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Inhibition of hepatic cholesterol synthesis by the alpha1-adrenoceptor blocker doxazosin in the hypercholesterolemic golden hamster
Life Sciences, Vol. 44, pp. 1013-1017 Printed in the U.S.A.
Pergamon Press
INHIBITION OF HEPATIC CHOLESTEROL SYNTHESIS BY THE ALPHAI-ADRENOCEPTOR BLOCKER DOXAZOSIN IN THE HYPERCHOLESTEROLEMIC GOLDEN HAMSTER.
H. Jansen*,**, R. Lammers**, M.G.A. Baggen**, J.M.A Penders*** and J.C. BirkenhHger**.
Departments of *Biochemistry I and ** Internal Medicine III, Medical Faculty, Erasmus University Rotterdam, ***Medical Department Pfizer Inc, Rotterdam, The Netherlands. (Received in final form February 7, 1989) Summary The effect of treatment with the alphal-speclfic adrenoceptor blocker, Doxazosln, on lipid parameters was studied in male Golden hamsters fed a cholesterol-enrlched diet. Within I week the Doxazosin-treated animals had a lower plasma (-12 %) and hepatic (-30 %) cholesterol content than the cholesterol-fed controls. De novo cholesterol synthesis in the liver was lowered by 39% in the Doxazosin-treated animals. These data indicate that the reported beneficial effect of alphal-blockade on plasma cholesterol levels may be due to lowering of the hepatic cholesterol synthesis.
The use of adrenoceptor blockers in the treatment of hypertension leads to side effects on plasma lipid- and llpoproteln concentrations. Beta-blockers provoke, generally, an undesirable shift in the lipid profile (a higher plasma total cholesterol and triglycerlde level and a lowering in HDL-cholesterol), while opposite effects of the alphal-blocker , Prazosln, have been reported (I-4). The mechanisms involved are only partly known. The inverse changes in plasma trlglyceride and HDL-cholesterol may be due to effects of the adrenoceptor blockers on lipoproteln llpase activity. Alpha-blockers tend to increase and beta-blockers to decrease this enzyme activity (5-7). The basis of the beneficial effect of alpha-blockade on plasma cholesterol is not known. To study a possible involvement of hepatic cholesterol synthesis Golden hamsters with dlet-induced hypercholesterolemla were used. It was found that treatment with the alphal-specific blocker, Doxazosln, leads within one week to a lowering in plasma- and liver cholesterol content, and a decrease in hepatic cholesterol synthesis. Methods and Materials Animals Male Golden hamsters were used (85-100 g). They were housed under controlled conditions (lights on 7.00-19.00 h, temp. 21 oc), with free access to food and water unless noted otherwise. 0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
1014
Cholesterol Synthesis and Alpha-Blockade
Vol. 44, No. 15, 1989
Animal treatment procedures The animals received during 3 weeks a normal lab chow or the same chow supplemented with 0.25 % cholesterol. During the following drug-treatment period of 1 week Doxazosin (I000 mg/kg) was added to the food of the experimental groups. The hamsters consumed about I0 g chow daily, which amount was not affected by the Doxazosin treatment (cholesterol-fed controls 10.5 + 1.3 g/d (n=6), cholesterol plus Doxazosin-fed animals 9.9 + 1.0 (n=6). The a ~ m a l s were fasted overnight before they were killed by decapitation. Biochemical methods Blood was collected in cold tubes and allowed to clot on ice. The liver was weighed and part of it (95-100 mg) immediately incubated for determination of de novo cholesterol synthesis, by following the incorporation of [14C]-acetate into cholesterol, as described before (8). Incorporation of 14C into cholesterolesters was negligible small in comparison to u n e s t e r i f i e d cholesterol (not shown). Another part of the liver was frozen for lipid analysis and stored at -20 °C. Within 1 week the cholesterol content in the livers was determined as follows. About I00 mg of the livers was homogenized in 2 ml 0.9 % NaCI. To 1.6 ml homogenate 6 ml methanol/chloroform (2/I) containing 3H-cholesterol (8000 dpm) and 14C-cholesteryloleate (8000 dpm) were added. After mixing, the samples were left for 30 min and centrifuged. Chloroform (2 ml) and distilled water (2 ml) were added. After centrifugation the lower, chloroform, phase was taken to dryness under a stream of N 2. The residue was dissolved in 50 ul chloroform/methanol (2/1). The lipids were separated on TLC-silica plates with heptane/diethylether/acetic acid (60/40/ I). The spots were identified in Iodine vapor, scraped from the plates and extracted with 1 ml chloroform/methanol (2/i). From the unesterified cholesterol extract I00 ul was in a counting vial dried under a stream of N 2 and counted with Instagel. 200 ul of the extract was, after drying, taken up in 50 ul isopropanol. In this mixture cholesterol was determined enzymatically. The 3H-cholesterol was used for calculation of the recovery of the extracted cholesterol. The extract containing cholesterol esters was dried and saponified during 30 min at 80 °C in a mixture of 0.95 ml ethanol, 0.05 ml 6 M KOH. After cooling, the mixture was neutralized and cholesterol was determined. 14Cradioactivity was determined in part of the neutralized mixture for calculation of the recovery of cholesterol esters. Triglycerides and cholesterol were determined with enzymic methods (Boehrlnger testkit combinations 701904 and 237574, respectively). 0.6 ml of the sera of 6 animals was pooled and subjected to density gradient ultracentrifugatlon to separate lipoprotein fractions at the following densities (9). Very low density lipoproteins (VLDL) d < 1.006, Intermediate density lipoprotelns (IDL) 1.006 < d < 1.019, Low density lipoproteins (LDL) 1.019 < d < 1.063 and High density lipoproteins (HDL) 1.0063 < d < 1.21 (d in g/ml). Statistical methods Differences between groups were evaluated with the student t-test. Results Effect of Doxazosin on lipids- and lipoproteins The cholesterol-rich diet led to more than a doubling (+ 115 %) of the serum cholesterol content, while trlglycerides were enhanced by 32 % (TABLE I). The increase in serum cholesterol was mainly due to the VLDL (+ 235 %) and LDL (+ 113 %) fractions. HDL-cholesterol was enhanced by 46 %. Doxazosin treatment had
Vol. 44, No. 15, 1989
Cholesterol Synthesis and Alpha-Blockade
1015
no s i g n i f i c a n t effects on s e r u m l i p i d e i n t h e a n i m a l s f e d a n o r m a l l a b chow (not shown). In the cholesterol-fed hamsters Doxazosin treatment lowered serum total cholesterol by 18 % (p < 0.025), serum trlglycerldes were nonslgnlflcantly lowered (- 13 %). The lowering In total cholesterol was due to a decrease in the LDL (- 35 %) and VLDL (- 20 %) fractions. HDL-cholesterol was e n h a n c e d by IOZ (TABLE II),
TABLE I Serum C h o l e s t e r o l and Trtglyceride C o n t e n t s i n G o l d e n H a m s t e r s on a N o r m a l o r Cholesterol-enrlched d i e t . Effect of Doxasosln Treatment.
Treatment
Cholesterol (~)
C o n t r o l chow (12) Cholesterol chow (12) Chol. chow plus Doxazosln (12) Means + s.e.m.. * denotes control--group (p < 0.025).
Trlglycerlde (~)
2.95÷0.t0 6.33 ~ 0 , 2 1 5.35 +_.0.32*
a statlst~cally
2.79 + 0.37 3.67 • 0.18 3.18 ~_ 0.29 signlflcant
difference
from the
TABLE I I
Distribution o f C h o l e s t e r o l
over Different
Lipop~otein
Fractions.
Treatment
VLDL (mS)
IDL (mM)
LDL (~)
HDL (mM)
C o n t r o l chow Cholesterol chow Chol. plus Doxazosin
0.43 1.87 I. 49
0.01 0.03 0.03
0.48 1.50 0.97
1.36 1.99 2.18
Means of 2 experiments, each carried out with pooled sera of 6 hamsters. Effect
of Doxazosin on hepatic
cholesterol
content
The cholesterol-enriched diet led not only t o accumulation of cholesterol in the plasma but also to high amounts of unestertfied cholesterol and cholesterol ester in the liver (Table 3). Within 1 week~ Doxazosln treatment lowered the hepatic cholesterol ester content per g tissue by 30 %. The d e c r e a s e In unesterlfled cholesterol was smaller ( - 1 2 %) but also statistically significant (p <0.02).
TABLE I I I Hepatic
Cholesterol
Treatment
Content
of Cholesterol-fed
Doxazosin-treated
Hamsters.
Cholesterol(ester) content (mg/g tissue) Unesterlfled cholesterol Cholesterol ester
Controls (12)
3.2 + 0.4
28.2 + 9.6
Doxazosin (12)
2.8 + 0.3*
19.8 + 2.2 **
Means + s.d.. * denotes a statistically significant cholesterol-fed control group (p < 0.02, ** p < 0.005).
difference
from
the
1016
Cholesterol Synthesis and Alpha-Blockade
Vol. 44, No. 15, 1989
Effect of Doxazosin on hepatic cholesterol synthesls Cholesterol-feeding had no significant effect on the de novo cholesterol synthesis in the liver, either expressed per gram wet tissue weight or per liver (Table 4). Doxazosin treatment led to a, statistically significant, 3 1 % lowering in the de novo cholesterol synthesis per gram of liver in comparison to the cholesterol-fed controls. If expressed per liver the decrease was somewhat larger (39 %) as Doxazosin tended to lower the liver weight.
TABLE IV Effect of Cholesterol-feeding and Doxazosin-treatment on [14C]-Acetate Incorporation in Cholesterol in the Golden Hamster. Treatment
Cholesterol synthesis (dpm/g tissue) (dpm/liver)
Control chow (12) I00.0 + 18.0 Cholesterol chow (22) 83.8 ~ 9.2 Chol. chow plus Doxazosin (21) 57.7 ~ 5.4*
I00.0 + 18.8 114.3 • 14.3 70.0 ~ 7.6*
Means + s.e.m.. The results are expressed as the percentage of the value in control-chow fed animals (I00 % equals 3040 dpm/g tissue and 10620 dpm/llver). * denotes a statistically significant difference from the cholesterol-fed control group (p < 0.02). Other groups were not significantly different from each other.
Discussion Feeding a cholesterol-enriched diet to Golden hamsters leads within 3 weeks to cholesterol accumulation in the plasma and liver. The blood cholesterol is mainly enhanced in the VLDL and LDL fractions. To the development of the hypercholesterolemia several mechanisms may contribute. Spady and Dietschy (I0) demonstrated a suppressed LDL-receptor activity and an enhanced LDL production rate in hamsters fed a cholesterol-enriched diet. We found that 14C-acetate incorporation into cholesterol during incubation of liver tissue in vitro was similar in the control-fed and cholesterol-fed animals. In other species, for example the rat, which are more resistant to high cholesterol diets hepatic cholesterol synthesis is greatly depressed during high cholesterol intake. The lowered endogenous cholesterol synthesls may than compensate, partly, the increased exogenous input of cholesterol; a mechanism that seems not to occur in hamsters. Therefore, we think that another factor that may play in the development of the hypercholesterolemia is the lack of suppression of the hepatic cholesterol synthesis by the cholesterol-rich diet. These results are quite unexpected as inhibition of cholesterol synthesis during cholesterol feeding in hamsters has been described (i0). However, in our experiments the animals were fasted overnight before use, while the inhibition of cholesterol synthesis was observed in fed rats at middark. At this period cholesterol synthesis is maximal. That the discrepancy with others would arise from the by us utilized methodology, for example because of contamination of the cholesterol with diglycerides, seems less likely but cannot be completely excluded. In control experiments it was found that lipid spots running in the vicinity of cholesterol on the thin layer plates were much lower labeled with 14C than the cholesterol spot (less than 10 %). Moreover, with the used methodology we found in hamsters at mlddark similar results as described by Spady and Dietschy (19), a lowering of hepatic cholesterol synthesis by 7 1 % in the cholesterol-fed hamsters. In the light of the presumed contribution of the
Vol. 44, No. 15, 1989
Cholesterol Synthesis and Alpha-Blockade
1017
cholesterol synthesis to the accumulation of cholesterol in the liver and plasma, it seems well possibly that the lowering of the hepatic cholesterol synthesis during treatment with Doxazosln has greatly contributed to the lowering of the serum and hepatic cholesterol content. Whether the cholesterol synthesis is affected directly by the alpha blockade or is secondary to, for example, an increase in LDL-receptors is not clear. Liver cholesterolester stores were much more decreased (30 %) than plasma cholesterol by Doxazosln. Probably, accumulation of liver cholesterol is primarily affected, so that only after depletion of the liver stores larger effects on plasma cholesterol are found on plasma cholesterol levels. From this study it appears that alphal-adrenoceptor blockade may affect de novo cholesterol synthesis, which in the Golden hamster may contribute to the beneficial changes in the lipid profile. Whether the cholesterol lowering effect of alpha-blockers in humans is achieved via the same mechanisms is not known. De novo cholesterol synthesis is difficult to assess in humans, but in the light of the present results it seems important to study this point. In humans not necessarily high cholesterol diets have to be involved, but the alpha adrenerglc effect may than arise from enhanced catecholamlne levels, occurring, for example, during stress conditions.
Acknowledgements The authors are indebted to Pfizer Inc., New York for financial help and the supply of Doxazosln and Miss Cecile Hanson for preparing the manuscript.
References I. P. LEREN, A. HEGELAND, I. HOLME, P.O. FOS8, I. H J E R ~ , I. and P.G. LUND-LARSEN, Lancet ii 4-6 (1980). 2. J. L~WENSTEIN and A.J. NEUSY, Am. J. Med. 76 79-84 (1984). 3. R.H. GRIMM and D.B. HUNNINGHAKE, Am. J. Med. 80 (suppl. 2A) 56-63 (1986). 4. M.H. WEINBERGER, Am. J. Med. 80 (suppl. 2A) 64-70 (1986). 5. L.A. FERRARA, T. MAROTTA, P. RUBBA, B. DE SIMONE, S. SORO and M. MANCINI, Am. J. Med. 80 (suppl. 2A) 104-108 (1986). 6. H.R. HARTER, V.N. MELTZER, C.A. TINDIRA, A.D. NAUWOVICH and A.P. GOLDBERG, Am. J. Med. 80 (suppl. 2A) 82-89 (1986). 7. H. JANSEN, J. LAIRD-MEETER, J.D. BARTH, M.G.A. BAGGEN, B. VAN AGEN, G.A. VAN ES, P.G. HUGENHOLTZ and J.C. BIRKENHXGER, Vth Washington Spring Symp. 1986;abstract 299. 8. H. JANSEN, Biochem. Biophys. Res. Commun. 131 574-578 (1985). 9. T.G. REDGRAVE, D.C.K. ROBERTS, and C.E. WEST, Anal. Biochem. 65 42-49 (1975). 10.D.K. SPADY and J.M. DIETSCHY, J. Clin° Invest. 81 300-309 (1988).
Pergamon Press
INHIBITION OF HEPATIC CHOLESTEROL SYNTHESIS BY THE ALPHAI-ADRENOCEPTOR BLOCKER DOXAZOSIN IN THE HYPERCHOLESTEROLEMIC GOLDEN HAMSTER.
H. Jansen*,**, R. Lammers**, M.G.A. Baggen**, J.M.A Penders*** and J.C. BirkenhHger**.
Departments of *Biochemistry I and ** Internal Medicine III, Medical Faculty, Erasmus University Rotterdam, ***Medical Department Pfizer Inc, Rotterdam, The Netherlands. (Received in final form February 7, 1989) Summary The effect of treatment with the alphal-speclfic adrenoceptor blocker, Doxazosln, on lipid parameters was studied in male Golden hamsters fed a cholesterol-enrlched diet. Within I week the Doxazosin-treated animals had a lower plasma (-12 %) and hepatic (-30 %) cholesterol content than the cholesterol-fed controls. De novo cholesterol synthesis in the liver was lowered by 39% in the Doxazosin-treated animals. These data indicate that the reported beneficial effect of alphal-blockade on plasma cholesterol levels may be due to lowering of the hepatic cholesterol synthesis.
The use of adrenoceptor blockers in the treatment of hypertension leads to side effects on plasma lipid- and llpoproteln concentrations. Beta-blockers provoke, generally, an undesirable shift in the lipid profile (a higher plasma total cholesterol and triglycerlde level and a lowering in HDL-cholesterol), while opposite effects of the alphal-blocker , Prazosln, have been reported (I-4). The mechanisms involved are only partly known. The inverse changes in plasma trlglyceride and HDL-cholesterol may be due to effects of the adrenoceptor blockers on lipoproteln llpase activity. Alpha-blockers tend to increase and beta-blockers to decrease this enzyme activity (5-7). The basis of the beneficial effect of alpha-blockade on plasma cholesterol is not known. To study a possible involvement of hepatic cholesterol synthesis Golden hamsters with dlet-induced hypercholesterolemla were used. It was found that treatment with the alphal-specific blocker, Doxazosln, leads within one week to a lowering in plasma- and liver cholesterol content, and a decrease in hepatic cholesterol synthesis. Methods and Materials Animals Male Golden hamsters were used (85-100 g). They were housed under controlled conditions (lights on 7.00-19.00 h, temp. 21 oc), with free access to food and water unless noted otherwise. 0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
1014
Cholesterol Synthesis and Alpha-Blockade
Vol. 44, No. 15, 1989
Animal treatment procedures The animals received during 3 weeks a normal lab chow or the same chow supplemented with 0.25 % cholesterol. During the following drug-treatment period of 1 week Doxazosin (I000 mg/kg) was added to the food of the experimental groups. The hamsters consumed about I0 g chow daily, which amount was not affected by the Doxazosin treatment (cholesterol-fed controls 10.5 + 1.3 g/d (n=6), cholesterol plus Doxazosin-fed animals 9.9 + 1.0 (n=6). The a ~ m a l s were fasted overnight before they were killed by decapitation. Biochemical methods Blood was collected in cold tubes and allowed to clot on ice. The liver was weighed and part of it (95-100 mg) immediately incubated for determination of de novo cholesterol synthesis, by following the incorporation of [14C]-acetate into cholesterol, as described before (8). Incorporation of 14C into cholesterolesters was negligible small in comparison to u n e s t e r i f i e d cholesterol (not shown). Another part of the liver was frozen for lipid analysis and stored at -20 °C. Within 1 week the cholesterol content in the livers was determined as follows. About I00 mg of the livers was homogenized in 2 ml 0.9 % NaCI. To 1.6 ml homogenate 6 ml methanol/chloroform (2/I) containing 3H-cholesterol (8000 dpm) and 14C-cholesteryloleate (8000 dpm) were added. After mixing, the samples were left for 30 min and centrifuged. Chloroform (2 ml) and distilled water (2 ml) were added. After centrifugation the lower, chloroform, phase was taken to dryness under a stream of N 2. The residue was dissolved in 50 ul chloroform/methanol (2/1). The lipids were separated on TLC-silica plates with heptane/diethylether/acetic acid (60/40/ I). The spots were identified in Iodine vapor, scraped from the plates and extracted with 1 ml chloroform/methanol (2/i). From the unesterified cholesterol extract I00 ul was in a counting vial dried under a stream of N 2 and counted with Instagel. 200 ul of the extract was, after drying, taken up in 50 ul isopropanol. In this mixture cholesterol was determined enzymatically. The 3H-cholesterol was used for calculation of the recovery of the extracted cholesterol. The extract containing cholesterol esters was dried and saponified during 30 min at 80 °C in a mixture of 0.95 ml ethanol, 0.05 ml 6 M KOH. After cooling, the mixture was neutralized and cholesterol was determined. 14Cradioactivity was determined in part of the neutralized mixture for calculation of the recovery of cholesterol esters. Triglycerides and cholesterol were determined with enzymic methods (Boehrlnger testkit combinations 701904 and 237574, respectively). 0.6 ml of the sera of 6 animals was pooled and subjected to density gradient ultracentrifugatlon to separate lipoprotein fractions at the following densities (9). Very low density lipoproteins (VLDL) d < 1.006, Intermediate density lipoprotelns (IDL) 1.006 < d < 1.019, Low density lipoproteins (LDL) 1.019 < d < 1.063 and High density lipoproteins (HDL) 1.0063 < d < 1.21 (d in g/ml). Statistical methods Differences between groups were evaluated with the student t-test. Results Effect of Doxazosin on lipids- and lipoproteins The cholesterol-rich diet led to more than a doubling (+ 115 %) of the serum cholesterol content, while trlglycerides were enhanced by 32 % (TABLE I). The increase in serum cholesterol was mainly due to the VLDL (+ 235 %) and LDL (+ 113 %) fractions. HDL-cholesterol was enhanced by 46 %. Doxazosin treatment had
Vol. 44, No. 15, 1989
Cholesterol Synthesis and Alpha-Blockade
1015
no s i g n i f i c a n t effects on s e r u m l i p i d e i n t h e a n i m a l s f e d a n o r m a l l a b chow (not shown). In the cholesterol-fed hamsters Doxazosin treatment lowered serum total cholesterol by 18 % (p < 0.025), serum trlglycerldes were nonslgnlflcantly lowered (- 13 %). The lowering In total cholesterol was due to a decrease in the LDL (- 35 %) and VLDL (- 20 %) fractions. HDL-cholesterol was e n h a n c e d by IOZ (TABLE II),
TABLE I Serum C h o l e s t e r o l and Trtglyceride C o n t e n t s i n G o l d e n H a m s t e r s on a N o r m a l o r Cholesterol-enrlched d i e t . Effect of Doxasosln Treatment.
Treatment
Cholesterol (~)
C o n t r o l chow (12) Cholesterol chow (12) Chol. chow plus Doxazosln (12) Means + s.e.m.. * denotes control--group (p < 0.025).
Trlglycerlde (~)
2.95÷0.t0 6.33 ~ 0 , 2 1 5.35 +_.0.32*
a statlst~cally
2.79 + 0.37 3.67 • 0.18 3.18 ~_ 0.29 signlflcant
difference
from the
TABLE I I
Distribution o f C h o l e s t e r o l
over Different
Lipop~otein
Fractions.
Treatment
VLDL (mS)
IDL (mM)
LDL (~)
HDL (mM)
C o n t r o l chow Cholesterol chow Chol. plus Doxazosin
0.43 1.87 I. 49
0.01 0.03 0.03
0.48 1.50 0.97
1.36 1.99 2.18
Means of 2 experiments, each carried out with pooled sera of 6 hamsters. Effect
of Doxazosin on hepatic
cholesterol
content
The cholesterol-enriched diet led not only t o accumulation of cholesterol in the plasma but also to high amounts of unestertfied cholesterol and cholesterol ester in the liver (Table 3). Within 1 week~ Doxazosln treatment lowered the hepatic cholesterol ester content per g tissue by 30 %. The d e c r e a s e In unesterlfled cholesterol was smaller ( - 1 2 %) but also statistically significant (p <0.02).
TABLE I I I Hepatic
Cholesterol
Treatment
Content
of Cholesterol-fed
Doxazosin-treated
Hamsters.
Cholesterol(ester) content (mg/g tissue) Unesterlfled cholesterol Cholesterol ester
Controls (12)
3.2 + 0.4
28.2 + 9.6
Doxazosin (12)
2.8 + 0.3*
19.8 + 2.2 **
Means + s.d.. * denotes a statistically significant cholesterol-fed control group (p < 0.02, ** p < 0.005).
difference
from
the
1016
Cholesterol Synthesis and Alpha-Blockade
Vol. 44, No. 15, 1989
Effect of Doxazosin on hepatic cholesterol synthesls Cholesterol-feeding had no significant effect on the de novo cholesterol synthesis in the liver, either expressed per gram wet tissue weight or per liver (Table 4). Doxazosin treatment led to a, statistically significant, 3 1 % lowering in the de novo cholesterol synthesis per gram of liver in comparison to the cholesterol-fed controls. If expressed per liver the decrease was somewhat larger (39 %) as Doxazosin tended to lower the liver weight.
TABLE IV Effect of Cholesterol-feeding and Doxazosin-treatment on [14C]-Acetate Incorporation in Cholesterol in the Golden Hamster. Treatment
Cholesterol synthesis (dpm/g tissue) (dpm/liver)
Control chow (12) I00.0 + 18.0 Cholesterol chow (22) 83.8 ~ 9.2 Chol. chow plus Doxazosin (21) 57.7 ~ 5.4*
I00.0 + 18.8 114.3 • 14.3 70.0 ~ 7.6*
Means + s.e.m.. The results are expressed as the percentage of the value in control-chow fed animals (I00 % equals 3040 dpm/g tissue and 10620 dpm/llver). * denotes a statistically significant difference from the cholesterol-fed control group (p < 0.02). Other groups were not significantly different from each other.
Discussion Feeding a cholesterol-enriched diet to Golden hamsters leads within 3 weeks to cholesterol accumulation in the plasma and liver. The blood cholesterol is mainly enhanced in the VLDL and LDL fractions. To the development of the hypercholesterolemia several mechanisms may contribute. Spady and Dietschy (I0) demonstrated a suppressed LDL-receptor activity and an enhanced LDL production rate in hamsters fed a cholesterol-enriched diet. We found that 14C-acetate incorporation into cholesterol during incubation of liver tissue in vitro was similar in the control-fed and cholesterol-fed animals. In other species, for example the rat, which are more resistant to high cholesterol diets hepatic cholesterol synthesis is greatly depressed during high cholesterol intake. The lowered endogenous cholesterol synthesls may than compensate, partly, the increased exogenous input of cholesterol; a mechanism that seems not to occur in hamsters. Therefore, we think that another factor that may play in the development of the hypercholesterolemia is the lack of suppression of the hepatic cholesterol synthesis by the cholesterol-rich diet. These results are quite unexpected as inhibition of cholesterol synthesis during cholesterol feeding in hamsters has been described (i0). However, in our experiments the animals were fasted overnight before use, while the inhibition of cholesterol synthesis was observed in fed rats at middark. At this period cholesterol synthesis is maximal. That the discrepancy with others would arise from the by us utilized methodology, for example because of contamination of the cholesterol with diglycerides, seems less likely but cannot be completely excluded. In control experiments it was found that lipid spots running in the vicinity of cholesterol on the thin layer plates were much lower labeled with 14C than the cholesterol spot (less than 10 %). Moreover, with the used methodology we found in hamsters at mlddark similar results as described by Spady and Dietschy (19), a lowering of hepatic cholesterol synthesis by 7 1 % in the cholesterol-fed hamsters. In the light of the presumed contribution of the
Vol. 44, No. 15, 1989
Cholesterol Synthesis and Alpha-Blockade
1017
cholesterol synthesis to the accumulation of cholesterol in the liver and plasma, it seems well possibly that the lowering of the hepatic cholesterol synthesis during treatment with Doxazosln has greatly contributed to the lowering of the serum and hepatic cholesterol content. Whether the cholesterol synthesis is affected directly by the alpha blockade or is secondary to, for example, an increase in LDL-receptors is not clear. Liver cholesterolester stores were much more decreased (30 %) than plasma cholesterol by Doxazosln. Probably, accumulation of liver cholesterol is primarily affected, so that only after depletion of the liver stores larger effects on plasma cholesterol are found on plasma cholesterol levels. From this study it appears that alphal-adrenoceptor blockade may affect de novo cholesterol synthesis, which in the Golden hamster may contribute to the beneficial changes in the lipid profile. Whether the cholesterol lowering effect of alpha-blockers in humans is achieved via the same mechanisms is not known. De novo cholesterol synthesis is difficult to assess in humans, but in the light of the present results it seems important to study this point. In humans not necessarily high cholesterol diets have to be involved, but the alpha adrenerglc effect may than arise from enhanced catecholamlne levels, occurring, for example, during stress conditions.
Acknowledgements The authors are indebted to Pfizer Inc., New York for financial help and the supply of Doxazosln and Miss Cecile Hanson for preparing the manuscript.
References I. P. LEREN, A. HEGELAND, I. HOLME, P.O. FOS8, I. H J E R ~ , I. and P.G. LUND-LARSEN, Lancet ii 4-6 (1980). 2. J. L~WENSTEIN and A.J. NEUSY, Am. J. Med. 76 79-84 (1984). 3. R.H. GRIMM and D.B. HUNNINGHAKE, Am. J. Med. 80 (suppl. 2A) 56-63 (1986). 4. M.H. WEINBERGER, Am. J. Med. 80 (suppl. 2A) 64-70 (1986). 5. L.A. FERRARA, T. MAROTTA, P. RUBBA, B. DE SIMONE, S. SORO and M. MANCINI, Am. J. Med. 80 (suppl. 2A) 104-108 (1986). 6. H.R. HARTER, V.N. MELTZER, C.A. TINDIRA, A.D. NAUWOVICH and A.P. GOLDBERG, Am. J. Med. 80 (suppl. 2A) 82-89 (1986). 7. H. JANSEN, J. LAIRD-MEETER, J.D. BARTH, M.G.A. BAGGEN, B. VAN AGEN, G.A. VAN ES, P.G. HUGENHOLTZ and J.C. BIRKENHXGER, Vth Washington Spring Symp. 1986;abstract 299. 8. H. JANSEN, Biochem. Biophys. Res. Commun. 131 574-578 (1985). 9. T.G. REDGRAVE, D.C.K. ROBERTS, and C.E. WEST, Anal. Biochem. 65 42-49 (1975). 10.D.K. SPADY and J.M. DIETSCHY, J. Clin° Invest. 81 300-309 (1988).