Double Bond Position Plays an Important Role in Delta-9 Desaturation and Lipogenic Properties of Trans 18:1 Isomers in Mouse Adipocytes.

The objective of this research was to study the delta-9 desaturation of individual trans (t) fatty acids that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on lipogenic gene expression in adipocytes. It was hypothesized that delta-9 desaturation and lipogenic properties of t-18:1 isomers depend on the position of double bond. Differentiated 3T3-L1 adipocytes were treated with 200 µM of t6-18:1, t9-18:1, t11-18:1, t13-18:1 or t16-18:1, cis (c)-9 18:1 or bovine serum albumin (BSA) vehicle control for 48 h. Cells were then harvested for fatty acid and gene expression analyses using gas chromatography and quantitative PCR respectively. Among t-18:1 isomers, t13-18:1 and t11-8:1 had the greatest percent delta-9 desaturation (44 and 41 % respectively) followed by t16-18:1 and t6-18:1 (32 and 17 % respectively), while c9-18:1 and t9-18:1 did not undergo delta-9 desaturation. Trans9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including fatty acid synthase and stearoyl-CoA desaturase-1 (P < 0.05), whereas the expression of these genes were not affected with other t-18:1 isomers (P > 0.05). Consistent with gene expression results, t9-18:1 increased the de novo lipogenic index (16:0/18:2n-6) compared with control cells and increased delta-9 desaturation index (c9-16:1/18:0) compared to other t-18:1 isomers (P < 0.05). The current study provides further evidence that the predominant trans fatty acid in PHVO (t9-18:1) has isomer specific lipogenic properties.

Isolation of α-linolenic acid biohydrogenation products by combined silver ion solid phase extraction and semi-preparative high performance liquid chromatography.

Polyunsaturated fatty acids typically found in cattle feed include linoleic (LA) and α-linolenic acid (ALA). In the rumen, microbes metabolize these resulting in the formation of biohydrogenation products (BHP), which can be incorporated into meat and milk. Bioactivities of LA-BHP, including conjugated linoleic acid (cis (c) 9,trans (t) 11-18:2 and t10,c12-18:2) and trans fatty acid isomers (t9-, t10- and t11-18:1) have been investigated, but effects of several BHP unique to ALA have not been extensively studied, and most ALA-BHP are not commercially available. The objective of the present research was to develop methods to purify and collect ALA-BHP using silver ion (Ag(+)) chromatography in sufficient quantities to allow for convenient bioactivity testing in cell culture. Fatty acid methyl esters (FAME) were prepared from perirenal adipose tissue from a cow enriched with ALA-BHP by feeding flaxseed. These were applied to Ag(+)-solid phase extraction, and eluted with hexane with increasing quantities of acetone (1, 2, 10, 20%) or acetonitrile (2%) to pre-fractionate FAME based on degree of unsaturation and double bond configuration. Fractions were collected, concentrated and applied to semi-preparative Ag(+)-high performance liquid chromatography (HPLC) for the isolation and collection of purified isomers, which was accomplished using isocratic elutions with hexane containing differing amounts of acetonitrile (from 0.015 to 0.075%). Purified trans-18:1 isomers collected ranged in purity from 88 to 99%. Purity of the ALA-BHP dienes collected, including c9,t13-18:2, t11,c15-18:2 and t10,c15-18:2, exceeded 90%, while purification of other dienes may require the use of other complementary procedures (e.g. reverse phase HPLC).

Individual trans 18:1 isomers are metabolised differently and have distinct effects on lipogenesis in 3T3-L1 adipocytes.

The objective of this research was to study the metabolism of individual trans fatty acids (FAs) that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on FA composition and lipogenic gene expression in adipocytes. Differentiated 3T3-L1 adipocytes were treated with 200 µM of either trans-9-18:1, trans-11-18:1, trans-13-18:1, cis-9-18:1 or BSA vehicle control for 120 h. Trans-9-18:1 increased total cell FA content (µmole/well) compared to other FA treatments, which was mainly related to the accumulation of trans-9-18:1 in the cells. Adipocytes were able to desaturate a significant proportion of absorbed trans-11-18:1 and trans-13-18:1 (~20 and 30% respectively) to cis-9,trans-11-18:2 and cis-9,trans-13-18:2, whereas trans-9-18:1 was mostly incorporated intact resulting in a greater lipophilic index (i.e. decreased mean FA fluidity) of adipocytes. Trans-9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including acetyl-CoA carboxylase (1.65 fold), FA synthase (1.45 fold), FA elongase-5 (1.52 fold) and stearoyl-CoA desaturase-1 (1.49 fold), compared to the control, whereas trans-11-18:1 and trans-13-18:1 did not affect the expression of these genes compared to control. Our results suggest that the metabolism and lipogenic properties of trans-11-18:1 and trans-13-18:1, typically the most abundant trans FA in beef from cattle fed forage-based diets, are similar and are different from those of trans-9-18:1, the predominant trans FA in PHVO.

Mycobacterium avium subsp. paratuberculosis in dairy products, meat, and drinking water.

Mycobacterium avium subsp. paratuberculosis (Map) is the cause of Johne's disease, a chronic infection of the gut, in ruminant animals that provide milk and/or meat for human consumption. Map also may be involved in Crohn's disease and type 1 diabetes in humans. Although the role of Map in human diseases has not been established, minimizing the exposure of humans to the organism is considered desirable as a precautionary measure. Infected animals can shed Map in feces and milk, and the organism can become disseminated in tissues remote from the gut and its associated lymph nodes. The presence of at least some Map in raw milk and meat and in natural waters is likely, but the numbers of Map in those foods and waters should be reduced through cooking or purification. The available information relating to Map in milk and dairy products, meats, and drinking water is reviewed here for assessment of the risks of exposure to Map from consumption of such foods and water.

Prevalence on beef carcasses of Mycobacterium avium subsp. paratuberculosis DNA.

Fifty samples were collected from each of skinned and dressed carcasses, from each of culled beef breeding cows and fed beef cattle <18 months old at two beef packing plants A and B, and from culled dairy cows at a packing plant C. The 450 samples were collected by swabbing an area of about 1000 cm2 in the anal region of each carcass. DNA extracted from each swab was tested for the IS900 and F57 sequences of the Mycobacterium avium subsp. paratuberculosis (MAP) genome by two stage, nested polymerase chain reaction (PCR) procedures. An internal amplification control (IAC) was detected in 45 or more of each group of 50 DNA preparations. IS900 and F57 were detected in some IAC-positive preparations from all and all but one of the groups of carcasses, respectively. Of the IAC-positive preparations in each group, between 6 and 54% were positive for IS900, and between 4 and 20% were positive for F57. When preparations were tested by single stage, quantitative PCR procedures, IS900 was detected in two samples but F57 was detected in none. The MAP DNA on carcasses was probably derived from small numbers of MAP from the environment that contaminated the animals' hides.

A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression.

Conjugated linoleic acid (CLA) can activate (in vitro) the nuclear transcription factors known as the peroxisome proliferators activated receptors (PPAR). CLA was fed at 11 g CLA/kg of feed for 45d to castrated male pigs (barrows) to better understand long term effects of PPAR activation in vivo. The barrows fed CLA had lean muscle increased by 3.5% and overall fat reduced by 9.2% but intramuscular fat (IMF %) was increased by 14% (P < 0.05). To measure the effect of long term feeding of CLA on porcine muscle gene expression, a semi-quantitative RT-PCR method was developed using cDNA normalized against the housekeeping genes cyclophilin and beta-actin. This method does not require radioactivity or expensive PCR instruments with real-time fluorescent detection. PPARgamma and the PPAR responsive gene AFABP but not PPARalpha were significantly increased (P < 0.05) in the CLA fed pig's muscle. PPARalpha and PPARgamma were also quantitatively tested for large differences in gene expression by western blot analysis but no significant difference was detected at this level. Although large differences in gene expression of the PPAR transcriptional factors could not be confirmed by western blotting techniques. The increased expression of AFABP gene, which is responsive to PPAR transcriptional factors, confirmed that dietary CLA can induce a detectable increase in basal PPAR transcriptional activity in the live animal.

Prolonged dietary treatment with conjugated linoleic acid stimulates porcine muscle peroxisome proliferator activated receptor gamma and glutamine-fructose aminotransferase gene expression in vivo.

Peroxisome proliferator activated receptors (PPARs) represent a family of DNA binding proteins that are activated by a variety of dietary and endogenous fatty acids. The PPAR proteins are expressed throughout the body and are the target of a variety of lipidaemic and insulin sensitizing drugs. Conjugated linoleic acid (CLA) is a collective name for octadecadienoic acid isomers with conjugated double bonds, which can also act as ligands for some of the PPAR family. To gain better understanding of the long-term effects of PPAR activation, CLA was fed at 11 g/kg of feed for 45 days to castrated male pigs (barrows). These barrows had a significant repartitioning of subcutaneous fat to lean tissue in the carcass: fat was reduced by 9 x 2% and lean muscle was increased by 3 x 5%, but intramuscular fat content was also increased by 14% (P<0 x 05). PPARgamma, glutamine-fructose aminotransferase (GFAT), adipocyte fatty acid binding protein (AFABP), but not PPARalpha mRNA levels were significantly increased (P<0 x 05) in the CLA-fed pigs. The increased expression of PPARgamma and AFABP indicates that CLA induced the development of preadipocytes from stromal-vascular (s-v) stem cells to promote intramuscular fat content. The increase in the expression of GFAT mRNA indicates that the glucose supply of the muscle cells had been increased with the CLA diet, possibly sparing intramuscular fatty acid reserves.

Testing for the RN(-) gene in retail pork chops.

A random sample of pork chops were purchased from local retail outlets to determine if the frequency of the RN(-) phenotype could be roughly estimated by GP measurements in fresh raw pork products or by genotyping for the nearest DNA microsatellite markers. Glycolytic potential (GP) is the estimated sum of glycogen, the intermediate metabolites of glycogenolysis, and the end product, lactate. GP has been used to identify a genetic mutation known as the RN(-) or Hampshire gene. Currently, there is no genetic test for the RN(-) allele and flanking DNA microsatellite markers were not useful at predicting the RN(-) phenotype in the random samples. Excessively high GP was found in 25% of the samples which correlated with a significant (P>0.05) drop in pH (5.8 to 5.7), a paler (L* value; 54.1 to 57.5) more yellowish (b* value; 9.6 to 11.6) color, and an increased cooking loss (9 to 18%), typical of the RN(-) phenotype. A genetic test for skin colour in swine proved that the majority (79%) of high GP pork sampled were from phenotypically white pigs. Analysis of glucose levels in post-rigor samples may be useful in progeny testing for the RN gene until a true genetic marker can be identified.

Effect of phospholipids and organic solvents on the formation of 5,16-androstadien-3 beta-ol from pregnenolone in adrenal and testicular microsomes.

The formation of 5,16-androstadien-3 beta-ol from pregnenolone (andien-beta synthase activity) is catalyzed by cytochrome P450c17, which also catalyzes C-17-hydroxy/lyase activity in the biosynthesis of androgens. Andien-beta synthase is very active in porcine Leydig cells, but it is almost undetectable in porcine and bovine adrenal, although the adrenal gland also expresses P450c17. We have treated microsomal preparations with lipids and organic solvents to examine if the andien-beta synthase and C-17-hydroxy/lyase activities of P450c17 were affected by these agents. The addition of some phospholipids to the microsomal preparations inhibited both P450c17 activities. Phospholipids with different fatty acids had no effect on the ratio of andien-beta synthase to C-17-hydroxy/lyase activity. The addition of solvents to the microsomal preparations generally inhibited both P450c17 activities. However, the addition of acetyl acetone up to 5% (v/v) preferentially increased the andien-beta synthase activity while decreasing, the C-17-hydroxy/lyase activity. The effect was dose-dependent, specific to acetyl acetone and was seen in both testis and adrenal microsomes. The exact nature of the stimulation of andien-beta synthase activity is unknown, but the andien-beta synthase activity obtained after treatment with acetyl acetone was directly correlated to total P450c17 activity in the untreated microsomes. The inhibition of C-17-hydroxy/lyase activity by acetyl acetone was particularly apparent with the C-17,20-lyase reaction rather than the 17 alpha-hydroxylase reaction. The addition of acetyl acetone can potentially be used to assess the total potential of P450c17 to catalyze andien-beta synthase activity in vitro.

The effects of immunization against luteinizing hormone-releasing hormone on performance, sexual development, and levels of boar taint-related compounds in intact male pigs.

The effect of a newly developed anti-LH-RH vaccine on the performance, sexual development, and incidence of boar taint-related compounds was investigated in young intact male pigs. At 29 kg BW, 40 crossbred intact males and 20 castrates were allocated to three groups. Castrates and half of the intact males were untreated. The remaining intact males were immunized against LH-RH at 29 kg and again at 89 kg BW. All pigs were slaughtered at 105 kg BW. Compared with control intact males, feed efficiency in castrates was decreased by 10%, muscle content was reduced by 5%, and carcass fat content was increased by 26%. Growth performance and carcass traits did not differ significantly between immunized and control intact males. Genital tract weight, measured at slaughter, was decreased (P < or = .002) by immunization. Plasma testosterone concentrations were not significantly affected at 89 kg BW, whereas they were sevenfold lower (P < .001) in immunized than in control intact males at 105 kg BW. Fat androsterone levels, measured at slaughter, were substantially reduced (P < .001) from .66 +/- .07 microgram/g in control to .21 +/- .01 microgram/g in immunized intact males. Rates of testicular steroid biosynthesis, measured in vitro, were decreased by immunocastration. Fat skatole levels were very low and did not differ significantly between the three groups. The present results demonstrate that anti-LHRH immunization was effective in reducing the level of androstenone, a boar taint-related compound, although having a limited effect on the performance of the animals.

Cytochrome P450c17 from porcine and bovine adrenal catalyses the formation of 5,16-androstadien-3 beta-ol from pregnenolone in the presence of cytochrome b5.

The synthesis of 5,16-androstadien-3 beta-ol from pregnenolone occurs via a cytochrome P450-dependent reaction (andien-beta synthase) that is analogous to the C17-hydroxylase/lyase reaction. It is not known whether the andien-beta synthase activity in adult porcine testis involves cytochrome P450c17 or is unique to porcine testis. Andien-beta synthase activity in testis microsomes was inhibited by high pH and concentration of salt, while C17-hydroxylase/lyase activity was stimulated under these conditions. Cytochrome P450c17 purified from adult porcine testis and adrenal glands and bovine adrenal glands had only C17-hydroxylase/lyase activity in the absence of cytochrome b5. However, when cytochrome b5 isolated from porcine testis was added, andien-beta synthase activity was detected in all three preparations of cytochrome P450c17, with the highest activity found in the porcine preparations. The andien-beta synthase activity was further increased from 2.5 to 6 times when NADH cytochrome b5 reductase was added along with cytochrome b5. Levels of mRNA for cytochrome b5 relative to cytochrome P450c17 mRNA were five times higher in porcine testis than in porcine adrenal. It appears that the andien-beta synthase activity is catalysed by cytochrome P450c17, which is not unique to the porcine testis and is dependent upon adequate levels of cytochrome b5.

The effect of inhibitors of DNA synthesis on 40-kilodalton polypeptide translation in rat L6 myoblasts.

Messenger RNA coding for a polypeptide of 40 kilodaltons (P40) was translated in proliferating rat L6 myoblasts but not in the terminally differentiated myotubes. The relationship between DNA synthesis, differentiation, and P40 mRNA translation was studied. Aphidicolin, a reversible inhibitor of DNA synthesis, was shown to block DNA synthesis in proliferating myoblasts without allowing these cells to differentiate. A second inhibitor, cytosine arabinoside, when added to dividing myoblasts also prevented differentiation. In the absence of biochemical differentiation P40 mRNA remained in the translated state. Translational repression of this mRNA was, therefore, linked to the biochemical differentiation of rat L6 myoblasts.

Cytoskeleton-bound mRNA for a 40-kDa polypeptide in rat L6 cells is not always translated.

The relationship between attachment of mRNA to the cytoskeletal framework and its translation was examined using the mRNA for a polypeptide of 40 kDa (P-40) which is translated in rat L6 myoblasts but not in the myotubes. In both myoblasts and myotubes this mRNA was found to be associated with the cytoskeletal framework. Furthermore, the stability of the association between P-40 mRNA and the cytoskeletal framework in absence of RNA and protein synthesis was examined by using actinomycin D and NaF to block RNA and protein synthesis, respectively. In absence of RNA synthesis portions of both nontranslated P-40 mRNA and translated actin mRNA of myotubes were released into the soluble fraction. In myoblasts, however, both mRNAs remained associated with the cytoskeletal framework following inhibition of RNA synthesis. Inhibition of protein synthesis, on the other hand, had a more dramatic effect on the association between the cytoskeletal framework and P-40 mRNA in myoblasts but not in myotubes. In contrast, the association between actin mRNA and cytoskeletal framework was unaffected by inhibition of protein synthesis in both myoblasts and myotubes. The results of these studies show that the molecular nature of association between cytoskeletal framework and mRNA may differ among mRNAs and may also depend on whether the cells are dividing or are terminally differentiated. Furthermore, no direct relationship between the translation of mRNA and its attachment to the cytoskeletal framework was observed.

Regulation of translation and stability of an mRNA coding for a 40-kDa polypeptide in rat L6 muscle cells.

The mRNA coding for a 40-kDa polypeptide (P-40) was previously cloned and sub-cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59-67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non-dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non-polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600-base-pair PstI fragment of the recombinant clone was 3'-end-labeled with cordycepin 5'-[alpha-32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600-nucleotide-long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3' ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P-40 mRNA. The results of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P-40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P-40 mRNA in myotubes. The relationship between the translation of P-40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady-state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of 3H-labeled P-40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P-40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P-40 mRNA was synthesized in both myoblasts and myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)