Low dietary protein increases vitamin A absorption and deposition into milk in periparturient rats.

Our objective was to study the effect of differing dietary crude protein and vitamin A on retinoid metabolism in a periparturient rat model. Sixty female rats, approximately 21 d before parturition, were fed rations containing either low protein (13%; LP) or high protein (22%; HP) crude protein and either low vitamin A (3 IU/g; LA) or high vitamin A (5 IU/g; HA), yielding treatments HPHA, HPLA, LPHA, and LPLA. Samples were collected at d -14, d +3, and +10 relative to parturition and analyzed for all-trans retinoid acid (RA), 13-Cis RA, and retinol. At d -14, serum all-trans RA concentrations decreased compared to baseline. At both d +3 and d +10, serum retinol increased and liver 13-Cis RA decreased. In the small intestine, 13-cis RA was higher in HPHA than HPLA pre-partum (0.93±0.12 vs. 0.40±0.12 ng/ml, P=0.04). Post-partum, 13-cis RA was lower in high vitamin HPHA and LPHA groups (0.35±0.06 and 0.38±0.06 ng/ml) than in low vitamin A HPLA and LPLA treatments (0.50±0.06 and 1.32±0.06 ng/ml, P<0.01). In rats fed LA diets, TNF-alpha expression tended to be lower in HPLA than LPLA groups on day +3 (0.69±0.34 vs 1.00±0.52, P=0.08), but not day +10 (0.56±0.25 vs. 1.00±0.49 fold change, P>0.10). Retinoids accumulated during pregnancy and were mobilized during lactation. The sequestration of retinoids was increased when dietary protein content was low. Further studies are needed to investigate how retinoid metabolism could be manipulated to improve vitamin A delivery to milk.

New Strains for Tissue-Specific RNAi Studies in Caenorhabditis elegans.

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1(ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1(ne219) and the reported germline only RNAi strain, rrf-1(pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1(ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

The biotin-ligating protein BPL-1 is critical for lipid biosynthesis and polarization of the Caenorhabditis elegans embryo.

Biotin is an essential cofactor for multiple metabolic reactions catalyzed by carboxylases. Biotin is covalently linked to apoproteins by holocarboxylase synthetase (HCS). Accordingly, some mutations in HCS cause holocarboxylase deficiency, a rare metabolic disorder that can be life-threatening if left untreated. However, the long-term effects of HCS deficiency are poorly understood. Here, we report our investigations of bpl-1, which encodes the Caenorhabditis elegans ortholog of HCS. We found that mutations in the biotin-binding region of bpl-1 are maternal-effect lethal and cause defects in embryonic polarity establishment, meiosis, and the integrity of the eggshell permeability barrier. We confirmed that BPL-1 biotinylates four carboxylase enzymes, and we demonstrate that BPL-1 is required for efficient de novo fatty acid biosynthesis. We also show that the lack of larval growth defects as well as nearly normal fatty acid composition in young adult worms is due to sufficient fatty acid precursors provided by dietary bacteria. However, BPL-1 disruption strongly decreased levels of polyunsaturated fatty acids in embryos produced by bpl-1 mutant hermaphrodites, revealing a critical role for BPL-1 in lipid biosynthesis during embryogenesis and demonstrating that dietary fatty acids and lipid precursors are not adequate to support early embryogenesis in the absence of BPL-1. Our findings highlight that studying BPL-1 function in C. elegans could help dissect the roles of this important metabolic enzyme under different environmental and dietary conditions.

Relationship between stearoyl-CoA desaturase 1 gene expression, relative protein abundance, and its fatty acid products in bovine tissues.

Stearoyl-CoA desaturase 1 (SCD1) greatly contributes to the unsaturated fatty acids present in milk and meat of cattle. The SCD1 enzyme introduces a double bond into certain saturated fatty acyl-CoAs producing monounsaturated fatty acids (MUFA). The SCD1 enzyme also has been shown to be active in the bovine mammary gland converting t11 18:1 (vaccenic acid) to c9 t11 conjugated linoleic acid (CLA). The objective of this study was to determine any association between the gene expression of SCD1 and occurrence of its products (c9 14:1, c9 16:1, c9 18:1, and c9 t11 18:2) in various bovine tissues. Tissue samples were obtained from lactating Holstein cows (n=28) at slaughter, frozen in liquid nitrogen and stored at -80 °C. Total RNA was extracted and converted to complementary DNA for quantitative real time polymerase chain reaction (PCR) analysis of the SCD1 gene. Extracted lipid was converted to fatty acid methyl esters and analysed by GC. Tissues varied in expression of SCD1 gene with mammary, cardiac, intestinal adipose, and skeletal muscle expressing greater copy number as compared with lung, large intestine, small intestine and liver (371, 369, 328, 286, 257, 145, 73, and 21 copies/ng RNA, respectively). Tissues with high mRNA expression of SCD1 contained greater SCD1 protein whereas detection of SCD1 protein in tissues with low SCD1 mRNA expression was very faint or absent. Across tissues, the desaturase indices for c9 18:1 (r=0.24) and sum of SCD products (r=0.20) were positively correlated with SCD1 gene expression (P<0.01 for both). Within each tissue, the relationship between SCD1 gene expression and the desaturase indices varied. No correlation was detected between SCD1 expression and desaturase indices in the liver, large and small intestines, lung, cardiac or skeletal muscles. Positive correlations, however, were detected between SCD1 expression and the desaturase indices in intestinal adipose tissue (P<0.02 for all) except 14:1, whereas only c9 18:1, c9 t11 18:2 and sum of all desaturase indices were positively correlated with SCD1 expression in mammary tissue (P < or = 0.03). Overall, the relationship between SCD1 gene expression and occurrence of its products seems to be tissue specific.

Short-term effects of dietary trans fatty acids compared with saturated fatty acids on selected measures of inflammation, fatty acid profiles, and production in early lactating Holstein dairy cows.

Feeding rations supplemented with fats may provide an opportunity to manipulate the health and performance of dairy cows; however, the relative effects of specific fats, such as trans fatty acids (TFA), are poorly understood. The objective of this study was to investigate the effects of a ration supplemented with TFA on the fatty acid (FA) profile of peripheral blood mononuclear cells (PBMC), plasma lipids, and milk; the gene expression of inflammatory markers; production of acute phase proteins; and production performance in early lactating dairy cows. Trans fat was fed at 0, 1.5, and 3% of dry matter, replacing (1:1 wt:wt) saturated fatty acids (SFA). Multiparous lactating Holstein cows at 7 d in milk (n=12) were randomly assigned to a treatment sequence in a 3 × 3 balanced Latin square design; each period lasted 14 d. Milk and heparinized blood were collected on d 0 (pretreatment) and on d 10 and 14 of each period. Plasma was collected and solid-phase extraction was used to isolate plasma phospholipids and nonesterified fatty acids. Additionally, PBMC were isolated for FA analysis and gene expression analysis by reverse transcription-PCR using bovine RPS9 as the endogenous control. The FA composition of PBMC, plasma lipid fractions, and milk were analyzed by gas chromatography. Data were analyzed using the MIXED procedure (SAS Institute Inc., Cary, NC). As dietary TFA increased, the percentage of some 18:1 trans isomers increased in PBMC, plasma lipids, and milk. Dietary TFA had no detectable effect on mRNA expression of proinflammatory TNFα or IL6. Expression of IL1ß and ICAM1 decreased with increasing TFA. In addition, supplementation of TFA did not affect percentages of milk fat, protein, lactose, or solids-not-fat, or somatic cell count. Overall, dietary TFA increased the trans FA present in PBMC, plasma lipids, and milk; however, dietary TFA decreased PBMC expression of some of the proinflammatory markers tested at the mRNA level compared with SFA in early lactating dairy cows. Together, these findings provide evidence that over short period of times, dietary TFA might be slightly less immune-stimulatory than dietary SFA.