Thyroid Hormone Responsive Protein Research Articles

Thyroid hormone-responsive protein mediates the response of chicken liver to fasting mainly through the cytokine-cytokine receptor interaction pathway

ABSTRACT 1. The objective of this study was to explore the mediating role of thyroid hormone-responsive protein (THRSP) in the response of chicken liver to fasting. 2. A batch of 7-d-old chicks with similar body weights were randomly divided into the control group and the fasting group (n = 10). The control group was fed ad libitum, while the test group fasted for 24 h. The liver and pectoral muscle tissues were collected. Chicken primary hepatocytes or myocytes were treated with different concentrations of thyroxine, glucose, insulin, oleic acid and palmitic acid, separately. Chicken primary hepatocytes were transfected with THRSP overexpression vector vs. empty vector, and the cells were used for transcriptome analysis. The mRNA expression of THRSP and other genes was determined by quantitative PCR. 3. The expression of THRSP in chicken liver and pectoral muscle tissues was significantly inhibited by fasting (P < 0.05). In chicken primary hepatocytes, the expression of THRSP was significantly induced by thyroxine (0.25, 0.5, 1 mmol/l), glucose (50, 100 mmol/l), and insulin (20 nmol/l), and was significantly inhibited by palmitic acid (0.125, 0.25 mmol/l). In the myocytes, expression of THRSP was significantly induced by thyroxine (0.25, 0.5, 1 mmol/l), glucose (50 mmol/l) and oleic acid (0.125, 0.25 mmol/l), was significantly inhibited by insulin (5 nmol/l) and was not significantly affected by palmitic acid. 4. Transcriptome analysis showed that overexpression of THRSP significantly affected the expression of 1411 DEGs, of which 1007 were up-regulated and 404 were down-regulated. The GO term and KEGG pathway enrichment analyses showed that these DEGs were mainly enriched in the interaction between cytokine and cytokine receptor and its regulation and signal transduction, cell growth and apoptosis and its regulation, immune response and retinol metabolism. 5. In conclusion, the THRSP gene mediates biological effects of fasting by influencing the expressional regulation of the genes related to biological processes such as cytokine-cytokine receptor interaction, cell growth and apoptosis, immune response, retinol metabolism, including TGM2, HSD17B2, RUNX3, IRF1, ANKRD6, UPP2, IKBKE, and PYCR1 genes, in chicken liver.

Hippocampal dentate gyri proteomics reveals Wnt signaling involvement in the behavioral impairment in the THRSP-overexpressing ADHD mouse model

Children with attention-deficit/hyperactivity disorder (ADHD) often struggle with impaired executive function, temporal processing, and visuospatial memory, hallmarks of the predominantly inattentive presentation (ADHD-PI), subserved by the hippocampus. However, the specific genes/proteins involved and how they shape hippocampal structures to influence ADHD behavior remain poorly understood. As an exploratory tool, hippocampal dentate gyri tissues from thyroid hormone-responsive protein overexpressing (THRSP OE) mice with defining characteristics of ADHD-PI were utilized in proteomics. Integrated proteomics and network analysis revealed an altered protein network involved in Wnt signaling. Compared with THRSP knockout (KO) mice, THRSP OE mice showed impaired attention and memory, accompanied by dysregulated Wnt signaling affecting hippocampal dentate gyrus cell proliferation and expression of markers for neural stem cell (NSC) activity. Also, combined exposure to an enriched environment and treadmill exercise could improve behavioral deficits in THRSP OE mice and Wnt signaling and NSC activity. These findings show new markers specific to the ADHD-PI presentation, converging with the ancient and evolutionary Wnt signaling pathways crucial for cell fate determination, migration, polarity, and neural patterning during neurodevelopment. These findings from THRSP OE mice support the role of Wnt signaling in neurological disorders, particularly ADHD-PI presentation.

Spermidine Ameliorates Nonalcoholic Steatohepatitis through Thyroid Hormone-Responsive Protein Signaling and the Gut Microbiota-Mediated Metabolism of Bile Acids

Spermidine, a natural polyamine and physiological autophagy inducer, is involved in various physiological processes. However, the impact and mechanism of spermidine on nonalcoholic steatohepatitis (NASH) remains unclarified. We found that daily spermidine intake was significantly lower in volunteers with liver dysfunction than the healthy controls, and the serum and fecal spermidine levels were negatively correlated with the NASH phenotypes. Spermidine supplementation significantly attenuated hepatic lipid accumulation, insulin resistance, hepatic inflammation, and fibrosis in NASH mice induced by a western diet. The ameliorating effect of spermidine on lipid accumulation might be partly regulated by thyroid hormone-responsive protein (THRSP) signaling and autophagy. Moreover, spermidine altered the profile of hepatic bile acids (BAs) and microbial composition and function. Furthermore, spermidine reversed the progression of hepatic steatosis, inflammation, and fibrosis in mice with preexisting NASH. Therefore, spermidine ameliorates NASH partly through the THRSP signaling and the gut microbiota-mediated metabolism of BAs, suggesting that spermidine might be a viable therapy for NASH.

Low striatal T3 is implicated in inattention and memory impairment in an ADHD mouse\xa0model overexpressing thyroid hormone-responsive protein

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder, potentially with a biological basis; however, its exact cause remains unknown. Thyroid hormone (TH) abnormalities are more prevalent in patients with ADHD than in the general population, indicating a shared pathogenetic mechanism for these conditions. Previously, we identified that overexpression of thyroid hormone-responsive protein (THRSP), a gene highly responsive to TH status, induced inattention in male mice. Herein, we sought to explore whether TH function in THRSP-overexpressing (THRSP OE) mice influences ADHD-like (inattention) behavior. We now confirm that THRSP overexpression in male mice reproduces behavioral features of ADHD, including sustained inattention and memory impairment, accompanied by excessive theta waves that were found normal in both the THRSP-knockout and hetero groups. Physiological characterization revealed low striatal T3 levels in the THRSP OE mice due to reduced striatal T3-specific monocarboxylate transporter 8 (MCT8), indicating brain-specific hypothyroidism in this transgenic mouse strain. TH replacement for seven days rescued inattention and memory impairment and the normalization of theta waves. This study further supports the involvement of the upregulated THRSP gene in ADHD pathology and indicates that THRSP OE mice can serve as an animal model for the predominantly inattentive subtype of ADHD.

Lipogenic Thrsp as a regulator of adipocyte metabolism

Background and Aims: Adipocyte lipogenesis is required in order for adipocytes to store excess energy and to maintain healthy energy homeostasis. Thyroid hormone responsive protein (THRSP) is a lipogenic nuclear protein. The function of THRSP in human has not yet been studied, hence we characterized its role in human adipocytes.

High-Throughput RNA Sequencing Reveals NDUFC2-AS lncRNA Promotes Adipogenic Differentiation in Chinese Buffalo (Bubalus bubalis L).

The buffalo (Bubalus bubalis L.) is prevalent in China and the increasing demand for meat production has changed its role from being a beast of burden to a meat source. The low fat deposition level has become one of the main barriers for its use in meat production. It is urgent to reveal factors involved in fat deposition in buffalo. This study performed RNA sequencing to investigate both long noncoding RNAs (lncRNAs) and mRNAs of adipose tissues in young and adult buffalos. A total of 124 lncRNAs and 2008 mRNAs showed differential expression patterns between young and adult samples. Coexpression analysis and functional enrichment revealed 585 mRNA–lncRNA pairs with potential function in fat deposition. After validation by qRT-PCR, we focused on a lncRNA transcribed from the ubiquinone oxidoreductase subunit C2 (NDUFC2) antisense (AS) strand which showed high correlation with thyroid hormone responsive protein (THRSP). NDUFC2-AS lncRNA is highly expressed in adipose tissue and maturation adipocytes and mainly exists in the nucleus. Functional assays demonstrated that NDUFC2-AS lncRNA promotes adipogenic differentiation by upregulating the expression levels of THRSP and CCAAT enhancer binding protein alpha (C/EBPα) in buffalo. These results indicate that NDUFC2-AS lncRNA promotes fat deposition in buffalo.

Recognition Impairment by Thrsp Overexpression in Brain

Thrsp is a genetic code for thyroid hormone responsive protein. Thrsp was reported to increase thyroid hormone induced neuronal cell death and to decrease proliferation of neural stem cell and progenitor cells. In this study we produced Thrsp overexpressed transgenic animals and evaluate its neuropharmacological behaviors. As a result, Thrsp transgenic animals showed increased locomotor activity, decreased attention and memory, and increased impulsivity. It also showed decreased cognitive function. Therefore Thrsp overexpression could affect on cognitive impairment and this animal might be used as an animal model for cognitive impairment. It needs further study to validate this animal model and to explore neuropharmacological activity of Thrsp in the brain.

Effects of protein and fat concentration in coproduct-based growing calf diets on adipogenic and lipogenic gene expression, blood metabolites, and carcass composition.

Crossbred calves ( = 30; age = 95 ± 1.7 d; BW = 179 ± 18 kg) were fed 1 of 5 growing diets: 1) corn-based control, 2) low-fat, low-protein coproduct blend, 3) high-fat, low-protein coproduct blend, 4) low-fat, high-protein coproduct blend, and 5) high-fat, high-protein coproduct blend for 112 d (growing phase) followed by a common corn-based finishing diet (additional 112 d; finishing phase). Calves were biopsied at 0, 112, and 224 d for transcriptional analysis via real-time quantitative PCR of 14 genes associated with adipogenesis and lipogenesis within the muscle. Serum was collected at d 0, 112, and 224 and analyzed for leptin, IGF-1, and GH concentration. Treatments were arranged in a 2 × 2 factorial. Data were analyzed using the MIXED procedures of SAS (SAS Inst. Inc., Cary, NC) to ascertain the effects of 2 protein levels, 2 fat levels, time, and any interactions. Increased protein and decreased fat in the growing diet resulted in a carryover effect that increased ( 0.01) gene expression of PPARγ, insulin-induced gene 1, thyroid hormone responsive SPOT14 protein, ATP citrate lyase, adiponectin, diacylglycerol O-acyltransferase homologue 2, fatty acid binding protein 4, fatty acid synthase, phosphoenolpyruvate carboxykinase 1, and stearoyl-CoA desaturase as well as serum leptin concentrations between d 112 and 224. Expression of sterol regulatory element binding transcription factor 1 was increased ( 0.01) at d 112 in steers fed high-protein, high-fat diets compared to those fed high-protein, low-fat diets. A fat × day interaction ( 0.01) occurred for the expression of adiponectin receptor 2 and CCAAT/enhancer binding protein alpha, resulting in a carryover effect wherein low-fat diets fed during the growing phase increased expression of both genes at the end of the finishing phase (d 224). After slaughter, cattle fed the control during the growing phase tended ( 0.09) to have greater marbling scores, whereas other carcass parameters were not different ( ≥ 0.13). These data indicate that feeding differing levels of dietary fat and protein during the growing phase does affect i.m. adipogenesis at the transcriptional level, but differences in gene expression were not sufficient to affect carcass quality among cattle fed coproducts.

Increased expression of thyroid hormone responsive protein (THRSP) is the result but not the cause of higher intramuscular fat content in cattle.

Thyroid hormone responsive protein (THRSP) is known to be involved in lipogenic processes in rodents. In cattle, THRSP could be a potential molecular marker for intramuscular fat (IMF) deposition since mRNA abundance was frequently found to be increased in skeletal muscle with high IMF content compared to those with low IMF. The aim of this study was to elucidate the background of this differential expression and to evaluate the role of THRSP as candidate for increased IMF content in cattle. By combination of mRNA and protein analyses, we could demonstrate that THRSP is present mainly in nuclei of adipose tissue, in intramuscular fat cells and associated cells, and in cells of the portal triad of liver, whereas muscle cells did not express THRSP. Cell culture analyses revealed furthermore that THRSP is expressed in mature adipocytes rather than in early stages of adipogenesis. Collectively, our data support the putative role of THRSP as transcriptional regulator and demonstrate that an increased expression of THRSP in M. longissimus is a consequence of but not the reason for a higher number of intramuscular adipocytes in cattle with enhanced IMF deposition.

Effects of protein and fat concentration in coproduct-based growing calf diets on adipogenic and lipogenic gene expression, blood metabolites, and carcass composition

Crossbred calves (n = 30; age = 95 ± 1.7 d; BW = 179 ± 18 kg) were fed 1 of 5 growing diets: 1) corn-based control, 2) low-fat, low-protein coproduct blend, 3) high-fat, low-protein coproduct blend, 4) low-fat, high-protein coproduct blend, and 5) high-fat, high-protein coproduct blend for 112 d (growing phase) followed by a common corn-based finishing diet (additional 112 d; finishing phase). Calves were biopsied at 0, 112, and 224 d for transcriptional analysis via real-time quantitative PCR of 14 genes associated with adipogenesis and lipogenesis within the muscle. Serum was collected at d 0, 112, and 224 and analyzed for leptin, IGF-1, and GH concentration. Treatments were arranged in a 2 × 2 factorial. Data were analyzed using the MIXED procedures of SAS (SAS Inst. Inc., Cary, NC) to ascertain the effects of 2 protein levels, 2 fat levels, time, and any interactions. Increased protein and decreased fat in the growing diet resulted in a carryover effect that increased (P < 0.01) gene expression of PPARγ, insulin-induced gene 1, thyroid hormone responsive SPOT14 protein, ATP citrate lyase, adiponectin, diacylglycerol O-acyltransferase homologue 2, fatty acid binding protein 4, fatty acid synthase, phosphoenolpyruvate carboxykinase 1, and stearoyl-CoA desaturase as well as serum leptin concentrations between d 112 and 224. Expression of sterol regulatory element binding transcription factor 1 was increased (P < 0.01) at d 112 in steers fed high-protein, high-fat diets compared to those fed high-protein, low-fat diets. A fat × day interaction (P < 0.01) occurred for the expression of adiponectin receptor 2 and CCAAT/enhancer binding protein alpha, resulting in a carryover effect wherein low-fat diets fed during the growing phase increased expression of both genes at the end of the finishing phase (d 224). After slaughter, cattle fed the control during the growing phase tended (P = 0.09) to have greater marbling scores, whereas other carcass parameters were not different (P ≥ 0.13). These data indicate that feeding differing levels of dietary fat and protein during the growing phase does affect i.m. adipogenesis at the transcriptional level, but differences in gene expression were not sufficient to affect carcass quality among cattle fed coproducts.

Differential Hepatic Gene Expression Profile and Histone Modification After Acute or Chronic Hyperthyroidism or During Remission.

In acute and chronic hyperthyroid patients, clinical symptoms may not always reflect their thyroid hormone status. After normalization of serum thyroid hormone concentrations in these patients, some symptoms caused by hyperthyroidism may remain unchanged (1). Even in subclinical hyperthyroid patients, functional recovery after restoration to euthyroidism is sometimes incomplete (2). The underlying mechanisms of such incomplete recovery are not yet fully understood. Indeed, functional adaptation or irreversible alteration may be induced during exposure to excess thyroid hormone. The liver is a representative target organ of thyroid hormone action, where T3 regulates various hepatic functions including cholesterol, triglyceride, and carbohydrate metabolism (3). To accomplish this, T3 regulates the expression of many rate-limiting enzymes of such metabolic pathways. In addition, thyroid hormone signaling activates autophagy, which plays a key role in regulating lipid metabolism, mitochondrial quality, and various proteins regulating metabolic pathways in hepatocytes (4). Thus, dysregulation of thyroid hormone action in liver may induce serious clinical conditions such as nonalcoholic fatty liver disease (5). To examine the role of thyroid hormone on the expression of hepatic genes, several groups have applied DNA microarray analysis to the hepatic gene expression profile (6). In these studies, it has become clear that the transcriptomic response is different between acute and chronic T3 treatment (7). However, the expression profile of T3-sensitive genes after amelioration of hyperthyroidism has not yet been studied. In this issue of Endocrinology, Ohba et al (8) examined the effects of a single T3 injection, chronic T3 injections (14 d), or chronic injections followed T3 withdrawal for 10 days on the hepatic gene expression profile of adult male mice using gene expression microarrays. Because they used a large dose of T3 (20 g/100 g body weight), all mice became hyperthyroid, which was confirmed by suppressed TSH levels. TSH levels then increased transiently 5 days after the last T3 injection, before normalizing by 10 days. They identified 680 genes that were induced by either a single or chronic injection of T3. Interestingly, only 10% of these genes were induced by both treatments, indicating that most acutely induced T3-sensitive genes became desensitized to the administration dose over time. Furthermore, 10% of genes did not return to normal levels of expression despite normalization of thyroid hormone concentrations after T3 withdrawal. Although Ohba et al (8) found many genes that were altered by T3 treatment or its withdrawal, such genes may not be directly regulated by thyroid hormone. Thus, they studied further the expression of 8 genes (B cell lymphoma 3 [Bcl3], cytochrome P450 17 -hydroxylase [Cyp17a1], Cyp 17,20-lyase [Cpt1a], fatty acid synthase [Fasn], fibroblast growth factor 21 [Fgf21], isocitrate dehydrogenase 3 [Idh3a], thyroid hormone-responsive protein [Thrsp], and type-1 iodothyronine deiodinase [Dio1]) thought to have functional thyroid hormone response elements. Interestingly, despite this, the expression pattern was not always the same. Among 6 genes that were induced by acute T3 treatment (Bcl3, Cpt1a, Dio1, Fasn, Idh3a, and Thrsp), the expression of 4 genes (Bcl3, Cpt1a, Fasn, and Thrsp) was not elevated after 14 days of T3 treatment, indicating desensitization by chronic treatment, whereas both Dio1 and Idh3a were induced by both

Thyroid hormone responsive protein spot 14 enhances lipogenesis in bovine mammary epithelial cells.

Thyroid hormone responsive spot 14 (THRSP, Spot14, S14) is a nuclear protein that regulates milk fat synthesis. To investigate the role of THRSP in lipogenesis in the dairy cow mammary gland, first, we examined the association between milk fat concentration and THRSP expression in the mammary gland. We found that the dairy cow mammary glands that produced milk with high fat had high THRSP mRNA and protein levels. Additionally, the study described the consequences of overexpression or depletion of THRSP on lipogenesis in cultured bovine mammary epithelial cells (BMECs). We found that BMECs with overexpressed THRSP increased triacylglycerol levels and enhanced the expression of FAS, PPARγ, and SREBP1, compared with the control BMECs. Depletion of THRSP produced the opposite effects. Overall, increased mammary expression of THRSP can be a marker of high fat. In addition, our results provide evidence that THRSP may regulate expression of PPARγ and SREBP1 and can regulate milk fat synthesis by directly affecting the activity of some classical lipogenic enzymes.

Modulation of tumor fatty acids, through overexpression or loss of thyroid hormone responsive protein spot 14 is associated with altered growth and metastasis.

IntroductionSpot14 (S14), encoded by the THRSP gene, regulates de novo fatty acid synthesis in the liver, adipose, and lactating mammary gland. We recently showed that S14 stimulated fatty acid synthase (FASN) activity in vitro, and increased the synthesis of fatty acids in mammary epithelial cells in vivo. Elevated de novo fatty acid synthesis is a distinguishing feature of many solid tumors compared with adjacent normal tissue. This characteristic is thought to be acquired during tumor progression, as rapidly proliferating cells have a heightened requirement for membrane phospholipids. Further, overexpression of FASN is sufficient to stimulate cell proliferation. While many studies have focused on the FASN enzyme in cancer biology, few studies have addressed the roles of proteins that modify FASN activity, such as S14.MethodsTumor fatty acids were modulated using two mouse models, mouse mammary tumor virus (MMTV)-neu mice overexpressing S14 and MMTV-polyomavirus middle T antigen (PyMT) mice lacking S14, and associations between elevated or impaired fatty acid synthesis on tumor latency, growth, metastasis, and signaling pathways were investigated. We evaluated S14-dependent gene expression profiles in mouse tumors by microarray and used publicly available microarray datasets of human breast tumors.ResultsS14 overexpression in the MMTV-Neu transgenic model is associated with elevated medium-chain fatty acids, increased proliferation and a shorter tumor latency, but reduced tumor metastasis compared to controls. Loss of S14 in the MMTV-PyMT model decreased FASN activity and the synthesis of medium-chain fatty acids but did not alter tumor latency. Impaired fatty acid synthesis was associated with reduced solid tumor cell proliferation, the formation of cystic lesions in some animals, and decreased phosphorylation of Src and protein kinase B (Akt). Analysis of gene expression in these mouse and human tumors revealed a relationship between S14 status and the expression of genes associated with luminal epithelial differentiation.ConclusionsThis study demonstrates a potential role for S14 in regulating mammary tumor growth and fatty acid synthesis in vivo. Furthermore, these results suggest that modulating the amount of medium chain fatty acids, by changing the levels of S14, has the potential to impact malignant mammary tumor phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0481-z) contains supplementary material, which is available to authorized users.

Abstract 1417: Stimulation of fatty acid synthesis by Spot14 enhances tumor cell proliferation but decreases metastasis in vivo

Abstract We have recently observed that Spot14 (S14), also known as Thyroid Hormone Responsive Protein, stimulates de novo fatty acid biosynthesis in mammary epithelial cells by increasing Fatty Acid Synthase (FASN) activity. In the presence of S14, FASN had increased Vmax, decreased Km, and shifted product formation toward medium chain fatty acids. Numerous studies have suggested that high expression of FASN is associated with higher tumor grade and poor clinical outcome, and likewise elevated expression of S14 has been linked to a poor clinical outcome for patients with invasive disease. In contrast, our analysis of S14 expression in databases of human breast tumors representing a variety of stages, grades, and subtypes suggests that high S14 is associated with ER-positive status, the luminal intrinsic subtype, and consequently, a favorable patient outcome. To determine the effect of S14 on mammary tumorigenesis in vivo, we crossed S14 null mice to MMTV-PyMT mice. Tumor latency was not altered in mice lacking S14; however, tumor cell proliferation was significantly decreased. Tumors arising in MMTV-PyMT mice lacking S14 were less dense and had a cystic morphology. There was a decrease in the phosphorylation of Src at Y416 and Akt at S473, indicating that activation of both signaling molecules was decreased in tumors lacking S14. Furthermore, tumors lacking S14 had reduced levels of de novo synthesized fatty acids. We also crossed MMTV-S14 transgenic mice expressing S14 in the mammary epithelium, to MMTV-Neu line 202 mice and observed that S14 overexpression significantly shortened tumor latency, increased tumor cell proliferation and increased concentration of fatty acids in tumor cells. Despite the increased proliferative index observed in the MMTV-Neu tumors expressing S14, there was a decrease in metastatic lesions detected in the lungs compared to MMTV-Neu mice. Gene expression profiling of mouse tumors revealed that tumors overexpressing S14 displayed features of lobular differentiation including increased expression of Elf5, a proposed master regulator of mammary differentiation, and expression of milk protein genes. Elevated fatty acid synthesis is a distinguishing feature of many solid tumors compared to adjacent normal tissue and this activity is thought to be acquired during tumor progression, as rapidly proliferating cancer cells have a heightened requirement for membrane phospholipid precursors. Our studies suggest that increased de novo fatty acid biosynthesis is sufficient to stimulate tumor cell proliferation; however, this is not correlated with increased metastasis, and in fact, tumors that expressed high levels of S14 appeared to have a more differentiated phenotype that may correlate with improved clinical outcome. Citation Format: Elizabeth A. Wellberg, Michael C. Rudolph, Andrew Lewis, Steven M. Anderson. Stimulation of fatty acid synthesis by Spot14 enhances tumor cell proliferation but decreases metastasis in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1417. doi:10.1158/1538-7445.AM2014-1417

Identification of exonic nucleotide variants of the thyroid hormone responsive protein gene associated with carcass traits and Fatty Acid composition in korean cattle.

The thyroid hormone responsive protein (THRSP) gene is a functional gene that can be used to indicate the fatty acid compositions. This study investigates the relationships of exonic single nucleotide polymorphisms (SNPs) in the THRSP gene and fatty acid composition of muscle fat and marbling score in the 612 Korean cattle. The relationships between fatty acid composition and eight SNPs in the THRSP gene (g.78 G>A, g.173 C>T, g.184 C>T, g.190 C>A, g.194 C>T, g.277 C>G, g.283 T>G and g.290 T>G) were investigated, and according to the results, two SNPs (g.78 G>A and g.184 C>T) in exon 1 were associated with fatty acid composition. The GG and CC genotypes of g.78 G>A and g.184 C>T had higher unsaturated fatty acid (UFA) and monounsaturated fatty acid (MUFA) content (p<0.05). In addition, the ht1*ht1 group (Val/Ala haplotype) in a linkage disequilibrium increased MUFAs and marbling scores for carcass traits (p<0.05). As a result, g.78 G>A and g.184 C>T had significantly relationships with UFAs and MUFAs. Two SNPs in the THRSP gene affected fatty acid composition, suggesting that GG and CC genotypes and the ht1*ht1 group (Val/Ala haplotype) can be markers to genetically improve the quality and flavor of beef.

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [(13)C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed.

Spot14/Mig12 heterocomplex sequesters polymerization and restrains catalytic function of human acetyl‐CoA carboxylase 2

Acetyl-CoA carboxylase 2 (ACC2) is an isoform of ACC functioning as a negative regulator of fatty acid β-oxidation. Spot14, a thyroid hormone responsive protein, and Mig12, a Spot14 paralog, have recently been identified as regulators of fatty acid synthesis targeting ACC1, a distinctive subtype of ACC. Here, we examined whether Spot14/Mig12 modulates ACC2. Nanoscale protein topography mapped putative protein–protein interactions between purified human Spot14/Mig12 and ACC2, validated by functional assays. Human ACC2 displayed consistent enzymatic activity, and homogeneous particle distribution was probed by atomic force microscopy. Citrate-induced polymerization and enzymatic activity of ACC2 were restrained by the addition of the recombinant Spot14/Mig12 heterocomplex but only partially by the oligo-heterocomplex, demonstrating that the heterocomplex is a designated metabolic inhibitor of human ACC2. Moreover, Spot14/Mig12 demonstrated a sequestering role preventing an initial ACC2 nucleation step during filamentous polymer formation. Thus, the Spot14/Mig12 heterocomplex controls human ACC2 polymerization and catalytic function, emerging as a previously unrecognized molecular regulator in catalytic lipid metabolism. © 2013 The Authors. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our goal was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation. Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6 h to 42 days postpartum in seven normal women. Over the first 96 h postpartum, daily milk fat output increased severalfold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production, including lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor 1 (SREBF1), increased 2.0-fold by 36 h and remained elevated over the study duration. Expression of genes for estrogen receptor 1, thyroid hormone-responsive protein, and insulin-induced 2 increased progressively to plateau by 96 h. In contrast, mRNA of peroxisome proliferator-activated receptor-γ decreased severalfold. With onset of lactation, increased de novo synthesis of FA was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. In conclusion, milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role.

Expression, purification and preliminary crystallographic analysis of human thyroid hormone responsive protein

Thyroid hormone responsive protein (Thrsp, also known as Spot 14 and S14) is a carbohydrate-inducible and thyroid-hormone-inducible nuclear protein specific to liver, adipose and lactating mammary tissues. Thrsp functions to activate genes encoding fatty-acid synthesis enzymes. Recent studies have shown that in some cancers human Thrsp (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumourigenesis. Thrsp, a member of the Spot 14 superfamily, is an acidic homodimeric protein with no sequence similarity to other mammalian gene products and its biochemical function is elusive. To shed light on the structure-function relationship of this protein, human Thrsp was crystallized. Recombinant human Thrsp (hThrsp), the N-terminally truncated human Thrsp(10-146) (hThrsp9) and their selenomethionyl (SeMet) derivatives were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 293 K using Li(2)SO(4) as a precipitant. Using synchrotron radiation, data for the hThrsp SeMet derivative, hThrsp9 and its SeMet derivative were collected to 4.0, 3.0 and 3.6 Å resolution, respectively, at 100 K. The crystals of full-length hThrsp and its SeMet derivative belonged to space group P4(1)2(1)2, with approximate unit-cell parameters a = b = 123.9, c = 242.1 Å, α = β = γ = 90.0°. In contrast, the crystals of the truncated hThrsp9 and its SeMet derivative belonged to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 91.6, b = 100.8, c = 193.7 Å, α = β = γ = 90.0°. A molecular-replacement solution calculated using a murine Spot 14 structure as a search model indicated the presence of six molecules per asymmetric unit, comprising three hThrsp homodimers.

Novel Theranostic Opportunities Offered by Characterization of Altered Membrane Lipid Metabolism in Breast Cancer Progression

Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many cancers. However, the precise molecular composition of lipids in tumors remains generally poorly characterized. The aim of the present study was to analyze the global lipid profiles of breast cancer, integrate the results to protein expression, and validate the findings by functional experiments. Comprehensive lipidomics was conducted in 267 human breast tissues using ultraperformance liquid chromatography/ mass spectrometry. The products of de novo fatty acid synthesis incorporated into membrane phospholipids, such as palmitate-containing phosphatidylcholines, were increased in tumors as compared with normal breast tissues. These lipids were associated with cancer progression and patient survival, as their concentration was highest in estrogen receptor-negative and grade 3 tumors. In silico transcriptomics database was utilized in investigating the expression of lipid metabolism related genes in breast cancer, and on the basis of these results, the expression of specific proteins was studied by immunohistochemistry. Immunohistochemical analyses showed that several genes regulating lipid metabolism were highly expressed in clinical breast cancer samples and supported also the lipidomics results. Gene silencing experiments with seven genes [ACACA (acetyl-CoA carboxylase α), ELOVL1 (elongation of very long chain fatty acid-like 1), FASN (fatty acid synthase), INSIG1 (insulin-induced gene 1), SCAP (sterol regulatory element-binding protein cleavage-activating protein), SCD (stearoyl-CoA desaturase), and THRSP (thyroid hormone-responsive protein)] indicated that silencing of multiple lipid metabolism-regulating genes reduced the lipidomic profiles and viability of the breast cancer cells. Taken together, our results imply that phospholipids may have diagnostic potential as well as that modulation of their metabolism may provide therapeutic opportunities in breast cancer treatment.