Shih-Torng Ding

Expression of porcine adipocyte transcripts: tissue distribution and differentiation in vitro and in vivo.

Transcription factor transcripts implicated in adipocyte differentiation (peroxisome proliferator-activated receptor gamma (PPAR gamma), retinoid x receptor alpha (RXR alpha), adipocyte determination and differentiation-dependent factor 1 (ADD1), and CCAAT/enhancer binding protein alpha (C/EBP alpha)) and adipocyte-characteristic protein transcripts (lipoprotein lipase (LPL) and adipocyte fatty acid binding protein (aP2)) were measured in pig tissues. Transcripts for PPAR gamma, ADD1, and aP2 were localized in porcine subcutaneous and perirenal adipose tissues; transcripts for C/EBP alpha and LPL were detected in other tissues, but the greatest concentrations were in the adipose tissues. In porcine stromal-vascular cells (S/V cells) differentiating in vitro, transcripts for PPAR gamma and aP2 increased gradually, transcripts for ADD1, and LPL increased early and transcripts for C/EBP alpha increased late. In pigs, adipose tissue transcripts for PPAR gamma, ADD1, and LPL were minimal at birth and increased to 28 days postpartum, transcripts for C/EBP alpha were low until 28 days and transcripts for aP2 were at high levels, regardless of age. Although transcript development was somewhat different in vitro and in vivo, the data suggest PPAR gamma (and ADD1 are involved in regulation of transcripts for LPL and that there may be more partially differentiated precursor cells in S/V cells at day 0 than in adipose tissue at birth.

Expression of porcine adipocyte transcripts during differentiation in vitro and in vivo.

Transcript concentrations for the transcription factors, CCAAT enhancer binding protein beta and alpha (C/EBPbeta and C/EBPalpha), plus the adipocyte-characteristic proteins, fatty acid synthase (FAS), glucose transporter 4 (Glut 4), hormone-sensitive lipase (HSL), insulin receptor (InsR), lipoprotein lipase (LPL), and leptin were measured during differentiation of porcine stromal-vascular (S/V) cells in vitro. These same transcripts, excluding FAS and InsR, were measured in porcine adipose tissue from birth to 7 weeks of age. In S/V cells, C/EBPbeta and InsR were continuously elevated. At day 0, C/EBPalpha was approximately 20% of the day 9 value. The LPL increased gradually from day 0 to 9, whereas most other transcripts had a lag period of several days. In tissue, C/EBPbeta was substantial at birth and increased gradually. The C/EBPalpha was relatively low at birth and increased at day 17. The LPL and leptin increased continuously. The Glut 4 was low at birth and increased at day 28. The HSL was relatively low at birth, increased at day 10, and plateaued at day 28. Transcripts in porcine S/V cells develop somewhat differently from adipocyte differentiation models established in clonal cells, but the porcine cells represent a model that should be more applicable to pigs.

Fatty acids modulate porcine adipocyte differentiation and transcripts for transcription factors and adipocyte-characteristic proteins*

Porcine stromal-vascular cells (S/V cells) differentiate into adipocytes in vitro when presented with appropriate hormones and growth factors. Porcine S/V cells were differentiated in vitro in serum-free media with or without fatty acids to determine the effect of fatty acids on differentiation and on transcripts for peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL) and adipocyte fatty acid binding protein (aP2). Differentiation was measured by Oil Red O staining and transcript concentrations were measured by Northern analysis using porcine riboprobes. Addition of 100 µM oleic acid (C18:1) for 5 days increased differentiation and the mRNA levels for PPARgamma, C/EBPalpha, LPL and aP2. Other medium- and long-chain fatty acids were less active. Adipocyte differentiation and transcript concentrations for PPARgamma, C/EBPalpha, LPL and aP2 were increased by C18:1 in a dose-related manner. Differentiation was greater at 10 days than at 5 days than at 1 day, and C18:1 increased differentiation at each time. Transcript concentrations were increased by C18:1 at 1 and 5 days, but not at 10 days. These results suggest that the main effect of C18:1 is on regulating gene expression (an acute or drug-like effect) rather than changing the membrane fluidity as a result of changing membrane fatty acid composition (a chronic or nutrient-like effect). Taken together, these results indicate that selected fatty acids modulate porcine adipocyte differentiation and transcripts for adipocyte differentiation-related proteins such as PPARgamma, C/EBPalpha, LPL and aP2.

Adiponectin receptor 1 regulates bone formation and osteoblast differentiation by GSK-3β/β-Catenin signaling in mice

Adiponectin and its receptors are expressed in bone marrow-derived osteoblasts. Previous studies in vivo and in vitro have produced controversial results. The purpose of this study was to use porcine adiponectin receptor 1 transgenic mice (pAdipoR1) as a model to evaluate the role of AdipoR1 on bone physiology at different ages. pAdipoR1 transgenic mice had higher bone mineral density than wild-type mice in both genders at 56 weeks of age. The bone volume and trabecular number, measured by micro-computed tomography (μCT) was significantly greater in transgenic than in wild-type female mice at both 8 and 56 weeks of age. ELISA analysis revealed that both serum osteocalcin and osteoprotegerin (OPG) were significantly increased in 8-week old pAdipoR1 transgenic mice of both genders. Furthermore, serum OPG was elevated at 32 and 56 weeks of age in female and male pAdipoR1 transgenic mice. Serum TRAP5b concentration was reduced in 8 and 56 weeks old male pAdipoR1 mice compared with wild-type male mice. Knock-down of AdipoR1 significantly decreased gene expression of osteocalcin, OPG, alkaline phosphatase and msh homeobox 2 and the mineralization in MC3T3-E1 cells and mesenchymal stem cells. In addition, pathscan analysis and real-time PCR analysis suggest AdipoR1 regulates osteoblast differentiation through GSK-3 β and β-Catenin signaling. Consequently, the lack of AdipoR1 impaired osteoblast differentiation and bone formation. We conclude that AdipoR1 is a critical factor for the osteoblast differentiation and bone homeostasis.

MODULATION OF ADIPOCYTE DETERMINATION AND DIFFERENTIATION-DEPENDENT FACTOR 1 BY SELECTED POLYUNSATURATED FATTY ACIDS

SummaryThe transcription factor, sterol regulatory binding protein 1c (also called adipocyte determination and differentiation-dependent factor 1), stimulates transcription of the messenger ribonucleic acids (mRNAs) for lipid synthesis enzymes. Hepatic ADD1 transcripts are reduced by polyunsaturated fatty acids (PUFAs). The ADD1 transcripts are expressed to a considerable extent in porcine adipocytes. Consequently, it was of interest to examine the effects of several PUFAs on ADD1 in a tissue wherein several long-chain fatty acids (FAs) increase adipocyte differentiation. The effects of arachidonic acid (C20∶4), docosahexaenoic acid (C22∶6), and cis 9, trans 11-conjugated linoleic acid (9,11-CLA) on differentiating preadipocyte ADD1 mRNA and protein and on preadipocyte differentiation were determined. Porcine stromal-vascular cells were plated in serum-containing medium and differentiated in serum-free medium containing insulin, hydrocortisone, and transferrin ± an individual FA. After 24-h differentiation ± FA, plates were stained with Oil Red O as an indicator of differentiation or total RNA was extracted or a nuclear fraction was isolated for protein measurement. Addition of C20∶4 or 9,11-CLA increased the number of Oil Red O-stained cells or the Oil Red O-stained material, whereas C22∶6 did not. Addition of C20∶4, C22∶6, or 9,11-CLA decreased the concentration of the mRNA and protein for ADD1. Thus, although all three FAs decreased the ADD1 mRNA and protein concentrations, C20∶4 and 9,11-CLA increased differentiation, measured by Oil Red O staining, whereas C22∶6 did not. The data suggest that the regulation of differentiation and mRNAs by individual FAs may involve distinct mechanisms.

Docosahexaenoic acid suppresses the expression of FoxO and its target genes

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, has previously been shown to ameliorate obesity-associated metabolic syndrome. To decipher the mechanism responsible for the beneficial effects of DHA on energy/glucose homeostasis and the metabolic syndrome, 30 weaned cross-bred pigs were randomly assigned to three groups and fed ad libitum with a standard diet supplemented with 2% of beef tallow, soybean oil or DHA oil for 30 days, and the gene expression profile of various tissues was evaluated by quantitative real-time polymerase chain reaction. The DHA-supplemented diets reduced the expression of forkhead box O transcription factor (FoxO) 1 and FoxO3 in the liver and adipose tissue. DHA treatments also decreased the expression of FoxO1 and FoxO3 in human hepatoma cells, SK-HEP-1 and human and porcine primary adipocytes. In addition, DHA also down-regulated FoxO target genes, such as microsomal triacylglycerol transfer protein (MTP), glucose-6-phosphatase, apolipoprotein C-III (apoC-III) and insulin-like growth factor binding-protein 1 in the liver, as well as reduced total plasma levels of cholesterol and triacylglycerol in the pig. Transcriptional suppression of FoxO1, FoxO3, apoC-III and MTP by DHA was further confirmed by reporter assays with each promoter construct. Taken together, our study indicates that DHA modulates lipid and glucose homeostasis in part by down-regulating FoxO function. The down-regulation of genes associated with triacylglycerol metabolism and very low density lipoprotein assembly is likely to contribute to the beneficial effects of DHA on the metabolic syndrome.

EGF-induced Grb7 Recruits and Promotes Ras Activity Essential for the Tumorigenicity of Sk-Br3 Breast Cancer Cells

Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.

Docosahexaenoic acid regulates adipogenic genes in myoblasts via porcine peroxisome proliferator-activated receptor γ

The nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) triggers adipocyte differentiation by regulating lipogenic genes. A ligand for PPARgamma is necessary to activate PPARgamma function. Fatty acids are potential ligands for PPARgamma activation. The current experiment was designed to determine the potential for individual fatty acids to activate porcine PPARgamma ectopically expressed in myoblasts. The expression of adipocyte fatty acid binding protein (aP2) and adiponectin in myoblasts stably expressing porcine PPARgamma was increased when docosahexaenoic acid (DHA) was added to the adipogenic medium. The response was positively related to DHA concentration and suggests that DHA may bind to and activate porcine PPARgamma, leading to increased expression of aP2 and adiponectin. The conditioned media collected from myoblasts expressing PPARgamma between d 3 and 6 or between d 6 and 9, but not DHA itself, activated the aP2 gene promoter-driven luciferase activity. These results suggest that a metabolite of DHA is the ligand binding to and activating porcine PPARgamma. The metabolite and pathway for its production are currently unknown.

CONJUGATED LINOLEIC ACID INCREASES THE DIFFERENTIATION OF PORCINE ADIPOCYTES IN VITRO

Abstract Individual long-chain fatty acids can modulate adipocyte differentiation. Conjugated linoleic acids (CLAs) either stimulate or inhibit 3T3-L1 clonal cell differentiation. We studied the effects of cis-9, trans-11 CLA (9,11-CLA), trans-10, cis-12 CLA (10, 12-CLA), and linoleic acid (LA) on differentiation of porcine stromal-vascular cells in vitro and on mRNA concentrations for adipocyte transcription factors and adipocyte-characterisitc proteins. Fatty acids were added to the differentiation medium at 0, 50, 100, or 300 μM, for 24 hours. The LA tended to, and 9,11-CLA increased differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA concentration was not changed by LA, 9,11-CLA, or 10,12-CLA. Lipoprotein lipase mRNA concentration was not changed by LA, but was increased by both CLA isomers. Adipocyte fatty acid binding protein (aP2) mRNA concentration was increased by LA and both isomers of CLA. In summary, CLA and LA increased differentiation of porcine stromal-vascular cells after 24 hours, but differentiation was not accompanied by increased PPARγ mRNA suggesting, at least in these experiments, that the primary action of the fatty acids was as ligands for PPARγ, rather than as inducers for PPARγ transcripts. The aP2 mRNA concentration was increased to a large extent by the various fatty acids.

The function of porcine PPARγ and dietary fish oil effect on the expression of lipid and glucose metabolism related genes

Peroxisome-proliferator-activated receptor γ (PPARγ) plays a critical role in regulation of adipocyte differentiation and insulin sensitivity. To become functional, PPARγ must be activated by binding an appropriate ligand. Polyunsaturated fatty acids (PUFA) are potential ligands for PPARγ. The current experiment was designed to determine the potential for PUFA, particularly eicosapentaenoic acid and docosahexaenoic acid, to activate the function of porcine PPARγ in vivo. Transgenic mice, expressing porcine PPARγ in skeletal muscle were generated and fed with a high-saturated fat (beef tallow) or high-unsaturated fat (fish oil) diet for 4 months. When transgenic mice were fed a fish oil supplemented diet, the expression of adipogenic and glucose uptake genes was increased, leading to reduced plasma glucose concentration. The PPARγ transgene increased the expression of Glut4 in the muscle. This result suggests that there was increased glucose utilization and, therefore, a reduced blood glucose concentration in the transgenic mice. Also, the plasma adiponectin was elevated by fish oil treatment, suggesting a role of adiponectin in mediating the PUFA effect. These results suggest that PUFA may serve as a natural regulator of glucose uptake in vivo and these effects are mainly through PPARγ function.