Infectious agents are not necessary for murine atherogenesis.

Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide. Atherogenesis was unaffected in doubly deficient animals. We further tested the role of infectious agents by creating a colony of germ-free apo E(-/)- mice. These animals are free of all microbial agents (bacterial, viral, and fungal). Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge. These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis.

The time-course development of the effects of ethanol ingestion on choline oxidase.

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumin resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.

Hepatic responses to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase: a comparison of atorvastatin and simvastatin.

We have compared the cellular responses to simvastatin (Simva) and atorvastatin (Atorva), two potent HMG-CoA reductase inhibitors. The two drugs exhibited similar IC50's for inhibition of either rat or human reductase, and single oral dosing in rats showed the compounds to be nearly equipotent at inhibiting hepatic cholesterol synthesis. Treatment of rats with Simva or Atorva in the feed for four days yielded comparable inductions of hepatic reductase activity and reductase protein. For example, 0.05% Simva induced reductase activity 27.3 +/- 9.1 fold and 0.05% Atorva induced activity 26.9 +/- 4.7 fold. This adaptive response was also studied in HepG2 cells, a human hepatoblastoma line, cultured for 24 h in delipidated serum and then for an additional 24 h with Simva or Atorva. Over a broad range (10 nM-10 microM), both drugs caused similar inductions of reductase activity, reductase protein, and reductase mRNA. Under all conditions, the drugs induced similar changes in the ratio of mRNA/protein suggesting that Simva and Atorva have similar effects on both transcriptional and post-transcriptional regulatory machinery. Moreover, reductase in cells treated with Simva or Atorva for 22 h responded similarly to subsequent challenge with 25-hydroxycholesterol. Finally, we measured the ability of the two reductase inhibitors to reduce ApoB secretion by HepG2 cells. Simva and Atorva at 0.5 microM inhibited ApoB secretion nearly identically, 38% and 42% respectively. We conclude that these two drugs induce similar adaptive responses in cells and that their actions are qualitatively and mechanistically identical. Human studies have shown that plasma is cleared of Atorva much more slowly than it is of Simva. The large pharmacokinetic difference in man, rather than some difference in mechanism, is the most likely explanation for the finding that the equipotent dose ratio for cholesterol lowering in humans of Simva to Atorva is about 2/1.

Selective protection and relative importance of the carboxylic acid groups of zaragozic acid A for squalene synthase inhibition.

Chemistry that allows selective modification of the carboxylic acid groups of the squalene synthase inhibitor zaragozic acid A (1) was developed and applied to the synthesis of compounds modified at the 3-,4-,5-,3,4-,3,5-, and 4,5-positions. A key step in this procedure is the selective debenzylation by transfer hydrogenolysis in the presence of other olefinic groups. These compounds were tested in the rat squalene synthase assay and in vivo mouse model. Modification at C3 retains significant enzyme potency and enhances oral activity, indicating that C3 is not essential for squalene synthase activity. Modification at C4 and C5 results in significant loss in enzyme activity. In contrast, substitution at C3 or C4 enhances in vivo activity. Furthermore, disubstitution at the C3 and C4 positions results in additive in vivo potency.

Clavaric acid and steroidal analogues as Ras- and FPP-directed inhibitors of human farnesyl-protein transferase.

We have identified a novel fungal metabolite that is an inhibitor of human farnesyl-protein transferase (FPTase) by randomly screening natural product extracts using a high-throughput biochemical assay. Clavaric acid [24, 25-dihydroxy-2-(3-hydroxy-3-methylglutaryl)lanostan-3-one] was isolated from Clavariadelphus truncatus; it specifically inhibits human FPTase (IC50 = 1.3 microM) and does not inhibit geranylgeranyl-protein transferase-I (GGPTase-I) or squalene synthase activity. It is competitive with respect to Ras and is a reversible inhibitor of FPTase. An alkaline hydrolysis product of clavaric acid, clavarinone [2,24,25-trihydroxylanostan-3-one], lacking the 3-hydroxy-3-methylglutaric acid side chain is less active as a FPTase inhibitor. Similarly, a methyl ester derivative of clavaric acid is also inactive. In Rat1 ras-transformed cells clavaric acid and lovastatin inhibited Ras processing without being overtly cytotoxic. Excess mevalonate reversed the effects of lovastatin but not of clavaric acid suggesting that the block on Ras processing by clavaric acid was due to inhibition of FPTase and not due to inhibition of HMG-CoA reductase. Despite these results, the possibility existed that clavaric acid inhibited Ras processing by directly inhibiting HMG-CoA reductase. To directly examine the effects of clavaric acid and clavarinone on HMG-CoA reductase, cholesterol synthesis was measured in HepG2 cells. No inhibition of HMG-CoA reductase was observed indicating that the inhibition of Ras processing by this class of compounds is due to inhibition of FPTase. To date, clavaric acid is the second reported nitrogen-free compound that competes with Ras to inhibit FPTase activity. A series of related compounds derived from computer-based similarity searches and subsequent rational chemical synthetic design provided compounds that exhibited a range of activity (0.04 --> 100 microM) against FPTase. Modest changes in the structures of these inhibitors dramatically change the inhibitory activity of these inhibitors.

Impaired response to hepatocyte growth factor in FRTL-5 rat thyroid cells expressing a functional hepatocyte growth factor receptor.

The FRTL-5 rat thyroid cell line is widely used for studies of thyrocyte function and growth. The objective of the present study was to investigate the effects of hepatocyte growth factor (HGF) on FRTL-5 cells. HGF has previously been known to be a potent regulator of thyrocyte growth and differentiation. Met, the receptor for HGF was expressed in FRTL-5 cells, as well as in primary cultures of porcine thyrocytes included in the study as control. On HGF stimulation Met was tyrosine phosphorylated in both porcine and FRTL-5 cells, indicating an activation of the receptor. Addition of HGF induced changes of cell shape, scattering and proliferation of the porcine thyrocytes, but not in the FRTL-5 cells; yet, a functional coupling of Met to the p85 subunit of the phosphatidylinositol-3'-kinase (PI3'-K) in coprecipitation experiments, formation of focal adhesions detected in immunofluorescence staining with an antivinculin antibody, and induction of c-fos mRNA in Northern blot analysis was observed in FRTL-5 cells, showing a transduction of the HGF/Met signal. In summary, despite the expression of apparently functional Met, the FRTL-5 cells are unable to properly respond to HGF.

Acetoacetyl-CoA ligase activity in the isolated rat hepatocyte: effects of 25-hydroxycholesterol and high density lipoprotein.

The activity of acetoacetyl-CoA (AcAc-CoA) ligase (E.C.6.2.1.16) in hepatocytes from rats was shown to be the same as the activity in homogenates of their livers. In hepatocytes treated with 25-hydroxycholesterol, AcAc-CoA ligase, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and rates of sterol synthesis were substantially decreased. Hepatocytes treated with high density lipoprotein (HDL) exhibited a 2 to 4 fold induction of HMG-CoA reductase activity; however an accompanying increase in AcAc-CoA ligase activity and the rate of cholesterol synthesis was not observed. We conclude (a) that increases in the activity of HMG-CoA reductase when mediated by HDL in hepatocytes do not result in a corresponding change in the capacity for sterol synthesis and (b) that changes in the activity state of HMG-CoA reductase can be dissociated from that of AcAc-CoA ligase.

The preparation of zaragozic acid A analogues by directed biosynthesis.

Zaragozic acid A analogues are produced by an unidentified sterile fungus when it is exogenously supplied with 2-thiophenecarboxylic acid, 3-thiophenecarboxylic acid, 2-furoic acid, 2-fluorobenzoic acid, 3-fluorobenzoic acid, or 4-fluorobenzoic acid. The analogues carry 2-thiophenyl, 3-thiophenyl, 2-furyl, o-fluorophenyl, m-fluorophenyl, or p-fluorophenyl group, respectively, at C-6' of the C-1 alkyl side chain replacing the phenyl group of natural zaragozic acid A. All the new analogues of zaragozic acid A possess picomolar inhibitory activity against squalene synthase in vitro.

A radiochemical assay for acetoacetyl-CoA synthetase.

A sensitive radiochemical assay is described for the assay of acetoacetyl-CoA synthetase activity in cytosolic extracts. Enzyme activity is measured by the incorporation of 14C from acetoacetate into acetyl carnitine as mediated by acetoacetyl-CoA synthetase, endogenous acetoacetyl-CoA thiolase, and exogenous carnitine acetyl transferase. Separation of 14C-labeled reactants from 14C-labeled acetyl carnitine is achieved by cation-exchange chromatography. The assay is sensitive with less than 10 pmol of product readily detected. Acetoacetyl-CoA synthetase activity was measured in human fibroblasts, 0.12 nmol min-1 mg cytosolic protein-1, and was found to be more than two orders of magnitude below the activity level of acetoacetyl-CoA synthetase in rat liver cytosol, 18.4 nmol min-1 mg cytosolic protein-1. An HPLC method is also described for the purification of [3-14C]acetoacetate.

Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells.

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.

Discovery, biosynthesis, and mechanism of action of the zaragozic acids: potent inhibitors of squalene synthase.

The zaragozic acids (ZAs), a family of fungal metabolites containing a novel 4,6,7-trihydroxy-2,8-dioxobicyclo[3.2.1]octane-3,4,5-tricarboxylic acid core, were discovered independently by two separate groups screening natural product sources to discover inhibitors of squalene synthase. This family of compounds all contain the same core but differ in their 1-alkyl and their 6-acyl side chains. Production of the ZAs is distributed over an extensive taxonomic range of Ascomycotina or their anamorphic states. The zaragozic acids are very potent inhibitors of squalene synthase that inhibit cholesterol synthesis and lower plasma cholesterol levels in primates. They also inhibit fungal ergosterol synthesis and are potent fungicidal compounds. The biosynthesis of the zaragozic acids appears to proceed through alkyl citrate intermediates and new members of the family have been produced through directed biosynthesis. These potent natural product based inhibitors of squalene synthase have potential to be developed either as cholesterol lowering agents and/or as antifungal agents.

Massive production of farnesol-derived dicarboxylic acids in mice treated with the squalene synthase inhibitor zaragozic acid A.

The zaragozic acids are potent inhibitors of squalene synthase. In vivo studies in mice confirmed our earlier observations that inhibition of squalene synthase by zaragozic acid A was accompanied by an increase in the incorporation of label from [3H]mevalonate into farnesyl-diphosphate (FPP)-derived isoprenoic acids (J. D. Bergstrom et al., 1993, Proc. Natl. Acad. Sci. USA 90, 80-84). Farnesyl-diphosphate-derived metabolites appear transiently in the liver. We were unable to detect any farnesol formation in the zaragozic acid-treated animals which indicates that FPP is readily converted to farnesoic acid and dicarboxylic acids in the liver. These metabolites were found to be produced only in the liver and not in the kidney. trans-3,7-Dimethyl-2-octaen-1,8-dioic acid and 3, 7-dimethyloctan-1,8-dioic acid were identified as the major end products of farnesyl-diphosphate metabolism in the urine of mice treated with zaragozic acid A. Quantitative analysis of these FPP-derived dicarboxylic acids by gas-liquid chromatography revealed that approximately 11 mg of total dicarboxylic acids is excreted per day into the urine of a mouse after 3 days of treatment with zaragozic acid A.

Non-autocrine, constitutive activation of Met in human anaplastic thyroid carcinoma cells in culture.

Activation of Met by its ligand HGF has been shown to elicit both mitogenic and motogenic responses in thyrocytes in vitro. In the present study we have investigated the expression of Met in human anaplastic thyroid carcinoma cells in culture. There was a variation in expression level and size of Met in the different cell lines; high Met expression was found in four cell lines, compared to non-neoplastic human thyrocytes. Treatment with glucoproteinase F showed that the size differences observed were due to variances in the degree of glycosylation. Interestingly, in cell lines with high expression of Met, the receptor proteins were found to be constitutively tyrosine phosphorylated. None of these cell lines expressed HGF mRNA, and addition of suramin did not affect the level of tyrosine phosphorylation of Met in unstimulated cells, suggesting the absence of autocrine stimulatory pathways. Furthermore, we did not observe MET gene amplification, activating mutations or phosphatase defects. The tyrosine phosphorylated receptors appeared functionally active since the receptors associated with the adaptor molecule Shc. In summary, we have found ligand-independent constitutively activated Met in four out of six anaplastic thyroid carcinoma cell lines.

Ketone body metabolism in the neonate: development and the effect of diet.

In the course of mammalian development milk has evolved with unique characteristics as has the capacity of the neonatal rat to process this nutrient source. The primary carbon source in milk is fat, which provides two readily utilized metabolites, acetoacetate and D(-)-3-hydroxybutyrate (ketone bodies), as well as free fatty acids and glycerol. Carbohydrate provides less than 12% of the caloric content of rat milk and glucose has to be produced by the suckling rat to maintain glucose homeostasis. One would predict that glucose would be used sparingly and in pathways that cannot be satisfied by other readily available metabolites. Studies of the uptake of metabolites and the development of key enzymes for the utilization of glucose and ketone bodies by developing brain support the concept that ketone bodies are preferred substrates for the supply of carbon to respiration and lipogenesis. Astrocytes, oligodendrocytes, and neurons from developing brain all have an excellent capacity to use ketone bodies for respiration. By contrast, glucose is utilized preferentially in the hexose monophosphate shunt by all three cell populations. We are examining the requirement for ketone bodies by developing brain with the application of a system to rear rat pups artificially on a milk substitute that promotes a hypoketonemia.

Metabolism in the artificially reared rat pup: effect of an atypical rat milk substitute.

A substitute for rat milk [Messer et al., 1969 (1)] has been evaluated as a nutrient source to artificially feed rat pups from 4 days after birth. The rat milk substitute has a normal fat concentration, suboptimal protein concentration and a high carbohydrate concentration when compared to natural rat milk. Rat pups artificially reared on the mild substitute by intermittent infusion via miniaturized intragastric cannulae have: 1) atypical ketone body metabolism: lower than normal concentration and turnover of D-(--)-3-hydroxybutyrate in blood and less than normal amounts of D-(--)-3-hydroxybutyrate used for respiration, 2) atypical carbohydrate metabolism: higher than normal insulin and galactose concentrations in blood and a greater than normal amount of glucose used for respiration, and 3) atypical amino acid levels: the concentrations of several amino acids in blood were 60% or less than normal, and the concentration of taurine in plasma was negligible. We observed frequent head tremors, hyperreactivity to handling and about a 20% incidence of cataracts in rat pups reared on the milk substitute. We conclude this rat milk substitute is not suitable as a nutrient source for the developing rat pup.

Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture.

Neuron, astrocyte, and oligodendrocyte cultures which were established from developing rat brain were examined for their utilization of glucose, ketone bodies, and free fatty acids by oxidative processes. 14CO2 production was measured in these cells from [1-14C] or [6-14C]glucose; [1-14C]octanoate and [1-14C], [6-14C], or [16-14C]palmitate; and [3-14C]acetoacetate and D(-)-3-hydroxy[3-14C]butyrate. Pyruvate dehydrogenase (EC 1.2.4.1.) and 3-oxoacid-CoA transferase (EC 2.8.3.5) activities were found at high levels in each of the cell populations. Astrocytes and oligodendrocytes produced much more 14CO2 from [1-14C]glucose than from [6-14C]glucose, indicating substantial hexose monophosphate shunt activity. This process was not as active in neurons. All three cell populations readily utilized the ketone bodies for oxidative metabolism at rates 7-9 times greater than they utilized glucose. Only astrocytes were able to utilize fatty acids for 14CO2 production, and the rate of utilization was greater than that of the ketone bodies. We found that the metabolic patterns of these brain cells which were derived from the developing brain complement the nature of the diet of the suckling animal which is rich in fat and low in carbohydrate. They readily utilized the ketone bodies or fatty acids and spared glucose for processes that metabolites of fat cannot fulfill.

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat.

1. Procedures are described to prepare nutritionally adequate rat milk-substitutes by modifying commercially available processed cow's milk, rich in carbohydrate and low in protein and fat compared with rat's milk. 2. Premilk formulas, prepared as intermediates in the preparation of rat milk-substitutes, are rich in protein but low in their concentration of fat, carbohydrate, and minerals when compared with rat's milk. 3. Premilks were supplemented with lactose, vitamins, minerals, fat as oil mixtures, certain amino acids and other constituents to yield rat milk-substitutes which resemble the known composition of rat's milk in their properties and composition. 4. Detailed analyses of the milk-substitutes show them to be comparable to rat's milk in energy content, pH, osmolarity, the concentration of the macronutrients, fat, protein and carbohydrate, and the major minerals. 5. Rat pups were artificially reared from postnatal day 4 or 5 until days 16-18 by fitting them with gastric cannulas through which the milk-substitutes could be infused automatically. 6. The nutritional impact of the milk-substitutes was assessed by a comparison of growth and metabolic characteristics for artificially reared rats with age-matched sucking rats reared by their mother. 7. Indices which were taken to be appropriate included (a) body-weight gain; (b) the concentration in blood of protein, amino acids, ketone bodies, carnitine, glucose, galactose, lactate, insulin, and the electrolytes calcium, sodium, potassium and chloride; (c) the turnover of glucose and 3-hydroxybutyrate; (d) the concentration in brain of protein, cholesterol, cerebroside sulphate and the activities of the enzymes pyruvate dehydrogenase (EC 1.2.4.1), 3-oxo-acid-CoA transferase (EC 2.8.3.5) and acetoacetyl-CoA ligase (EC 6.2.1.16). 8. The studies suggest that milk-substitutes approximating to rat's milk in composition promote acceptable metabolism in the artificially reared rat pup.