Stimulation of bile acid synthesis by dibutyryl cyclic AMP in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to examine the effects of dibutyryl cyclic AMP on the incorporation of¹⁴C-acetate and¹⁴C-cholesterol into bile acids. After an initial lag period, both precursors were incorporated into cholic and chenodeoxycholic acids at a linear rate for the subsequent 60 min. An apparent stimulation of bile acid formation from¹⁴C-acetate by dibutyryl cyclic AMP was complicated by the concomitant inhibition of cholesterol synthesis. In experiments with¹⁴C-cholesterol, dibutyryl cyclic AMP (1 mM) increased the labeled cholic and chenodeoxycholic acids in the medium by 83 and 224%, respectively, but cellular levels of labeled bile acids were unchanged. As a result, the nucleotide stimulated the overall incorporation of¹⁴C-cholesterol into cholic acid by 39% and into chenodeoxycholic acid by 123%. The mean ratio of labeled cholic to chenodeoxycholic acid declined from 55∶45 in control cells to 41∶59 in cells incubated with dibutyryl cyclic AMP. The results demonstrate that label incorporation can be used to study the regulation of bile acid synthesis in isolated hepatocytes. We propose that dibutyryl cyclic AMP enhances bile acid production by phosphorylating, and thus stimulating the activity of, cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis.     

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl₂ and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca²⁺/calmodulin; this latter effect was blocked by the chelator ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca²⁺, Ca²⁺/calmodulin, Mg²⁺ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca²⁺/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca²⁺/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.     

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg²⁺ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg²⁺. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg²⁺. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg²⁺ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg²⁺. This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North Carolina.     

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver

The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Effects of phorbol esters,A23187 and vasopressin on oleate metabolism in isolated rat hepatocytes

Studies were conducted to compare the metabolic effects of vasopressin, 4β-phorbol-12-myristate-13-acetate (PMA) and A23187 on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats. Vasopressin inhibited the formation of acid-soluble products from [1-¹⁴C]oleate (0.25 mM, 0.5 mM and 1 mM), the inhibition being most marked at low (0.25 mM) concentration of oleate. Conversion of [1-¹⁴C]oleate into¹⁴CO₂ and esterified products was stimulated by vasopressin. The stimulatory effect of this hormone on¹⁴CO₂ production was most marked at high (1 mM) concentration of oleate, whereas that on [1-¹⁴C]-oleate esterification was most marked at low (0.25 mM) concentration of oleate. These vasopressin actions were abolished when hepatocytes were incubated in the absence of calcium in the medium. Our results strongly suggest that both increase in esterification and increase in oxidation to CO₂ contribute to the anti-ketogenic action of vasopressin when oleate is added as substrate, although the relative extent of their contribution varies according to the oleate concentration. The anti-ketogenic action of vasopressin was mimicked by PMA but not by A23187. PMA also caused a stimulation of [1-¹⁴C]oleate esterification although the effect was diminished at 1 mM [1-¹⁴C]oleate. A23187 failed to affect [1-¹⁴C]oleate esterification. The metabolic effects of PMA were elicited in the absence of extracellular calcium, too. Conversion of [1-¹⁴C]oleate into¹⁴CO₂ was only slightly increased by both PMA and A23187 when 1 mM [1-¹⁴C]oleate was added as substrate. The marked stimulatory effect of vasopressin on¹⁴CO₂ production from [1-¹⁴C]oleate was not reproduced even by the combination of PMA and A23187. The possible involvement of protein kinase C and calcium mobilization in the regulation of oleate metabolism is discussed.     

Comparative effect of glucagon,dibutyryl cyclic AMP,and epinephrine on the desaturation and elongation of linoleic acid by rat liver microsomes

The effect of glucagon, dibutyryl cyclic adenosine 3′,5′-monophosphate, and epinephrine on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied. The incubations were performed with rat liver microsomes and labeled linoleic acid under desaturating and elongating conditions. Under desaturating conditions, linoleic acid was converted to γ-linolenic acid, whereas under elongating conditions it was converted to 20∶2ω6. Glucagon, dibutyryl cyclic AMP, and epinephrine decreased the oxidative desaturation of linoleic acid to γ-linolenic acid while the elongating reaction was not modified in the experimental conditions tested. Consequently, the results support the hypothesis that the oxidative desaturation of linoleic acid to γ-linolenic acid is the main controllable step in the biosynthesis of polyunsaturated fatty acids of the linoleic acid family in the microsomes.     

Stimulation of fatty acid synthesis by 4β-phorbol-12-myristate-13-acetate in isolated rat hepatocytes

The tumor-promoting agent 4β-phorbol-12-myristate-13-acetate (TPA) is shown to be a potent stimulator of fatty acid synthesis in isolated rat hepatocytes. The maximal effect of TPA is seen at 10⁻⁶ M, and the concentration for half-maximal effect is ca. 10⁻⁸ M. Stimulation of fatty acid synthesis by TPA is shown not to require the presence of extracellular Ca⁺⁺. TPA produces a significant increase in lactate and pyruvate accumulation. The possible involvement of protein kinase C in short-term regulation of fatty acid synthesis in the liver is discussed.     

Stimulation of a neutral triacylglycerol hydrolase from rat heart by phosphatidylethanolamine and lysophosphatidylethanolamine

Triacylglycerol hydrolase activity measured at pH 7.5 in a pH 5.2 precipitate fraction from rat heart was increased two-to three-fold by the presence of phosphatidylethanolamine (PE) or lysophosphatidylethanolamine (LPE). This stimulatory effect also could be obtained in assays with particulate and soluble subcellular fractions and was observed with two different methods of preparing triolein substrate emulsions. Ethanolamine and glycerophosphorylethanolamine had no effect on hydrolase activity, whereas phosphatidylcholine (PC) and acidic phospholipids such as cardiolipin were inhibitory. Palmitic acid, palmityl CoA and palmityl carnitine inhibited PE-stimulated hydrolase activity, but ethyl esters of palmitate had no effect. The preparation of acetone-ether powders resulted in a marked reduction of triacylglycerol hydrolase activity, but PE and LPE now stimulated hydrolase activity by ten-fold or greater, suggesting that these phospholipids may have an obligatory role in modulating triacylglycerol hydrolase activity. Triton X-100 also stimulated hydrolase activity in acetone-ether powders.     

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase,triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 ×g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the three activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.     

Purification of lysosomal cholesteryl ester hydrolase from rat liver by preparative isoelectric focusing

Ion-exchange chromatography and preparative isoelectric focusing (PIEF) were compared to produce a stable rat liver lysosomal cholesteryl ester hydrolase of high specific activity. The PIEF purification method proved to be more rapid and easier to perform. PIEF purification involved the following steps: i) osmotic shock of the lysosome fraction, ii) (NH₄)₂ SO₄ precipitation (10–70%, w/v), iii) Sepharose CL-6B gel filtration, and iv) PIEF. The enzyme was purified 60–120-fold with a yield of 2–4%. The activity of the purified enzyme was best restored by stabilizing with a 0.5% (w/v) albumin solution. The purified enzyme produced one major band on SDS-polyacrylamide gel electrophoresis having a MW of 58,500 daltons. Gel filtration showed a MW of 58,000 daltons. The optimum pH of the enzyme was 4.5, and the isoelectric point was 6.0–6.2. The specific activity of hydrolysis of cholesteryl oleate and triolein increased by similar rates during purification.     

Inhibition by 5-(tetradecyloxy)-2-furoic acid of fatty acid and cholesterol synthesis in isolated rat hepatocytes

Fatty acid and cholesterol synthesis in isolated rat hepatocytes were strongly inhibited by 5-(tetradecyloxy)-2-furoic acid. With either³H₂O or [2-¹⁴C]acetate as the labeled precursor, the concentrations of inhibitor causing 50% decrease in fatty acid and cholesterol synthesis were, respectively, <0.005 mM and 0.020 mM. At 0.1 mM inhibitor, citrate concentration in cells from fed rats was increased by 75%; lactate and pyruvate concentrations were decreased by 30%; ethanol oxidation was decreased by 20%; with cells from starved rats, the mitochondrial [NAD⁺]/[NADH] was decreased. Other parameters were unaffected. Both its potency and its specificity indicate that 5-(tetradecyloxy)-2-furoic acid will be useful in studies on the regulation of lipid biosynthesis.     

Cholesterol ester hydrolase activity in mammary tissue of the lactating rat

Cholesterol ester hydrolase activity has been studied in mammary glands of rats. Subcellular fractionation of the glands obtained in mid-lactation indicated that around 80% of the recovered activity was associated with particulate fractions. Two distinct cholesterol ester hydrolase activities were identified, one with an optimum pH of 7.5–9.0 and the second (approximately 5% of the total activity) with a more acidic pH optimum. Although the neutral cholesterol ester hydrolase had some properties in common with the lipoprotein lipase in mammary tissue, it was shown to be a separate entity by several criteria. Its activity could be increased following treatment with Mg-ATP and cAMP-dependent protein kinase, suggesting identity with the hormone sensitive lipase of adipose tissue. The cholesterol ester hydrolase activity in mammary glands just after parturition was greater than in glands obtained either from late-pregnant or midlactating animals. The subcellular distribution of the neutral cholesterol ester hydrolase suggested that it may have a different function to the neutral cholesterol ester hydrolase of adrenals and other tissues. Nevertheless the fact that the activity of the enzyme can be modulated by cAMP-dependent protein kinase suggests the possibility that hormonal control of this enzyme may be involved in the regulation of cholesterol metabolism in the mammary gland.     

Cholesterol metabolism in human monocyte-derived macrophages: Stimulation of cholesteryl ester formation and cholesterol excretion by serum lipoproteins

The role of lipoproteins and serum in the formation and accumulation of cholesteryl esters in human monocyte-derived macrophages (HMD macrophages) was investigated; studies were also carried out with IC21 cells (a cell line derived from mouse peritoneal macrophages). Following preincubation of HMD macrophages with lipoprotein-depleted serum (LPDS), both native and acetylated low density lipoprotein (LDL and AcLDL, respectively) stimulated the formation of cholesteryl esters with a resultant increase in cellular cholesteryl ester content. Cholesteryl ester formation and accumulation was also stimulated in macrophages exposed continuously to 25-hydroxycholesterol. However, the stimulation of cholesterol esterification by either lipoproteins or 25-hydroxycholesterol was not inhibited by progesterone in HMD macrophages, but was in the IC21 cells. Cholesterol efflux and the hydrolysis of cellular cholesterol ester, promoted by serum components, were studied in HMD macrophages preloaded with cholesteryl ester by incubation with 25-hydroxy cholesterol. Replacement of the medium with one devoid of 25-hydroxycholesterol resulted within 24 hr in at least a 30% decrease in the cholesteryl ester content of the HMD macrophages; replacement with a medium high in cholesterol acceptor content (LPDS or high density lipoprotein) and incubation for three days led to the most marked decreases in cellular cholesterol content. Thus, hydrolysis of the cholesteryl esters by HMD macrophages was not dependent on the presence of cholesterol acceptors in the medium, but cellular cholesterol content was.     

A diurnal variation of hepatic acid cholesteryl ester hydrolase activity in the rat

The diurnal variation in lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) has been examined in fed, fasted and adrenalectomized rats. The Acid CEH activity of normal rat liver exhibits a diurnal rhythm with maxima at 06.00 hours and minima at 18.00 hours, but such a rhythm was not observed in spleen and brain. This rhythm was abolished after fasting for two days, and the resulting Acid CEH activity remained constant at the minimum level. However, adrenalectomy did not abolish the diurnal rhythm. These results indicate that the Acid CEH activity varies according to a diurnal rhythm with maxima and minima separated by approximately 12 hr. Further, it is evident that the appearance of this rhythm is dependent upon dietary, but not adrenal hormone influence.     

Characterization of a cytosolic protein in rat liver inhibiting neutral cholesteryl ester hydrolase

Neutral cholesteryl ester hydrolase activity (EC 3.1.1.13) present in microsomes isolated from lactating rat mammary glands was found to be inhibited by a factor (or factors) occurring in the cytosolic fraction of male rat liver. The inhibitor was heat-labile, non-dialyzable, destroyed by proteolysis, and was stable following preparation of an acetone/diethyl ether powder of the cytosolic fraction. The protein also inhibited the activity of hormone-sensitive lipase (HSL) (from bovine adipose tissue) and esterase fromCandida cylindracea, but seemed to be more active against the neutral hydrolase found in rat liver microsomes. For the mammary gland microsomal cholesteryl ester hydrolase, the extent of the inhibitory effect was dependent on the concentration of the cytosolic protein, 50% inhibition being achieved by about 100 μg of cytosolic protein, and on the method of initiating the enzyme assay. Kinetic analysis indicated that, under circumstances where the reaction was initiated by the addition of substrate, the inhibition was characterized as “uncompetitive”. When an inhibitor/substrate complex was allowed to form in the absence of enzyme, an element of “competitive” inhibition was introduced into the reaction. Food withdrawal reducted the activity of the inhibitor in live by 56%, but activity was fully restored by short-term re-feeding. In contrast, feeding a diet high in fat led to a 34% increase in activity. The present findings suggest that the inhibitory factor(s) may be involved in the regulation of the hydrolysis of cholesteryl esters in the liver and also in other cell types.     

Levels of cyclic 3′-5′-adenosine monophosphate (cAMP) in maintenance cultures of rat hepatocytes in response to insulin and glucagon

Under conditions of short-term stimulation of lipogenesis by insulin in maintenance cultures of hepatocytes from starved rats, basal levels of cyclic 3′-5′-adenosine monophosphate (cAMP) are invariant. Glucagon signals increased levels of cAMP and severely diminishes lipogenesis. Insulin partially counteracts both glucagon effects.     

Evidence that cholesteryl ester hydrolase and triglyceride lipase are different enzymes in rat liver

Studies on intracellular cholesteryl ester hydrolase (CEH) and triglyceride lipase (TGL) from rat adipose tissue and adrenal cortex have suggested that a single protein is responsible for both activities. To determine whether one hepatic protein catalyzes both reactions, we studied several properties of CEH and TGL in rat liver. During liver perfusion with heparin, perfusate peaks of TGL and CEH did not consistently coincide, and TGL activity was considerably higher and less heat-stable than that of CEH. Significant TGL, but not CEH, activity was released during incubation of isolated hepatocytes. Although microsomes isolated from hepatocytes contained both activities, the specific activities of CEH and TGL in cytosol from hepatocytes were 95% and 3%, respectively, of those found in cytosol from whole liver. Preincubation of liver cytosol with 5 mM Mg²⁺ decreased CEH, but not TGL, activity. Intracellular CEH and TGL activities were completely separated by prep-disc gel electrophoresis. Finally, both cytosolic and microsomal TGL, but not CEH, activities were inhibited by antiserum against rat hepatic TGL. We conclude that extracellular TGL does not have CEH activity and intracellular CEH differs from TGL.     

Dual action of neutral sphingomyelinase on rat hepatocytes: Activation of cholesteryl ester metabolism and biliary cholesterol secretion and inhibition of VLDL secretion

To address the role of cell membrane neutral sphingomyelinase (EC 3.1.4.12; SMase) in the regulation of cholesterol metabolism in the liver parenchymal cell, we examined the effect of exogenous neutral SMase on the metabolism of cholesteryl esters and the secretion of VLDL and biliary lipids in isolated rat hepatocytes. We show that treatment of hepatocytes with SMase (20 mU/mL) resulted in the intracellular buildup of cholesteryl esters, increased ACAT (EC 2.3.1.26) activity without affecting the ACAT2 mRNA level, and increased cytosolic and microsomal cholesteryl ester hydrolase (EC 3.1.1.13) activity. This was accompanied by increases in the secretion of biliary. bile acid, phospholipid, and cholesterol and in increased cholesterol 7α-hydroxylase (EC 1.14.13.17) activity and levels of mRNA, as well as decreased levels of apoB mRNA and a decreased secretion of VLDL apoB (apoB-48, ∼45%; apoB-100, ∼32%) and lipids (∼55%). Moreover, the VLDL particles secreted had an abnormal size and lipid composition; they were larger than controls, were relatively enriched in cholesteryl ester, and depleted in TG and cholesterol. Cell-permeable ceramides did not replicate any of the reported effects. These findings demonstrate that the increased cholesteryl ester turnover, oversecretion of biliary cholesterol and bile acids, and undersecretion of VLDL cholesterol and particles are concerted responses of the primary hepatocytes to exogenous neutral SMase brought about by regulation at several levels. We suggest that plasma membrane neutral SMase may have a specific, ceramide-independent effect in the regulation of cholesterol out-put pathways in hepatocytes.