Acetoacetate: a major substrate for the synthesis of cholesterol and fatty acids by isolated rat hepatocytes

The diuretic drug amiloride antagonises the insulin-dependent increase in phosphorylation of ATP-citrate lyase in hepatocytes isolated from rats that had been starved and refed a fat-free diet. Studies with a range of protein kinases and protein phosphatases that have been shown to phosphorylate or dephosphorylate purified ATP-citrate lyase in vitro revealed that amiloride was a non-specific inhibitor of all protein kinases tested, but did not significantly affect any of the protein phosphatases. These results cast doubt on previous claims that growth factors stimulate phosphorylation of ribosomal protein S6 by activating an amiloride-sensitive Na+/H+ exchange system, and that insulin inhibits a protein phosphatase that is activated by amiloride.     

The effects of lactate and acetate on fatty acid and cholesterol biosynthesis by isolated rat hepatocytes

1. The present study demonstrates that lactate and acetate stimulate fatty acid synthesis and inhibit cholesterogenesis by isolated rat hepatocytes. 2. Exposure of the intact cells to lactate increases the activity of acetyl-CoA carboxylase, as can be measured in homogenates of these cells. A similar effect by acetate was not observed. 3. Both acetate and lactate drastically increase the cellular level of citrate. 4. Possible mechanisms underlying the difference in response of fatty acid and cholesterol synthesis to an increase in substrate availability are discussed. Futhermore, a mechanism is proposed for the lactate effect on acetyl-CoA carboxylase.     

The role of substrate supply in the regulation of cholesterol biosynthesis in rat hepatocytes.

1. Compactin, (-)-hydroxycitrate and dexamethasone gave rise to a decrease in the rate of cholesterol production in hepatocytes from fed rats by interfering with the flow of substrate into the sterol biosynthetic pathway. The cells responded to the deficit of biosynthetic sterol by increasing the activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase). 2. Compactin and (-)-hydroxycitrate gave similar results in hepatocytes from rats starved for 24 h but in this case dexamethasone had no significant effect. 3. Exogenous oleate interferes with the production of carbohydrate-derived acetyl-CoA and also gives rise initially to opposing effects on the rate of sterol synthesis and HMG-CoA reductase activity. Over a longer period, however, oleate itself was capable of replacing carbohydrate as the major source of carbon for sterol synthesis. 4. The increase in HMG-CoA reductase activity observed when liver cells were incubated in the presence of compactin, (-)-hydroxycitrate or oleate could be partially reversed by the simultaneous presence of glucagon. 5. Under some physiological conditions, a deficiency of biosynthetic cholesterol or of a related precursor may lead to an increase in the activity of HMG-CoA reductase.     

Control of cholesterol biosynthesis,uptake and storage in hepatocytes by Cideb

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.     

Formation of cyclic AMP from exogenous ATP by isolated hepatocytes and adipocytes

Suspensions of isolated rat hepatocytes and adipocytes converted exogenous ATP to cyclic AMP at a rate which was about 30--50% of that observed with homogenates of isolated cells. Formation of cyclic AMP was stimulated by hormones (isoprenaline in the case of adipose tissue and glucagon in the case of liver) and sodium fluoride. Experiments with [alpha-32P]ATP indicated that the conversion of exogenous ATP to cyclic AMP did not occur within the cells. It is proposed that in isolated hepatocytes ad adipocytes some catalytic units of adenylate cyclase are exposed on the outer surface of the cell membrane.     

Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate

The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.     

Immunohistochemical identification of endogenous sources of cardiac glycoside biosynthesis

Using immunohistochemical technique, digoxin-containing cells were detected in some brain areas, liver, pancreas and heart. The possible existence of endogenous sources of cardiac glycoside biosynthesis is suggested. The substances under study are termed "endocorsides".     

Utilization of endogenous lipid by the isolated perfused rat heart

1. The lipids of the rat heart have been studied with regard to amount, classes present and fatty acid composition of free fatty acids, triglycerides and phospholipids. Myocardial lipid contained 300μmoles of total fatty acid/g. dry wt. of which only 2–4μmoles were free; the remainder was esterified, chiefly as phospholipid. Neutral esters, of which triglyceride was the principal form, made up 15% of the total fatty acids. 2. When normal hearts were perfused with a nutrient-free medium until exhaustion, the triglyceride concentration declined from 43 to 13μmoles/g. dry wt. The content of phospholipids, partial glycerides and cholesteryl esters did not change. When the lipids of the rat heart were labelled with [1-¹⁴C]palmitate before perfusion with non-nutrient medium, radioactivity disappeared from the triglyceride, diglyceride and free fatty acid fractions, but not from the phospholipid or other ester classes. 3. These experiments support the view that only a small fraction of the total cardiac lipid, principally triglycerides and to a smaller extent diglycerides, is available as a source of fuel in the absence of exogenous substrate.     

Purine metabolism and urate biosynthesis in isolated chicken hepatocytes

The metabolism of some purine compounds to urate and their effects on de novo urate synthesis in chicken hepatocytes were investigated. The purines, listed in descending order of rates of catabolism to urate, were hypoxanthine, xanthine, inosine, guanosine, guanine, IMP, GMP, adenosine, AMP, and adenine. During a 1-h incubation period, conversion to urate accounted for more than 80% of the total quantities of guanine, guanosine, and inosine metabolized, but only 42% of the adenosine and 23% of the adenine metabolism. Adenine, adenosine, and AMP inhibited de novo urate synthesis [( 14C]formate incorporation into urate), whereas the other purines, especially guanine, guanosine, and GMP, stimulated de novo urate synthesis. When hepatocytes were incubated with glutamine and adenosine, AMP, guanine, guanosine, or GMP, the rates of de novo urate synthesis were lower than the additive effects of glutamine and the purine in separate incubations. Increasing phosphate concentrations had no effect on urate synthesis in the absence of added purines but, in combination with adenosine, AMP, guanosine, or GMP, increased urate synthesis. These results indicate that the ratio of adenine to guanine nucleotides and the interaction between substrates and purine nucleotides are involved in the regulation of urate biosynthesis in chicken liver.     

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol to taurocholic acid; however, taurochenodeoxycholic acid synthesis was stimulated. 25-30% of the cholesterol synthesized from mevalonolactone was converted to taurochenodeoxycholic, taurocholic and two so-far unidentified bile acids. All bile acids were secreted into the incubation medium. When cholesterol was added as mixed liposomes with phosphatidylcholine, it was taken up by the cells and converted to bile acids. At low concentrations of liposomes, the greater part of the cholesterol which was taken up by the cells was converted to bile acids. At higher concentrations, considerable amounts of cholesterol and cholesterol ester accumulated inside the cells. When mevalonolactone and cholesterol liposomes was added together, both substrates were used simultaneously for bile acids synthesis. HDL cholesterol was the best substrate tested, yielding large amounts of two, so-far, unidentified bile acids (possibly allo-bile acids) and smaller amounts of taurocholic and taurochenodeoxycholic acid. Addition of HDL suppressed the conversion of endogenous cholesterol to taurocholic acid; taurochenodeoxycholic acid synthesis, however, was stimulated.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.     

cAMP levels from endogenous and exogenous arachidonic acid in isolated dog lung

We infused exogenous arachidonic acid (AA) into salt-perfused isolated dog lungs. This led to elevations in adenosine 3',5'-cyclic monophosphate (cAMP) which were from conversion of the AA to cyclooxygenase products. The maximal levels of cAMP occurred at far less than maximal levels of cyclooxygenase products. Next, we infused A 23187 to release endogenous pulmonary AA. This led to elevations in cAMP that were from conversion of this endogenous AA to cyclooxygenase products. The level of these products was far less than maximal levels from exogenous AA. However, maximal levels of cAMP from conversion of endogenous AA were similar to maximal levels of cAMP from conversion of exogenous AA. We conclude that maximal levels of pulmonary cAMP from endogenous or exogenous AA are from conversion of the AA to far less than maximal levels of pulmonary cyclooxygenase products. This indicates that levels of cAMP rather than levels of cyclooxygenase products are a potential rate-limiting step in cAMP-linked pulmonary actions of such products from pulmonary conversion of endogenous or exogenous AA.     

Secretion of lecithin: cholesterol acyltransferase from isolated rat hepatocytes.

1. Lecithin:cholesterol acyltransferase is secreted from isolated rat heptocytes. 2. The secretion is stimulated when serum is added to the incubation medium. 3. Optimal conditions for secretion are: 5-10(6) hepatocytes per ml, 5 h incubation, pH 7.3-7.4 and 25% serum in the incubation medium. 4. Concomitantly with the secretion of lecithin:cholesterol acyltransferase there is a secretion of unesterified cholesterol and triacylglycerol. 5. Colchicine or cycloheximide in the incubation medium inhibits secretion of lecithin:cholesterol acyltransferase.     

外源IBA对无花果扦插苗生理特性及内源IAA生物合成途径的影响

【目的】随着区域栽培环境的变化,扦插苗生根难问题逐渐凸显。探讨适宜浓度吲哚丁酸(IBA）对无花果插穗生根萌芽、抗氧化性及生长素生物合成途径相关基因表达的影响,可为其应用于无花果的育种、繁殖、推广和种植提供理论依据。【方法】以‘波姬红’无花果品种硬枝为插穗,分析不同质量浓度IBA(0,30,45,60,90 mg/L)处理对插穗生根性状、抗氧化特性的影响,并对45 mg/L IBA处理及对照组的扦插枝条中段的腋芽进行转录组分析。【结果】（1）无花果插穗萌芽率和生根率在45 mg/L IBA处理时达到最大值,并与其他处理和对照差异显著。（2）随着IBA浓度的增加,插穗SOD和CAT活性先下降后上升,并均在45,60 mg/LIBA处理下显著低于对照,而POD活性无显著变化;各浓度IBA处理插穗中MDA和H2O2含量均显著高于对照,且45 mg/L IBA处理MDA显著低于其余处理。（3）45 mg/L IBA处理及对照组中共存在6 879个差异表达基因,KEGG富集显示有10个差异途径,GO富集分析表明生物学过程和分子功能为主要的生物学途径;与CAT、SOD相关的基因集中富集在过氧化物酶体通路上,POD相关基因则富集在苯丙烷生物合成通路中;IAA生物合成途径中代谢相关基因FcGH3显著上调表达,与信号转导相关基因FcAUX1、FcARG7和FcARF等显著下调表达。【结论】外源IBA处理会导致无花果插穗抗氧化酶和IAA生物合成途径中相关基因表达的差异变化,增强插穗抗逆性,促进插穗生根、萌芽、成苗,并以外源45 mg/L IBA促进效果最好。