Phosphatase and tensin homolog (PTEN) suppresses triacylglycerol accumulation and monounsaturated fatty acid synthesis in goat mammary epithelial cells.

Phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor in nonruminants and regulates various cellular processes including growth through dephosphorylation of phosphoinositide substrates. Although studies with bovine mammary tissue suggested a role for PTEN during lactation, its potential role in lipid metabolism remains unknown. Objectives of the present study were to determine PTEN abundance in goat mammary tissue at 2 stages of lactation (n = 6 Xinong Saanen dairy goats per stage), and to use gene-silencing and adenoviral transfections in vitro with isolated goat mammary epithelial cells (GMEC) to evaluate the role of PTEN abundance of lipid metabolism-related genes. Abundance of PTEN decreased by 51.5% at peak lactation compared with the dry period. The PTEN was overexpressed in isolated GMEC through adenoviral transfection using an adenovirus system with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and silenced via targeted small interfering RNA (siRNA) transfection with a scrambled small interfering RNA as a negative control. Cell culture was performed for 48 h before RNA extraction, triacylglycerol (TAG) analysis, and fatty acid analysis. Overexpression of PTEN downregulated abundance of acetyl-coenzyme A carboxylase α (ACACA), fatty acid synthase (FASN), sterol regulatory element binding transcription factor1 (SREBF1), stearoyl-coenzyme A desaturase 1 (SCD1), diacylglycerol acytransferase 1 (DGAT1), 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) coupled with an increase in patatin-like-phospholipase domain containing 2 (PNPLA2), hormone-sensitive lipase (LIPE), and carnitine palmitoyltransferase 1 ß (CPT1B). Furthermore, overexpressing PTEN in vitro resulted in a significant decrease in TAG concentration and concentration of C16:1. In contrast, interference of PTEN led to an opposite effect on lipid metabolism in GMEC. These changes suggested a shift from lipogenesis and esterification to lipolysis and fatty acid oxidation. Collectively, PTEN seems to play a role in monounsaturated fatty acids synthesis and lipid accumulation in GMEC.

Evaluation of the inhibitory effects of drugs on the growth of Babesia gibsoni using relative quantification real-time PCR.

To evaluate the inhibitory effects of drugs on the growth of Babesia gibsoni, relative quantification real-time PCR method was developed in this study. The 18S rRNA gene was used as a target gene for the 2-ΔΔCt method analysis. Additionally, chicken RNA was added to the parasitized blood before total RNA extraction. The chicken ß-actin gene was selected as an internal control gene for the 2-ΔΔCt method analysis. The 100 µL parasitized blood samples with different percentages of parasitized erythrocytes (PPEs) (3%, 1.5%, 0.75%, 0.375% and 0.1875%) were prepared for relative quantification of B. gibsoni. Regression analysis results revealed significant linear relationships between the relative quantification value and parasitemia. 18S rRNA gene expression was significantly decreased after treatment with diminazene aceturate and artesunate in vitro drug sensitivity test. This result suggested that this relative quantification real-time PCR method can be used to evaluate the effects of drug inhibition.

Purification of Plasmodium and Babesia- infected erythrocytes using a non-woven fabric filter.

The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.

Purification of Plasmodium and Babesia- infected erythrocytes using a non-woven fabric filter

@#The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.

Evaluation of the inhibitory effects of drugs on the growth of Babesia gibsoni using relative quantification real-time PCR

@#To evaluate the inhibitory effects of drugs on the growth of Babesia gibsoni, relative quantification real-time PCR method was developed in this study. The 18S rRNA gene was used as a target gene for the 2–ΔΔCt method analysis. Additionally, chicken RNA was added to the parasitized blood before total RNA extraction. The chicken β-actin gene was selected as an internal control gene for the 2–ΔΔCt method analysis. The 100 µL parasitized blood samples with different percentages of parasitized erythrocytes (PPEs) (3%, 1.5%, 0.75%, 0.375% and 0.1875%) were prepared for relative quantification of B. gibsoni. Regression analysis results revealed significant linear relationships between the relative quantification value and parasitemia. 18S rRNA gene expression was significantly decreased after treatment with diminazene aceturate and artesunate in vitro drug sensitivity test. This result suggested that this relative quantification real-time PCR method can be used to evaluate the effects of drug inhibition.

TTK contributes to tumor growth and metastasis of clear cell renal cell carcinoma by inducing cell proliferation and invasion.

The aim of this study was to determine the expression levels of TTK in clear cell renal cell carcinoma (ccRCC) tissues and its possible link with the clinical pathologic characteristics and the prognosis of patients suffering this disease, and to further explore the potential role of TTK in the progression of ccRCC. Immunohistochemical (IHC) assays were performed to detect the expression levels of TTK in 112 samples of ccRCC tissues and corresponding non-tumor tissues. According to the results of IHC assays, patients were divided into TTK high expression and low expression group. Clinical analysis related to the clinical features (age, gender, T stage), and the potential link between TTK expression levels and clinical features were analyzed. In addition, the effects of TTK on the proliferation and invasion of ccRCC cells were detected through colony formation assay and transwell assays, respectively. The possible effects of TTK on tumor growth and metastasis were measured in mice. We found a high expression level of TTK in human ccRCC tissues from patients who received surgical treatment. We also found its expression level was obviously associated with clinical characteristics, such as T stage (p=0.008) and lymphatic metastasis (p=0.023). We further confirmed that knockdown of TTK suppressed cell proliferation and invasion in 2 types of ccRCC cells, HTB-47 and CRL-1932 cells. Furthermore, TTK contributes to tumor growth and metastasis of ccRCC in mice. We found that TTK affected the progression of ccRCC and further mechanically confirmed it could be a novel therapeutic target for ccRCC treatment.

Observation of SOA tracers at a mountainous site in Hong Kong: Chemical characteristics, origins and implication on particle growth.

Secondary organic aerosol (SOA) is an important constituent of airborne fine particles. PM2.5 (particles with aerodynamic diameters≤2.5µm) samples were collected at a mountainous site in Hong Kong in autumn of 2010, and analyzed for SOA tracers. Results indicated that the concentrations of isoprene SOA tracers (54.7±22.7ng/m3) and aromatics SOA tracers (2.1±1.6ng/m3) were on relatively high levels in Hong Kong. Secondary organic carbon (SOC) derived from isoprene, monoterpenes, sesquiterpenes and aromatics was estimated with the SOA tracer based approach, which constituted 0.35±0.15µg/m3 (40.6±5.7%), 0.20±0.03µg/m3 (30.4±5.5%), 0.05±0.02µg/m3 (5.6±1.7%) and 0.26±0.20µg/m3 (21.3±8.2%) of the total estimated SOC. Biogenic SOC (0.60±0.18µg/m3) dominated over anthropogenic SOC (0.26±0.20µg/m3) at this site. In addition to the total estimated SOC (17.8±4.6% of organic carbon (OC) in PM2.5), primary organic carbon (POC) emitted from biomass burning also accounted for a considerable proportion of OC (11.6±3.2%). Insight into the OC origins found that regional transport significantly (p<0.05) elevated SOC from 0.37±0.17 to 1.04±0.39µg/m3. Besides, SOC load could also increase significantly if there was influence from local ship emission. Biomass burning related POC in regional air masses (0.81±0.24µg/m3) was also higher (p<0.05) than that in samples affected by local air (0.29±0.35µg/m3). Evidences indicated that SOA formation was closely related to new particle formation and the growth of nucleation mode particles, while biomass burning was responsible for some particle burst events in Hong Kong. This is the first SOA study in afforested areas of Hong Kong.

Fatty acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat mammary epithelial cells.

In nonruminants, it is well established that elongation of very long-chain fatty acid-like fatty acid elongase 6 (ELOVL6) catalyzes the synthesis of C18:0 from C16:0 in lipogenic tissues like adipose and liver. However, the role of ELOVL6 in regulating lipid metabolism in ruminant mammary gland remains unknown. In the present study, ELOVL6 was overexpressed or knocked down via adenoviral transfection to assess its role in goat mammary epithelial cells. Results revealed that ELOVL6 overexpression had a weak effect on the expression of genes related to triacylglycerol (TAG) synthesis and desaturation. Overexpression of ELOVL6 increased the content of C18:0 at the expense of C16:0, and increased the elongation index of C16:0. Overexpression of ELOVL6 had no significant effect on the elongation index of C16:1n-7 and the desaturation indices of C16:0 and C18:0. Knockdown of ELOVL6 had a negative effect on mRNA expression of the esterification genes GPAM and diacylglycerolacyltransferase 2 (DGAT2) and TAG concentration; however, it increased the concentration of C16:0 and decreased C18:1n-7 and C18:1n-9 in goat mammary epithelial cells. Accordingly, downregulation of ELOVL6 significantly decreased the elongation indices of C16:0 and C16:1n-7. The lack of change in the desaturation indices of C16:0 and C18:0 upon knockdown of ELOVL6 was consistent with the minor change in SCD1 expression. In conclusion, these are the first results highlighting an important role of ELOVL6 in long-chain fatty elongation and TAG synthesis in ruminant mammary cells.

Liver X receptor α promotes the synthesis of monounsaturated fatty acids in goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase 1 in an SREBP-1-dependent manner.

Stearoyl-coenzyme A desaturase 1 (SCD1) is a pivotal enzyme in the biosynthesis of monounsaturated fatty acids (MUFA). It is tightly regulated by transcription factors that control lipogenesis. In nonruminants, liver X receptor α (LXRα) is a nuclear receptor and transcription factor that acts as a key sensor of cholesterol and lipid homeostasis. However, the mechanism whereby LXRα regulates the expression and transcriptional activity of SCD1 in ruminant mammary cells remains unknown. In this study with goat mammary epithelial cells (GMEC), the LXRα agonist T 4506585 (T09) markedly enhanced the mRNA expression of SCD1 and sterol regulatory element binding factor 1 (SREBF1). The concentrations of C16:1 and C18:1 and their desaturation indices also were increased by LXRα activation. However, knockdown of LXRα did not alter the mRNA expression of SCD1. Although SCD1 was repressed by SREBF1 knockdown, T09 significantly increased SCD1 expression. Further analysis revealed that the SCD1 promoter activity was activated by LXRα overexpression. The goat SCD1 promoter contains 2 LXR response elements (LXRE), 1 sterol response element (SRE), and 1 nuclear factor Y (NF-Y) binding site. Site-directed mutagenesis of LXRE1, LXRE2, or SRE alone did not eliminate the upregulation of SCD1 when LXRα was overexpressed. In contrast, when NF-Y alone or in combination with SRE was mutated simultaneously, the basal transcriptional activity of the SCD1 promoter was markedly decreased and did not respond to LXRα overexpression. Furthermore, when SREBF1 was knocked down, overexpression of LXRα did not affect the promoter activity of SCD1. Together, these data suggest that LXRα regulates the expression of SCD1 through increasing SREBP-1 abundance to promote interaction with SRE and NF-Y binding sites. The present study provides evidence that LXRα is involved in the synthesis of MUFA in the goat mammary gland through an indirect mechanism.

Characterization of the liver X receptor-dependent regulatory mechanism of goat stearoyl-coenzyme A desaturase 1 gene by linoleic acid.

Stearoyl-coenzyme A desaturase 1 (SCD1) is a key enzyme in the biosynthesis of palmitoleic and oleic acid. Although the transcriptional regulatory mechanism of SCD1 via polyunsaturated fatty acids (PUFA) has been extensively explored in nonruminants, the existence of such mechanism in ruminant mammary gland remains unknown. In this study, we used goat genomic DNA to clone and sequence a 1,713-bp fragment of the SCD1 5' flanking region. Deletion assays revealed a core region of the promoter located between -415 and -109 bp upstream of the transcription start site, and contained the highly conserved PUFA response region. An intact PUFA response region was required for the basal transcriptional activity of SCD1. Linoleic acid reduced endogenous expression of SCD1 and sterol regulatory element binding factor-1 (SREBF1) in goat mammary epithelial cells. Further analysis indicated that both the sterol response element (SRE) and the nuclear factor Y (NF-Y) binding site in the SCD1 promoter were responsible for the inhibition effect by linoleic acid, whereas the effect was abrogated once NF-Y was deleted. In addition, SRE and NF-Y were partly responsible for the transcriptional activation induced via the liver X receptor agonist T 4506585 (Sigma-Aldrich, St. Louis, MO). When goat mammary epithelial cells were cultured with linoleic acid, addition of T 4506585 markedly increased SCD1 transcription in controls, but had no effect on cells with a deleted SRE promoter. These results demonstrated that linoleic acid can regulate SCD1 expression at the transcriptional level through SRE and NF-Y in a liver X receptor-dependent fashion in the goat mammary gland.

Thyroid hormone responsive (THRSP) promotes the synthesis of medium-chain fatty acids in goat mammary epithelial cells.

In nonruminants, thyroid hormone responsive (THRSP) is a crucial protein for cellular de novo lipogenesis. However, the role of THRSP in regulating the synthesis of milk fatty acid composition in goat mammary gland remains unknown. In the present study, we compared gene expression of THRSP among different goat tissues. Results revealed that THRSP had the highest expression in subcutaneous fat, and expression was higher during lactation compared with the dry period. Overexpression of THRSP upregulated the expression of fatty acid synthase (FASN), stearoyl-coenzyme A desaturase 1 (SCD1), diacylglycerol acyltransferase 2 (DGAT2), and glycerol-3-phosphate acyltransferase (GPAM) in goat mammary epithelial cells. In contrast, overexpression of THRSP led to downregulation of thrombospondin receptor (CD36) and had no effect on the expression of acetyl-coenzyme A carboxylase α (ACACA) and sterol regulatory element binding transcription factor1 (SREBF1). In addition, overexpressing THRSP in vitro resulted in a significant increase in triacylglycerol (TAG) concentration and the concentrations of C12:0 and C14:0. Taken together, these results highlight an important role of THRSP in regulating lipogenesis in goat mammary epithelial cells.

Sterol regulatory element binding protein-1 (SREBP-1)c promoter: Characterization and transcriptional regulation by mature SREBP-1 and liver X receptor α in goat mammary epithelial cells.

Sterol regulatory element binding protein-1 (SREBP-1) is a key transcription factor that regulates lipogenesis in rodent liver. Two isoforms (SREBP-1a and SREBP-1c) of SREBP-1 are transcribed by an alternative promoter on the same gene (SREBF1), and the isoforms differ only in their first exon. Although the regulatory effects of SREBP-1 on lipid and milk fat synthesis have received much attention in ruminants, SREBP-1c promoter and its regulatory mechanisms have not been characterized in the goat. In the present study, we cloned and sequenced a 2,012-bp fragment of the SREBP-1c 5'-flanking region from goat genomic DNA. A luciferase reporter assay revealed that SREBP-1c is transcriptionally activated by the liver X receptor α (LXRα) agonist T0901317, and is decreased by SREBP-1 small interfering (si)RNA. A 5' deletion analysis revealed a core promoter region located -395 to +1 bp upstream of the transcriptional start site (TSS). Site-directed mutagenesis of LXRα binding elements (LXRE1 and LXRE2) and sterol regulatory elements (SRE1 and SRE2) revealed that the full effects of T 4506585 require the presence of both LXRE and SRE. We also characterized a new SRE (SRE1) and demonstrated a direct role of SREBP-1 (auto-loop regulation) in maintaining its basal transcription activity. Results suggest that goat SREBP-1c gene is transcriptionally regulated by mature SREBP-1 (auto-loop circuit regulation) and LXRα in goat mammary epithelial cells.

Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells.

Milk fat originates from the secretion of cytosolic lipid droplets (CLD) synthesized within mammary epithelial cells. Adipocyte differentiation-related protein (ADRP; gene symbol PLIN2) is a CLD-binding protein that is crucial for synthesis of mature CLD. Our hypothesis was that ADRP regulates CLD production and metabolism in goat mammary epithelial cells (GMEC) and thus plays a role in determining milk fat content. To understand the role of ADRP in ruminant milk fat metabolism, ADRP (PLIN2) was overexpressed or knocked down in GMEC using an adenovirus system. Immunocytochemical staining revealed that ADRP localized to the surface of CLD. Supplementation with oleic acid (OA) enhanced its colocalization with CLD surface and enhanced lipid accumulation. Overexpression of ADRP increased lipid accumulation and the concentration of triacylglycerol in GMEC. In contrast, morphological examination revealed that knockdown of ADRP decreased lipid accumulation even when OA was supplemented. This response was confirmed by the reduction in mass of cellular TG when ADRP was knocked down. The fact that knockdown of ADRP did not completely eliminate lipid accumulation at a morphological level in GMEC without OA suggests that some other compensatory factors may also aid in the process of CLD formation. The ADRP reversed the decrease of CLD accumulation induced by adipose triglyceride lipase. This is highly suggestive of ADRP promoting triacylglycerol stability within CLD by preventing access to adipose triglyceride lipase. Collectively, these data provide direct in vitro evidence that ADRP plays a key role in CLD formation and stability in GMEC.

Peroxisome proliferator-activated receptor γ1 and γ2 isoforms alter lipogenic gene networks in goat mammary epithelial cells to different extents.

In nonruminants, the alternative splicing of peroxisome proliferator-activated receptor γ (PPARG) generates PPARG1 and PPARG2 isoforms. Although transcriptional control differences between isoforms have been reported in human adipose tissue, their roles in ruminant mammary cells are not well known. To assess which of these isoforms is more closely associated with the regulation of mammary lipogenic pathways, their tissue distribution was analyzed and the expression of key genes regulating lipogenic gene networks was measured after overexpression of the 2 isoforms in goat mammary epithelial cells (GMEC). The expression of PPARG2 was markedly greater in adipose tissue, whereas PPARG1 is the main isoform in goat mammary tissue (ratio of PPARG1:PPARG2 was close to 37:1). As was reported in previous work, PPARG1 upregulated the transcription regulators SREBF1 and PPARG and the lipogenic genes FASN, ACACA, and SCD. Along with a tendency for greater expression of AGPAT6, DGAT1, and PLIN2, these data suggest that PPARG1 is the isoform controlling lipogenesis in mammary cells. Addition of the PPARG ligand rosiglitazone (ROSI) to GMEC overexpressing both isoforms upregulated the expression of LPL and CD36, which help control uptake of long-chain fatty acids into mammary cells. Other responses to ROSI addition to GMEC overexpressing PPARG1 and PPARG2 included upregulation of AGPAT6, DGAT1, INSIG1, SREBF1, and NR1H3. Although the data suggest that both PPARG1 and PPARG2 could affect mammary lipogenesis via control of gene expression when stimulated (e.g., by ROSI), the fact that PPARG1 is more abundant in mammary tissue and that its overexpression alone upregulated key lipogenic gene networks suggest that it is the more important isoform in goat mammary cells.

Peroxisome proliferator-activated receptor-γ stimulates the synthesis of monounsaturated fatty acids in dairy goat mammary epithelial cells via the control of stearoyl-coenzyme A desaturase.

In rodents, peroxisome proliferator-activated receptor-γ (PPARG) plays a crucial role in fatty acid (FA) metabolism through regulation of gene expression, including stearoyl-coenzyme A desaturase (SCD), which is the rate-limiting enzyme for the biosynthesis of monounsaturated FA. However, whether or how PPARG regulates the activity of mammary SCD in ruminants is unknown. This study explored the potential role of PPARG isoforms in regulating SCD mRNA expression in lactating goat mammary epithelial cells (GMEC). Using quantitative real-time PCR, we observed a positive correlation between PPARG and SCD expression in the goat mammary gland at peak lactation. Overexpression of both PPARG1 and PPARG2 in GMEC increased markedly the expression of SCD, the concentration of 16:1 and 18:1, and the desaturation indices of 16:1 and 18:1. The PPARG ligand rosiglitazone further increased SCD expression and desaturation indices in GMEC, overexpressing PPARG1 and PPARG2. Incubation with rosiglitazone alone increased the expression of SCD, but did not alter the concentration of 16- to 18-carbon FA or their desaturation indices. The results provide evidence that PPARG regulates the expression and activity of SCD in GMEC. As such, PPARG may contribute to regulation of SCD and monounsaturated FA synthesis during lactation.