Derivation of the equations that describe the effects of unstirred water layers on the kinetic parameters of active transport processes in the intestine.
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Animals↗
Biomedical subjects
J M Dietschy
Publications and source records attributed to J M Dietschy.
Comparison of the effects of cholic acid and chenic acid feeding on rates of cholesterol synthesis in the liver of the rat.
To compare the ability of cholic acid and chenic acid to suppress cholesterol synthesis in the liver, these two bile acids were fed in varying amounts to rats for either 66 hr or 6 weeks. In both instances there were significant changes in the bile acid pool in the small intestine and suppression of the rate of cholesterol synthesis in the liver. The administration of cholic acid, however, consistently produced greater suppression of the rate of cholesterol synthesis from octanoate or of microsomal HMG CoA reductase activity than did the administration of a similar amount of chenic acid. Furthermore, this difference was present whether the rates of cholesterogenesis were measured at high-substrate concentrations, ie, under conditions where Vmax was apparently achieved, or under circumstances where there was essentially no extracellular substrate present. These findings do not support the view that the superiority of chenic acid for dissolution of gallstones is secondary to its greater effect as an inhibitor of hepatic cholesterol synthesis.
Animals↗
Regulation of sterol synthesis in adrenal gland of the rat by both high and low density human plasma lipoproteins.
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Acetates↗
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.
Adenine↗
Dissociation of beta-hydroxy-beta-methylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis in the liver following the intravenous administration of lipid.
After the intravenous administration to the intact rat of triglyceride carried in either intestinal lipoproteins or in an artificial fat emulsion, the enzymatic activities of microsomal beta-hydroxy-beta-methylglutaryl-CoA reductase activity in the liver assayed in vitro became markedly elevated. This elevation of enzyme activity was not associated with a corresponding change in the overall rate of cholesterol synthesis in the rat liver slice as measured by the incorporation of either [3H]water or [1-14C]octanoate into nonsaponifiable lipids or into digitonin-precipitable sterols. The degree of dissociation of hydroxymethylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis correlated closely with the amount of lipid administered to the animal, the level of circulating lipids, and the level of ketone synthesis manifest in the liver cell suggesting that this phenomenon might be the consequence of a detergent effect of elevated cellular levels of fatty acids. In any event, under these experimental circumstances hydroxymethylglutaryl-CoA redutase activity no longer reflects the rate at which the liver cell is synthesizing cholesterol.
Acetates↗
Enzymatic measurement of free and esterified cholesterol levels in plasma and other biological preparations using the oxygen electrode in a modified glucose analyzer.
A method is described for assaying free and esterified cholesterol using the oxygen electrode in a modified glucose analyzer to measure the relative amount of oxygen utilization taking place during oxydation of free cholesterol by the enzyme, cholesterol oxidase. A second enzyme, cholesterol ester hydrolase, is utilized to generate free cholesterol from cholesterol esters. This assay procedure is rapid, specific, reproducible and applicable to the measurement of free and esterified cholesterol carried in the major plasma lipoprotein fractions of man and the rat and, in addition, it can be utilized for the assay of sterols in subcellular fractions of cells.
Animals↗
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.
Animals↗
Ability of six different lipoprotein fractions to regulate the rate of hepatic cholesterogenesis in vivo.
Two in vivo assay procedures were used to study the inhibitory activity of cholesterol carried in three intestinal lymph and three serum lipoprotein fractions on the rate of cholesterol synthesis in the liver. In the first preparation, different lipoproteins were injected intravenously as a bolus into rats at the mid-light phase of the diurnal light cycle, following which they were killed 12 hours later in the mid-dark phase of the cycle. Using this assay, three intestinal lymph lipoprotein fractions of varying Sf values all produced a similar degree of inhibition which averaged approximately 11%/mg of cholesterol injected. The serum lipoprotein fractions caused only about one-third this amount of inhibition. Detailed analysis of events occurring within the liver during this 12-hour assay period revealed that there were marked differences in the rate of net cholesterol uptake into the liver and in the rate of new removal of cholesterol esters from the liver following injection of each of these different lipoprotein fractions. The amount of inhibition of sterol synthesis produced by any fraction was proportional to the product of the incremental increase in hepatic cholesterol ester content and the time over which this increase in esters occurred. In the second type of assay where the lipoprotein fractions were administered to the animals as a continuous intravenous infusion over 24 hours the largest increase in hepatic cholesterol ester content and the greatest inhibition of cholesterol synthesis was found with intestinal lipoproteins having Sf values larger than 8000. Intestinal lipoprotein fractions with lower Sf values and all serum lipoprotein fractions were significantly less effective in bringing about an increase in hepatic cholesterol ester content and in producing inhibition of cholesterol synthesis by the liver. These studies emphasize the primary role of cholesterol carried in lipoproteins of intestinal origin in regulating hepatic sterol synthesis. The inhibitory activity of these fractions appears to correlate with the ability of these lipoproteins to bring about a maximal increase in hepatic cholesterol ester content which, in turn, appears to relate to the capacity of these fractions to transfer cholesterol rapidly into the hepatocyte while, at the same time, slowing the rate of cholesterol mobilization from the liver.
Animals↗
The interaction of various control mechanisms in determining the rate of hepatic cholesterogenesis in the rat.
In these studies rats were subjected to diurnal light-cycling, stress, fasting and the feeding of cholestyramine, beta-sitosterol and cholesterol in various combinations. In control animals exposed to light cycling for 2 weeks the rate of hepatic cholesterogenesis was 3.7 fold higher in the mid-dark than in the mid-light phase of the light cycle. The magnitude of this difference varied with the duration of light cycling and the size of the animals. Similarly, enhanced rates of cholesterol synthesis were seen in the mid-dark phase relative to the mid-light phase of the light cycle in rats where the base-line level of hepatic cholesterogenesis was increased by feeding cholestyramine (1.6-fold) or beta-sitosterol (2.9-fold) or was depressed by fasting (19-fold) or cholesterol feeding (2.1-fold). Restraining animals for 48 h also increased the rate of cholesterol synthesis in the liver; in control animals, this stress enhanced the level of cholesterogenesis seen at both the mid-light and mid-dark phases of the light cycle. In addition, both the effects of stress and of diurnal light cycling could be identified in groups of animals where base-line cholesterogenic activity was varied by fasting or by feeding cholestyramine, beta-sitosterol or cholesterol. These studies illustrate the complexity of the control of hepatic cholesterol synthesis and suggest that the final rate of cholesterogenesis may be the result of several different effectors modifying by different mechanisms the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase.
Animals↗
The kinetic characteristics of inhibition of hepatic cholesterogenesis by lipoproteins of intestinal origin.
In these studies intestinal lipoproteins were injected intravenously into recipient rats in order to study the kinetic characteristics of cholesterol uptake by the liver cell and inhibition of the cholesterol synthetic pathway. Net cholesterol uptake from circulating intestinal lipoproteins took place only in the liver, and only this tissue manifested inhibition of cholesterol syntheses. Cholesterol uptake by the liver, quantified by a rise in the cholesterol ester content, was a linear function of time and of the amount of lipoprotein cholesterol administered to the animals. Using groups of rats that were either fed cholesterol or injected intravenously with intestinal lipoproteins as a bolus or as a continuous infusion, there was generally a correlation between inhibition of the rate of cholesterol synthesis and the cholesterol ester content of the liver. However, there was no consistent quantitative relationship between these two variables suggesting either that cholesterol ester was not the immediate effector of the inhibition or, alternatively, that there was intracellular localization of the effector at the site of control of the rate-limiting enzyme in the cholesterogenic pathway.
Animals↗
Permeability characteristics of the adipocyte cell membrane and partitioning characteristics of the adipocyte triglyceride core.
The unidirectional rates of passive permeation of a homologous series of saturated fatty acids and bile acids into rat epididymal adipocytes were measured to determine the permeability characteristics of this mammalian cell membrane. For fatty acids containing 5 to 12 carbon atoms the logarithm of the permeability coefficient was a linear function of the number of carbons in the fatty acid chain: fatty acids with less than five carbon atoms showed anomalously high permeabilities. Using the data for the fatty acids with 5 to 12 carbon atoms, the incremental free energy of transfer (delta delta F w leads to l) of the -CH2 moiety from the aqueous environment into the fat cell was calculated to equal -547 cal mole-1. The delta delta F w leads to l of the -OH moiety calculated from data using bile acids as the probe molecules was +1,225 cal mole-1. After rupturing the fat cells by freeze-thawing, partition ratios also were measured between bubber and the lipid phase of the adipocyte core using both the fatty acid series and a series of terminal diols as probe molecules. Using these partition ratios delta delta F w leads to l for the -CH2 and -OH substituent groups was calculated to equal -830 and +2,070 cal mole-1, respectively. On the basis of these studies, two conclusions were drawn. First, like many epithelial surfaces and the erythrocyte membrane, the fat cell membrane exhibits anomalously high permeabilities to small molecular weight, polar compounds. Since this behavior in the adipocyte, as in the erythrocyte, cannot be attributed to structures such as tight junctions, it must be explained on the basis of some physico-chemical feature of the cell membrane itself. Secondly, the values of the delta delta F w leads to l indicate that the adipocyte membrane is less polar than the intestinal and gallbladder membranes but more polar than the membranes of Nitella and the erythrocyte.
Adipose Tissue↗
The intestinal unstirred layer: its surface area and effect on active transport kinetics.
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Animals↗
Limitations of acetate as a substrate for measuring cholesterol synthesis in liver.
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Acetates↗
Bile acids: their absorption from the gastrointestinal tract and role during fat absorption.
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Bile Acids and Salts↗
Normal mechanisms of fat absorption and derangements induced by various gastrointestinal diseases.
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Bile Acids and Salts↗
Adaptive responses in hepatic and intestinal cholesterogenesis following ileal resection in the rat.
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Animals↗
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.
Action Potentials↗
Effect of alterations of the specific activity of the intracellular acetyl CoA pool on apparent rates of hepatic cholesterogenesis.
We have previously shown significant dilution of the specific activity of the intracellular acetyl CoA pool when radiolabeled acetate is used as the precursor in liver slice experiments. In the present study, using liver from animals subjected to various manipulations known to alter the rate of cholesterogenesis, the specific activity of the intramitochondrial acetyl CoA pool was 27-49% of the theoretical specific activity expected if no endogenous dilution occurred. Because the cytosolic acetyl CoA pool that gives rise to cholesterol is not in equilibrium with the intramitochondrial pool, these values cannot be used to correct the flux of labeled carbon from [(14)C]acetate into cholesterol. However, because [(14)C]octanoate is rapidly oxidized intramitochondrially to acetyl CoA, which feeds both the intra- and extramitochondrial metabolic pathways, [(14)C]octanoate can be utilized to determine true flux rates of C(2) units into cholesterol and other products. Using this substrate in liver slices from animals subjected to a variety of experimental manipulations, the specific activity of the intracellular acetyl CoA pool was 54-71% of the expected specific activity. After correction for endogenous dilution, the C(2) flux into cholesterol varied from 335 to 459 nmoles.g(-1).hr(-1) in control animals, was suppressed 10-40-fold in animals subjected to fasting and cholesterol feeding, and increased into the range of 1500 nmoles.g(-1).hr(-1) after derepression with cholestyramine feeding or biliary diversion. Data also are presented that show very good agreement between the corrected C(2) flux rate from octanoate into cholesterol and microsomal HMG CoA reductase activity in the same liver under conditions in which the synthetic rates were varied over a 100-fold range.
Acetates↗