7 E. L. HESS AND S. E. LAGG, Himhem Biophys. Res. Cowwnzcz,

Received July zznd, 1966 Revised manuscript received Biochinz.

Hioph?s.

Acta,

November

1.37 (1967) 4oo-402

zand, 1966

Institute

Service.

IL (1963) 320.

4 (1965)