Changes in gene expression with iron loading and chelation in cardiac myocytes and non-myocytic fibroblasts.

Iron overload is associated with long-term cardiac iron accumulation and tissue changes such as fibrosis. To determine short-term iron-dependent changes in expression of genes associated with iron homeostasis and fibrosis we measured mRNA on Northern blots prepared from cultured rat neonatal cardiomyocytes and non-myocytes (fibroblasts) as a function of iron loading and chelation. Transferrin receptor mRNA was reduced in myocytes exposed to various concentrations of iron for 3 days and this decline was associated with a 63% decline in iron-response element (IRE) binding of iron regulatory protein-1, indicating that myocytes utilize IRE-dependent mechanisms to modulate gene expression. In myocytes iron caused a dose-dependent decline in mRNAs coding for transforming growth factor- beta(1)(TGF- beta(1)), biglycan, and collagen type I while plasminogen activator inhibitor-1 mRNA was unaffected by iron loading and decorin mRNA doubled. Total TGF- beta bioactivity was also decreased by iron loading. Thus, the effects of iron loading on genes related to cardiac fibrosis are gene-specific. Addition of deferoxamine for 1 day did not have any significant effect on any of these genes. Parallel changes in gene expression were exhibited by non-myocytes (fibroblasts), where chelation also decreased TGF- beta(1)mRNA and activity, and mRNA for collagen type I and biglycan, and collagen synthesis. In addition to these changes in transcripts associated with matrix formation the mRNA of the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase was unaffected by iron loading but doubled in both cell types upon treatment with deferoxamine. These findings suggest that in both cardiac myocytes and non-myocyte fibroblasts gene expression is coupled to intracellular iron pools by gene-specific and IRE-dependent and idependent mechanisms. This linkage may influence matrix deposition, a significant component of cardiac injury.

Characterization of Fe2+ and Fe3+ transport by iron-loaded cardiac myocytes.

Plasma iron overload causes cardiac iron accumulation leading to toxicity and organ failure. In order to understand the basis of iron acquisition, we examined mechanisms of Fe3+ and Fe2+ uptake in control and iron-loaded cardiomyocyte cultures. Iron loading increased rates of Fe3+ and Fe2+ uptake, primarily by increasing Vmax. Inhibition of Fe3+ transport by impermeable Fe2+ chelators and the presence of a cell surface ferricyanide reductase activity are consistent with a role for redox cycling in Fe3+ uptake. However, flavoproteins and copper-dependent oxidases known to be required for redox-active iron transport in yeast do not appear to be involved in iron uptake by cardiac myocytes, nor do the abundant cardiac L-type Ca2+ channels. The data suggest that both redox states of iron contribute to cardiac iron accumulation in iron overload.

Beyond alcohol: beverage consumption and cardiovascular mortality.

This paper reviews epidemiological investigations which have identified an inverse relationship between alcohol consumption and death from coronary heart disease: evidence from studies of mixed populations as well as of single-sex populations have, on the whole, demonstrated that this relationship is independent of sex or age. This 'cardioprotective effect' of alcohol can be explained, at least in part, by ethanol-related increases in high density lipoprotein cholesterol and reduced platelet coagulability. With certain beverages, especially red wine, phenolic compounds may provide additional protection by altering eicosanoid metabolism in favour of increased prostacyclin and decreased thromboxane synthesis, as well as antioxidant functions which prevent the peroxidation of low-density lipoprotein. Trans-resveratrol, a tri-hydroxy stilbene present in the skins of specific grape cultivars, is a constituent of certain red wines which may play a crucial role in modulating lipoprotein metabolism, eicosanoid synthesis, oxidation and coagulation. Preliminary studies using the human hepatoma cell line HepG2 are described, demonstrating that this compound has no effect upon cell viability or overall protein synthesis in these cells, and at high concentrations DNA synthesis as measured by radioactive thymidine incorporation is enhanced. Reduced intracellular concentration and secretion of apolipoprotein B have been shown to occur in response to resveratrol although a clear dose-dependency has not yet been demonstrated. The mechanisms underlying these changes as well as the effects upon the synthesis and secretion of other apolipoproteins are under active investigation in our laboratory.

Modulation by iron loading and chelation of the uptake of non-transferrin-bound iron by human liver cells.

Hepatic non-transferrin-bound Fe (NTBI) flux and its regulation were characterized by measuring the uptake of Fe from [59Fe]/nitrilotriacetate (NTA) complexes in control and Fe-loaded cultures of human hepatocellular carcinoma cells (HepG2). Exposure to ferric ammonium citrate (FAC) for 1 to 7 days resulted in a time- and dose-dependent increase in the rate of NTBI uptake. In contrast to previous studies showing a dependence of the rate of Fe uptake on extracellular Fe, this was positively correlated with total cellular Fe content. The Fe3+ chelating agents deferoxamine (DFO), 1,2-dimethyl-3-hydroxypyrid-4-one (CP 020) and 1,2-diethyl-3-hydroxypyrid-4-one (CP 094) prevented or diminished the increase in NTBI transport when present during Fe loading and reversed the stimulation in pre-loaded cells in relation to their abilities to decrease intracellular iron. Although saturation of the Fe uptake process was not achieved in control cells, kinetic modelling to include linear diffusion-controlled processes yielded estimated parameters of Km = 4.3 microM and Vmax = 2.6 fmol/micrograms protein/min for the underlying process. There was a significant increase in the apparent Vmax (31.2 fmol/micrograms protein per min) for NTBI uptake in Fe-loaded cells, suggesting that Fe loading increases the number of a rate-limiting carrier site for Fe. Km also increased to 15.2 microM, comparable to values reported when whole liver is perfused with FeSO4. We conclude that HepG2 cells possess a transferrin-independent mechanism of Fe accumulation that responds reversibly to a regulatory intracellular Fe pool.

Iron transport and subcellular distribution in Hep G2 hepatocarcinoma cells.

Thalassemic patients with iron overload are presently treated with deferoxamine or the experimental chelator deferiprone. To understand how these agents remove iron from the liver, cultured human hepatoma cells loaded with iron were previously used as a model for hepatic iron overload. The present study was undertaken to characterize further the pathways of iron transport and distribution in these cells. The activation energy for Fe2+ transport is 19 kJ/mol greater than for Fe3+, and the rate of Fe2+ transport--but not that of Fe3(+)--decreases with temperature above 25 degrees C, suggesting distinct uptake processes for different redox states of iron. Iron loading, which promotes a greater rate of Fe3+ transport, also caused a proportionally greater deposition of iron in the microsomal and cytosolic compartments and specifically lowered the activities of succinate-cytochrome c reductase and 5'-nucleotidase, representative markers of the mitochondria and plasma membrane, respectively. Both deferiprone and deferoxamine decreased total cellular iron and iron in each fraction except cytosol, indicating mobilization of iron for clearance from the cell via the cytosol. This model may be useful in characterizing the determinants of effective chelation in patients.

Uptake of non-transferrin-bound iron by both reductive and nonreductive processes is modulated by intracellular iron.

Non-transferrin-bound iron (NTBI) uptake occurs in a variety of cells by a saturable, specific and temperature-sensitive process. Our previous studies indicated that NTBI uptake by cardiac myocytes and Hep G2 cells was reversibly up-regulated by iron deposition. In the present work we have characterized this up-regulation and examined its mechanism by comparing the uptake of oxidized (Fe3+) and ascorbate-reduced (Fe2+) forms of iron. Iron loading markedly enhanced the uptake of iron both in the presence and absence of ascorbate, but the increment was greater when ascorbate was absent. This up-regulation is partially inhibited by actinomycin D and cycloheximide, indicating a requirement for protein synthesis. Uptake by the iron-loaded cells was less sensitive to thiol-alkylating agents and competing metal ions, but was more sensitive to proteolysis. Iron loading causes an increase in both Km and Vmax for uptake of both Fe2+ and Fe3+, although the values differ, suggesting distinct rate-limiting steps for uptake of Fe2+ and Fe3+. Consistent with this idea, uptake of the two ions showed differential sensitivity to thiol reagents, competing metal ions and monensin. The Fe(2+)-specific chelators bathophenanthroline disulfonate and ferrozine markedly inhibited iron uptake whether ascorbate was present or not, indicating that Fe3+ uptake is dependent on reduction to the ferrous state. This requirement for reduction was independent of the iron status of the cells, demonstrating that the process of up-regulation is not due to the appearance of a new mechanism for translocation of Fe3+ without reduction. Taken together, the evidence favors a model of NTBI transport where an obligate and rate-determining reduction of Fe3+ occurs prior to or during uptake, followed by translocation through an Fe2+ carrier. The distinct translocation mechanisms of uptake in the presence and absence of ascorbate suggest that exogenous Fe2+ does not access the carrier available to the nascent ferrous ion derived from the reductase and is consistent with close coupling between the reduction and the translocation processes. In iron-loaded cells with increased rates of NTBI transport, a similar mechanism prevails.

Effects of iron loading on uptake, speciation, and chelation of iron in cultured myocardial cells.

Accumulation of Fe in the myocardium in circumstances of transferrin saturation is associated with heart failure in Fe-loaded patients. To characterize the underlying causes of this phenomenon, we measured the flux as well as the speciation of Fe in normal and Fe-loaded cultures of rat myocardiocytes. Fe loading with low-molecular-weight Fe (ferric ammonium citrate) promoted a dose- and time-dependent increase in the rate of uptake of non-transferrin-bound Fe (NTBI) that was positively correlated (R = 0.9, p < 0.005) with cellular iron content. At concentrations sufficient to produce this up-regulation, membrane integrity was unaffected but the rate of spontaneous beating of the cells was decreased by 60%. The enhanced rate of NTBI uptake in Fe-loaded cells reverted to control rates after treatment with therapeutic concentrations of Fe chelators deferoxamine, 1,2-dimethyl-3-hydroxypyrid-4-one and 1,2-diethyl-3-hydroxypyrid-4-one under conditions where approximately 80% of the cellular Fe was removed by chelation. Fe loading of cultured myocytes also induced shifts in Fe speciation. Thus the ratio of Fe bound in hemosiderin-like precipitates to ferritin-bound Fe increased twofold, from a range of 0.84 to 1.44 in control cells to 1.96 to 3.3 in iron-loaded cells. This increased ratio was similar to that measured in the heart and liver of a thalassemic patient who underwent a double transplant for the failure of both organs, even though the Fe content of the heart (mean, 5.8 mg Fe/gm dry weight) was much less than that of the liver (28.1 mg/gm dry weight). These results suggest that increased rates of uptake of NTBI may exacerbate iron loading of the heart and contribute to iron-mediated cardiotoxicity, whereas the clinical benefits of chelation therapy may be enhanced by the down-regulation of NTBI uptake.

Apolipoprotein synthesis and secretion in Hep G2 cells: effects of monensin and cycloheximide.

Hep G2 cells were used to study the relationship between apolipoprotein synthesis and secretion, as revealed by their interaction with agents modulating these processes. Cycloheximide inhibited the secretion of both apolipoproteins (apo) AI and B, but the reduction in apo AI secretion was evident at earlier times. Monensin also inhibited secretion of apo AI and apo B, but only apo AI accumulated intracellularly. Pulse-chase studies showed that, at concentrations of monensin that had no effect on total protein synthesis, apo B synthesis was specifically inhibited. Triacylglycerol synthesis was inhibited to the same extent as apo B synthesis, but this preceded the latter inhibition and unlike apo B there was an accumulation of intracellular triglyceride. These results suggest that distinctive mechanisms modulate the synthesis and secretion of apo AI and apo B, and that apo B synthesis can be specifically inhibited by mechanisms that initially block triglyceride production.

Speciation of tissue and cellular iron with on-line detection by inductively coupled plasma-mass spectrometry.

Iron accumulating to excess in tissues of humans and animal models occurs mainly as complexes with transferrin, ferritin, other hemoproteins, and insoluble hemosiderin particles. To determine the distribution of Fe amongst these molecular species, we have used inductively coupled plasma-mass spectrometry as a means of on-line, isotope-specific detection for their liquid chromatographic separation. The stable isotope 57Fe is a suitable isotope for monitoring the Fe content of each fraction, and its availability at high isotopic enrichment makes it an attractive choice for tracer studies when the use of a radioisotope is undesirable, e.g., in human subjects. The detection system offers the advantages of high sensitivity (detection limits in the parts per billion range), a wide dynamic range (linearity of the calibration curve over several orders of magnitude), and on-line analysis facilitating real-time evaluation of the chromatographic separation, in addition to isotope-specific information. The Fe distributions in healthy rat livers, liver and heart tissue from Fe-loaded human subjects, and human hepatocyte cultures are reported. The ferritin:hemosiderin ratio in these samples is shown to be an indicator of the degree of Fe loading and correlates well with that determined by Zeeman-corrected electrothermal atomic absorption as an alternative means of detection.

Enzyme-linked immunosorbent assay to measure apolipoproteins AI and B secreted by a human hepatic carcinoma cell line (Hep G2).

We describe an enzyme-linked immunosorbent assay (ELISA) to measure apolipoproteins AI and B secreted by Hep G2 cells and in cell homogenates. These assays utilize commercially available polyclonal antibodies, affinity-purified to improve their specificity, thereby achieving a dramatic increase in the sensitivity of the assay. These affinity-purified antibodies were also more sensitive than a series of monoclonal antibodies tested. We achieved a sensitivity of 0.4 ng in the apo AI assay, and a sensitivity of 5 ng in the apo B assay. By these methods, we measured secretion rates by Hep G2 cells of 358 +/- 41 ng/mg cell protein/hr for apo B and 137 +/- 8 ng/mg cell protein/hr for apo AI. These assays also allowed the measurement of intracellular apolipoproteins and thus can be used to facilitate investigations of human lipoprotein metabolism in cell culture systems.

Dietary fish oils modify adipocyte structure and function.

Dietary fish oils, enriched with omega-3 fatty acids (e.g., MaxEPA fish oil), inhibit lipogenesis and have a marked hypotriglyceridemic effect in man and experimental animals. Dietary omega-3 fatty acids also reduce adipose tissue trophic growth in rats. To understand the metabolic basis for this, we measured the effect of fish oil feeding upon rat plasma triglyceride concentration, fat pad mass, fat cell size, fat cell lipolysis, as well as lipoprotein binding to adipocyte plasma membranes. In adolescent (250 g) male Wistar rats fed 20% (w/w) fish oil supplemented diets for 3 weeks, plasma triglyceride levels and epididymal and perirenal fat pad mass were significantly (P less than 0.005) reduced compared to pair-fed controls given 20% lard diets. These differences in fat pad mass between the diets were greater than differences in whole animal mass or in the mass of livers, testes, kidneys, spleens, or hearts. Isoproterenol-stimulated lipolysis was significantly (P less than 0.005) higher in fish oil fed rats than in pair-fed controls. In young (100 g) rats plasma triglyceride levels were 10 times lower in the fish oil fed group after 5 weeks as compared to the lard-fed controls. This was accompanied by a reduction in epididymal and perirenal fat pad mass as well as a 2-3-fold decrease in adipocyte volumes; there was no significant difference between the two groups in fat cell number in each region. Plasma membranes of epididymal adipocytes from fish oil fed rats bound significantly (P less than 0.001) less HDL1 than the lard-fed rats, possibly as a result of a reduction in fat cell size and/or alteration of plasma membrane structure. Thus in both young and old rats, the reduction in plasma triglyceride concentration in conjunction with increased hormone-stimulated lipolysis may explain in part the selective reduction in adipose tissue trophic growth accompanying fish oil consumption.

The biological significance of lipoprotein lipase modulation by phenobarbital and heparin.

When confluent cultures of 3T3 F442A cells were treated with insulin, differentiation occurred within 6 days as indicated by LPL secretion followed by increased intracellular levels of protein and triacylglycerol. PB increased LPL secretion 2- to 3-fold and intracellular LPL 3- to 10-fold in a time-dependent manner; these increments were less in proportion to the length of the time interval between confluence and initiation of PB treatment. These results are consistent with the notion that PB promotes conversion of adipocyte precursors to mature adipocytes by increasing the proportion of the former that become susceptible to the differentiating stimulus. Human subjects treated with heparin by continuous i.v. infusion over 4 days showed an initial decrease in serum triacylglycerol concentration in response to the initial bolus injection, accompanied by sharp increases in circulating LPL and HTGL, but the triacylglycerol concentration returned to normal within 24 hr. Rats infused with heparin by means of peritoneal implantation of osmotic minipumps demonstrated dose-dependent increases in circulating LPL, accompanied by reduction in heart muscle LPL but inconsistent changes in other tissues examined. Heparin had no effect on the clearance of circulating LPL but did reduce the total body pool of endothelial-bound enzyme. No changes in fasting triacylglycerol and free glycerol were observed, but exogenous VLDL were cleared at a faster rate in heparinized animals. Since the latter also manifested a decrease in de novo fatty acid synthesis, it seems that the heparinized rat is able to maintain circulating levels of triacylglycerol by efficient re-esterification of preformed fatty acids despite the enhanced lipolysis consequent upon higher plasma LPL activity.

Selective uptake of cholesteryl ester from high density lipoproteins by plasma membranes of adipose tissue.

The interaction between high density lipoproteins (HDL) and adipose tissue is an important pathway for cholesterol and cholesteryl ester flux. In intact fat cells, a disproportionately greater net uptake of cholesteryl ester occurs subsequent to lipoprotein binding than would have been predicted from a consideration of holoparticle uptake alone. To characterize the early events in this process, cholesteryl hexadecyl ether, a nonmetabolizable, accumulative marker of cholesteryl ester, was incorporated into canine HDL2, and its uptake by omental adipocyte plasma membranes was measured in relation to the binding of HDL2, which in this animal species is enriched in apolipoprotein A-I and free of apolipoprotein E. The dose-response profile for HDL2 binding was consistent with a single lipoprotein binding site at all concentrations of HDL2, whereas uptake of cholesteryl ester from HDL2 was biphasic, suggesting a high affinity site at low HDL2 concentrations and a low affinity site at high lipoprotein concentrations. Pronase treatment stimulated binding twofold and this was accompanied by a parallel twofold stimulation of cholesteryl ester uptake. EDTA, on the other hand, reduced binding and uptake of cholesteryl ester by 20%, indicating partial dependence upon divalent cations. The proportion of HDL2 cholesteryl ester accumulated by plasma membranes relative to HDL2 protein bound was not altered by either pronase or EDTA, despite the fact that these agents had opposite effects upon binding. In dissociation studies, a portion of membrane-associated HDL2 did not equilibrate with exogenous HDL2 and a greater proportion of the cholesteryl ester failed to dissociate. A stepwise mechanism for cholesteryl ester uptake, involving (i) saturable, high affinity HDL2 binding to cell surface sites, (ii) vectoral, HDL2 concentration-dependent delivery of cholesteryl ester to the membrane, and (iii) cholesteryl ester sequestration into a nonexchangeable membrane compartment, appears to be independent of metabolic energy or cell processing.

Effect of alcohol on lipoprotein metabolism. II. Lipolytic activities and mixed function oxidases.

The mechanism by which alcohol increases plasma total high density lipoproteins (HDLs) and HDL-cholesterol is unknown, but it may involve modulation of the lipolytic enzymes, hepatic triglyceride lipase (HTGL) and/or lipoprotein lipase (LPL) in hepatic and extrahepatic tissues. The modulation of HDL metabolism by alcohol may also be related to its potential to induce mixed function oxidases in liver microsomes. These possibilities were examined by a pair-feeding protocol in which rats were fed diets with 35% of the caloric content as ethanol; control groups received a diet with an isocaloric amount of sucrose or were fed chow ad libitum. Alcohol caused a significant decrease in HTGL activity of liver microsomes, but there was no significant effect of alcohol upon the activities of LPL in adipose tissue and heart muscle. The relative rates of mixed function oxidases, assayed in control liver microsomes using ethoxy-,pentoxy- and benzyloxy-resorufin as substrates, were benzyloxy greater than ethoxy greater than pentoxy. This order was not affected by alcohol, but the oxidation of ethoxy- and pentoxyresorufin was reduced in liver microsomes from the ethanol-fed group. HTGL synthesis and secretion were also measured using primary rat hepatocyte cultures isolated from animals on the above dietary regimes and maintained for up to 3 days in basal medium alone or supplemented with 10 mmol/l ethanol. In basal media the order of activity of extracellular HTGL, released by the addition of heparin, was sucrose-fed greater than chow-fed greater than ethanol-fed. The rate of HTGL secretion from hepatocytes was stimulated in ethanol-containing medium, and was greater in hepatocytes from the sucrose-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Microsomal induction, alcohol, and lipoprotein metabolism: is there a three-way relationship?

The role of ethanol as a microsomal enzyme-inducing agent and as a modulator of lipid metabolism is reviewed. In an attempt to ascertain the mechanisms underlying the latter effects we examined the changes in hepatic triglyceride lipase (HTGL), hepatic high density lipoprotein (HDL) binding, and apolipoprotein secretion mediated by ethanol in a variety of experimental situations. Chronic administration of ethanol to rats decreased the ability of the liver to secrete HTGL, but primary liver cultures prepared from both ethanol and sucrose-fed rats secreted more HTGL when acutely exposed to ethanol over a 3-day period than when grown in a control medium. Hep G2 cells when grown in ethanol-containing medium for 14-28 days manifested increased HDL-binding capacity; apolipoprotein-A1 secretion was increased by ethanol but apolipoprotein B secretion was not affected. These findings suggest that increased plasma HDL concentrations which follow chronic ethanol ingestion may be due, at least in part, to increased hepatic secretion and reduced intravascular conversion of the lipoprotein despite enhanced reuptake by the liver; they are not consistent with an ethanol-mediated alteration in very low density lipoprotein secretion by the liver.

Effect of alcohol on lipoprotein metabolism. I. High density lipoprotein binding.

The effects of ethanol upon the binding of [125I]-labelled human high density lipoprotein 3 (HDL3) was examined in rat liver microsomes and monolayer cultures of human hepatoma (Hep G2) cells. Alcohol feeding to rats (35% caloric content) caused a significant (p less than 0.05) increase in serum cholesterol concentrations relative to pair-fed controls, but HDL3 binding to rat liver microsomes was unaffected by alcohol consumption. By contrast, addition of 10 mM ethanol to Hep G2 cells increased HDL3 binding, and this increase was observed after 14, 28 and 40 days of exposure. This alcohol-dependent rise in HDL3 binding was associated with a 2.3- to 5-fold rise in receptor number (Bmax), and a 2- to 6-fold increase in the dissociation constant (Kd). The data suggest that the net effect of increased receptor number and lower receptor affinity is to increase the capacity of hepatocytes to metabolize circulating high density lipoproteins, and that this increase in the face of elevated plasma high density lipoprotein cholesterol consequent upon alcohol consumption would facilitate greater mobilization of cholesterol from peripheral tissues to the liver.

Lipolytic enzymes as markers of induction and differentiation.

Factors leading to microsomal enzyme induction are associated with hypertriglyceridemia in man. Phenobarbital (PB) increases hepatic synthesis of triglyceride but lowers its serum concentration in rats due to increased postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL); these changes are accompanied by increased activity of these lipolytic enzymes in adipose tissue and liver. The present work explores the cellular mechanisms whereby PB increases the tissue content of these enzymes, using primary cultures of rat liver hepatocytes and a continuous cell line of mouse fibroblasts (preadipocytes) that undergo differentiation into mature fat cells. Secretion and synthesis of HTGL in primary rat hepatocytes increased 50% with insulin; when PB was added with insulin, activity was enhanced an additional 50%. By contrast, insulin inhibited HTGL secretion from the well differentiated rat hepatoma cell line, FU-5-5, C8, and this inhibition was partly overcome by PB. These results suggest that different control mechanisms govern the synthesis and secretion of HTGL in normal rat liver cells and hepatoma. In cultured pre-adipocytes (3T3-F442A) insulin promoted differentiation when added to confluent cultures. PB (0.5 mM) resulted in marked enhancement of conversion of adipocytes characterized by a two- to threefold increase in extracellular LPL and a 10-fold increase in intracellular enzyme. These results suggest that PB promotes conversion of uncommitted cells into pre-adipocytes at an early stage in the differentiation of adipose tissue.

Secretion of triglyceride lipase from rat hepatocytes in culture: modulation by insulin and phenobarbital.

Hepatic triglyceride lipase (HTGL) was measured in primary rat hepatocytes maintained for 3 days under three different culture conditions: basal medium, basal medium plus insulin, and basal medium plus insulin and phenobarbital. The activity of HTGL secreted by these cells was measured by treating intact cells with heparin; intracellular enzyme was subsequently measured in cell homogenates. Insulin stimulated intracellular triglyceride lipase activity by 48% and extracellular lipase by 30%. Phenobarbital, an enzyme-inducing drug, caused a further 15% increase in extracellular hepatic triglyceride lipase; whereas, the intracellular activity was reduced. The presence of insulin greatly stimulated the rate of enzyme secretion, and this rate was not notably affected by the presence of phenobarbital. After 3 days in culture, the short term (2-8 h) synthesis and secretion of enzyme from cultures treated with insulin or insulin plus phenobarbital were equally inhibited by cycloheximide. Monensin also inhibited enzyme secretion in both cultures and caused a similar increase in intracellular lipase activities. Insulin did not significantly affect the proportion of intracellular enzyme (17.7% basal vs. 15.8% insulin). On the other hand phenobarbital produced a 20-30% reduction in the proportion of intracellular enzyme (12.5 vs. 17.7% basal or 15.8% insulin). These findings suggest a drug-induced redistribution of triglyceride lipase.

Modulation by phenobarbital of the differentiation of 3T3 preadipocytes.

The effect of phenobarbital upon the differentiation of two preadipocyte cell lines, 3T3 F442A and 3T3 L-1, was examined by measuring the synthesis and secretion of lipoprotein lipase. Extracellular enzyme was measured by treating intact cells with heparin, and the intracellular enzyme was subsequently assayed in cell homogenates. When confluent cultures of 3T3 F442A cells were treated with insulin, the cells underwent differentiation as indicated by increased activity of lipoprotein lipase within 6 days, followed in turn by increased levels of protein and triglyceride. Addition of phenobarbital with insulin enhanced total lipoprotein lipase, protein, and triglyceride content. The activity of lipoprotein lipase accumulated in the heparin-releasable fraction during differentiation was increased 2- to 3-fold and the intracellular enzyme was enhanced 15- to 20-fold by the addition of phenobarbital. The ability of phenobarbital to modulate differentiation was dependent upon the time of addition. When added early in the postconfluent period, there was a greater increase in lipoprotein lipase activity than when the drug was added at later times. Phenobarbital also stimulated lipoprotein lipase in differentiating 3T3 L-1 cells in the presence of insulin, although lipoprotein lipase activity was moderately enhanced by phenobarbital alone in these cells. These results suggest that phenobarbital may affect the conversion of adipoblasts into preadipocytes and thereby increase the proportion of cells susceptible to the differentiating stimulus.

Effect of cholesterol upon the conformation of band 3 and its transmembrane fragment.

Vesicles enriched in the anion transport protein band 3 and its transmembrane domain were prepared, and the cysteine residues were labelled with an extrinsic fluorescent probe, monobromobimane. Fluorescence energy transfer was demonstrated between intrinsic tryptophans and monobromobimane, and an average interchromophoric distance, Rav, was defined. Rav values and fluorescence emission wavelengths were used to monitor the conformation of band 3 and its transmembrane domain as a function of cholesterol content. The vesicles were treated with ovolecithin liposomes to reduce the cholesterol concentration, and there was an increase in Rav from 17.25 to 20.70 A (1 A = 0.1 nm) in intact band 3. A somewhat smaller increase in Rav for the transmembrane domain was observed (18.03-19.04). The tryptophan fluorescence emission wavelength was also blue shifted in the cholesterol-depleted preparations relative to the untreated samples. Combining the effects of cholesterol depletion upon Rav and the fluorescence emission maxima, it is suggested that the conformation of band 3 is influenced by the level of cholesterol in the bilayer.