Effects of gender on hepatic HMG-CoA reductase, cholesterol 7
-hydroxylase, and LDL receptor in hereditary analbuminemia
Youngshin Shin,1
Nosratola D. Vaziri,1
Nel Willekes,2
Choong H. Kim,1 and
Jaap A. Joles2
1Division of Nephrology and Hypertension, Departments of Medicine, Physiology and Biophysics, University of California, Irvine, California; and 2Department of Nephrology and Hypertension, University Medical Center, Utrecht, The Netherlands
Submitted 28 January 2005
; accepted in final form 15 July 2005
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ABSTRACT
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Hypoalbuminemia is accompanied by hypercholesterolemia in both nephrotic syndrome and hereditary analbuminemia. Hypercholesterolemia is more severe in the female than in the male Nagase analbuminemic rats (NAR). The sex difference in plasma cholesterol diminishes after ovariectomy (OVX) and reappears after estrogen replacement in the NAR. The molecular mechanism responsible for the sex difference in severity of hypercholesterolemia in NAR is not known and was investigated here. To this end, hepatic hydroxylmethylglutaryl (HMG)-CoA reductase, cholesterol 7
-hydroxylase, and LDL receptor were determined in male, female, and OVX female NAR and Sprague-Dawley (SD) rats. Plasma cholesterol, triglycerides, and hepatic HMG-CoA reductase activities were greater in both female and male NAR than in SD rats. This was coupled with upregulation of cholesterol 7
-hydroxylase in both male and female NAR compared with SD controls. LDL receptor in male NAR was similar to that in male SD rats but was significantly reduced in female NAR. OVX partially, but significantly, reduced plasma cholesterol and triglyceride levels in female NAR. This was coupled with a significant rise in hepatic cholesterol 7
-hydroxylase and a modest increase in hepatic LDL receptor. In contrast, OVX resulted in a mild elevation of plasma cholesterol and no significant changes in total hepatic HMG-CoA reductase, cholesterol 7
-hydroxylase, or LDL receptor in female SD rats. Thus the greater severity of hypercholesterolemia in the female NAR appears to be due, in part, to a combination of the constrained compensatory upregulation of cholesterol 7
-hydroxylase and LDL receptor deficiency.
hyperlipidemia; hypercholesterolemia; nephrotic syndrome; proteinuria; low-density lipoprotein; hydroxymethylglutaryl-CoA reductase; bile acids
HEAVY GLOMERULAR PROTEINURIA (nephrotic syndrome) results in hypoalbuminemia, diminished plasma oncotic pressure, and a marked rise in plasma total cholesterol concentration (14). Hypercholesterolemia, albeit less severe, is also present the Nagase rats (2, 3, 11, 13, 14) and humans (1) with hereditary analbuminemia in whom proteinuria is absent. These observations suggest that hypoalbuminemia, per se, can raise plasma cholesterol concentration. Hypercholesterolemia is more severe in the female Nagase analbuminemic rat (NAR) than in the male NAR (11, 14). The difference in plasma cholesterol concentration between the male and female NAR diminishes after ovariectomy (OVX) and reappears with estrogen replacement (710). Similarly, estrogen administration aggravates hypercholesterolemia in rats with adriamycin-induced nephrotic syndrome, wherein hypoalbuminemia is due to proteinuria (4). In contrast, orchiectomy does not change plasma cholesterol in the male NAR (8). These observations suggest that the effect of hypoalbuminemia on plasma cholesterol concentration is amplified by an ovarian factor, most likely estrogen.
In an earlier study (6), we found a significant increase in extrahepatic cholesterol synthesis in NAR. In a subsequent study (13), we observed a marked increase in the abundance of immunodetectable 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (the rate-limiting enzyme for cholesterol biosynthesis) in the liver of male NAR compared with the male Sprague-Dawley (SD) control rats. This was accompanied by a significant increase in the abundance of cholesterol 7
-hydroxylase, the rate-limiting enzyme in cholesterol conversion to bile acids (13). The functional relevance of the latter observation was validated by the results of a previous study (21), which demonstrated a significant increase in fecal excretion of bile acids in NAR. However, we (13) found no significant difference in LDL receptor protein abundance between the male NAR and SD control rats. Taken together, these observations suggest that hypercholesterolemia in NAR is due primarily to increased cholesterol biosynthetic capacity that is mitigated, in part, by increased cholesterol catabolism. In contrast, increased cholesterol production capacity in nephrotic syndrome is compounded by acquired LDL receptor deficiency and a lack of compensatory rise in hepatic cholesterol catabolic capacity (cholesterol 7
-hydroxylase) (12, 1620). These compounding factors account for the greater severity of hypercholesterolemia in nephrotic rats than in male rats with hereditary analbuminemia. Although our previous studies (13) provided the molecular basis of hypercholesterolemia in the male NAR, comparative studies aimed at uncovering the mechanism(s) responsible for differences in lipid levels between the male and female NAR are lacking. The present study was designed to address this issue.
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METHODS
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Animals.
Sixteen female and eight male analbuminemic (NAR) and 16 female and 8 male Sprague-Dawley (SD) rats were used in the study. The SD rats were purchased from Harlan-Olac (Blackthorn, UK). The analbuminemic rats were obtained from our own pathogen-free colony (which was founded with animals generously donated by Dr. S. Nagase, Tokyo, Japan). The female NAR and SD rats were randomly assigned to ovariectomized (OVX, n = 8) and sham-operated (n = 8) groups. The surgical procedures were carried out under general anesthesia and aseptic conditions. Rats were observed for 78 wk after surgery.
Animals were housed behind protective barriers to prevent infection were and fed regular chow (Special Diets Services, Witham, Essex, UK) containing 77% cereal products, 20% protein (soybean and fish meal), and 3% supplements (vitamins, minerals, and amino acids). Sentinel animals were monitored regularly for possible infection by nematodes and pathogenic bacteria, as well as antibodies to a large number of rodent viral pathogens (ICLAS, Nijmegen, The Netherlands). They remained consistently negative throughout the course of the experiment. The animals were provided with water and food ad libitum. The Utrecht University Board for study of experimental animals approved the protocol.
Tissue processing.
At the end of the observation period, the rats were exsanguinated in the nonfasting state under general anesthesia (pentobarbital sodium 60 mg/kg ip). The abdomen was opened, blood was collected (via abdominal aorta puncture), and the liver was removed, blotted dry, weighed, snap-frozen in liquid nitrogen, and stored at 80°C until analyzed. Lysosome-free microsomes were isolated as previously described (19).
Lipid and protein assays.
Plasma cholesterol and triglycerides and total and microsomal liver cholesterol were determined enzymatically (Boehringer, Mannheim, Germany). Total and microsomal liver protein was determined by the Bradford method using an albumin/globulin standard (Sigma, St. Louis, MO).
Lipoprotein isolation by density-gradient ultracentrifugation.
Plasma lipoproteins were separated by density-gradient ultracentrifugation. The subdivision of LDL into LDL1 and LDL2 was performed in order to separate the apolipoprotein (apo)B-containing lipoproteins (LDL1) from the other particles present in the density range of 1.0191.063 g/ml (711). Lipoprotein cholesterol was measured as described above.
Measurements of HMG-CoA reductase activity and protein.
Liver microsomal HMG-CoA reductase enzymatic activity in each rat was determined in a manner identical to that described in our earlier study (19). Hepatic HMG-CoA reductase protein abundance in each rat was quantified by Western analysis, as described in our previous study (13), using an antibody generously supplied by Prof. G. C. Ness (Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, FL).
Measurement of cholesterol 7
-hydroxylase and LDL receptor protein.
Immunodetectable hepatic cholesterol 7
-hydroxylase in each rat was quantified by Western blot, as described in our earlier study (12), using an antibody generously supplied by Prof. John Y. L. Chiang. Hepatic LDL receptor protein abundance in each rat was quantified by Western blot with a mouse antibovine LDL receptor antibody, as previously described (18). In each case, protein abundance was expressed relative to the abundance of
-actin to correct for potential differences in protein loading. The
-actin antibody used in the study was purchased from Chemicon International (Temecula, CA).
Data presentation and statistical analysis.
Two-way analysis of variance and the Student-Neuman-Keuls test for multiple comparisons were applied as appropriate. P values
0.05 were considered significant. To gain insight into the possible effects of the liver and body size, the enzyme values were given in both an absolute and a normalized (multiplied by liver weight and divided by body weight) manner.
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RESULTS
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Plasma total cholesterol, triglyceride, and lipoprotein cholesterol fractions.
Data are shown in Table 1. As expected, plasma total cholesterol and triglyceride concentrations in the NAR groups were significantly higher than in the SD controls. This was also the case for all lipoprotein cholesterol fractions in the female and male NAR, with the exception of intermediate-density lipoprotein (IDL) cholesterol, which was not significantly elevated in the male NAR. The most striking difference in lipoprotein cholesterol fractions involved LDL1, which was nine times greater in female NAR than in female SD rats. Plasma total cholesterol and all lipoprotein cholesterol fractions, as well as triglyceride concentrations, in the female NAR were significantly greater than in the male NAR. In contrast, plasma total cholesterol, all lipoprotein cholesterol fractions, and triglyceride concentration were similar among the male and female SD rats. In confirmation of the earlier studies (7, 9, 10), plasma total, VLDL, IDL, and HDL cholesterol, as well as triglyceride concentrations, were significantly lowered by OVX in the female NAR. It is of interest that the cholesterol-lowering effect of OVX in NAR did not involve LDL1 and LDL2 fractions. The opposite effect was noted in female SD rats, which showed a mild but significant rise in plasma total and LDL2 cholesterol concentrations after OVX.
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Table 1. Plasma concentrations of total cholesterol, triglycerides, and cholesterol contents in various lipoprotein fractions
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Liver weight and chemical composition.
Data are summarized in Table 2. Body weight was generally higher in the male than in the female rats and greater in the male (but not female) SD rats than in the NAR. OVX resulted in a moderate rise in body weight. Liver weight (corrected for body weight) was characteristically increased in the NAR (strain effect P < 0.001; Table 2). Hepatomegaly was most conspicuous in the female NAR. OVX led to a significant increase in liver mass in the SD rats, but a significant decline in liver mass in the NAR. The changes in liver mass paralleled those of plasma cholesterol among the female NAR and SD rats. There were no differences in liver protein concentration (mg protein/g liver) and only a slight, albeit significant, difference in liver cholesterol concentrations. Microsomal protein and cholesterol concentrations (per g liver) were high in male NAR, but low in female NAR.
HMG-CoA reductase data.
Data are shown in Figs. 1 and 2 and in Table 3. Total liver HMG-CoA reductase activity normalized for body size was significantly greater in all of the NAR groups than in the corresponding SD groups. There were no significant effects of gender or OVX in either NAR or SD rats. HMG-CoA reductase protein abundance (HMG-CoA reductase-to-
-actin protein ratio) was similar among the male and female NAR and significantly greater in both than in male and female SD rats. OVX did not significantly change HMG-CoA reductase protein abundance in female NAR but did insignificantly raise it in female SD rats. Similarly, when normalized for liver and body size, total HMG-CoA reductase protein abundance was higher in all NAR groups than in SD rats.

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Fig. 1. Top: total hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (R) activity, representative Western blots for 2 rats/group. Bottom: group data depicting hepatic HMG-CoA reductase protein abundance in male and female Nagase analbuminemic rats (NAR) and Sprague-Dawley (SD) rats. #P < 0.01 vs. SD of same sex.
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Fig. 2. Top: total hepatic HMG-CoA reductase activity, representative Western blots for 2 rats/group. Bottom: group data depicting hepatic HMG-CoA reductase protein abundance in sham-operated and ovariectomized (OVX) female NAR and SD rats. #P < 0.01 vs. SD of same sex.
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Table 3. Protein abundance of HMG-CoA reductase, cholesterol 7 -hydroxylase and LDL receptor in the liver, normalized for liver size and body weight
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Cholesterol 7
-hydroxylase data.
Data are shown in Figs. 3 and 4 and in Table 3. Cholesterol 7
-hydroxylase protein abundance (cholesterol 7
-hydroxylase protein-to-
-actin protein ratio) was significantly higher in the livers of male and female NAR than in the corresponding SD groups. Although the measurements were similar among the male and female NAR, they were higher in the male than in the female SD groups. OVX resulted in a marked rise in hepatic cholesterol 7
-hydroxylase protein abundance in female NAR and a modest rise in the female SD rats. When normalized for liver size and body weight, cholesterol 7
-hydroxylase protein abundance was higher in all NAR groups than in the SD groups but lower in intact female NAR than in OVX female NAR. Although the value found in the OVX SD group was higher than that in the intact female SD rats, the difference did not reach statistical significance. Interestingly,
-actin abundance seemed to decline with OVX in the NAR animals. Although not clear, this phenomenon might be linked to the reversal of hepatomegaly in this model.

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Fig. 4. Representative Western blots for 2 rats/group and group data depicting hepatic CH 7 H in sham-operated and OVX female NAR and SD rats. *P < 0.01 vs. female of same strain; #P < 0.01 vs. SD of same sex.
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LDL receptor data.
Data are shown in Figs. 5 and 6 and in Table 3. LDL receptor abundance (LDL receptor-to-
-actin protein ratio) was reduced in female NAR, compared with both male NAR and female SD rats. OVX partially restored hepatic LDL receptor protein abundance in the female NAR. Hepatic LDL receptor protein abundance in the male NAR was not different from that found in the male SD rats. It is of note that after an adjustment for liver and body weight, there were no significant differences in hepatic LDL receptor protein abundance among the study groups.

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Fig. 5. Representative Western blots for 2 rats/group and group data depicting hepatic LDL receptor (LDL-r) in male and female NAR and SD groups. *P < 0.05 vs. male of same strain; #P < 0.01 vs. SD of same sex.
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Fig. 6. Representative Western blots for 2 rats/group and group data depicting hepatic LDL-r in sham-operated and OVX female NAR and SD rats. #P < 0.01 vs. SD of same sex.
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DISCUSSION
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Hypercholesterolemia in NAR was accompanied by a significant increase in hepatic HMG-CoA reductase protein abundance and enzyme activity. These findings point to increased hepatic cholesterol biosynthetic capacity in NAR. Increased hepatic cholesterol generating capacity, shown here in all NAR, and increased extrahepatic cholesterol production reported previously (6) work in concert to raise plasma cholesterol concentration. In addition, the LDL receptor abundance was reduced in female NAR. OVX resulted in a fall in plasma cholesterol levels in the female NAR to a value that was intermediate between female and male NAR. The reduction in plasma cholesterol concentration by OVX in the female NAR was accompanied by an increase in hepatic cholesterol 7
-hydroxylase abundance and a mild rise in LDL receptor abun-dance. These observations, in part, account for the effect of gender on and the role of ovaries in regulation of plasma cholesterol concentration in NAR, shown here and reported in previous studies (7, 911, 14).
The primary pathway of cholesterol catabolism is its conversion to bile acids for secretion in the bile and eventual excretion in the feces. The rate-limiting enzyme for cholesterol conversion to bile acids is cholesterol 7
-hydroxylase, which plays an important part in cholesterol homeostasis and regulation of plasma cholesterol concentration. Increased cholesterol production capacity in NAR groups was coupled with a compensatory rise in hepatic cholesterol 7
-hydroxylase protein abundance. This observation confirms the results of our earlier studies (13) of the male NAR and provides the molecular basis for increased fecal bile acid excretion in this model (21). Together, these findings exclude a reduction in cholesterol catabolism as a potential mechanism of hypercholesterolemia in NAR. In contrast to hereditary analbuminemia, increased cholesterol production capacity in nephrotic syndrome is accompanied by no rise in hepatic cholesterol 7
-hydroxylase (12, 20). This can, in part, account for the greater elevation of plasma cholesterol in rats with nephrotic syndrome compared with that seen in the male NAR.
Hypercholesterolemia and elevated plasma LDL cholesterol in nephrotic syndrome are associated with and, in part, are due to acquired hepatic LDL receptor deficiency (18, 20). However, in confirmation of our earlier studies (13), hepatic LDL receptor protein abundance was normal in the male NAR employed in the present study. In contrast to the male NAR, hepatic LDL receptor abundance in the female NAR was significantly reduced and partially restored by OVX. The presence of LDL receptor deficiency in female but not male NAR accounts for the greater severity of hypercholesterolemia and elevation of plasma LDL1 levels in the female than in the male NAR. Partial restoration of LDL receptor abundance by OVX points to the contribution of ovaries to the gender differences in expression of this receptor in the NAR. It should be noted that the gender difference in hepatic LDL receptor abundance in the NAR cannot be explained by differences in hepatocellular cholesterol concentration. This is because intracellular cholesterol in female NAR was similar to, and microsomal cholesterol was actually lower than, that found in the male NAR.
The greater VLDL and IDL cholesterol levels in female NAR compared with male and OVX NAR are most likely related to the previously reported higher secretion rate and lower clearance of the triglyceride-rich lipoproteins in female than in male and OVX NAR (5, 15). The lower clearance of VLDL and chylomicrons in the female than male NAR was due primarily to the lower activities of lipoprotein lipase and hepatic lipase that were restored by OVX in the female NAR (5, 15). Together, greater production and lower clearance of the apoB-containing lipoproteins in the female than in the male NAR play an important part in gender differences in plasma cholesterol and triglyceride levels in these animals. Moreover, the reversal of these abnormalities by OVX points to the contribution of ovaries to these processes. The partial reduction of HDL cholesterol levels after OVX in female NAR to levels observed in male NAR suggests differential effects of gender on HDL metabolism in NAR. However, the mechanism(s) responsible for this gender related effect is unknown and awaits further investigation. Interestingly, the lower plasma cholesterol concentration after OVX was coupled with a significant increase in cholesterol 7
-hydroxylase abundance. It thus appears that the reduction in severity of hypercholesterolemia by OVX in female NAR might be due primarily to a rise in cholesterol catabolism rather than reduced cholesterol production.
In conclusion, the results suggest that an increase in hepatic cholesterol biosynthetic capacity, coupled with LDL receptor deficiency and insufficient hepatic cholesterol catabolism, contribute to the greater severity of hypercholesterolemia in female than in male NAR. These effects are compounded by previously reported greater secretion and lower clearance of apoB-containing lipoproteins in female than in male NAR (5, 15). The latter abnormalities are reversed by OVX.
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FOOTNOTES
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Address for reprint requests and other correspondence: N. D. Vaziri, UCI Medical Center, Division of Nephrology and Hypertension, Bldg. 53, Rm. 125, 101 The City Drive, Rt. 81, Orange, CA 92868 (e-mail: ndvaziri{at}uci.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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