Tumor growth reduction is regulated at the gene level in Walker 256 tumor-bearing rats supplemented with fish oil rich in EPA and DHA.

We investigated the effect of fish oil (FO) supplementation on tumor growth, cyclooxygenase 2 (COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and RelA gene and protein expression in Walker 256 tumor-bearing rats. Male Wistar rats (70 days old) were fed with regular chow (group W) or chow supplemented with 1 g/kg body weight FO daily (group WFO) until they reached 100 days of age. Both groups were then inoculated with a suspension of Walker 256 ascitic tumor cells (3 × 10(7) cells/mL). After 14 days the rats were killed, total RNA was isolated from the tumor tissue, and relative mRNA expression was measured using the 2(-ΔΔCT) method. FO significantly decreased tumor growth (W=13.18 ± 1.58 vs WFO=5.40 ± 0.88 g, P<0.05). FO supplementation also resulted in a significant decrease in COX-2 (W=100.1 ± 1.62 vs WFO=59.39 ± 5.53, P<0.001) and PPARγ (W=100.4 ± 1.04 vs WFO=88.22 ± 1.46, P<0.05) protein expression. Relative mRNA expression was W=1.06 ± 0.022 vs WFO=0.31 ± 0.04 (P<0.001) for COX-2, W=1.08 ± 0.02 vs WFO=0.52 ± 0.08 (P<0.001) for PPARγ, and W=1.04 ± 0.02 vs WFO=0.82 ± 0.04 (P<0.05) for RelA. FO reduced tumor growth by attenuating inflammatory gene expression associated with carcinogenesis.

Tumor growth reduction is regulated at the gene level in Walker 256 tumor-bearing rats supplemented with fish oil rich in EPA and DHA

We investigated the effect of fish oil (FO) supplementation on tumor growth, cyclooxygenase 2 (COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and RelA gene and protein expression in Walker 256 tumor-bearing rats. Male Wistar rats (70 days old) were fed with regular chow (group W) or chow supplemented with 1 g/kg body weight FO daily (group WFO) until they reached 100 days of age. Both groups were then inoculated with a suspension of Walker 256 ascitic tumor cells (3×107 cells/mL). After 14 days the rats were killed, total RNA was isolated from the tumor tissue, and relative mRNA expression was measured using the 2-ΔΔCT method. FO significantly decreased tumor growth (W=13.18±1.58 vs WFO=5.40±0.88 g, P<0.05). FO supplementation also resulted in a significant decrease in COX-2 (W=100.1±1.62 vs WFO=59.39±5.53, P<0.001) and PPARγ (W=100.4±1.04 vs WFO=88.22±1.46, P<0.05) protein expression. Relative mRNA expression was W=1.06±0.022 vs WFO=0.31±0.04 (P<0.001) for COX-2, W=1.08±0.02 vs WFO=0.52±0.08 (P<0.001) for PPARγ, and W=1.04±0.02 vs WFO=0.82±0.04 (P<0.05) for RelA. FO reduced tumor growth by attenuating inflammatory gene expression associated with carcinogenesis.

Crystal structure of a class I ubiquitin conjugating enzyme (Ubc7) from Saccharomyces cerevisiae at 2.9 angstroms resolution.

Ubiquitin-conjugating enzymes are a family of related proteins that participate in the ubiquitination of proteins. Previous studies on the crystal structures of Saccharomyces cerevisiae Ubc4 and Arabidopsis thaliana Ubc1 indicated that the smallest enzymes (class I), which consist entirely of the conserved core domain, share a common tertiary fold. Here we report the three-dimensional structure of the S. cerevisiae class I enzyme encoded by the UBC7 gene. The crystal structure has been solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.183 at 2.93 A resolution. Bond lengths and angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 2.3 degrees, respectively. Ubc7 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet. With the exception of two regions where extra residues are present, the tertiary folding of Ubc7 is similar to those of the other two enzymes. The ubiquitin-accepting cysteine is located in a cleft between two loops. One of these loops is nonconserved, as this region of the Ubc7 molecule differs from the other two enzymes by having 13 extra residues. There is also a second single amino acid insertion that alters the orientation of the turn between the first two beta-strands. Analysis of the 13 ubiquitin-conjugating enzyme sequences in S. cerevisiae indicates that there may be two other regions where extra residues could be inserted into the common tertiary fold. Both of these other regions exhibit significant deviations in the superposition of the three structures and, like the two insertion regions in Ubc7, may represent hypervariable regions within a common tertiary fold. As ubiquitin-conjugating enzymes interact with different substrates or other accessory proteins in the ubiquitination pathway, these variable surface regions may confer distinct specificity to individual enzymes.

Bacterial expression of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Ubc7.

The coding sequence for the yeast ubiquitin-conjugating enzyme Ubc7 was obtained by PCR from Saccharomyces cerevisiae genomic DNA. This sequence was placed in a plasmid containing the lambdaPL promoter and was used for temperature-regulated expression in Escherichia coli. The expressed 18-kDa protein was isolated in the inclusion body fraction from bacterial lysates, in contrast to the soluble nature of other yeast ubiquitin-conjugating enzymes expressed in E. coli. Selective solubilization of the protein using 5 M urea followed by dialysis, MonoQ FPLC, and Superdex-75 FPLC yielded electrophoretically pure Ubc7 protein. The purified protein was enzymatically active as determined by formation of enzyme-linked thiolester with ubiquitin. The ability of Ubc7 protein to regain enzymatic activity after urea denaturation appears to be attributable to the stable core alpha/beta folded structure common to the ubiquitin-conjugating enzymes whose structures have been determined to date.

Peroxisomal fatty acyl-CoA oxidase is not regulated by triiodothyronine.

In studies using primary cultures of adult rat hepatocytes in serum-free medium, peroxisomal fatty acyl-CoA oxidase activity was not altered by the presence of 3,5,3'-triiodothyronine, whereas time- and dose-dependent increases in the thyroid hormone-responsive enzyme mitochondrial glycero-3-phosphate dehydrogenase were seen. Activity of peroxisomal oxidase was stimulated with clofibric acid in the absence of 3,5,3'-triiodothyronine. The results demonstrate that hepatic peroxisomal fatty acyl-CoA oxidase activity is not directly regulated by 3,5,3'-triiodothyronine and that stimulation of peroxisomal fatty acyl-CoA oxidase activity by clofibric acid does not require thyroid hormone.

The oxidation of dicarboxylic acid CoA esters via peroxisomal fatty acyl-CoA oxidase.

Evidence supporting a common peroxisomal beta-oxidation pathway for the coenzyme A thioesters of medium-chain-length dicarboxylic acids (DCn-CoA) and monocarboxylic acids (MCn-CoA) has been obtained. Using the mono-CoA esters of dodecanedioic acid (DC12-CoA) and lauroyl-CoA (MC12-CoA) as substrates, parallel inductions of activities and parallel increases in specific activities during purification of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) from rat liver after di(2-ethylhexyl)phthalate treatment were seen. The purified enzyme was used for antiserum production in rabbits; antiserum specificity was verified by immunoblot analysis. Coincident losses of oxidase activities with MC12-CoA and DC12-CoA were found in immunotitration experiments with rat liver homogenates, supporting the hypothesis that peroxisomal fatty acyl-CoA oxidase is solely responsible for the oxidation of medium-chain length dicarboxylic acid substrates. Kinetic studies with purified enzyme using the mono-CoA esters of sebacic (DC10-CoA), suberic (DC8-CoA), and adipic (DC6-CoA) acids along with DC12-CoA revealed substrate inhibition. Although these substrates exhibited similar calculated Vmax values, with decreasing chain length, the combination of increasing Km values and decreasing substrate inhibition constant (Ki) caused the maximum obtainable velocity to decrease. These studies offer an explanation for the previously observed limit of the ability of peroxisomes to chain-shorten dicarboxylates and increased urinary excretion of adipic acid when peroxisomal oxidation of dicarboxylic acids is enhanced.

A diet rich in (n-3) fatty acids increases peroxisomal beta-oxidation activity and lowers plasma triacylglycerols without inhibiting glutathione-dependent detoxication activities in the rat liver.

By using comparisons with a safflower oil diet (15% w/w) and a control, low-fat diet, the ability of a fish oil diet (15% MaxEPA) rich in the (n-3) fatty acids, eicosapentaenoic acid and docosahexaenoic acid, to alter hepatic activities has been determined in adult, male rats. Compared with the safflower diet, treatment for 2 weeks with the fish oil diet caused significant increases in the ratio of liver weight/body weight and the specific activities in liver homogenates of peroxisomal enzymes fatty acyl-CoA oxidase (263%) and catalase (149%) and caused a significant lowering of plasma triacylglycerol levels. Fish oil diets rich in (n-3) fatty acids should thus be placed in the category of hypotriglyceridemic agents which stimulate peroxisomal beta-oxidation activity. In contrast to the effects seen with the other hypotriglyceridemic, peroxisomal proliferating agents such as clofibrate, hepatic glutathione peroxidase and glutathione S-transferase activities are unchanged or are increased rather than inhibited with the fish oil diet.

Determination of peroxisomal fatty acyl-CoA oxidase activity using a lauroyl-CoA-based fluorometric assay.

A simple, sensitive fluorometric method for the determination of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) activity has been developed. Studies of enzyme activity relative to subcellular distribution and to clofibrate induction indicate that this assay is specific for peroxisomal fatty acyl-CoA oxidase. The lauroyl-CoA-dependent production of H2O2 is quantitated by measuring the oxidation of 4-hydroxyphenyl-acetic acid to a fluorescent product in a horseradish peroxidase-coupled assay. Assays can be performed in either a fixed time or continuous mode. In either mode, H2O2 production is related to a change in fluorescence intensity through use of a standard curve generated with known amounts of H2O2. The use of lauroyl-CoA (12:0), rather than the more generally used substrate palmitoyl-CoA (16:0), provides significant advantages. Much of the substrate inhibition problem associated with palmitoyl-CoA has been avoided, and a greater than 4.5-fold higher specific activity has been achieved compared with a palmitoyl-CoA-based assay. In the fixed-time mode, linearity relative to time and to the amount of enzyme added has been established without resorting to the use of bovine serum albumin as a substrate binding medium. Sensitivity is estimated to be at least equal to that of the most sensitive methods reported, while reliability, versatility and range have been improved. Use of this method should greatly facilitate the study of peroxisomal beta-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.

Subcellular localization of adrenal cortical glutathione peroxidase and protective role of the mitochondrial enzyme against lipid peroxidative damage.

In view of the physiological importance of adrenocortical lipid peroxidation, we have carried out subcellular fractionation to determine the location of glutathione peroxidase, an enzyme which protects against lipid peroxidation. Glutathione peroxidase is present in both cytosolic (92%) and mitochondrial (8%) fractions. The small activity in mitochondria is not due to contamination by the cytosolic activity as evidenced by several rigorous approaches. The mitochondrial enzyme is located in the matrix and appears to be effective in protection from NADPH-dependent lipid peroxidative damage of cytochrome P-450 and succinic dehydrogenase, which are located exclusively in the inner membrane.

Glucagon and fasting do not activate peroxisomal fatty acid beta-oxidation in rat liver.

In a study of the endocrine control of peroxisomes, the effects of acute glucagon treatment and fasting on hepatic peroxisomal beta-oxidation in rats have been investigated. The activity of the rate-limiting peroxisomal beta-oxidation enzyme, fatty acyl-CoA oxidase, was measured to determine whether activation of peroxisomal beta-oxidation could account for the increase in total hepatic fatty acid oxidation following acute glucagon exposure. Catalase, a peroxisomal enzyme not directly involved in beta-oxidation, was also measured as a control for total peroxisomal activity. No changes with acute glucagon treatment of intact animals were observed with either activity as measured in liver homogenates or partially purified peroxisomal fractions. These observations indicate the lack of acute control by glucagon of peroxisomal function at the level of total enzyme activity. Previous work on the effects of fasting on hepatic fatty acid beta-oxidation [H. Ishii, S. Horie, and T. Suga (1980) J. Biochem. 87, 1855-1858] suggested an enhanced role for the peroxisomal beta-oxidation pathway during starvation. It was found that the peroxisomal beta-oxidation system, as measured by fatty acyl-CoA oxidase activity, does increase with duration of fast when expressed on a per gram wet weight liver basis. However, when this activity is expressed as total liver capacity, a decline in activity with increasing duration of fast is observed. Furthermore, this decline in peroxisomal capacity parallels the decline in total liver capacity for citrate synthase, a mitochondrial matrix enzyme, and total liver protein. These data indicate that peroxisomal beta-oxidation activity is neither stimulated nor even preferentially spared from proteolysis during fasting.

Rapid action of glucagon on hepatic mitochondrial calcium metabolism and respiratory rates.

The time course for the effects of acute, in vivo glucagon treatment on energy-linked functions of isolated hepatic mitochondria has been studied. After 1 min of glucagon treatment, two changes are observed in mitochondrial function. State 4 (nonphosphorylating) respiratory rates with L-glutamate as substrate are decreased. No significant change is observed in the State 4 respiratory rates with succinate as substrate at 1 min of treatment. Concurrent with the change in nonphosphorylating respiratory rates is a decrease in the half time of spontaneous calcium release from mitochondria preloaded with calcium in a phosphate-containing medium. After 2-4 min of treatment, the previously reported stimulations in rates of State 3 (phosphorylating) respiration and calcium influx into mitochondria are observed. After approximately 6 min of treatment, these changes have reached their maxima. The combined effects of increased calcium uptake rate and decreased calcium efflux rate leads to a decrease in the calcium cycling rate of mitochondria. This decrease in the cycling rate should lead to the increased efficiency of mitochondrial energy transduction and may be responsible, in part, for the increased functional capability of mitochondria isolated from glucagon-treated animals. A correlate of the reduced cycling rate is a decrease in the steady-state concentration at which the mitochondria can buffer the calcium concentration of the incubation medium. The changes observed in calcium efflux rates and respiratory rates exhibit a time course consistent with possible intermediates in the glucagon-induced stimulation of hepatic gluconeogenesis.

Glucagon treatment stimulates the metabolism of hepatic submitochondrial particles.

Hepatic submitochondrial particles, prepared at neutral pH from rats pretreated with glucagon, exhibited stimulated rates of State 3 and uncoupled respiration when succinate or NADH were the substrates, but not when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine were employed. Measurements of 8-anilino-1-naphthalenesulfonic acid fluorescence in the particles indicated that glucagon treatment resulted in a stimulation of energization supported by succinate respiration or ATP hydrolysis. Similarly, the energy-linked pyridine nucleotide transhydrogenase and reverse electron flow reactions driven by succinate oxidation or ATP were also stimulated. The results indicate that mitochondrial substrate transport is not the prime locus of glucagon action. It is suggested that the increased level of energization in particles prepared from glucagon-treated rats is a reflection of a stimulation of the respiratory chain, possibly between cytochromes b and c, and the ATP-forming reactions.

Glucagon stimulation of mitochondrial respiration.

Acute glucagon treatment of intact rats has been found to cause a stimulation of hepatic mitochondrial respiration as measured by monitoring oxygen uptake polarographically. Rates of State 3 respiration with several NAD-linked substrates and succinate were increased significantly after hormonal treatment and isolation of mitochondria. This stimulation cannot be ascribed to a partial uncoupling effect since State 4 respiration as measured by monitoring oxygen uptake polarographically. Rates of State 3 respiration with either slightly increased or unchanged. Furthermore, rates of uncoupled respiration with these substrates were also stimulated after hormonal treatment. On the other hand, respiratory rates (State 3, 4, and uncoupled) with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine as substrate were unaffected by glucagon treatment. The hormonally stimulated rates of respiration produced a corresponding increase in the rate of generation of high energy state as indicated in measurements of Ca2+ uptake by isolated mitochondria. Rates of Ca2+ uptake were monitored by two methods: measurement of initial rates of proton ejection following CaCl2 additions and measurement of disappearance of Ca2+ from the suspension medium using murexide as indicator in a dual wavelength spectrophotometer. A significant stimulation in the initial rate of succinate-dependent Ca2+ uptake was noted after glucagon treatment of animals and isolation of hepatic mitochondria. No effect of the hormonal treatment was seen on the extent of Ca2+ uptake or the stoichiometry of H+ ejected per Ca2+ taken up. That the hormonal effect on Ca2+ transport is at the level of the substrate-induced generation of high energy state is indicated by the observation that no effect of glucagon treatment is seen on ATP-dependent Ca2+ uptake. Glucagon-induced changes in the activities of substrate-metabolizing enzymes are considered unlikely for the following reasons: (a) previously published data showed a lack of a hormonal effect on pyruvate-metabolizing enzymes and (b) data in this study showing no effect of glucagon treatment on the activity of NAD-malate dehydrogenase as measured in mitochondrial lysates. All of these observations are consistent with either an activation of mitochondrial substrate transport and/or a stimulation of mitochondrial electron transport by glucagon treatment. Regardless of the exact mechanism involved, the effect of the hormonal treatment is to produce an increase in ATP synthetic and ion-pumping capability during a period of increased energy demand, i.e. increased gluconeogenesis.