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Dietary protein regulates in vitro lipogenesis and lipogenic gene expression in broilers.

The purpose of this experiment was to determine the possible relationship between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 21 or 30% protein ad libitum. In addition, another group of birds was fed on a regimen consisting of a daily change in the dietary protein level (12 or 30%). This latter group was further subdivided such that one-half of the birds received each level of protein on alternating days. Birds were sampled from 28 to 30 days of age. Measurements taken included in vitro lipogenesis, malic enzyme activity the expression of the genes for malic enzyme, fatty acid synthase and acetyl coenzyme carboxylase. In vitro lipogenesis and malic enzyme activity were inversely related to dietary protein levels (12-30%) and to acute changes from 12 to 30%. In contrast, expression of malic enzyme, fatty acid synthase and acetyl CoA carboxylase genes were constant over a dietary protein range of 12-21%, but decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out, however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.

Acetyl-CoA Carboxylase↗

[Automation of chemical analysis in enology].

Automatic dosages took place a short time ago in oenology laboratories. First researchs about automation of usual manual analysis have been completed by I.N.R.A. Station of Dijon during 1969--1972 years. Then, other researchs were made and in 1974 the first automatic analyser appeared in application laboratories. In all cases continuous flow method was used. First dosages which has been carried out are volatic acidity, residual sugars, total SO2. The rate of work is 30 samples an hour. Then, an original way for free SO2 was suggested. At present, about a dozen of laboratories in France use these dosages. The ethanol dosage automation, very important in oenology, is very difficult to carry out. A new method using a thermometric analyzer is tested. Research about many dosages as tartaric, malic, lactic acids, glucose, fructose, glycérol, have been performed especially by I.N.R.A. Station in Narbonne. But these dosages are not current and at present no laboratory apply them. Now, equipments price and redemption, change of tradionnal dosages for automatical methods and the level of knowledge required for operators are well known. The reproducibility and the accuracy of the continuous flow automatic dosages allow, for enough important laboratories, to make an increasing number of analysis necessary for wine quality control.

Autoanalysis↗

Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids.

A study was done to determine if various organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted Escherichia coli O157:H7 cells. E. coli O157:H7 strain EO139, isolated from venison jerky, was grown in tryptic soy broth (TSB) and in TSB supplemented with 1% glucose (TSBG) for 18 h at 37 degrees C, then plated on tryptic soy agar (TSA) acidified with malic, citric, lactic, or acetic acid at pH 5.4, 5.1, 4.8, 4.5, 4.2, and 3.9. Regardless of whether cells were grown in TSB or TSBG, visible colonies were not formed when plated on TSA acidified with acetic, lactic, malic, or citric acids at pH values of < or =5.4, < or =4.5, < or =4.2, or < or =4.2, respectively. Cells not adapted to reduced pH did not form colonies on TSA acidified with lactic acid (pH 3.9) or acetic acid (pH 3.9 and 4.2); however, a portion of acid-adapted cells remained viable on TSA containing lactic acid (pH 3.9) or acetic acid (pH 4.2) and could be recovered in TSB. Inactivation of acid-adapted cells was less than that of unadapted cells in TSB acidified at pH 3.9 with citric, lactic, or acetic acid and at pH 3.4 with malic acid. Significantly (P< or =0.05) higher numbers of acid-adapted cells, compared with unadapted cells, were detected 12 h after inoculation of TSB acidified with acetic acid at pH 3.9; in TSB containing lactic acid (pH 3.9), the number of acid-adapted cells was higher than the number of unadapted cells after 5 h. In TSB acidified at pH 3.9 with citric acid or pH 3.4 with malic acid, significantly higher numbers of acid-adapted cells survived. This study shows that organic acids differ in their inhibitory or lethal activity against acid-adapted and unadapted E. coli O157:H7 cells, and acid-adapted cells are more tolerant than unadapted cells when subsequently exposed to reduced pH caused by these acids.

Adaptation, Physiological↗

Unidirectional inhibition and activation of "malic' enzyme of Solanum tuberosum by meso-tartrate.

A kinetic study of "malic' enzyme (EC 1.1.1.40) from potato suggests that the mechanism is Ordered Bi Ter with NADP+ binding before malate, and NADPH binding before pyruvate and HCO3-. The analysis is complicated by the non-linearity that occurs in some of the plots. meso-Tartrate is shown to inhibit the oxidative decarboxylation of malate but to activate the reductive carboxylation of pyruvate. To explain these unidirectional effects it is suggested that the control site of "malic' enzyme binds organic acids (including meso-tartrate) which activate the enzyme. meso-Tartrate, however, competes with malate for the active site and thus inhibits the oxidative decarboxylation of malate. Because meso-tartrate does not compete effectively with pyruvate for enzyme-NADPH, its binding at the control site leads to a stimulation of the carboxylation of pyruvate. A similar explanation is advanced for the observation that malic acid stimulates its own synthesis.

Bicarbonates↗

Feed restriction significantly alters lipogenic gene expression in broiler breeder chickens.

Broiler breeder pullets were divided into two groups at 21 wk of age. One group was given free access to feed (ad libitum) and the other fed a limited amount of feed (restricted). At 22 wk, all birds were photostimulated and maintained throughout an egg-laying cycle ending at 36 wk. Samples of liver and abdominal fat pad were collected just before photostimulation (prelight), after photostimulation at first egg and at peak egg production (plateau). Hepatic expression of sterol regulatory element binding protein-1, ATP-citrate lyase, fatty acid synthase, malic enzyme, acetyl-CoA carboxylase and stearoyl-CoA (Delta9) desaturase 1 genes in ad libitum birds declined from their highest levels just before photostimulation as the birds came into and maintained egg production. In contrast, the restricted birds had significant (P < 0.05) increases in the expression of these genes after photostimulation at first egg with a subsequent decline as they reached peak egg production. Hepatic expression of fatty acid binding protein, VLDL apolipoprotein (apoVLDL-II) and apoB genes increased significantly (P < 0.05) in both ad libitum and restricted breeders after photostimulation, whereas apoA1 gene expression declined during this time. Abdominal fat pad weights were significantly (P < 0.05) higher in the ad libitum compared with restricted birds after photostimulation. Lipoprotein lipase in this tissue showed a pattern of expression similar to that observed for the hepatic lipogenic enzyme genes. In conclusion, feed restriction during the pullet-to-breeder transition period significantly (P < 0.05) altered hepatic lipogenic gene expression in broiler breeders.

ATP Citrate (pro-S)-Lyase↗

Effect of dietary energy on lipogenesis in turkey hens prior to and after sexual maturity.

An experiment was conducted with 208 Diamond Hybrid Small White turkey hens to determine the role of dietary energy on in vitro lipid metabolism. Young turkey hens were fed diets containing either a high (3100 kcal metabolizable energy) or low (2400 kcal metabolizable energy) energy level from 12 to 30 weeks of age. Both diets contained 12% protein and were fed on an ad libitum basis. Hens were then maintained under a constant (21 degrees C) or cyclic (12 to 27 degrees C) daily temperature regime and fed either a high (47% of the total energy) or low (8% of the total energy) fat diet for a 100-day egg collection period. Hens were killed at the end of the growing phase, after 1 week of receiving the breeder diets and after 2 weeks of stimulatory light (14 hr/day). Hens were also killed following a 100-day egg collection period. In vitro lipid metabolism and enzyme activities were determined at these periods. In vitro fatty acid synthesis was greater (P less than .05) in hens fed the high-energy prebreeder diet although total energy intakes for both groups of birds were similar. Transferring both groups of hens to individual cages in the breeder house decreased (P less than .05) fatty acid synthesis; however, lipogenic rates were higher (P less than .05) after 2 and 18 weeks of stimulatory light. The high-fat diet decreased (P less than .05) in vitro lipogenesis, fatty acid synthesis, malic enzyme and esterification of palmitate. Acetate utilization in the presence of pyruvate was one-half the rate in the absence. Diets fed prior to sexual maturity did not affect egg production or in vitro lipogenesis of mature hens.

Animals↗

High pressure liquid chromatographic determination of major organic acids in cranberry juice.

A reverse phase high pressure liquid chromatographic method is presented for the simultaneous separation and determination of quinic, malic, and citric acids in single strength, undiluted cranberry juice. After a 1 : 10 dilution and cleanup through a disposable column, major organic acids in cranberry juice are separated on a Bondapak/C18 column and quantitated by using a differential refractometer. Twenty-seven samples of different single strength cranberry juice were analyzed using this method; the mean content of quinic, malic, and citric acids were 1.32 (std dev. 0.150), 0.92 (std dev. 0.079), and 1.08% (std dev. 0.111), respectively. Mean percent recoveries of each acid were quinic 95.4 (std dev. 6.8), malic 96.6 (std dev. 5.8), and citric 94.0% (std dev. 4.8).

Acids↗

Energy and protein relations in the broiler chicken. 2. Effect of varied protein and constant carbohydrate levels on body composition and lipid metabolism.

Male broiler chicks were fed diets containing 18, 23 or 30% protein on an ad libitum basis for 3 weeks. Chicks were also fed these diets on a restriction regime consisting of seven 3-day cycles (2 days with and 1 day without feed). Another group of chicks were fed diets containing 12, 23 or 30% protein on a 3-day rotation consisting of a daily change in dietary protein. Ad libitum. Feed intakes of chicks were inversely (P less than .05) related to the level of the dietary protein. Body composition data seemed to support a positive (P less than .05) relationship between dietary protein and total carcass protein and an inverse (P less than .05) relationship between dietary protein and carcass fat. In vitro lipogenesis was greater (P less than .05) in chicks fed an 18% protein diet than in chicks fed either 23 or 30% protein diets. Restricted. Protein levels had no effect on lipogenesis in chicks fed on a restriction regime but regulated lipogenesis in chicks fed on the rotational regime. Fatty acid synthetase, malic enzyme and isocitrate dehydrogenase activities, when measured during the rotational regime, were greatest following the 12% diet and least following the 30% diet. Enzyme activities, determined during the restriction regime, were greatest (P less than .05) after 1 day of refeeding and least (P less than .05) after 1 day of feed withdrawal. Measurement of net glucose production in vitro indicated that high-protein diets and feed withdrawal stimulated production of glucose.

Animals↗

Lipogenic enzyme gene expression in rat liver during development after birth.

The time courses and the regulation of lipogenic enzyme gene expression during development after birth have been investigated. The mRNA concentrations and activities of liver lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase, and ATP-citrate lyase) were very low in all the suckling rats, regardless of dietary fat of the mothers. After weaning to the same diet as the mothers, the mRNA and enzyme levels were greatly increased by the fat-free or hydrogenated fat diet but not so greatly increased by the corn or fish oil diet. The mRNA concentrations of all the groups reached maximum at 4-6 weeks old and then decreased, usually to 40-60% of the maximal levels. It appeared that the gene expression after weaning is subject to strong nutritional regulation, as well as developmental regulation. The plasma levels of triiodothyronine and insulin were low during suckling. Malic enzyme mRNA level was increased by triiodothyronine treatment even during suckling, but the absolute increase was much less than after weaning. Thus, the gene expression of lipogenic enzymes during suckling appeared to be suppressed by nutritional and hormonal regulation, or may not be sufficiently developed. On the other hand, the hepatic triacylglycerol levels were increased slightly at 2 weeks old and greatly at 3 weeks. As the gene expression of lipogenic enzymes was still low at that time, the major triacylglycerols appear to be obtained from milk and accumulated in preparation for weaning.

ATP Citrate (pro-S)-Lyase↗

The limitations of paradigms: studies on the intermediary metabolism of Trypanosoma cruzi.

We review the development of our knowledge and interpretations of the intermediary metabolism of Trypanosoma (Schizotrypanum) cruzi. Already in the 1950's it was clearly established that when this organism was exposed to large external concentrations of carbohydrates it was unable to catabolize them completely, even in the presence of oxygen, producing a mixture of CO2, dicarboxylic acids (succinic, malic) and alanine as end products. However, subsequent work tended to emphasize such paradigmatic features as a full complement of glycolytic enzymes in all stages of the life cycle of the parasite, a functional Kreb's cycle, a cytochrome-dependent electron transport chain and phosphorylative oxidation which suggested that T. cruzi had the basic metabolic properties of classical glucose-utilizing cells, in contrast with the degenerate glycolytic metabolism of bloodstream African trypanosomes. Only in the 1980's interest revived on the how and why of the incomplete carbohydrate catabolism by this parasite. The primary reason for this anomaly was found to be the presence of a constitutive phospho-enol-pyruvate carboxykinase (PEPCK, ATP-dependent, E.C.4.1.1.49), present in all stages of the parasite's life cycle, and the lack of regulation of the glycolytic route at its classical control points, hexokinase and phosphofructokinase. On the other hand, the presence of two distinct glutamate dehydrogenases (NAD+ and NADP(+)-dependent), the former being strictly regulated by the energy charge of the cell and the Krebs' cycle activity, indicated that amino acids can be a primary source of energy for this organism.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Metabolism of cis-12-octadecenoic acid and trans-9,trans-12-octadecadienoic acid and their influence on lipogenic enzyme activities in mouse liver.

High carbohydrate (65% glucose) diets containing cis-12-octadecenoic acid (12c-18:1) or trans-9,trans-12-octadecadienoic acid (9t,12t-18:2) were fed to weanling mice to investigate the influence of fatty acid structure on six hepatic enzyme activities involved in lipid metabolism. Results with these diets were compared to those with diets containing no fatty acids, saturated fatty acids; cis-9-octadecenoic acid (9c-18:1) and cis-9,cis-12-octadecadienoic acid (9c,12c-18:2). These comparisons show saturated fatty acids, 9c-18:1, 12c-18:1, and 9t,12t-18:2, had little or no influence on the activity levels of fatty acid synthetase, malic enzyme (EC 1.1.1.40)citrate cleavage enzyme (EC 4.1.3.8), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and acetyl-CoA carboxylase (EC 6.4.1.2). Neither 12c-18:1 nor 9t,12t-18:2 produced the dramatic enzyme-lowering effect exhibited by the diet containing 9c,12c-18:2 when compared to the diet devoid of fat. Thus, both the 9 and 12 bonds must be present in the same molecule. Also, at least one and probably both bonds must be in the cis configuration to depress liver enzyme activities. Capillary gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were both used for analysis of the methyl esters derived from the hepatic lipids. The GC and GC-MS data provided (a) direct evidence for incorporation of both isomers into hepatic lipids and (b) indirect evidence that 9t,12t-18:2 lowered liver delta 9-desaturase activity. In addition, since these products were found in the complex liver lipids, there is no doubt that the various enzymes concerned with activation and acylation utilize both of these isomeric fatty acids as substrates.

Animals↗

Effects of fructans-type prebiotics on lipid metabolism.

Several nondigestible but fermentable dietary carbohydrates are able to regulate lipemia and triglyceridemia in both humans and animals. The mechanism of their serum lipid-lowering effect remains to be elucidated. Oligofructose, which is a mixture of nondigestible and fermentable fructans, can decrease triacylglycerol in VLDL when given to rats. The triacylglycerol-lowering action of oligofructose is due to a reduction of de novo fatty acid synthesis in the liver through inhibition of all lipogenic enzymes, namely acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthase, malic enzyme (EC 1.1.1.40), ATP citrate lyase (EC 4.1.3.8), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Our results suggest that oligofructose decreases lipogenic enzyme gene expression. Postprandial insulin and glucose concentrations are low in the serum of oligofructose-fed animals and this could explain, at least partially, the metabolic effect of oligofructose. Moreover, some events occurring in the gastrointestinal tract after oligofructose feeding could be involved in the antilipogenic effect of this fructan: the production of propionate through fermentation, a modulation of the intestinal production of incretins (namely glucose-dependent insulinotropic peptide and glucagon-like peptide-1), or the modification of the availability of digestible carbohydrates. Recent studies showed that the hypotriglyceridemic effect of fructans also occurs in humans.

Animals↗

Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I.

Rat brown adipocytes at day 22 of foetal development showed greater size, higher mitochondria content and larger amounts of lipids, as determined by flow cytometry, than 20-day foetal cells. Simultaneously, an inhibition on the percentage of brown adipocytes into S+G2/M phases of the cell cycle was observed between days 20 and 22 of foetal development. The expression of several adipogenesis-related genes, such as fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase and insulin-regulated glucose transporter, increased at the end of foetal life in brown adipose tissue. In addition, the lipogenic enzyme activities and the lipogenic flux increased during late foetal development, resulting in mature brown adipocytes showing a multilocular fat droplet phenotype. Concurrently, brown adipocytes induced the expression of the uncoupling protein (UP) mRNA and UP protein, as visualized by immunofluorescence. The three isoforms of CCAAT enhancer-binding proteins (C/EBPs) were expressed at the mRNA level in brown adipose tissue at day 20. C/EBP alpha decreased and C/EBP beta and delta increased their expression between days 20 and 22 of foetal development, respectively. Brown adipose tissue constitutively expressed insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNAs. Moreover, IGF-IR mRNA content increased between days 20 and 22 in parallel with the occurrence of tissue differentiation.

Adipocytes↗

Pyruvate stimulates hormonal induction of lipogenic enzymes in primary cultured rat hepatocytes.

Hormonal inductions of lipogenic enzyme activities (fatty acid synthetase, malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and ATP-citrate lyase) were studied in primary cultured rat hepatocytes. Insulin, triiodothyronine and dexamethasone markedly stimulated the inductions of the enzymes (particularly G6PD and ME) in the presence of pyruvate. Lactate also induced their activities. The activities of these enzymes in the presence of appropriate hormone combinations and a substrate amount of pyruvate were as high as, or higher than those in the liver of rats on high-carbohydrate, low-fat diet. The aldolase and glucokinase activities induced by these hormones were not enhanced by the addition of pyruvate. The induction by pyruvate was inhibited by actinomycin D or cycloheximide. The ATP content of rat hepatocytes was maintained without increase during culture with pyruvate for 6 days. These results indicate that the additions of pyruvate, or its metabolites to cultures of isolated hepatocytes have specific effects on the inductions of certain hepatic enzymes, possibly acting at the level of transcription. Their effects are similar to those of feeding a high-carbohydrate, low-fat diet to intact animals.

ATP Citrate (pro-S)-Lyase↗

Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme.

DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for 13 strains of lactic acid bacteria, representing Pediococcus, Leuconostoc, Lactobacillus and Oenococcus genera. The sequences were aligned with the corresponding region of mleS in Lactococcus lactis. The phylogenetic distance matrix tree of all mle sequences was compared with the 16S rRNA phylogenetic tree. The analysis showed that the mle fragment evolved more rapidly than the 16S gene and differently. Pediococcus and Lactobacillus species were intermixed in the 16S rRNA tree whereas they were separated in the mle tree. Leuconostoc mesenteroides and Oenococcus oeni were distinct from other species in the 16S rRNA tree, whereas they were intermixed with Lactobacillus species and Lactococcus lactis in the mle tree. The amino acid sequences deduced from partial mle genes were aligned with 22 malic enzyme sequences and the corresponding phylogenetic tree was constructed. Malic and malolactic enzymes were distinct at the phylogenetic level, except for malic enzymes of yeast and Escherichia coli which were nearer the malolactic enzymes than the other malic enzymes. The analysis of conserved sites showed several interesting amino acids specific to either malic enzyme or malolactic enzyme.

Amino Acid Sequence↗

Meal-feeding specifically induces obese mRNA expression.

Meal-feeding caused a rapid and specific induction of ob mRNA levels in rat adipose tissue. A high-carbohydrate meal caused a mean 5-fold increase in ob mRNA within 3 h after feeding and almost reached the levels of expression seen in rats fed ad lib. After the meal ob mRNA declined with a half-life of less than 2 h. The increase in ob mRNA in response to the meal was prevented by prior injection of actinomycin-D, an inhibitor of transcription. None of the mRNAs for other genes involved in lipid metabolism (lipoprotein lipase, fatty acid synthase, malic enzyme, hormone-sensitive lipase, S14 or adipsin) showed any significant difference between pre-meal and post-meal levels.

Actins↗

Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium.

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N-3 readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 microM. The enzyme is also stimulated to varying degrees by alpha-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl-6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.

Basidiomycota↗

mRNA levels of SREBP-1c do not coincide with the changes in adipose lipogenic gene expression.

Physiological differences in lipid metabolism exist according to adipose sites. To delineate at which step such gene regulation could occur, mRNA levels of various proteins involved in the overall lipogenic process were determined in subcutaneous (SC) and retroperitoneal (RP) adipose tissues. Fatty acid synthase, malic enzyme, ATP citrate lyase, insulin-sensitive glucose transporter, and glucose-6-phosphate dehydrogenase mRNA levels were coordinately reduced (by up to 50-fold) during fasting in RP and in SC relative to fed rats, and restored or overexpressed (by up to 5- to 6-fold) during refeeding. The response was most often delayed and lower in SC compared to RP. This could contribute to site-specific differences. Interestingly, SREBP-1c mRNA levels were markedly decreased by fasting in SC but remained unchanged in RP. Refeeding tended to restore levels close to fed group values. We conclude that mRNA levels of SREBP-1c do not coincide with the expected changes in adipose lipogenic gene expression of fasted/refed rats.

Adipose Tissue↗