Cholesterogenesis: derepression in extrahepatic tissues with 4-aminopyrazolo (3,4-d) pyrimidine.

Administration of 4-aminopyrazolo [3,4-d] pyrimidine decreased serum cholesterol levels in the rat to less than 5 milligrams per deciliter. Coincident with this change, there was a 2.1 - to 16.0-fold increase in the rate of sterol synthesis in seven extrahepatic tissues. This suggests that cholesterol carried in serum lipoproteins plays a major role in regulating sterol synthesis in many extrahepatic tissues.

Unstirred water layers in intestine: rate determinant of fatty acid absorption from micellar solutions.

Bile acid and fatty acid uptake from micellar solutions by intestinal cells fails to reflect the incremental free energy changes expected for permeation that is rate limited by cell membranes. However, altering the size of the diffulsing particle or the thickness of tle unstirred water layer does change uptake. These observations show that the unstirred water layer is rate limiting for intestinal absorption of lipids from micellar solutions.

Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers.

Several nuclear hormone receptors involved in lipid metabolism form obligate heterodimers with retinoid X receptors (RXRs) and are activated by RXR agonists such as rexinoids. Animals treated with rexinoids exhibited marked changes in cholesterol balance, including inhibition of cholesterol absorption and repressed bile acid synthesis. Studies with receptor-selective agonists revealed that oxysterol receptors (LXRs) and the bile acid receptor (FXR) are the RXR heterodimeric partners that mediate these effects by regulating expression of the reverse cholesterol transporter, ABC1, and the rate-limiting enzyme of bile acid synthesis, CYP7A1, respectively. Thus, these RXR heterodimers serve as key regulators of cholesterol homeostasis by governing reverse cholesterol transport from peripheral tissues, bile acid synthesis in liver, and cholesterol absorption in intestine.

The role of bile salts in controlling the rate of intestinal cholesterogenesis.

According to current concepts, the liver and gastrointestinal tract are considered to be the major, if not the sole, sources of circulating serum cholesterol. While several mechanisms have been described which control the rate of hepatic cholesterogenesis, only biliary diversion is known to alter the rate of sterol synthesis in the intestine. The present study was designed to identify the inhibitory constituent of bile and to define its anatomic and biochemical sites of action. After either biliary diversion or cholestyramine feeding, there is a marked enhancement of cholesterogenesis at every level of the small intestine; this effect is specific for sterol synthesis since acetate incorporation into fatty acids and CO(2) is unaffected by these experimental manipulations. In the present investigation bile salt has been shown to be the constituent of whole bile responsible for the inhibited rate of sterol synthesis found in the intact animal, and in addition, an inverse relationship has been shown to exist between the steady-state intraluminal bile salt concentration and the rate of cholesterogenesis in the adjacent bowel wall. The inhibitory effect of bile salt is directed at the cells of the intestinal crypt, the major anatomic site for sterol synthesis in the small bowel. This feedback inhibition has been localized in the biosynthetic sequence to a step between acetyl CoA and mevalonic acid and, presumably, is at the enzymatic step mediated by hydroxymethylglutaryl reductase. These studies emphasize the close interrelationship which exists between the mechanisms of control of cholesterogenesis in the liver and small intestine. Sterol synthesis in the liver is regulated by exogenous cholesterol intake, whereas the rate of intestinal sterol synthesis is controlled by bile salt, the major end product of the hepatic catabolism of cholesterol.

Sterol synthesis and low density lipoprotein clearance in vivo in the pregnant rat, placenta, and fetus. Sources for tissue cholesterol during fetal development.

Whereas the greatest relative increase in body mass occurs during the third trimester of fetal life, the source of the cholesterol that supports this growth is uncertain. These studies used [3H]water and 125I-cellobiose-labeled low density lipoproteins to quantitate absolute rates of cholesterol acquisition in vivo by the fetus of the rat. Preliminary studies demonstrated that [3H]water administered intravenously to the mother rapidly equilibrated with the body pool of water in the fetus and that 22-microgram atoms of H from the water pool were incorporated into each micromole of newly synthesized cholesterol. After administration of [3H]water to pregnant rats, the rates of sterol synthesis per 100 g of whole body weight were severalfold higher in the fetus than in the dams. Individual organs of the dam such as the liver, however, had much higher synthetic rates than those in the fetus. When maternal hepatic cholesterol synthesis was suppressed by cholesterol feeding, newly synthesized cholesterol disappeared from the maternal blood yet there was essentially no change in the rate of appearance of newly synthesized sterol in the fetus, placenta, and fetal membranes. The placenta did take up low density lipoproteins at rates equal to about one-third of that seen in the maternal liver, but none of the apolipoprotein or cholesterol was transferred to the fetus. These studies indicate that the rat fetus receives little or no cholesterol from the mother but, rather, satisfies its need for cholesterol during fetal development through local synthesis. Furthermore, the fetal membranes appear to be an important site for sterol synthesis in the fetal compartment.

Cholesterol synthesis in the squirrel monkey: relative rates of synthesis in various tissues and mechanisms of control.

Cholesterol synthesis has been extensively investigated in various tissues of lower mammals; however, there is little specific information concerning cholesterologenesis in the primate. Furthermore, experiments in whole animals suggest that important differences may exist in the features of cholesterologenesis in the dog and rat versus the monkey and man. Using the new world squirrel monkey, therefore, we performed the present studies to determine the rates of cholesterologenesis in various tissues per unit weight, to define the relative rates of whole organ synthesis, and to evaluate the operation of control mechanisms in these tissues.In control animals fed a low cholesterol chow diet, the liver and ileum were the two most active sites for cholesterologenesis followed, in order, by the colon, esophagus, and proximal small bowel. Rates of synthesis in 10 other tissues tested were considerably lower than these found in the gastrointestinal tract. When rates of whole organ synthesis were calculated, three tissues, i.e., liver, bowel, and skin, accounted for 92% of the total demonstrable synthetic activity.Following cholesterol feeding utilizing either a solid chow or liquid formula diet, marked suppression of hepatic cholesterologenesis occurred while synthesis in other organs remained essentially unaltered. Similarly, fasting animals for periods up to 96 hr resulted in suppression of synthesis in the liver, but not in various levels of the intestine. Finally, biliary diversion for 48 hr caused a twofold increase in hepatic cholesterologenesis and a six- to eightfold increase in sterol synthesis in the small but not the large intestine.

Interaction of dietary cholesterol and triglycerides in the regulation of hepatic low density lipoprotein transport in the hamster.

These studies report the effects of dietary cholesterol and triglyceride on rates of receptor-dependent and receptor-independent LDL transport in the liver of the hamster. In animals fed diets enriched with 0.1, 0.25, or 1% cholesterol for 1 mo, receptor-dependent LDL transport in the liver was suppressed by 43, 63, and 77%, respectively, and there were reciprocal changes in plasma LDL-cholesterol concentrations. In addition, dietary triglycerides modified the effect of dietary cholesterol on hepatic LDL transport and plasma LDL concentrations so that at each level of cholesterol intake, polyunsaturated triglycerides diminished and saturated triglycerides accentuated the effect of dietary cholesterol. When animals were raised from weaning on diets containing small amounts of cholesterol, the decline in receptor-dependent LDL transport was nearly abolished by the addition of polyunsaturated or monounsaturated triglycerides, but was markedly augmented by the addition of saturated lipids. When animals raised on diets containing cholesterol and saturated triglycerides were returned to the low cholesterol, low triglyceride control diet, hepatic receptor-dependent LDL transport and plasma LDL-cholesterol concentrations returned essentially to normal within 2 wk. Neither receptor-independent LDL transport nor the receptor-dependent uptake of asialofetuin was significantly altered by dietary cholesterol or triglyceride suggesting that the effect of these lipids on hepatic LDL receptor activity was specific and not due to a generalized alteration in the physiochemical properties of hepatic membranes. These studies demonstrate the important role of saturated triglycerides in augmenting the effect of cholesterol in suppressing hepatic LDL receptor activity and elevating LDL-cholesterol levels.

The mechanisms of and the interrelationship between bile acid and chylomicron-mediated regulation of hepatic cholesterol synthesis in the liver of the rat.

Hepatic cholesterol synthesis is controlled by both the size of the bile acid pool in the enterohepatic circulation and by the amount of cholesterol reaching the liver carried in chylomicron remnants. These studies were undertaken to examine how these two control mechanisms are interrelated. When the size of the pool was systematically varied, the logarithm of the rate of hepatic cholesterol synthesis varied in an inverse linear fashion with the size of the taurocholate pool between the limits of 0 and 60 mg of bile acid per 100 g of body weight. The slope of this relationship gave the fractional inhibition of cholesterol synthesis associated with expansion of the taurocholate pool and was critically dependent upon the amount of cholesterol available for absorption from the gastrointestinal tract. Furthermore, the degree of inhibition of cholesterol synthesis in the liver seen with taurocholate feeding was reduced by partially blocking cholesterol absorption with beta-sitosterol even though the bile acid pool was still markedly expanded. In rats with diversion of the intestinal lymph from the blood, a five-fold expansion of the taurocholate pool resulted in only slight suppression of the rate of hepatic cholesterol synthesis, and even this inhibition was shown to be attributable to small amounts of cholesterol absorbed through collateral lymphatic vessels and (or) to a fasting effect. Similarly, the infusion of either taurocholate or a combination of taurocholate and taurochenate into rats with no biliary or dietary cholesterol available for absorption caused no suppression of hepatic cholesterol synthesis. Finally, the effect of changes in the rate of bile acid snythesis on hepatic cholesterol synthesis was examined. The fractional inhibition of cholesterol synthesis found after administration of an amount of cholesterol sufficient to raise the hepatic cholesterol ester content by 1 mg/g equalled only --0.36 when bile acid snythesis was increased by biliary diversion but was --0.92 when bile acid synthesis was suppressed by bile acid feeding. It is concluded that (a) bile acids are not direct effectors of the rate of hepatic cholesterol synthesis, (b) most of the inhibitory activity seen with bile acid feeding is mediated through increased cholesterol absorption, and (c) bile acids do have an intrahepatic effect in that they regulate hepatic cholesterol synthesis indirectly by altering the flow of cellular cholesterol to bile acids.

Delineation of the dimensions and permeability characteristics of the two major diffusion barriers to passive mucosal uptake in the rabbit intestine.

THE RATE OF PASSIVE ABSORPTION INTO THE INTESTINAL MUCOSAL CELL IS DETERMINED BY AT LEAST TWO MAJOR DIFFUSION BARRIERS: an unstirred water layer and the cell membrane. This study defines the morphology and permeability characteristics of these two limiting structures. The unstirred water layer was resolved into two compartments: one behaves like a layer of water overlying the upper villi while the other probably consists of solution between villi. The superficial layer is physiologically most important during uptake of highly permeant compounds and varies in thickness from 115 to 334 mum as the rate of mixing of the bulk mucosal solution is varied. From data derived from a probe molecule whose uptake was limited by the unstirred layer, the effective surface area of this diffusion barrier also was determined to vary with stirring rate and equaled only 2.4 cm(2).100 mg(-1) in the unstirred condition but increased to 11.3 cm(2).100 mg(-1) with vigorous mixing. This latter value, however, was still only 1/170 of the anatomical area of the microvillus membrane. With these values, uptake rates for a number of passively absorbed probe molecules were corrected for unstirred layer resistance, and these data were used to calculate the incremental free energy changes associated with uptake of the -CH(2)- (-258 cal.mol(-1)), -OH (+564), and taurine (+1,463) groups. These studies, then, have defined the thickness and area of the unstirred layer in the intestine and have shown that this barrier is rate-limiting for the mucosal uptake of compounds such as fatty acids and cholesterol; in addition, the lipid membrane of the microvillus surface has been shown to be a relatively polar structure.

Failure of bile acids to control hepatic cholesterogenesis: evidence for endogenous cholesterol feedback.

Studies were undertaken to define the role of bile acids in the control of hepatic cholesterogenesis from acetate. Both biliary diversion and biliary obstruction increase the rate of sterol synthesis by the liver 2.5- to 3-fold. After biliary diversion, however, the bile acid content of the liver is decreased, whereas after biliary obstruction, it is markedly increased. Thus, there is no relationship between the tissue content of bile acid and the rate of hepatic cholesterol synthesis. Furthermore, restoration of the enterohepatic circulation of bile acid in animals with biliary diversion fails to prevent the rise in synthetic activity seen after this manipulation. These data indicate that bile acid plays no direct inhibitory role in the regulation of cholesterol synthesis by the liver. Other experiments were therefore undertaken to evaluate the possibility that changes in cholesterogenic activity observed after manipulation of the enterohepatic circulation of bile acid actually are the result of changes in the enterolymphatic circulation of cholesterol. In support of this thesis it was found that intestinal lymphatic diversion causes the same specific enhancement of cholesterol synthetic activity as biliary diversion and that both of these operative procedures increase enzymatic activity at the step mediated by beta-hydroxy-beta-methyl glutaryl reductase. Furthermore, the increase in the rate of sterol synthesis by the liver seen in animals with biliary diversion can be prevented by the infusion of approximately 7 mg of cholesterol/24 hr in the form of chylomicrons. This is an amount of cholesterol circulating normally in the enterolymphatic circulation of the intact rat.These results indicate that bile acid plays no direct role in the control of hepatic cholesterogenesis, but rather, it is the enterohepatic circulation of endogenous cholesterol that determines directly the rate at which cholesterol is synthesized by the liver.

Cholesterol synthesis in the intestine of man: regional differences and control mechanisms.

Cholesterol in the circulating serum pool is derived either from absorption of dietary cholesterol or from endogenous synthesis principally in the liver and gastrointestinal tract. While the control of intestinal cholesterogenesis has been elucidated in several lower animal species, no data currently are available in the case of man. In the present study using tissue specimens obtained by suction biopsy in 29 normal subjects, we have shown the rate of cholesterogenesis is low in the stomach (25 +/-6 mmumoles/g per 2 hr) and rectum (40 +/-8 mmumoles/g per 2 hr); in the small bowel the rate progressively decreases in the proximal duodenum (90 +/-16 mmumoles/g per 2 hr); distal duodenum (80 +/-11 mmumoles/g per 2 hr); and distal jejunum (35 +/-5 mmumoles/g per 2 hr); but abruptly increases in the distal ileum (280 +/-33 mmumoles/g per 2 hr). Indirect evidence is provided that the intestinal crypt epithelium is the main site of this sterol synthesis. Fasting for 48 hr suppressed the rate of cholesterogenesis in the distal duodenum from a control value of 80 +/-11 mmumoles/g per 2 hr to 40 +/-8 mmumoles/g per 2 hr while cholesterol feeding for 7 days did not alter the rate of cholesterol synthesis (75 +/-12 mmumoles/g per 2 hr). This resistance to cholesterol feeding also was present in the distal ileum where control and cholesterol-fed subjects had comparable rates of cholesterogenesis (280 and 261 mmumoles/g per 2 hr, respectively). Interruption of the enterohepatic circulation, in contrast, resulted in greatly enhanced sterol synthesis with a mean rate of 259 +/-29 mmumoles/g per 2 hr being found in the duodenum of four patients with biliary obstruction as compared with the rate of 80 +/-11 mmumoles/g per 2 hr in control subjects. These studies indicate that the mechanisms of control of cholesterol synthesis by the human intestine are similar to those described for the intestine of lower animals; this also appears to be true for the human liver. Thus, the marked differences in over-all cholesterol metabolism between various lower mammalian species and man cannot be explained by fundamental differences in control mechanisms; rather, these differences must reflect variations in some other parameter of cholesterol metabolism.

The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell.

Studies were undertaken to define the mechanism whereby bile acid facilitates fatty acid and cholesterol uptake into the intestinal mucosal cell. Initial studies showed that the rate of uptake (Jd) of several fatty acids and cholesterol was a linear function of the concentration of these molecules in the bulk phase if the concentration of bile acid was kept constant. In contrast, Jd decreased markedly when the concentration of bile acid was increased relative to that of the probe molecule but remained essentially constant when the concentration of both the bile acid and probe molecule was increased in parallel. In other studies Jd for lauric acid measured from solutions containing either 0 or 20 mM taurodeoxycholate and saturated with the fatty acid equaled 79.8+/-5.2 and 120.8+/-9.4 nmol.min(-1).100 mg(-1), respectively: after correction for unstirred layer resistance, however, the former value equaled 113.5+/-7.1 nmol.min(-1).100 mg(-1). Maximum values of Jd for the saturated fatty acids with 12, 16, and 18 carbons equaled 120.8+/-9.4, 24.1+/-3.2, and 13.6+/-1.1 nmol.min(-1).100 mg(-1), respectively. These values essentially equaled those derived by multiplying the maximum solubility times the passive permeability coefficients appropriate for each of these compounds. The theoretical equations were then derived that define the expected behavior of Jd for the various lipids under these different experimental circumstances where the mechanism of absorption was assumed to occur either by uptake of the whole micelle, during interaction of the micelle with an infinite number of sites on the microvillus membrane or through a monomer phase of lipid molecules in equilibrium with the micelle. The experimental results were consistent both qualitatively and quantitatively with the third model indicating that the principle role of the micelle in facilitating lipid absorption is to overcome unstirred layer resistance while the actual process of fatty acid and cholesterol absorption occurs through a monomer phase in equilibrium with the micelle.

Characterization of bile acid absorption across the unstirred water layer and brush border of the rat jejunum.

We have examined the rate-limiting steps involved in bile acid absorption across the unstirred water layer and lipid cell membrane of the jejunal mucosa. Uptake of the polar bile acid taurocholate is limited solely by the cell membrane since this compound permeates the unstirred water layer more rapidly than the lipid cell membrane and stirring does not enhance uptake. With less polar bile acids which permeate the cell membrane relatively more rapidly, however, the unstirred water layer does exert resistance to mucosal uptake of these compounds. That the unstirred water layer is even more rate limiting to uptake from micellar solutions is indicated by the facts that the rate of bile acid absorption from such solutions is lower than from corresponding monomer solutions, stirring markedly enhances uptake from micellar solutions while increases in viscosity of the incubation media depress uptake and expansion of the micelle size further depresses absorption rates. We also have examined the important question of whether the micelle crosses the brush border intact once it reaches the aqueous-lipid interface. The observations that the calculated permeation rate of the micelle should be extremely low, the rate of mucosal cell uptake plateaus at a constant value when the critical micelle concentration is reached at the aqueous-lipid interface, and the different components of a mixed micelle are taken up at different rates indicate that uptake of the intact micelle does not occur; rather, bile acid absorption must be explained in terms of monomers in equilibrium with the micelle. Finally, after correction of the permeability coefficients of the various bile acids for the unstirred layer resistance the incremental partial molar free energy of solution of the hydroxyl group in the brush border membrane was calculated to equal -6126 cal.mole(-1) indicating that passive diffusion of these compounds occurs through a very polar region of the cell membrane.

Mechanisms by which saturated triacylglycerols elevate the plasma low density lipoprotein-cholesterol concentration in hamsters. Differential effects of fatty acid chain length.

These studies were designed to elucidate how shorter (MCT) and longer (HCO) chain-length saturated triacylglycerols and cholesterol interact to alter steady-state plasma LDL-cholesterol levels. When either MCT or HCO was fed in the absence of cholesterol, there was little effect on receptor-dependent LDL transport but a 36-43% increase in LDL-cholesterol production. Cholesterol feeding in the absence of triacylglycerol led to significant suppression of receptor-dependent LDL transport and a 26-31% increase in LDL-cholesterol production. However, when the longer chain-length saturated triacylglycerol was fed together with cholesterol there was a marked increase in the suppression of receptor-dependent LDL transport and an 82% increase in production rate. Together, these two alterations accounted for the observed eightfold increase in plasma LDL-cholesterol concentration. In contrast, feeding the shorter chain-length saturated triacylglycerol with cholesterol actually enhanced receptor-dependent LDL transport while also causing a smaller increase (52%) in the LDL-cholesterol production rate. As a result of these two opposing events, MCT feeding had essentially no net effect on plasma LDL-cholesterol levels beyond that induced by cholesterol feeding alone.

Regulatory effects of the saturated fatty acids 6:0 through 18:0 on hepatic low density lipoprotein receptor activity in the hamster.

The plasma concentration of cholesterol carried in low density lipoproteins is principally determined by the level of LDL receptor activity (Jm) and the LDL-cholesterol production rate (Jt) found in animals or man. This study delineates which saturated fatty acids alter Jm and Jt and so increase the plasma LDL-cholesterol level. Jm and Jt were measured in vivo in hamsters fed a constant level of added dietary cholesterol (0.12%) and triacylglycerol (10%), where the triacylglycerol contained only a single saturated fatty acid varying in chain length from 6 to 18 carbon atoms. After feeding for 30 d, the 12:0, 14:0, 16:0, and 18:0 fatty acids, but not the 6:0, 8:0, and 10:0 compounds, became significantly enriched in the liver total lipid fraction of the respective groups fed these fatty acids. However, only the 12:0, 14:0, and 16:0 fatty acids, but not the 6:0, 8:0, 10:0, and 18:0 compounds, suppressed Jm, increased Jt, and essentially doubled plasma LDL-cholesterol concentrations. Neither the 16:0 nor 18:0 compound altered rates of cholesterol synthesis in the extrahepatic organs, and both lowered the hepatic total cholesterol pool. Thus, the different effects of the 16:0 and 18:0 fatty acids could not be attributed to a difference in cholesterol delivery to the liver. Since these changes in LDL kinetics took place without an apparent alteration in external sterol balance, the regulatory effects of the 12:0, 14:0, and 16:0 fatty acids presumably are mediated through some change in a putative intrahepatic regulatory pool of sterol in the liver.

Sites of tissue binding and uptake in vivo of bacterial lipopolysaccharide-high density lipoprotein complexes: studies in the rat and squirrel monkey.

When gram-negative bacterial lipopolysaccharides (LPS) are injected intravenously into the rabbit or rat, they bind to plasma lipoproteins, particularly high density lipoproteins (HDL). The present studies were performed to examine the mechanisms by which LPS-HDL complexes are removed from the circulation and taken up by various tissues. Our approach was to compare the sites of specific tissue binding and uptake of HDL and of LPS-HDL complexes in the rat and squirrel monkey. In the rat, binding of homologous 125I-HDL was demonstrated principally in the adrenal gland, ovary, liver, and spleen. [3H]LPS-HDL complexes (produced in vitro by incubating Salmonella typhimurium [3H]LPS with rat HDL and lipoprotein-free plasma) bound to the same tissues, but with apparently lower affinities. The specificity of binding of both 125I-HDL and [3H]LPS-HDL to these organs was demonstrated in two ways. First, tissue binding of both radiolabeled preparations was swamped out by raising the circulating levels of HDL-cholesterol from 32 to 140 mg/dl. Second, treatment of the animals with dexamethasone abolished specific binding of both HDL preparations to the adrenal gland while administration of adrenocorticotropin increased the specific adrenal binding of the two preparations. The steady-state plasma clearance rate for 125I-HDL equaled 774 +/- 29 microliters/h and was significantly lower (557 +/- 39 microliters/h) for the LPS-HDL complex, a finding that presumably reflected the lesser ability of the various tissues to bind the LPS-HDL complex. Binding studies were also done in the squirrel monkey, an animal that has the same level of circulating HDL cholesterol as the rat, but nearly three times more cholesterol in low density lipoproteins. Specific binding of homologous 125I-HDL and [3H]LPS-HDL was again found principally in the adrenal gland and liver. The results indicate that the sites of tissue uptake of bacterial LPS are strongly influenced by binding of LPS to HDL. In particular, LPS-HDL binding may be an important determinant of the extent to which LPS are taken up by the adrenal gland during bacterial sepsis.

Kinetic constants for receptor-dependent and receptor-independent low density lipoprotein transport in the tissues of the rat and hamster.

In this study, carried out in the rat and hamster, the receptor-dependent low density lipoprotein (LDL) transport process in each organ was characterized in terms of its maximal uptake rate (Jm) and Michaelis constant (Km), while the rate of receptor-independent uptake was defined in terms of its proportionality constant (P). The highest Jm values of 50-126 micrograms/h per g were found in the liver and endocrine glands in both species and receptor-dependent uptake also was detected in other organs like spleen, kidney, and intestine. The Km values were essentially the same in all of the organs and equaled approximately 90 mg/dl in both species. The receptor-independent uptake constants also were similar in the two species and were highest in the spleen, liver, and intestine. From these values for Jm, Km, and P, it was possible to construct theoretical curves that predict the plasma LDL-cholesterol concentration and fractional catabolic rate given any alteration in LDL-cholesterol production or the magnitude of receptor-dependent LDL transport in any organ of the rat or hamster.

Receptor-independent low density lipoprotein transport in the rat in vivo. Quantitation, characterization, and metabolic consequences.

Receptor-independent low density lipoprotein (LDL) transport plays a critical role in the regulation of plasma cholesterol levels; hence, these studies were done to characterize this process in the tissues of the rat. High rates of receptor-independent clearance were found in the spleen, but other organs, like liver, gastrointestinal tract, and endocrine glands manifested lower clearance rates that varied from 3 to 9 microliter/h per g, while the rates in nervous tissue, muscle, and adipose tissue were less than 1 microliter/h per g. Receptor-dependent uptake was much higher in liver (85 microliter/h per g) and adrenal gland (219 microliter/h per g), but was also low in most other tissues. At normal plasma LDL concentrations, 67% of the receptor-dependent transport in the whole animal was accounted for by LDL uptake in the liver. In contrast, the receptor-independent uptake found in the whole animal took place in many organs, including skeletal muscle (20%), liver (16%), small bowel (15%), skin (10%), and spleen (7%). Furthermore, in liver, the rate of cholesterol synthesis could be varied 11-fold, yet the rate of receptor-independent LDL clearance remained constant at approximately 8 microliter/h per g. When the circulating levels of LDL were systematically increased, receptor-independent LDL clearance also remained constant, so that hepatic LDL-cholesterol uptake by this mechanism increased linearly, from 1 to 20 micrograms/h per g, as the plasma LDL-cholesterol level was increased from 10 to 250 mg/dl. Finally, when equal amounts of LDL-cholesterol were delivered into the liver by either the receptor-dependent or receptor-independent mechanism, there was significant suppression of cholesterol synthesis and an increase in cholesteryl esters. Thus, in any situation in which receptor-dependent LDL degradation is lost, cholesterol balance in the whole animal and across individual organs is maintained by receptor-independent mechanisms, although when the new steady state is achieved, circulating levels of LDL must necessarily be very much increased.

Characterization of the kinetics of the passive and active transport mechanisms for bile acid absorption in the small intestine and colon of the rat.

Bile acid uptake occurs via passive diffusion in all regions of the intestine and via active absorption in the ileum. Determination of the passive permeability coefficient for ionized monomers ((*)P(-)) demonstrated that permeability decreased by a factor of 3.4, 6.8, and 8.1 for the addition of a hydroxyl, glycine, or taurine group, respectively, to the steroid nucleus. Removal of the negative charge increased permeation by a factor of 4.4; however, permeability coefficients for the protonated monomers showed the same relative decrease with addition of a hydroxyl group. The calculated incremental free energies of solution (deltaDeltaF(W-->1)) associated with these additions equaled + 757 (hydroxyl), + 1178 (glycine), and + 1291 (taurine) cal/mole. Passive permeability coefficients for the transverse colon showed the same relative relationships among the various bile acids. After making appropriate corrections for passive permeability across the ileum, apparent values for the maximal transport velocity ((*)V(max)) and Michaelis constant ((*)K(m)) of the active transport system were measured. (*)V(max) depended upon the number of hydroxyl groups on the steroid nucleus; values for the trihydroxy bile acids were high (1543-1906 pmoles/min per cm) while those for the dihydroxy (114-512 pmoles/min per cm) and monohydroxy (45-57 pmoles/min per cm) acids were lower. In contrast, (*)K(m) values were related to whether the bile acid was conjugated; unconjugated bile acids had values ranging from 0.37 to 0.49 mM, while values for the conjugated bile acids were approximately half as high (0.12-0.23 mM).

Inappropriate hepatic cholesterol synthesis expands the cellular pool of sterol available for recruitment by bile acids in the rat.

These studies test the hypothesis that a major determinant of excessive biliary cholesterol secretion is a level of hepatic sterol synthesis that is inappropriately high relative to the needs of the liver cell for preserving cholesterol balance. Biliary cholesterol secretion was measured in vivo in two models after loading the hepatocyte with sterol by two different mechanisms. In the first model, cholesterol was delivered physiologically to the liver in chylomicron remnants. This resulted in a sixfold increase in cholesteryl ester content and marked suppression of cholesterol synthesis, but biliary cholesterol secretion remained essentially constant. In the second model, 3-hydroxy-3-methyl-glutaryl CoA reductase levels in the liver were markedly increased by chronic mevinolin (lovastatin) administration. Withdrawal of the inhibitor resulted in a sudden fivefold increase in the rate of sterol synthesis in the liver of the experimental animals that was inappropriately high for cellular needs. This excessive synthesis, in turn, was accompanied by a fivefold increase in the cholesteryl ester content, enrichment of microsomal membranes with cholesterol and, most importantly, by a threefold increase in the rate of biliary sterol secretion. As the rate of sterol synthesis gradually returned to normal over 48 h, the cholesterol ester content, the lipid composition of the microsomal membranes, and rate of cholesterol secretion into bile also returned to baseline values. These results further support the concept of functional compartmentalization of cholesterol in the hepatocyte. Derangements that cause an inappropriately high rate of sterol synthesis in the endoplasmic reticulum may lead to an expansion of that pool of cholesterol that is recruitable by bile acids and, hence, to greater situation of the bile.