Zinc status alters growth and oxidative stress responses in rat hepatoma cells.

Zinc deficiency and excess influence cellular homeostasis and are believed to modulate apoptosis. Zinc also regulates cell growth and proliferation. Understanding of the role of zinc in the mechanisms associated with these changes is limited because of its diverse, complex, and cell-specific effects. Therefore, we investigated the oxidative stress responses and the underlying molecular mechanisms associated with the disruption of intracellular zinc homeostasis in H4IIE rat hepatoma cells. We found that zinc excess (100 µM) and DTPA (diethylenetriaminepentaacetic acid; 50-100 µM) induced zinc deficiency both generate reactive oxygen species (ROS) and decrease viability in H4IIE cells. However, cotreatment with the antioxidant, N-acetyl-L-cysteine (NAC) both reduced ROS production and protected cells from death. We additionally observed an increase in Bax mRNA and cytochrome c release from the mitochondria in DTPA-treated cells and an elevated expression of Fas/Fas ligand mRNA with zinc treatment. Both treatments increased p53 and MdM2 protein concentrations along with caspase 3/7 activity. These results suggest that zinc deficiency stimulates mitochondrial-dependent apoptosis whereas zinc activates the extrinsic-apoptotic pathway. Both decreasing and increasing cellular zinc concentrations modulate ROS mediated apoptosis and warrant further research on zinc mediated cancer chemoprevention in this and other cancer cell lines.

Zinc and cancer: implications for LIV-1 in breast cancer.

Zinc is a trace mineral which is vital for the functioning of numerous cellular processes, is critical for growth, and may play an important role in cancer etiology and outcome. The intracellular levels of this mineral are regulated through the coordinated expression of zinc transporters, which modulate both zinc influx as well as efflux. LIV-1 (ZIP6) was first described in 1988 as an estrogen regulated gene with later work suggesting a role for this transporter in cancer growth and metastasis. Despite evidence of its potential utility as a target gene for cancer prognosis and treatment, LIV-1 has received relatively little attention, with only three prior reviews being published on this topic. Herein, the physiological effects of zinc are reviewed in light of this mineral's role in cancer growth with specific attention being given to LIV-1 and the potential importance of this transporter to breast cancer etiology.

The effects of transformation and ZnT-1 silencing on zinc homeostasis in cultured cells.

We have previously demonstrated that reducing the availability of zinc with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) promotes efflux of (65)Zn from rat primary hepatocytes and pituitary cells, but increases retention of label in rat hepatoma (H4IIE) and anterior pituitary tumor (GH3) cell lines. To further understand this differential response between primary cells and the corresponding cancer cell lines, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Rat primary hepatocytes were electroporated with the SV40 large T-antigen-coding plasmid pSV3-neo and selected for neomycin resistance. This resulted in cell division of the normally quiescent hepatocytes. When these cells were prelabeled with (65)Zn, DTPA decreased efflux of (65)Zn, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of dividing cells and be mediated by alterations in the activity of zinc transporter ZnT-1, which is responsible for zinc efflux. To further understand the mechanism of DTPA-induced zinc retention, we down-regulated the expression of ZnT-1 in rat hepatoma cells using vector-based short hairpin RNA interference. Expression of ZnT-1 protein was reduced to approximately 50%. Down-regulation of ZnT-1 resulted in greater retention of (65)Zn in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT-1 is required for the decreased efflux in response to DTPA. We conclude that ZnT-1 expression is crucial for maintaining zinc homeostasis, in particular, for the enhanced retention of zinc in transformed cells when subjected to zinc deprivation.

Rapid homeostatic response of H4IIE cells to diethylenetriaminepentaacetic acid is not due to changes in the amount or localization of ZnT-1 protein.

We have demonstrated that reducing zinc availability with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) causes rapid inhibition of cellular zinc efflux in H4IIE hepatoma cells but increases zinc efflux in primary hepatocytes. Similar differences were also observed between the rat anterior pituitary cell line GH3 and primary anterior pituitary cells. We hypothesized that the difference between the transformed and primary cells is due to differential regulation of ZnT-1 or SLC-30A-1 because this is the only zinc efflux transporter localized to the plasma membrane. The effects of DTPA (50 µM) and zinc (100 µM) treatment on messenger RNA (mRNA) and protein expression and protein localization of ZnT-1 were studied in H4IIE cells and primary hepatocytes. Although zinc tended to increase ZnT-1 mRNA in H4IIE cells, DTPA had no effect on ZnT-1 mRNA and protein expression in either hepatoma cells or hepatocytes. Although ZnT-1 is thought to be localized on the plasma membrane, this localization was not seen in these liver cells where ZnT-1 was distributed throughout the cytoplasm. Vesicular localization of ZnT-1 appeared to increase with zinc treatment. Total zinc content was reduced by DTPA in H4IIE cells. Diethylenetriaminepentaacetic acid also reduced metallothionein 1 mRNA, reflecting this reduction in cellular zinc. We conclude that the rapid homeostatic response of cells to altered zinc availability must be attributed to a transporter other than ZnT-1 or to changes in the activity of ZnT-1 by a novel mechanism.

Acute energy deprivation affects skeletal muscle protein synthesis and associated intracellular signaling proteins in physically active adults.

To date, few studies have characterized the influence of energy deprivation on direct measures of skeletal muscle protein turnover. In this investigation, we characterized the effect of an acute, moderate energy deficit (10 d) on mixed muscle fractional synthetic rate (FSR) and associated intracellular signaling proteins in physically active adults. Eight men and 4 women participated in a 20-d, 2-phase diet intervention study: weight maintenance (WM) and energy deficient (ED; approximately 80% of estimated energy requirements). Dietary protein (1.5 g x kg(-1) x d(-1)) and fat (approximately 30% of total energy) were constant for WM and ED. FSR and intracellular signaling proteins were measured on d 10 of both interventions using a primed, constant infusion of [(2)H(5)]-phenylalanine and Western blotting techniques, respectively. Participants lost approximately 1 kg body weight during ED (P < 0.0001). FSR was reduced approximately 19% (P < 0.05) for ED (0.06 +/- 0.01%/h) compared with WM (0.074 +/- 0.01%/h). Protein kinase B and eukaryotic initiation factor 4E binding protein 1 phosphorylation were lower (P < 0.05) during ED compared with WM. AMP activated protein kinase phosphorylation decreased (P < 0.05) over time regardless of energy status. These findings show that FSR and associated synthetic intracellular signaling proteins are downregulated in response to an acute, moderate energy deficit in physically active adults and provide a basis for future studies assessing the impact of prolonged, and perhaps more severe, energy restriction on skeletal muscle protein turnover.

Zinc retention differs between primary and transformed cells in response to zinc deprivation.

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on (65)Zn uptake and retention. DTPA reduced the uptake of (65)Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with (65)Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of (65)Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of (65)Zn in all cell lines examined. DTPA increased the retention of (65)Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that (65)Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with (14)C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.

Carbohydrate restriction alters hepatic cholesterol metabolism in guinea pigs fed a hypercholesterolemic diet.

The current study was undertaken to evaluate the effect of carbohydrate restriction on hepatic cholesterol metabolism in guinea pigs fed a hypercholesterolemic diet. Hartley male guinea pigs (n = 10 per group) were fed 1 of 3 diets: a diet with a percent energy distribution of 42:23:35 carbohydrate:protein:fat and 0.04% cholesterol (control), a diet with the same macronutrient distribution but with 0.25% cholesterol (HChol), or a carbohydrate-restricted (CR) diet with a percent energy distribution of 11:30:59 carbohydrate:protein:fat and 0.25% cholesterol for 12 wk. There was more accumulation of hepatic cholesterol and triglycerides as well as lower 3-hydroxy-3-methyl glutaryl-CoA reductase messenger RNA abundance in guinea pigs fed the high-cholesterol diets (HChol and CR) (P < 0.01). Guinea pigs fed the CR diet had lower concentrations of hepatic total cholesterol and cholesteryl ester than those fed the HChol diet (P < 0.05). There was no diet effect on hepatic LDL receptor expression. Hepatic acyl CoA cholesteryl acyltransferase (ACAT) activity was lowest in guinea pigs fed the low-cholesterol diet (9.7 +/- 4.8 pmol.min(-1).mg(-1)), intermediate in those fed the CR diet (37.3 +/- 12.4 pmol.min(-1).mg protein(-1)), and highest in guinea pigs fed the HChol diet (55.9 +/- 11.2 pmol.min(-1).mg(-1)). ACAT activity was significantly correlated with hepatic cholesterol (r = 0.715; P < 0.01) and LDL cholesterol (r = 0.59; P < 0.01) for all dietary groups, suggesting a major role of this enzyme in hepatic cholesterol homeostasis and in lipoprotein concentrations. These results indicate that dietary cholesterol increases hepatic lipid accumulation and affects hepatic cholesterol homeostasis. Carbohydrate restriction in the presence of high cholesterol is associated with lower hepatic ACAT activity and an attenuation of hepatic cholesterol accumulation.

A combination of psyllium and plant sterols alters lipoprotein metabolism in hypercholesterolemic subjects by modifying the intravascular processing of lipoproteins and increasing LDL uptake.

We previously demonstrated that a diet therapy involving consumption of 7.28 g psyllium (PSY) and 2 g of plant sterols (PS) per day reduced LDL cholesterol from 3.6 +/- 0.7 to 3.1 +/- 0.8 mmol/L (P < 0.01) and decreased the number of intermediate density lipoprotein particles and the smaller LDL and HDL subfractions in hypercholesterolemic individuals (n = 33). The study design was a randomized double blind crossover. Subjects consumed either 2 test cookies containing PSY+PS or 2 placebo cookies for 1 mo with a 3-wk wash out between treatments. To explore mechanisms of the lipid-lowering effects of combined PSY+PS, we present data related to intravascular and molecular regulation. Intake of PSY+PS decreased the cholesterol concentration in LDL-1 from 2.46 +/- 0.66 to 2.26 +/- 0.46 mmol/L and in LDL-2 from 0.63 +/- 0.24 to 0.54 +/- 0.27 mmol/L (P < 0.05) in the test compared with the placebo period. An increase in LDL peak size from 27.3 +/- 0.8 to 27.5 +/- 0.6 nm (P < 0.05) and a decrease in the prevalence of LDL pattern B from 27 to 18% (P < 0.05) also occurred during the PSY+PS period. Cholesteryl ester transfer protein activity was 11% lower (P < 0.05) during the test period. Notably, the abundance of the LDL receptor in circulating mononuclear cells as measured by real time PCR was 26% higher during the test compared with the placebo period (P < 0.03). These results indicate that the hypocholesterolemic action of PSY and PS can be explained in part by modifications in the intravascular processing of lipoproteins and by increases in LDL receptor-mediated uptake.

Validation of using gene expression in mononuclear cells as a marker for hepatic cholesterol metabolism.

HMG-CoA reductase and the LDL receptor are ubiquitously expressed in major tissues. Since the liver plays a major role in regulating circulating LDL, it is usually of interest to measure the effects of drug or dietary interventions on these proteins in liver. In humans, peripheral blood mononuclear cells have been used as a surrogate for liver to assess regulation of these genes, although there is concern regarding the validity of this approach. The purpose of this study was to evaluate the relationship between liver and mononuclear cell expression of HMG-CoA reductase and the LDL receptor in guinea pigs, a well established model for human cholesterol and lipoprotein metabolism. We extracted RNA from liver and mononuclear cells of guinea pigs from a previous study where the effects of rapamycin, an immunosuppresant drug used for transplant patients, on lipid metabolism were evaluated. Guinea pigs were assigned to three different diets containing the same amount of fat (15 g/100 g) and cholesterol (0.08 g/100 g) for a period of 3 weeks. The only difference among diets was the concentration of rapamycin: 0, 0.0028 or 0.028 g/100 g. There were no differences in plasma LDL cholesterol (LDL-C) among groups. Values were 78.4 +/- 14.3, 65.8 +/- 17.2 and 68.4 +/- 45.4 mg/dL (P > 0.05) for guinea pigs treated with 0, low or high doses of rapamycin, respectively. The mRNA abundance for the LDL receptor and HMG-CoA reductase was measured both in liver (n = 30) and mononuclear cells (n = 22) using reverse transcriptase PCR. In agreement with the finding of no changes in plasma LDL-C, there were also no differences for the expression of HMG-CoA reductase or the LDL receptor among groups. However, a positive correlation was found between liver and mononuclear cells for both HMG-CoA reductase (r = 0.613, P < 0.01) and the LDL receptor (r = 0.622, P < 0.01). These correlations suggest that monocytes can be used in humans as an index for liver to assess diet and drug effects on the expression of HMG-CoA reductase and the LDL receptor.

Hormones and nutrients regulate acetyl-CoA carboxylase promoter I in rat primary hepatocytes.

This study investigated the regulation of acetyl-CoA carboxylase (ACC) promoter activity by hormones and nutrients. Genomic clones including promoter I (PI) of the ACC gene were isolated and sequenced. ACC PI fragments (-1,049/+100 or -220/+21 bp) were subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. The ACC PI/luciferase chimeric plasmids were transfected into primary rat hepatocytes using lipofectin. Insulin treatment increased the activity of -1,049/+ 100 and -220/+21 ACC PI by 3.0- and 3.5-fold, respectively, compared to the control. The activity of both constructs was also increased by dexamethasone (Dex) and triiodothyronine (T3), with the greatest effects seen with all three hormones present. With -1,049/+100 or -220/+21 ACC PI, the addition of glucose increased luciferase activity compared to glucose-free control (p<0.05). On the other hand, polyunsaturated fatty acids (PUFA) reduced the activity of the -1,049/+100 ACC PI construct, with eicosapentaenoic acid and docosahexaenoic acid showing the greatest effect (about 70% of the control). However, the addition of PUFA to the culture media did not affect the activity of -220/+21 ACC PI. Therefore, insulin, Dex, T3, glucose, and PUFA regulate ACC gene expression, at least in part, through the PI promoter.

Coumarin-based chemosensors for zinc(II): toward the determination of the design algorithm for CHEF-type and ratiometric probes.

The synthesis of a series of coumarin-based chemosensor assemblies for zinc is detailed, using established and novel synthetic pathways. Variations of the nature of the chelating unit (DPA or cyclen), position of the attachment point of the chelating unit (3- or 4-position), and nature of the 7-substituent (-OH, -OAc, or -NR2) on the coumarin play a crucial role in whether, and to what extent, a CHEF-type or ratiometric response of the chemosensor is observed. Solvent effects are also discussed. The chemosensors were shown to be competent for detecting zinc pools in cultured rat pituitary (GH3) and hepatoma (H4IIE) cell lines. The work further defines the design algorithms for zinc-selective CHEF-type and ratiometric chemosensors.

The lowering of plasma lipids following a weight reduction program is related to increased expression of the LDL receptor and lipoprotein lipase.

To determine whether changes in plasma lipids following a weight loss program were related to modifications in gene expression of the LDL receptor (LDL-R), lipoprotein lipase (LPL), and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, overweight/obese premenopausal women were recruited. The 10-wk, randomized, double-blind intervention consisted of a hypoenergetic diet, high in protein (30% energy) and low in carbohydrate (40% energy), increased physical activity (number of steps taken per day), and intake of a supplement (carnitine or placebo). Our initial hypothesis was that carnitine would enhance the beneficial effects of weight loss on plasma lipids and anthropometrics. Because the carnitine and placebo groups did not differ in any of the measured variables, data for all subjects were pooled and comparisons were made between baseline and postintervention. Mean weight loss was 4.4 kg (P < 0.001), and plasma triglycerides (TG), total, and LDL cholesterol (LDL-C) were reduced by 31.8, 9.9, and 11.9%, respectively (P < 0.001). The expression of the genes of interest was measured in RNA extracted from mononuclear cells at baseline and postintervention using a semiquantitative RT-PCR method. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. After 10 wk, there was a 25.7% increase in the abundance of LPL mRNA (P < 0.01) and a 27.7% increase in that of LDL-R mRNA (P < 0.01). The expression of HMG-CoA reductase was not altered by weight loss. The results suggest that the increased expression of the LDL-R and LPL after the intervention might have contributed to the lower plasma LDL-C and TG observed in these women.

Illuminating zinc in biological systems.

Zinc is the second most abundant transition metal in the human body, fulfilling a multitude of biological roles, but the mechanisms underlying its physiology are poorly understood. The lack of knowledge is, in part, due to the hitherto limited techniques available to track zinc in biological systems. The recent emergence of a number of zinc-specific molecular sensors has provided a new tool to image zinc in live cells and tissue samples. This contribution highlights the concepts behind using zinc-specific fluorescent molecular sensors to gain information about zinc action in biological samples, and provides representative examples of images recorded.

Diethylenetriaminepentaacetic acid enhances thyroid hormone action by a transcriptional mechanism.

Zinc is thought to be required as a structural component of the thyroid hormone (triiodothyronine, T3) receptor (TR). However, we have previously demonstrated that use of diethylenetriaminepentaacetic acid (DTPA) to restrict zinc availability to cultured cells actually potentiates rather than inhibits thyroid hormone action. In this article, the mechanisms underlying these effects of DTPA have been investigated. Treatment of GH3 rat pituitary tumor cells with DTPA in the presence of T3 resulted in twofold greater concentrations of growth hormone (GH) mRNA. Addition of actinomycin D to inhibit transcription showed that GH mRNA was actually less stable in the presence of DTPA, eliminating mRNA stabilization as a possible mechanism underlying this effect. Cycloheximide was able to block the induction by DTPA, showing a requirement for protein synthesis. Transient transfection of a GH promoter/luciferase reporter construct into GH3 cells revealed an inhibitory effect of DTPA on luciferase activity. However, when cells were stably transfected with the same construct, a T3-dependent stimulation of luciferase activity by DTPA was observed, mimicking the effects seen with the endogenous mRNA. Thus, the GH promoter does mediate the effects of DTPA, but stable integration into chromosomal material is required.

Synthesis of a fluorescent chemosensor suitable for the imaging of zinc(II) in live cells.

The synthesis of a coumarin-cyclen conjugate-based zinc-specific chemosensor and its ability to sense Zn(2+) in vitro is described. Using fluorescence microscopy, the chemosensor was shown to be capable of imaging Zn(2+) in live rat pituitary tumour cells.

Social research in an integrated science of nutrition: future directions.

This paper is one in a series from the American Society of Nutritional Sciences Long Range Planning Committee, in which we are attempting to map out the implications of future directions in nutritional sciences for ASNS. Here, we address the area of social nutrition research and identify a series of orientations that are now emerging and likely to shape future research in this area. As with other areas of nutrition, a key feature is the importance of an integrated approach, both across social science disciplines and between social and biological scientists.

Sex and hormonal status modulate the effects of psyllium on plasma lipids and monocyte gene expression in humans.

Psyllium (PSY) intake decreases plasma LDL cholesterol (LDL-C) in men and pre- and post-menopausal women while PSY effects on plasma triglycerides (TG) are sex related. A significant decrease in plasma TG was observed in men while postmenopausal women experienced an increase in plasma TG concentrations following PSY supplementation. To further explore the mechanisms by which sex and hormonal status influence the effects of PSY on plasma lipids, HMG-CoA reductase, LDL receptor and lipoprotein lipase (LPL) mRNA abundance were measured in mononuclear cells isolated from these subjects. The intervention followed a randomized crossover design in which participants were allocated to either 15 or 0 g (control) of PSY/d for 30 d. Compared to the control period, PSY intake induced a 20% increase in HMG-CoA reductase mRNA abundance (P < 0.05) while no significant changes in LDL receptor mRNA abundance were observed. In contrast, LPL mRNA abundance was 24% higher in men and 23% lower in postmenopausal women (P < 0.05) when comparing PSY with the control period. These results suggest that the LDL-C lowering induced by PSY was related to changes in HMG-CoA reductase gene expression in monocytes while the expression of LPL in this system was affected by sex and hormonal status.

Gender and hormonal status affect the regulation of hepatic cholesterol 7alpha-hydroxylase activity and mRNA abundance by dietary soluble fiber in the guinea pig.

Dietary soluble fiber (SF) consistently lowers plasma LDL cholesterol (LDL-C) concentrations, however, secondary mechanisms governing this reduction are not completely defined. Moreover, these mechanisms appear to differ with gender. Male, female and ovariectomized (to mimic menopause) guinea pigs were used to assess effects of gender, hormonal status and SF on activity and expression of hepatic cholesterol 7alpha-hydroxylase (CYP7). Diets were identical except for fiber source (control 10% cellulose, SF 5% psyllium/5% pectin). SF intake resulted in 44% lower plasma total cholesterol, 51% lower plasma LDL-C and 22% lower plasma triacylglycerol (TAG) concentrations. However, ovariectomized guinea pigs fed either the control or SF diets, had the highest plasma LDL-C and TAG levels (P<0.01). SF altered hepatic cholesterol metabolism by effectively reducing hepatic free cholesterol, TAG and microsomal free cholesterol, while activity of CYP7, the rate-limiting enzyme of cholesterol catabolism, was up-regulated. Hepatic CYP7 mRNA abundance paralleled the increase in enzyme activity. Ovariectomized guinea pigs had lowest activity and expression of hepatic CYP7 even after intervention with SF. These results suggest that induction of hepatic CYP7 activity may account, in large part, for the hypocholesterolemic effect of SF. Gender and hormonal status influence metabolic responses to dietary SF with estrogen deprivation leading to the most detrimental lipid profile.

Corn fiber oil lowers plasma cholesterol by altering hepatic cholesterol metabolism and up-regulating LDL receptors in guinea pigs.

To evaluate some of the mechanisms involved in the hypocholesterolemic effects of corn fiber oil (CFO), male Hartley guinea pigs were fed diets containing increasing doses of CFO [0 (control), 5, 10 or 15 g/100 g]. Total fat was adjusted to 15 g/100 g in all diets with regular corn oil. Diets contained 0.25 g/100 g cholesterol. A positive control group (LC) with low dietary cholesterol (0.04 g/100 g) was also included. Plasma LDL cholesterol concentrations were 32, 55 and 57% (P < 0.0005) lower with increasing doses of CFO. Compared with controls, intake of CFO resulted in 27-32% lower hepatic microsomal cholesterol (P < 0.0001), the regulatory pool of LDL receptor (LDL-R) expression. CFO intake resulted in favorable plasma and hepatic cholesterol concentrations, similar to those in guinea pigs fed the LC diet. Hepatic cholesterol 7alpha-hydroxylase (CYP7) activity was approximately 88% higher in guinea pigs fed the two higher dosages of CFO (P < 0.05). In parallel, CYP7 mRNA abundance was approximately 88% higher in guinea pigs fed all three CFO diets. CFO treatment also induced hepatic LDLR mRNA by 66-150% with significant differences at the highest CFO dose. These results suggest that CFO, as a result of decreased bile acid absorption, increased mRNA abundance and activity of CYP7. Because hepatic cholesterol is the substrate for CYP7, a lowering of cholesterol concentrations in the total and microsomal pools was observed. As a response to the depleted microsomal free cholesterol pool, the LDL receptor was up-regulated, drawing more cholesterol from plasma, thus leading to the observed decrease in plasma LDL cholesterol concentrations.