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PubMed1984

Capacity of lipoproteins to act as substrates for lecithin:cholesterol acyltransferase. Enhancement by pre-incubation with an artificial triacylglycerol emulsion.

Previous studies have shown that high concentrations of very low density lipoproteins (VLDL) stimulate the formation of cholesteryl esters in human plasma, possibly by acting as recipients of cholesteryl esters transferred from high density lipoproteins (HDL). To gain further insight into this phenomenon, experiments were performed to determine whether the triacylglycerol emulsion Intralipid, which acts as an artificial recipient of HDL cholesteryl esters, has an effect on cholesterol esterification similar to that of VLDL. Intralipid, in contrast to VLDL, is devoid of apolipoproteins which may stimulate cholesterol esterification. Human plasma, which had previously been depleted of VLDL, was pre-incubated in the presence or absence of Intralipid. After pre-incubation, the Intralipid was removed and rates of cholesterol esterification were measured in subsequent incubations of the Intralipid-depleted fractions. The presence of Intralipid during the pre-incubation had a marked stimulatory effect on cholesterol esterification, comparable to that of VLDL in earlier studies. The pre-incubation with Intralipid also markedly reduced the cholesteryl ester content, and increased the triacylglycerol content, of the HDL. These changes in composition coincided with two changes in the elution profile of HDL after density-gradient ultracentrifugation which were (i) a reduction in the density of particles in the HDL3 subfraction, which virtually disappeared as an identifiable peak, and (ii) the emergence of a discrete population of very dense lipoproteins consisting primarily of protein and phospholipid. Since all these changes were related to redistributions of lipids, the results highlight the importance of lipid transfers in the regulation of plasma cholesterol esterification.

Cholesterol Esters↗

PubMed1983

Oleate metabolism in isolated hepatocytes from lean and obese Zucker rats. Influence of a high fat diet and in vitro response to glucagon.

The uptake and metabolism of [1-14C]oleate (0.3 mmol/L) were studied in isolated hepatocytes from lean and obese Zucker rats fed either a control (low-fat) diet or a high-fat diet. With the control diet, [1-14C]oleate uptake was increased by 70% in the obese rats, and fat-feeding decreased this uptake to values comparable to that of their lean littermates. Interestingly, the hepatocyte mean surface area was increased in the obese mutants by 21% with the control diet and by 30% with the high-fat diet. The possible reasons for the differences in oleate uptake are discussed. With the control diet, cells from the obese rats showed a four-fold rise in [1-14C]oleate esterification, while ketogenesis (beta-hydroxybutyrate + acetoacetate production) as well as the radioactive acid-soluble products were greatly depressed. Production of CO2 was very low and similar in both groups of animals. Adaptation to the high-fat diet in the obese rats resulted in a reversal between esterification and oxidation of oleate: the latter became the major metabolic pathway as in the lean rats. The ketogenic capacity was greatly if not completely restored. In the lean animals, glucagon stimulated ketogenesis both in the presence or absence of oleate and decreased [1-14C]oleate esterification. In the obese rats, the hormone exerted a significant ketogenic effect only if oleate was present and did not influence its esterification. The data demonstrate the following abnormalities in the hepatocytes of obese Zucker rats: (1) an enlargement of cell size, (2) an increased oleate uptake, (3) a virtual absence of a ketogenic response to exogenous oleate, and (4) a markedly increased esterification of the latter. The metabolic defects, but not the cell size, appear to be largely corrected by an adaptation to a high-fat diet. The hepatic response to glucagon was decreased in the obese rats at the level of endogenous ketogenesis.

Animals↗

PubMed1994

Regulatory factors in the development of fatty infiltration of the liver during gram-negative sepsis.

To further understand the development of fatty liver during gram-negative sepsis, we measured fatty acid uptake in addition to esterification and secretion of lipids by freshly isolated hepatocytes from fasted and fed control and Escherichia coli-treated rats. Rats were made septic by intravenous (IV) injection of 8 x 10(7) live E coli colonies per 100 g body weight. For the fasted groups, food was removed after E coli injection. Fed rats received a nutritionally adequate diet intragastrically for 5 days before and 24 hours after inducing sepsis. Twenty-four hours after E coli injection, the esterification of newly synthesized fatty acids, as measured by 3H2O incorporation, and the esterification of exogenous fatty acids, measured from 14C-palmitate incorporation, into triglyceride (TG), total cholesterol, and total phospholipid phosphorus were significantly greater in hepatocytes from fasted septic rats compared with their control rats. In fed septic rats, esterification of 14C-palmitate into TG was fourfold greater than in the fed control rats. The increased rates of esterification in hepatocytes from fasted and fed septic rats were not accompanied by an increase in the labeled TG in the medium. This inability to secrete the additional TG that the hepatocytes produce resulted in a higher concentration of cellular TG in fasted and fed septic rats than in their controls. The enzymes glycerol-3-phosphate acyltransferase (GPAT) and phosphatidate phosphohydrolase (PPH) do not appear to be factors contributing to the increased TG synthesis, since the increase in enzyme activity was not accompanied by a similar increase in TG synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Antibodies, Monoclonal↗

PubMed2000

Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger.

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.

Aspergillus niger↗

PubMed1987

Enzymes involved in plasma cholesterol transport.

Regulation of plasma cholesterol transport is to a large extent a function of factors that regulate plasma cholesterol esterification and the transfers of cholesteryl esters between plasma lipoprotein fractions. Plasma cholesterol esterification is catalysed by the action of lecithin: cholesterol acyltransferase on lipids on the surface of HDL, while the transfers of cholesteryl esters require activity of a specific lipid transfer protein. Esterification of the cholesterol on the surface of HDL generates a concentration gradient down which unesterified cholesterol moves from tissues into the plasma. Once within the plasma and esterified, the newly formed cholesteryl esters are incorporated initially into the core of HDL particles before being redistributed to other classes of lipoproteins. The end result of these processes of esterification and transfer is that most of the cholesterol in human plasma is accommodated within the core of LDL, where its transport is a function of the highly regulated uptake by tissues of intact LDL particles. The capacity of HDL to act as substrates for lecithin: cholesterol acyltransferase varies inversely with HDL particle size. Thus, factors such as the concentration of triglyceride-rich lipoproteins and activities of the lipid transfer protein, hepatic lipase, lipoprotein lipase and the HDL conversion protein, which are known to influence HDL particle size, may also be important as regulators of plasma cholesterol esterification.

Animals↗

PubMed2000

Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution.

Previous studies have shown that scavenger receptor BI (SR-BI) stimulates the bidirectional flux of free cholesterol (FC) between HDL and SR-BI-expressing cells. A major component of the enhanced FC flux appears to occur independently of HDL binding to SR-BI and may be due to changes in membrane lipid domains resulting from SR-BI expression (1). In the present study, the impact of SR-BI on cellular cholesterol metabolism was determined by examining SR-BI-mediated changes in cellular cholesterol mass, the esterification of HDL-derived FC, and changes in membrane lipid pools. Growth of SR-BI-expressing cells in medium containing HDL led to increased cellular cholesterol mass, most of which accumulated as ester. The esterification of HDL-derived FC was enhanced by SR-BI-expression to a far greater extent than the SR-BI mediated increase in FC uptake, suggesting an SR-BI-mediated effect on cholesterol utilization in the cell. This observation was tested by comparing FC esterification rates in SR-BI positive and negative cells when equivalent amounts of extracellular FC were taken up via cyclodextrins or apolipoprotein AI/phospholipid disks, neither of which contained cholesteryl ester. Under these conditions, SR-BI did not preferentially stimulate cholesterol esterification. These results indicate that the enhanced esterification of HDL-derived FC in SR-BI-expressing cells is due to the expanded pool of cellular FC and not to a specific effect of SR-BI on cholesterol utilization. Two approaches were used to test the effects of SR-BI expression on membrane lipid organization. In the first, the sensitivity of cellular FC to exogenous cholesterol oxidase was tested under conditions in which there is a preferential oxidation of caveolar cholesterol. SR-BI-expression was found to greatly increase the fraction of cellular cholesterol available to the oxidase as compared to either vector-transfected cells or cells expressing the related class B scavenger receptor CD36. These results suggest that SR-BI expression alters the distribution of membrane-free cholesterol to a caveolar fraction or alters the accessibility of this membrane fraction to exogenous cholesterol oxidase. In the second approach, the efflux of cellular FC to high concentrations of cyclodextrins was monitored under conditions where desorption of FC from the plasma membrane is rate limiting for efflux. SR-BI-expressing cells showed a shift in the distribution of FC between two kinetic pools with more FC in the fast pool and less in the slow pool. These data support a model in which SR-BI expression leads to a redistribution of cholesterol to membrane domains that serve to facilitate the flux of FC between cells and lipoproteins.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1997

Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts.

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER) in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [14C]oleoyl-CoA converted approximately 0.1-2% of total cell-free cholesterol to [14C]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.

Cells, Cultured↗

PubMed1998

Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes.

The enzyme acyl coenzyme A:cholesterol acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial component of intracellular lipid homeostasis. Two enzymes catalyze this activity in Saccharomyces cerevisiae (yeast), and several lines of evidence suggest multigene families may also exist in mammals. Using the human ACAT1 sequence to screen data bases of expressed sequence tags, we identified two novel and distinct partial human cDNAs. Full-length cDNA clones for these ACAT related gene products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2) cDNA library. ARGP1 was expressed in numerous human adult tissues and tissue culture cell lines, whereas expression of ARGP2 was more restricted. In vitro microsomal assays in a yeast strain deleted for both esterification genes and completely deficient in sterol esterification indicated that ARGP2 esterified cholesterol while ARGP1 did not. In contrast to ACAT1 and similar to liver esterification, the activity of ARGP2 was relatively resistant to a histidine active site modifier. ARGP2 is therefore a tissue-specific sterol esterification enzyme which we thus designated ACAT2. We speculate that ARGP1 participates in the coenzyme A-dependent acylation of substrate(s) other than cholesterol. Consistent with this hypothesis, ARGP1, unlike any other member of this multigene family, possesses a predicted diacylglycerol binding motif suggesting that it may perform the last acylation in triglyceride biosynthesis.

Acylation↗

PubMed1999

Mechanisms involved in the regulation of free fatty acid release from isolated human fat cells by acylation-stimulating protein and insulin.

The effects of acylation-stimulating protein (ASP) and insulin on free fatty acid (FFA) release from isolated human fat cells and the signal transduction pathways to induce these effects were studied. ASP and insulin inhibited basal and norepinephrine-induced FFA release by stimulating fractional FFA re-esterification (both to the same extent) and by inhibiting FFA produced during lipolysis (ASP to a lesser extent than insulin). Protein kinase C inhibition influenced none of the effects of ASP or insulin. Phosphatidylinositol 3-kinase inhibition counteracted the effects of insulin but not of ASP. Phosphodiesterase 3 (PDE3) activity was stimulated by ASP and insulin, whereas PDE4 activity was slightly increased by ASP only. Selective PDE3 inhibition reversed the effects of both ASP and insulin on fractional FFA re-esterification and lipolysis. Selective PDE4 inhibition slightly counteracted the ASP but not the effect of insulin on fractional FFA re-esterification and did not prevent the action of ASP or insulin on lipolysis. Thus, ASP and insulin play a major role in regulating FFA release from fat cells as follows: insulin by stimulating fractional FFA re-esterification and inhibiting lipolysis and ASP mainly by stimulating fractional FFA re-esterification. For both ASP and insulin these effects on FFA release are mediated by PDE3, and for ASP PDE4 might also be involved. The signaling pathway preceding PDE is not known for ASP but involves phosphatidylinositol 3-kinase for insulin.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Rosiglitazone controls fatty acid cycling in human adipose tissue by means of glyceroneogenesis and glycerol phosphorylation.

Control of fatty acid homeostasis is crucial to prevent insulin resistance. During fasting, the plasma fatty acid level depends on triglyceride lipolysis and fatty acid re-esterification within fat cells. In rodents, Rosiglitazone controls fatty acid homeostasis by stimulating two pathways in the adipocytes, glyceroneogenesis and glycerol phosphorylation, that provide the glycerol 3-phosphate necessary for fatty acid re-esterification. Here, we analyzed the functionality of both pathways for controlling fatty acid release in subcutaneous adipose tissue samples from lean and overweight women before and after Rosiglitazone ex vivo treatment. In controls, pyruvate, used as a substrate of glyceroneogenesis, could contribute to the re-esterification of up to 65% of the fatty acids released after basal lipolysis, whereas glycerol phosphorylation accounted for only 14 +/- 9%. However, the efficiency of glyceroneogenesis diminished as body mass index (BMI) of women increased. After Rosiglitazone treatment, increase of either pyruvate- or glycerol-dependent fatty acid re-esterification was strictly correlated to that of phosphoenolpyruvate carboxykinase and glycerol kinase, the key enzymes of each pathway, but depended on BMI of the women. Whereas the Rosiglitazone responsiveness of glyceroneogenesis was rather constant according to the BMI of the women, glycerol phosphorylation was mostly enhanced in lean women (BMI < 27). Overall, these data indicate that, whereas glyceroneogenesis is more utilized than glycerol phosphorylation for fatty acid re-esterification in human subcutaneous adipose tissue in the physiological situation, both are solicited in response to Rosiglitazone but with lower efficiency when BMI is increased.

Adipose Tissue↗

PubMed1994

Photoregulation of alpha-chymotrypsin activity in organic media: effects of bioimprinting.

alpha-Chymotrypsin exhibits photoswitchable activities in an organic solvent after covalent modification of the protein backbone with thiophenefulgide active ester (2). The thiophenefulgide-modified alpha-chymotrypsin exhibits reversible photoisomerizable properties between states (3)-E and (3)-C. The modified alpha-chymotrypsin, where nine lysine residues are substituted by thiophenefulgide units, retains 60% of the activity of the native enzyme. The activities of thiophenefulgide-modified alpha-chymotrypsin toward esterification of N-acetyl-L-phenylalanine (4) by ethanol in cyclohexane are controlled by the configuration of the attached photoisomerizable component and by prior bioimprinting of the protein backbone with the reaction substrate (4). The esterification of (4) in cyclohexane using bioimprinted (3)-C is two-fold faster than in the presence of (3)-E. In the presence of a nonbioimprinted enzyme, esterification of (4) by (3)-C is five-fold faster than with (3)-E. The activity of bioimprinted (3)-E toward esterification of (4) is 4.5-fold higher than that of nonbioimprinted (3)-E. Switchable cyclic esterification of (4) is accomplished by sequential photoisomerization of the thiophenefulgide-modified alpha-chymotrypsin between states (3)-C and (3)-E.

Chymotrypsin↗

PubMed1984

Hepatic palmitate metabolism in fasting Zucker rats: effect of lactate on partitioning.

Hepatocytes were isolated from 48-h fasted lean and obese Zucker rats and incubated with [1-14C] palmitate. The partitioning of fatty acids to oxidation and esterification products and the effect of lactate or glucose on this partitioning were studied. With palmitate as the only substrate, the cells from the lean rats oxidized 80% of the palmitate that was metabolized compared with only 35% oxidation in the cells from the obese rats. Thus fatty acid esterification remained the predominate pathway of palmitate metabolism in the hepatocytes from fasting obese rats. The addition of lactate to the cells from the lean rats decreased oxidation and increased esterification to levels similar to those in the obese rat hepatocytes. Glucose had similar effects on esterification in lean rat hepatocytes but was less potent than lactate. The cells from the obese rats were insensitive to the addition of lactate or glucose. These studies indicate that the inability of hepatocytes from obese rats to induce ketogenesis from exogenous fatty acids after a 48-h fast and their insensitivity to added lactate or glucose may be related to the availability of endogenous energy sources in the obese liver that can be oxidized and thus inhibit ketogenesis and support fatty acid esterification.

Animals↗

PubMed1988

Stimulation of in vitro triglyceride synthesis in the rat hepatocyte by growth hormone treatment in vivo.

Hepatic fatty acid metabolism in the rat is sexually differentiated. Rates of esterification by the liver of fatty acid into triglyceride and other esterification products (phospholipid, diglyceride, cholesteryl esters) are higher in the female than in the male. There is evidence to suggest that GH feminizes other hepatic systems that exhibit sexual dimorphism, including hepatic steroid metabolism, PRL receptors, and estrogen binding. To investigate the role of GH in maintenance of the high rates of fatty acid esterification observed in the female, we assessed rates of [1-14C]oleic acid utilization by hepatocytes prepared from hypophysectomized (hypox) cortisol/T3-replaced female rats with an without continuous in vivo infusion of human (h) GH (5 micrograms/h). In addition, we assessed the effect of in vivo hGH treatment (5 micrograms/h) on [1-14C]oleic acid utilization in the normal male rat. Hypophysectomy was accompanied by a reduction in incorporation of [1-14C]oleic acid into products of esterification (triglyceride, phospholipid, diglyceride) and oxidation (CO2, ketone bodies). Continuous infusion of hGH (5 micrograms/h; 14 days) restored rates of fatty acid esterification in the hypox-cortisol/T3-replaced female rat, with the exception of cholesteryl esters. hGH infusion partially restored rates of fatty acid oxidation in the hypox cortisol/T3-replaced female rat. Treatment of the adult male rat with continuous infusion of hGH (5 micrograms/h; 7 days) resulted in increased rates of incorporation of [1-14C] oleic acid into triglyceride. In contrast, incorporation of oleic acid into phospholipid, diglyceride, and cholesteryl esters was unaltered. These results suggest that GH may be an important regulator of hepatic fatty acid metabolism.

Animals↗

PubMed1992

Estradiol-17 beta effects on lipid metabolism of adipose tissue in nutritionally induced lean and obese ovariectomized ewes.

Nutritionally manipulated lean (68 kg) and obese (87 kg) ovariectomized ewes were administered estradiol-17 beta (E2) or sham implants. Ewes individually had ad libitum access to corn silage. Rates of de novo lipogenesis, palmitate esterification, and glycerol and fatty acid release were determined with slices of subcutaneous adipose tissue at 0, 5, and 30 d after implantation. Condition and E2 interacted over time (linear, P less than .12; quadratic, P less than .05) to affect intake. Lean ewes implanted with E2 decreased intake initially after implantation, whereas obese ewes implanted with E2 decreased intake later after implantation. The linear effect of time x condition x E2 interacted (P less than .02) for lipogenesis. Lipogenesis was inhibited in both the lean and obese ewes implanted with E2. Lean compared with obese ewes without E2 had increased lipogenesis at a faster rate over time. Esterification increased (linear, P less than .01) in the lean ewes and decreased (quadratic, P less than .01) in the obese ewes over time. A time x E2 interaction occurred for esterification (P less than .02). Glycerol and fatty acid release were variable over time within condition and E2. A 48-h adipose tissue culture determined the effect of E2 on lipid metabolism. Estradiol-17 beta decreased (P less than .05) lipogenesis, decreased (P less than .08) esterification, and maintained fatty acid and glycerol release. Data in vivo and in culture indicated that E2 acted to decrease de novo lipogenesis and palmitate esterification and had little or no effect on lipolysis.

Adipose Tissue↗

PubMed1991

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.

Animals↗

PubMed1975

Analysis of pesticides by chemical derivatization. I. a new procedure for the formation of 2-chloroethyl esters of ten herbicidal acids.

A new esterification procedure was developed to form 2-chloroethyl esters for 10 common herbicidal acids, using dicyclohexyl carbodiimide and 2-chloroethanol. This reagent was found to be more desirable than the commonly used boron trichloride/2-chloroethanol reagent. Maximum esterification was given for picloram at a lower reaction temperature with a shorter reaction time. Also, maximum esterification of dicamba and 2,3,6-TBA was achieved. The boron trichloride/2-chlorethanol gave poor or little esterification with these 2 acids under the various conditions investigated. The effect of solvents on esterification with the 2 reagents was also explored.

Chemical Phenomena↗

PubMed1986

Sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase from the corn earworm, Heliothis zea.

The enzymatic activity and sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase (ACAT) were measured in microsomes of cells from Heliothis zea. Under standard assay conditions, the specific enzymatic activity of ACAT was highest in the intestine followed by the fat body and ovary (380.7, 30.7, 8.3 pmol/min per mg, respectively). The structure of the exogenous sterol used in the ACAT assay affected its rate of esterification. The relative rates of esterification of analogs of cholesterol with various modifications of the side chain were: 24-H greater than 24 alpha-CH3 greater than delta 22 greater than delta 24 greater than 24 alpha-C2H5 greater than 24 beta-CH3, delta 22-24 beta-CH3 and delta 22-24 alpha-C2H5. The number and position of double bonds in the B-ring of the sterol nucleus greatly affected the rate of esterification of sterols by ACAT. The average relative rates of esterification of sterols with differences in their B-rings were: delta 7 much greater than delta 8 greater than delta 0 greater than delta 5 greater than delta 5.7. The presence of a 9,14-cyclopropane group and/or methyl groups at the C-4 and 14 positions prevented significant esterification of such sterols. The formation of cholesteryl and lathosteryl esters was partially inhibited in microsomes from the intestine, fat body, and ovary by the addition of the ACAT inhibitor, 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]prop anamide (Sandoz Compound 58-035).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗