Genotype of bovine sterol regulatory element binding protein-1 (SREBP-1) is associated with fatty acid composition in Japanese Black cattle

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of D-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-g, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, D-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 mM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration ( P , 0.01). Expression of D-9 desaturase mRNA was decreased five-and seven-fold, respectively, following 50 and 100 mM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6-and 18-fold in cells supplemented with 50 and 100 mM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both D-9 desaturase ( P , 0.001) and SREBP-1c ( P , 0.001), while a significant positive relationship was observed between D-9 desaturase and SREBP-1c gene expression ( P , 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both D-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

Section: Discussionmentioning

confidence: 99%

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model

Waters

Kenny²,

Killeen³

et al. 2009

“…Lipogenic genes have been postulated as good markers for IMF content or its composition, such as fatty acid synthase, SREBP1 and stearoyl-CoA desaturase-1 (SCD1; e.g. Bourneuf et al, 2006;Hoashi et al, 2007 and2008 in cattle;Hé rault et al, 2008 in chickens). Genes involved in intracellular fatty-acid transport within skeletal muscles have been also notably proposed.…”

Section: Genetic Markersmentioning

confidence: 99%

Intramuscular fat content in meat-producing animals: development, genetic and nutritional control, and identification of putative markers

Hocquette

Gondret

Baéza³

et al. 2010

654

447

Intramuscular fat (IMF) content plays a key role in various quality traits of meat. IMF content varies between species, between breeds and between muscle types in the same breed. Other factors are involved in the variation of IMF content in animals, including gender, age and feeding. Variability in IMF content is mainly linked to the number and size of intramuscular adipocytes. The accretion rate of IMF depends on the muscle growth rate. For instance, animals having a high muscularity with a high glycolytic activity display a reduced development of IMF. This suggests that muscle cells and adipocytes interplay during growth. In addition, early events that influence adipogenesis inside the muscle (i.e proliferation and differentiation of adipose cells, the connective structure embedding adipocytes) might be involved in interindividual differences in IMF content. Increasing muscularity will also dilute the final fat content of muscle. At the metabolic level, IMF content results from the balance between uptake, synthesis and degradation of triacylglycerols, which involve many metabolic pathways in both adipocytes and myofibres. Various experiments revealed an association between IMF level and the muscle content in adipocyte-type fatty acid-binding protein, the activities of oxidative enzymes, or the delta-6-desaturase level; however, other studies failed to confirm such relationships. This might be due to the importance of fatty acid fluxes that is likely to be responsible for variability in IMF content during the postnatal period rather than the control of one single pathway. This is evident in the muscle of most fish species in which triacylglycerol synthesis is almost zero. Genetic approaches for increasing IMF have been focused on live animal ultrasound to derive estimated breeding values. More recently, efforts have concentrated on discovering DNA markers that change the distribution of fat in the body (i.e. towards IMF at the expense of the carcass fatness). Thanks to the exhaustive nature of genomics (transcriptomics and proteomics), our knowledge on fat accumulation in muscles is now being underpinned. Metabolic specificities of intramuscular adipocytes have also been demonstrated, as compared to other depots. Nutritional manipulation of IMF independently from body fat depots has proved to be more difficult to achieve than genetic strategies to have lipid deposition dependent of adipose tissue location. In addition, the biological mechanisms that explain the variability of IMF content differ between genetic and nutritional factors. The nutritional regulation of IMF also differs between ruminants, monogastrics and fish due to their digestive and nutritional particularities.

“…Investigations of MFD have highlighted key regulatory mechanisms in mammary lipid synthesis and this provides a platform for the development of methods to enhance milk fat yield and improve the fatty acid profile of milk fat. For example, SREBP1 and the SREBP1 regulatory proteins are being used as candidate genes for identification of singlenucleotide polymorphisms that may explain genetic differences in milk fat yield (Medrano and Rincon, 2007) and FA composition of bovine fat (Hoashi et al, 2007). Under certain marketing systems and management schemes, it may be advantageous to reduce milk fat yield , and in some feeding and management systems the reduction in milk fat yield has allowed for a repartitioning of nutrients to support increased milk and milk protein yield (e.g.…”

Section: Insights Gained From Milk Fat Depressionmentioning

confidence: 99%

Recent advances in the regulation of milk fat synthesis

Harvatine

Boisclair

Bauman

2009

268

272

In addition to its economic value, milk fat is responsible for many of milk's characteristics and can be markedly affected by diet. Diet-induced milk fat depression (MFD) was first described over a century ago and remains a common problem observed under both intensive and extensive management. The biohydrogenation theory established that MFD is caused by an inhibition of mammary synthesis of milk fat by specific fatty acids (FA) produced as intermediates in ruminal biohydrogenation. During MFD, lipogenic capacity and transcription of key lipid synthesis genes in the mammary gland are down-regulated in a coordinated manner. Our investigations have established that expressions of sterol response element-binding protein 1 (SREBP1) and SREBP-activation proteins are down-regulated during MFD. Importantly, key lipogenic enzymes are transcriptionally regulated via SREBP1. Collectively, these results provide strong evidence for SREBP1 as a central signaling pathway in the regulation of mammary FA synthesis. Spot 14 is also down-regulated during MFD, consistent with a lipogenic role for this novel nuclear protein. In addition, SREBP1c and Spot 14 knock-out mice exhibit reduced milk fat similar to the magnitude and pattern of MFD in the cow. Application of molecular biology approaches has provided the latest chapter in the regulation of milk fat synthesis and is reviewed along with a brief background in nutritional regulation of milk fat synthesis in ruminants.