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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 28, 2001 as doi:10.1096/fj.01-0653fje. |
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Department of Human Biology, Maastricht University, 6200 MD, Maastricht, The Netherlands
2Correspondence: Department of Human Biology, Maastricht University, PO Box 616, 6200 MD, Maastricht, The Netherlands. J.Plat@HB.UNIMAAS.NL
SPECIFIC AIMS
The decrease in cholesterol absorption caused by plant stanol esters leads to a compensatory increased endogenous cholesterol synthesis. The consequences of these metabolic changes at the molecular level are unknown, and so we analyzed in non-hypercholesterolemic subjects the effects of plant stanol ester consumption on changes in low density lipoprotein (LDL) receptor cell surface protein expression as well as mRNA levels of the LDL receptor and HMG-CoA reductase in mononuclear blood cells.
PRINCIPAL FINDINGS
1. Reduced serum LDL cholesterol concentrations
Consumption of the plant stanol esters for 8 wk decreased serum LDL cholesterol concentrations by 0.45 ± 0.33 mmol/l (P<0.001 vs. the control group; 95% confidence interval (CI) for the difference in changes between the two groups, -0.36 to -0.78 mmol/l). No effects on HDL cholesterol and triacylglycerol concentrations were found.
2. Reduced serum plant sterol and increased serum lathosterol concentrations
Compared with the control group, cholesterol-standardized serum campesterol concentrations were significantly lowered after plant stanol ester consumption (P<0.001; 95% CI for the difference in changes, -62.7 to -116.3 [102xµmol/mmol cholesterol]), suggesting a reduced cholesterol absorption. The cholesterol-standardized lathosterol concentration was significantly higher in the experimental than the control group (P=0.007; 95% CI for the difference in changes, 5.7 to 33.6 [102x µmol/mmol cholesterol]), indicating increased endogenous cholesterol synthesis.
3. Increased LDL receptor mRNA but unchanged HMGCoA reductase mRNA expression
At the end of the run-in period, LDL receptor mRNA concentrations in mononuclear blood cells correlated positively with those of HMG-CoA reductase mRNA (r=0.619; P<0.001), illustrating the coordinated expression of both genes. Plant stanol ester consumption increased LDL receptor mRNA concentrations by 55.1 ± 57.3% (or 17.9±13.5 mRNA copies/µg total RNA). This change was significantly different from the change of 11.9 ± 30.3% (or 4.3±13.3 mRNA copies/µg total RNA) in the control group (P=0.003; 95% CI for the difference in changes, 5.0 to 22.3 mRNA copies/µg total RNA). Moreover, there was no difference in changes of LDL receptor expression between the plant stanol ester mixtures (P=0.999; 95% CI for the difference in changes, -10.5 to 10.5 mRNA copies/µg total RNA). The change in LDL receptor mRNA concentrations correlated negatively with the change in serum LDL cholesterol concentrations (r=-0.361; P=0.015). Thus, the largest LDL cholesterol reductions were seen in those subjects with the largest increases in LDL receptor mRNA concentration.
HMG-CoA reductase mRNA concentrations increased by 66.4 ± 68.1% (or 183.9±182.4 mRNA copies/µg total RNA) in the plant stanol ester group, but did not reach statistical significance compared with the change of 32.5 ± 28.7% (or 105.1±90.8 mRNA copies/µg total RNA) in the control group (P=0.124; 95% CI for the difference in changes, -22.4 to 180.2 mRNA copies/µg total RNA). Effects on HMG-CoA reductase mRNA concentrations were similar during consumption of both plant stanol ester mixtures (P=0.619; 95% CI for the difference, -106.5 to 175.5 mRNA copies/µg total RNA).
Correlation coefficients were calculated to examine whether changes in LDL receptor or HMG-CoA reductase mRNA concentrations were related to changes in markers for cholesterol synthesis and intestinal absorption. As shown in Table 1
, there was a positive trend between changes in LDL receptor mRNA concentrations with changes in cholesterol-standardized lathosterol concentrations (r=0.292; P=0.054) and a negative trend with changes in cholesterol-standardized campesterol concentrations (r=-0.285; P=0.061). These findings may indicate that subjects with the largest reduction in campesterol concentrations had the largest increase in cholesterol synthesis and LDL receptor mRNA concentrations. There was no statistically significant relation between changes in HMG-CoA reductase mRNA with changes in serum LDL cholesterol concentrations (r=-0.095; P=0.541), in LDL receptor mRNA concentrations (r=0.087; P=0.576), in serum cholesterol-standardized lathosterol (r=0.018; P=0.908), or in campesterol concentrations (r=-0.042; P=0.789).
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4. Higher LDL receptor cell surface expression correlates with LDL cholesterol reduction
To examine whether changes in LDL receptor mRNA concentrations translated into an increased number of LDL receptor molecules, we determined the changes in LDL receptor cell surface expression on mononuclear blood cells. Compared with changes in LDL receptor protein expression in the control group, consumption of plant stanol esters significantly increased LDL receptor protein levels in monocytes and T lymphocytes by 37% (P=0.003) and 25% (P=0.013), respectively, whereas the 13% higher expression in B lymphocytes did not reach statistical significance (P=0.419). The change in cell surface LDL receptor protein expression in monocytes correlated with the change in LDL cholesterol (r=-0.440; P<0.001), illustrating the functionality of the higher LDL receptor protein expression with respect to LDL cholesterol reductions (Fig. 1
). This correlation was also significant for T lymphocytes (r=-0.307; P=0.018), whereas for B lymphocytes no significant relation was found (r=-0.018; P=0.898).
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CONCLUSIONS
The LDL cholesterol-lowering effect of plant stanol esters has been shown in many human intervention studies. This is caused by competition between cholesterol and plant stanols for incorporation into mixed micelles. The subsequent reduced solubility of cholesterol into the mixed micelles results in decreased intestinal cholesterol absorption. Consequently, less cholesterol enters the circulation, leading to an increase in cholesterol synthesis and a decrease in LDL synthesis. The underlying regulatory mechanisms of these two latter effects are unknown.
We have now found that plant stanol ester consumption increased LDL receptor mRNA concentrations in mononuclear blood cells. LDL receptor protein concentrations on the surface of mononuclear blood cells were also increased, suggesting an increased translation of its mRNA. This increased protein expression was functional, since the observed changes in LDL cholesterol correlated negatively with the changes in cell surface LDL receptor protein (Fig. 1)
. Mononuclear cells are a heterogeneous cell population; when cultured in lipoprotein-deficient serum, monocytes bind and degrade more 125I-LDL than do lymphocytes. Further, monocytes incorporate 
20-fold more [2-14C]mevalonate into sterols than lymphocytes do. These finding suggest that cholesterol metabolism is more active in monocytes than lymphocytes. In agreement with this, we found that at the end of the run-in period, LDL receptor expression on monocytes was twice as high as T lymphocytes and 12-fold higher than B lymphocytes. However, the relative increases in cell surface LDL receptor protein expression after plant stanol ester consumption were similar in monocytes and T lymphocytes. This suggests that even though monocytes showed higher values at baseline, both cell types are equally responsive to diet-induced changes in LDL receptor expression. B lymphocytes, however, were hardly responsive to plant stanol ester-induced changes in LDL receptor expression.
Using radiolabeled LDL, Gylling et al. showed that plant stanol ester consumption decreased LDL production in non-insulin-dependent diabetic men. It was speculated that this effect was due to a lower flux of intestinal cholesterol to the liver, resulting in lower very low density lipoprotein (VLDL) synthesis and subsequently reduced formation of LDL from VLDL and/or IDL. Our results now suggest that an alternative pathway is lowered LDL formation via a higher clearance of IDL from the circulation as explained by the higher expression of the LDL receptor. Gylling and co-workers further found a lowered production of IDL from VLDL. Since evidence for a specific VLDL receptor for the clearance of VLDL cholesterol in humans is scanty, it is debatable whether the decreased IDL production is caused by increased VLDL clearance. A more likely explanation may be a changed pathway for hepatic VLDL secretion. From in vitro studies with primary cells, Twisk et al. concluded that higher LDL receptor expression is related to an increased uptake of newly secreted apoB containing lipoprotein particles (nascent VLDL) and to an increased presecretory degradation of newly synthesized apoB. These pathways support the idea that the higher LDL receptor expression during plant stanol ester consumption indeed leads to a lower hepatic VLDL appearance into the circulation. In humans, however, VLDL production during plant stanol ester consumption has never been measured, and certainly warrants further study.
Plant stanol ester consumption not only up-regulates LDL receptor expression, but also increases whole body cholesterol synthesis, as indicated by elevated serum cholesterol-standardized lathosterol concentrations. Changes in HMG-CoA reductase mRNA concentrations in mononuclear blood cells, however, did not reach statistical significance despite a substantial increase of 34% in the plant stanol ester group. In addition, changes in serum cholesterol-standardized lathosterol concentrations did not correlate with those in HMG-CoA reductase mRNA concentrations. This finding may suggest that HMG-CoA reductase activity is not only regulated at a transcriptional level, but that post-transcriptional steps are also important. The lack of correlation between changes in HMG-CoA reductase and cholesterol-standardized lathosterol concentrations further suggests that changes in human HMG-CoA reductase mRNA do not necessarily reflect protein levels or activity. Indeed, it was found that cholesterol feeding lowered HMG-CoA reductase expression in rats at the level of translation and not at a transcriptional level. Others, however, have reported a clear correlation between HMG-CoA reductase mRNA and cholesterol synthesis after cholesterol feeding in rats. These contradictory findings illustrate that the mechanisms regulating HMG-CoA activity are complex and need further study.
In summary, we have shown that plant stanol ester consumption increases LDL receptor mRNA expression in human monocytes and T lymphocytes. This higher LDL receptor mRNA expression is translated into a greater amount of cell surface LDL receptor protein, which correlates with changes in serum LDL cholesterol. We therefore suggest that the increased LDL receptor expression contributes to a lowered LDL formation along the apoB cascade. The increase in HMG-CoA reductase activity, as shown by increased serum cholesterol-standardized serum lathosterol concentrations, may be regulated only partly at a transcriptional level (Fig. 2
). Although a positive relationship exists for LDL receptor and HMG-CoA reductase mRNA expression in mononuclear blood cells and the liver, it remains to be determined whether our results can be extrapolated to other tissues.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0653fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0653fje 
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) in serum LDL cholesterol and cholesterol-standardized campesterol and lathosterol concentrations with changes in LDL receptor mRNA and protein and in HMG-CoA reductase mRNA concentrations of mononuclear blood cellsa
