Andrea J. Lengi

Identification and characterization of a novel bovine stearoyl-CoA desaturase isoform with homology to human SCD5

Stearoyl-CoA desaturase (SCD) is an enzyme responsible for the production of cis-9, trans-11 conjugated linoleic acid in ruminant fats, and for the synthesis of palmitoleoyl-CoA and oleoyl-CoA. To date, only one SCD isoform has been described in ruminant species, although multiple isoforms have been found in many other mammalian species. In this paper, we describe for the first time a second SCD isoform in cattle, which appears to be an ortholog of human SCD5 rather than a homolog of bovine SCD1 or any of the described murine SCD isoforms. As described in other SCD proteins, the predicted amino acid sequence of bovine SCD5 includes four transmembrane domains and three conserved histidine motifs. The amino-terminus of the predicted protein sequence of SCD5 lacks the PEST sequences typically found in SCD1 homologs, which are thought to target proteins for rapid degradation. Similar to human SCD5, the bovine SCD5 gene is organized into five exons and four introns, and is highly expressed in the brain. In other tissues examined, mRNA expression of SCD5 was minimal. Furthermore, the expression levels of SCD5 between brain gray and white matter are not different. This is the first description of a homolog of human SCD5 in a non-primate species.

Effects of intravenous infusion of trans-10, cis-12 18:2 on mammary lipid metabolism in lactating dairy cows.

It has previously been established that supplementation of trans-10, cis-12 18:2 reduces milk fat content and fat deposition in several species. The objectives of the study were 1) to examine whether potential mechanisms by which trans-10, cis-12 18:2 is reported to affect lipid metabolism in adipose tissue of different species could be partly responsible for the inhibition in milk fat synthesis in lactating dairy cows; and 2) to investigate the effects of trans-10, cis-12 18:2 on the expression of a newly identified isoform of stearoyl-coenzyme A desaturase (SCD) in bovine mammary tissue. Four primiparous Holstein cows in established lactation, fitted with indwelling jugular catheters, were used in a balanced 2 x 2 crossover design. For the first 5 d of each period, cows were infused intravenously with a 15% lipid emulsion providing 10 g/d of either cis-9, cis-12 18:2 (control) or trans-10, cis-12 18:2 (conjugated linoleic acid; CLA). On d 5 of infusion, mammary gland biopsies were performed and tissues were analyzed for mRNA expression of acetyl-coenzyme A carboxylase, fatty acid synthetase, lipoprotein lipase, SCD1, SCD5, sterol regulatory element-binding protein-1, IL6, IL8, and tumor necrosis factor-alpha by real-time PCR. Compared with the control treatment, CLA reduced milk fat concentration and yield by 46 and 38%, respectively, and increased the trans-10, cis-12 18:2 content in milk fat from 0.05 to 3.54 mg/g. Milk yield, milk protein, and dry matter intake were unaffected by treatment. Infusion of the CLA treatment reduced the mRNA expression of acetyl-coenzyme A carboxylase and fatty acid synthetase by 46 and 57%, respectively, and tended to reduce the expression of SCD1 and lipoprotein lipase. Abundance of mRNA for sterol regulatory element-binding protein-1 was reduced by 59% in the CLA treatment group. However, infusing trans-10, cis-12 18:2 did not affect the expression of transcripts for SCD5, tumor necrosis factor-alpha, IL6, and IL8. Results from the current study corroborate the idea that effects of trans-10, cis-12 18:2 reported on adipose tissue in animal models and humans are not part of the response in the inhibition of milk fat synthesis in lactating dairy cows. They also support the hypothesis that SCD1 and SCD5 present important differences in their regulation and physiological roles.

Comparison of pig, sheep and chicken SCD5 homologs: Evidence for an early gene duplication event

Stearoyl-CoA desaturase (SCD) catalyzes the desaturation of saturated fatty acyl-CoA substrates at the delta-9 position. Multiple SCD isoforms are well characterized in rodents, especially in mice, where four isoforms have been described. In humans and cows, two SCD isoforms have been described: SCD1, which is a homolog of murine SCD1, and SCD5, which appears to be a distinct SCD gene rather than an ortholog of any of the four murine isoforms. In this paper, we describe for the first time SCD5 homologs in sheep, pigs and chickens. The SCD5 nucleotide sequences have notably higher GC content than SCD1 sequences, and the predicted protein sequences lack N-terminal PEST sequences typically found in SCD1 proteins. Similar to humans and bovines, the mRNA expression of sheep and pig SCD5 is greatest in the brain, and the mRNA expression of chicken SCD5 is greatest in the pancreas and brain. In contrast, SCD1 expression was found to be highest in adipose tissue in pigs and sheep, and liver in the chicken. This is the first report of an SCD5 homolog in a non-mammalian species, and suggests that SCD5 may be the result of an early gene duplication event that occurred before the divergence of mammals.

Short communication: Identification of the bovine sterol regulatory element binding protein-1c promoter and its activation by liver X receptor1

Sterol regulatory element binding proteins (SREBP) are a family of transcription factors that regulate cholesterogenesis and lipogenesis. Sterol regulatory element binding proteins-1a and -1c are transcribed from the same gene by the use of alternate promoters, and only differ at their first exon. Sterol regulatory element binding protein-1c is hypothesized to be an important regulator of genes involved in milk fat synthesis in the lactating dairy cow. However, the bovine SREBP-1c promoter has not been previously characterized, and studies to date that have investigated the role of SREBP-1 in the bovine mammary gland have not distinguished between isoforms 1a and 1c. The purpose of this study was to characterize the bovine SREBP-1c promoter and to investigate the DNA elements involved in the regulation of SREBP-1c expression by the liver X receptor agonist T0901317 in 2 different bovine mammary epithelial cell lines. Luciferase reporter constructs containing the wild-type SREBP-1c promoter or constructs with mutated liver X receptor response elements or sterol response element were transfected into MacT cells and bovine mammary epithelial (BME-UV) cells. We have demonstrated that the liver X receptor response elements sites in the SREBP-1c promoter are necessary for mediating the T0901317 response, and that stimulation through the sterol response element site plays only a minor role in this pathway. This report describes the bovine SREBP-1c promoter and its regulation by liver X receptor in bovine mammary epithelial cells.

Short communication: Effect of trans-10,cis-12 conjugated linoleic acid on activation of lipogenic transcription factors in bovine mammary epithelial cells

The objective of this study was to examine the effect of trans-10,cis-12 conjugated linoleic acid (t10c12CLA) on the activation of transcription factors that potentially regulate lipid synthesis in a bovine mammary epithelial cell line (MAC-T). Cells were transfected with luciferase reporter constructs containing sterol response element (SRE and SRE complex) for sterol regulatory element binding protein-1, peroxisome proliferator response element for peroxisome proliferator-activated receptor γ, or liver X receptor response element for liver X receptor. Different concentrations of t10c12CLA (0, 25, 50, 75, or 100μM) were applied to cells to determine the activation of transcription factors. The influence of t10c12CLA bond structure on transcription factor activation was also investigated by treating cells with different 18:1 fatty acid isomers (trans-10 18:1 or cis-12 18:1) at 100μM. Cells were harvested for luciferase assay after 24h of treatment. Compared with linoleic acid and cis-9,trans-11 CLA controls, the SRE reporters had significantly lower activity in t10c12CLA-treated cells at 50 and 75μM for SRE complex and SRE, respectively. Lower SRE and SRE complex activation was observed in t10c12CLA treatment at 25, 50, and 75μM compared with 0μM. The peroxisome proliferator response element and liver X receptor response element reporters did not respond differently between the t10c12CLA treatment and controls. Compared with t10c12CLA, both trans-10 18:1 and cis-12 18:1 increased the activities of SRE and SRE complex reporters by 1.3- to 4.2-fold. In conclusion, t10c12CLA has an inhibitory role in lipogenic transcription factor activation of SRE, and this negative effect is due to the conjugation of trans-10 and cis-12 double bonds in the fatty acid. Furthermore, we found no support for a regulatory role of response elements for peroxisome proliferator-activated receptor γ or liver X receptor in the t10c12CLA inhibition of mammary lipid synthesis.

Regulation of the bovine SCD5 promoter by EGR2 and SREBP1.

In rodents, the transcription factors early growth response 2 (EGR2) and sterol regulatory element binding protein 1a (SREBP1a) regulate transcription of the stearoyl-CoA desaturase 2 (SCD2) gene during peripheral nerve myelination, which may be important for synthesis of the lipid component of myelin. Most non-rodent genomes do not contain the SCD2 gene, but rather express SCD5 in brain and nervous tissues. In this paper, we asked whether bovine SCD5 is regulated in a similar manner to rodent SCD2. Expression of EGR2 did not result in an increase in endogenous SCD5 mRNA expression in JEG3 cells, but did result in activation of truncated bovine SCD5 promoter luciferase reporter constructs. Similar results were obtained with expression of the active form of SREBP1a; however, unlike rodent SCD2, there was no synergistic activation of the bovine SCD5 promoter reporters when EGR2 and SREBP1a were co-expressed. Mutation of the putative EGR2 binding site in the SCD5 promoter abolished activation by SREBP1a, suggesting that EGR2 and SREBP1a bind to the same site in the SCD5 promoter. Finally, we have identified a region of the bovine SCD5 promoter between 505 and 305 base pairs upstream of the transcriptional start site that appears to be important for maintaining basal levels of transcription of this gene. While it appears that there are some differences between the regulation of rodent SCD2 and bovine SCD5, the promoters of both genes can be activated by EGR2 and SREBP1a. This is the first report of potential regulators of SCD5 transcription.

Bovine brain region-specific stearoyl-CoA desaturase expression and fatty acid composition.

In this study, we sought to determine the relationship between stearoyl-CoA desaturase (SCD) gene isoform expression in the bovine brain and the accumulation of 18:1n-9. Two SCD gene isoforms are found in cows—SCD1 and SCD5. Samples of six brain regions (cerebellum, frontal cortex, hippocampus, hypothalamus, midbrain, and pons) were collected from animals at four different ages (neonates, weanlings, yearlings, and adults) for mRNA isolation and fatty acid analysis. Expression of SCD1 and SCD5 mRNA was compared across age groups to determine its developmental regulation. Fatty acid composition and SCD isoform mRNA expression were compared to examine the correlation of SCD1 and SCD5 with 18:1n-9 content in different brain regions. We found statistically significant correlations between SCD1 and SCD5 mRNA expression and the ratio of 18:1n-9 to 18:0 across age groups, with stronger correlations observed for SCD5. Similarly, there was a significant correlation between the ratio of 18:1n-9 to 18:0 and SCD5 mRNA expression across brain regions. SCD1 mRNA and the 18:1n-9 to 18:0 ratio were negatively correlated in the hippocampus. There was no trend of increasing 18:1n-9 content or SCD expression with age. Correlations indicated a stronger relationship between SCD5 mRNA expression and the 18:1n-9 to 18:0 ratio, potentially indicating a strong contribution of the SCD5 isoform to brain 18:1n-9 content. This is the first study examining a potential role for SCD5 in providing 18:1n-9 for brain lipids.