Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non-ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP-1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP-1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF-Y)-binding sites within a -1713 to +65-base pair region of the SCD1 promoter completely abolished SREBP-1-induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP-1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. J. Cell. Physiol. 232: 635-649, 2017. © 2016 Wiley Periodicals, Inc.

Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates the interface between cellular lipid metabolism, redox status and organelle differentiation. Conditional prostatic epithelial knockout of PPARγ in mice resulted in focal hyperplasia which developed into mouse prostatic intraepithelial neoplasia (mPIN). The grade of PIN became more severe with time. Electron microscopy (EM) showed accumulated secondary lysosomes containing cellular organelles and debris suggestive of autophagy. Consistent with this analysis the autophagy marker LC3 was found to be upregulated in areas of PIN in PPARγ KO tissues. We selectively knocked down PPARγ2 isoform in wild-type mouse prostatic epithelial cells and examined the consequences of this in a tissue recombination model. Histopathologically grafted tissues resembled the conditional PPARγ KO mouse prostates. EM studies of PPARγ- and PPARγ2-deficient epithelial cells in vitro were suggestive of autophagy, consistent with the prostatic tissue analysis. This was confirmed by examining expression of beclin-1 and LC3. Gene expression profiling in PPARγ-/γ2-deficient cells indicated a major dysregulation of cell cycle control and metabolic signaling networks related to peroxisomal and lysosomal maturation, lipid oxidation and degradation. The putative autophagic phenotypes of PPARγ-deficient cells could be rescued by re-expression of either γ1 or γ2 isoform. We conclude that disruption of PPARγ signaling results in autophagy and oxidative stress during mPIN pathogenesis.

Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.

MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post-transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid-lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs, and 821 pre-miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR-145) had significant differences in expression over the lactation cycle. Further studies revealed that miR-145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR-145. Overexpression of miR-145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR-145 impaired fatty acid synthesis. Inhibition of miR-145 increased methylation levels of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR-145. These findings underscore the need for further studies to evaluate the potential for targeting miR-145 for improving beneficial milk components in ruminant milk. J. Cell. Physiol. 232: 1030-1040, 2017. © 2016 Wiley Periodicals, Inc.

Akt serine/threonine kinase acts as a central mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, regulating a series of biological processes. In lipid metabolism, Akt activation regulates a series of gene expressions, including genes related to intracellular fatty acid synthesis. However, the regulatory mechanisms of Akt in dairy goat mammary lipid metabolism have not been elaborated. In this study, the coding sequences of goat Akt1 gene were cloned and analyzed. Gene expression of Akt1 in different lactation stages was also investigated. For in vitro studies, a eukaryotic expression vector of Akt1 was constructed and transfected to goat mammary epithelial cells (GMECs), and specific inhibitors of Akt/mammalian target of rapamycin (mTOR) signaling were applied to GMECs. Results showed that Akt1 protein was highly conserved, and its mRNA was highly expressed in midlactation. In vitro studies indicated that Akt1 phosphorylation activated mTOR and subsequently enhanced sterol regulatory element binding protein 1 (SREBP1), thus increasing intracellular triacylglycerol content. Inhibition of Akt/mTOR signaling down-regulated the gene expression of lipogenic genes. Overall, Akt1 plays an important role in regulating de novo fatty acid synthesis in goat mammary epithelial cells, and this process probably is through the mTOR/SREBP1 axis.