Digital expression analysis of the genes associated with salinity resistance after overexpression of a stress-responsive small GTP-binding RabG protein in peanut.

The Rab protein family is the largest family of the small GTP-binding proteins. Among them, the RabG genes are known to be responsive to abiotic stresses, but the molecular mechanisms of the stress responses mediated by RabG genes in plants is poorly understood. To investigate the molecular mechanism of AhRabG gene in peanut, transgenic plants overexpressing the AhRabG gene (S6) with relatively higher salinity resistance than the non-transgenic plants (S7) were obtained. Digital gene expression (DGE) sequencing was performed with the leaves of S6 and S7 plants before and after salinity-stress treatment. The AhRabG gene in peanut was found to be involved in a few pathways such as "photosynthesis", "oxidative phosphorylation", "AMPK signaling pathway", "plant hormone signal transduction", etc. A total of 298 differentially expressed genes (DEGs) were found to be upregulated or downregulated at five sampling time points based on the comparison between S6 and S7 plants. Among them, 132 DEGs were responsive to salinity stress in S6 and/or S7 after salinity-stress treatment. These 132 DEGs included genes encoding various transcription factors and proteins involved in resistance to salinity stress such as MYB, AP2, RING-H2 zinc finger proteins, late embryogenesis abundant (LEA) proteins, dehydration-responsive protein RD22, peroxidases, CBL-interacting protein kinases, calcium-binding proteins, and others. The information from this study will be useful for further studies on elucidating the mechanism of salinity resistance conferred by RabG genein peanut.

Effects of atosiban on stress-related neuroendocrine factors.

Atosiban, an oxytocin/vasopressin receptor antagonist, is used to decrease preterm uterine activity. The risk of preterm delivery is undoubtedly associated with stress, but potential side effects of atosiban on neuroendocrine functions and stress-related pathways are mostly unknown. These studies were designed to test the hypothesis that the chronic treatment of rats with atosiban modulates neuroendocrine functions under stress conditions. Male rats were treated (osmotic minipumps) with atosiban (600 µg/kg per day) or vehicle and were restrained for 120 min/day for 14 days. All animals were treated with a marker of cell proliferation 5-bromo-2-deoxyuridine. Anxiety-like behavior was measured using an elevated plus-maze. Treatment with atosiban failed to modify plasma concentrations of the stress hormones ACTH and corticosterone, but led to a rise in circulating copeptin. Atosiban increased prolactin levels in the non-stressed group. Oxytocin receptor mRNA levels were increased in rats exposed to stress. Treatment with atosiban, in both control and stressed animals, resulted in a decrease in oxytocin receptor gene expression in the hypothalamus. No changes were observed in vasopressin receptor 1A and 1B gene expression. The decrease in hippocampal cell proliferation induced by stress exposure was not modified by atosiban treatment. This study provides the first data, to our knowledge, revealing the effect of atosiban on gene expression of oxytocin receptors in the brain. Atosiban-induced enhancement of plasma copeptin indicates an elevation in vasopressinergic tone with potential influence on water-electrolyte balance.

Differential gene expression between the porcine morula and blastocyst.

The survival and development of pre-implantation embryos are determinant factors affecting the outcome of animal reproduction. It is essential to transfer the expression of the genetic material from maternal sources, that is the ovum to the zygote before implantation to ensure successful development. Differentiation and transformation of blastomeres initiated during the morula and blastocyst stages is an important step of the embryonic development prior to implantation. We collected morula and early blastocyst samples from pure-bred Landrace pigs in vivo to study the differential gene expression patterns at these two stages. Total RNA was extracted from individual embryos and two rounds of amplification were employed. Two micrograms of antisense RNA, targets, were prepared and hybridized with each of four custom made oligo microarrays representing 24,000 porcine genes. The analyses of replicate hybridizations showed that among the 24,000 genes, 162 genes were expressed fivefold or greater in the morula compared to early blastocysts and 2126 genes were expressed fivefold or greater in early blastocysts compared to the morula. Of these differentially expressed genes, 1429 genes were functionally annotated with related human Gene Ontology terms. In addition to basic metabolic processes, genes related to signal transduction, transportation and cell differentiation were found in both stages and were up-regulated as embryo development proceeded. Real time polymerase chain reaction was utilized to quantify 12 genes differentially expressed in the 2 embryonic stages and validated the reliability of major evidences shown in microarrays. In conclusion, we have obtained a preliminary landscape of genes differentially expressed during the transition from morula to early blastocysts in pigs and showed a generally increased transcriptional activity, perhaps in preparation for implantation. Our results provide an opportunity to study the functions of these genes in relation to the development and survival of pre-implantation porcine embryos.

Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene.

The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

Regulation of the expression of angiotensin-converting enzyme 2 by polyunsaturated fatty acids in porcine adipocytes.

Using suppression subtractive hybridization technique, we found that 2 novel genes (AEUG1 and AEUG3) were highly expressed in the adipocytes compared with preadipocytes. We then identified that these 2 genes were both angiotensin-converting enzyme 2 (ACE2). We applied 3'RACE (rapid amplification of cDNA end), 5'RACE, and PCR to obtain the full-length porcine ACE2 cDNA sequence. Because eicosanoids derived from PUFA are involved in regulating blood pressure, we hypothesized that PUFA can regulate the expression of the vasodilator ACE2. Preadipocytes from Landrace pigs were induced to differentiate for 4 d, then treated with 50 µM of different PUFA, CLA, arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or stearic acid (18:0). Addition of EPA or 18:0 for 48 h did not change the ACE2 mRNA abundance, whereas the treatments of arachidonic acid, CLA, and DHA significantly decreased ACE2 mRNA abundance after 48 h (P ≤ 0.05). Treatment with PUFA did not change (P > 0.05) type I and type II angiotensin receptor mRNA abundance. To further understand how PUFA metabolites affect ACE2 mRNA expression, we inhibited individual enzymes that are involved in eicosanoid production. We found that 3 individual eicosanoid pathway enzyme inhibitors recovered the PUFA effect on the expression of ACE2, indicating these pathways are involved in mediating the PUFA function. In conclusion, we obtained the full-length porcine ACE2 cDNA sequence and found PUFA could downregulate the expression of ACE2 through its metabolites without changing the expression of their receptor in porcine adipocytes.

Visfatin regulates genes related to lipid metabolism in porcine adipocytes.

Visfatin is a visceral adipose tissue-specific adipocytokine that plays a positive role in attenuating insulin resistance by binding to the insulin receptor. Visfatin has been suggested to play a role in the regulation of lipid metabolism and inflammation; however, the mechanism remains unclear. We investigated the effects of visfatin on the regulation of gene expression in cultured porcine preadipocytes and differentiated adipocytes. In preadipocytes, the mRNA abundance of lipoprotein lipase and PPARgamma were significantly increased by visfatin or insulin treatment after 8 d (all P < 0.05). In the presence of insulin, the mRNA abundance of adipocyte fatty acid-binding protein was 24.7-fold greater than in the untreated group (P < 0.05), whereas visfatin alone had no effect on adipocyte fatty acid-binding protein mRNA abundance. Adipocyte differentiation was induced by insulin treatment for 8 d. In differentiated porcine adipocytes, exposure to insulin or visfatin for 24 h increased (P < 0.05) fatty acid synthase mRNA abundance but had no effect on the expression of sterol regulatory element binding-protein 1c mRNA. We also found a 5.8-fold upregulation of IL-6 expression in porcine adipocytes after 24 h of treatment with visfatin (P < 0.05). These results demonstrated that visfatin upregulated lipoprotein lipase expression in preadipocytes, potentially facilitating lipid uptake, and increased the gene expression of fatty acid synthase in differentiated adipocytes to potentially enhance lipogenic activity. Furthermore, visfatin can upregulate IL-6 expression in differentiated porcine adipocytes. The information presented in this study provides insights into the roles of visfatin in lipid metabolism in pigs.

Porcine peroxisome proliferator-activated receptor delta mediates the lipolytic effects of dietary fish oil to reduce body fat deposition.

Peroxisome proliferator-activated receptor delta promotes fatty acid catabolism and energy expenditure in skeletal muscle and adipose tissues. A ligand for PPARdelta is required to activate PPARdelta function. Polyunsaturated fatty acids are potential ligands for PPARdelta activation. The current experiment was designed to determine the potential for PUFA, particularly from dietary fish oil, to activate porcine PPARdelta in vivo. Transgenic mice were generated to overexpress porcine PPARdelta in the adipose tissue. Mice were fed a high-saturated fat (13% beef tallow), or high-unsaturated fat (13% fish oil) diet, or a diet containing 4 mg/kg of a PPARdelta ligand (L165041) for 4 mo. Compared with beef tallow feeding, fish oil feeding reduced fat mass and decreased (P < 0.05) plasma triacylglycerol and FFA concentrations in the transgenic mice. Adipose tissue expression of genes involved in adipogenesis (i.e., lipoprotein lipase and adipocyte fatty acid-binding protein) was decreased in transgenic mice fed fish oil or the PPARdelta ligand. In the same mice, expression of the lipolytic gene, hormone-sensitive lipase was increased (P < 0.05). Fish oil feeding also stimulated expression of genes participating in fatty acid oxidation in the liver of transgenic mice compared with wild-type mice. Overall, these results indicate that PUFA may serve as natural and effective regulators of lipid catabolism in vivo and many of these effects may be generated from activation of PPARdelta.

Abundantly expressed hepatic genes and their differential expression in liver of prelaying and laying geese.

Geese have a short egg-laying period and a low egg production rate. To induce and maintain egg laying, genes related to generating hepatic lipid for yolk deposition should be adequately expressed. Liver mRNA from 6 laying geese was extracted and used for construction of a full-length enriched cDNA library. About 2,400 clones containing gene sequences were determined and National Center for Biotechnology Information Gallus gallus Gene Index databases were used to compare and analyze these sequences. Ten highly expressed genes were selected to determine the differential expression between laying and prelay goose liver. Tissue distribution data showed that very low density apolipoprotein II, liver type fatty acid binding protein, vitellogenin I, and vitellogenin II transcripts were specifically expressed in the liver of laying geese. Ovoinhibitor, preproalbumin, alpha-2-hs-glycoprotein, and vitamin D binding protein mRNA were highly expressed in the liver and to a lesser extent in other tissues. Ovotransferrin mRNA was expressed in liver, ovary, oviduct, shell gland, brain, and adipose tissues. The concentration of transthyretin mRNA was high in the liver and brain. The mRNA concentrations of liver type fatty acid binding protein, alpha-2-hs-glycoprotein, and transthyretin in the livers of laying and prelay geese were not different. The concentrations of hepatic ovotransferrin, ovoinhibitor, preproalbumin, very low density apolipoprotein II, vitellogenin I, vitellogenin II, and vitamin D binding protein mRNA were higher in the liver of laying geese than in prelay geese, suggesting that these genes may be involved in laying function or lipid metabolism related to egg formation.

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk.

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.

The effect of feed restriction on expression of hepatic lipogenic genes in broiler chickens and the function of SREBP1.

To study the role of sterol regulatory element-binding proteins (SREBP) in lipogenesis and cholesterol synthesis in the chicken, two experiments were carried out. In the first study, seven-week-old broilers (n=16) were allocated into 2 groups, fasted for 24 h or refed for 5 h after a 24 h fasting. The mRNA concentrations for SREBPs and other lipogenic genes in the liver were determined by quantitative real time PCR. The hepatic mRNA relative abundance of lipogenic genes and genes involved in cholesterol synthesis were significantly greater (p<0.001) in the refed broilers. Similar results were demonstrated with Northern analysis. The data suggest that in the liver of fasted broilers, genes associated with lipogenesis and cholesterol biosynthesis were inhibited. Indeed, the mRNA concentrations for fatty acid synthase (FAS), malic enzyme, and stearoyl coenzyme A desaturase were almost undetectable after the 24 h fasting. The data also demonstrated that the expression of lipogenic genes coordinate well as a group during the refeeding period. Second, three small interfering RNA (siRNA) oligonucleotides against SREBP1 were designed to be used in transfecting a chicken hepatocarcinoma cell line LMH. One of the three siRNAs effectively reduced SREBP1 mRNA concentration (p<0.01). The acetyl coenzyme A carboxylase(alpha) (ACC(alpha)) mRNA was also significantly reduced by the SREBP1 siRNA treatment, suggesting that SREBP1 can upregulate the expression of this lipogenic gene. This siRNA, however, did not affect the mRNA for FAS. Taken together, the RNA interference study showed that SREBP1 has the ability to regulate the expression of ACC(alpha). This study has helped us understand more about the function of SREBP1 and the physiology of the broiler chickens.

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

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.

Fasting regulates the expression of adiponectin receptors in young growing pigs.

Adiponectin is an adipocyte-derived hormone that can improve insulin sensitivity. Its functions in regulating glucose utilization and fatty acid metabolism in mammals are mediated by 2 subtypes of adiponectin receptors (AdipoR1 and AdipoR2). This study was conducted to determine the effect of fasting on the expression of adiponectin and its receptors. The expression of adiponectin was not affected in s.c. adipose tissue, but adiponectin expression increased in visceral adipose tissue after fasting. In contrast, expression of both AdipoR mRNA was increased in the liver and s.c. adipose tissue of 24-h-fasted pigs compared with fed pigs, but the mRNA in muscle and visceral adipose tissue was not affected by fasting. A third putative adiponectin receptor, T-cadherin, was cloned and the mRNA expression was determined. T-Cadherin has been recognized to act as a vascular adiponectin receptor in vascular endothelial and smooth muscle cells. Our data showed that the expression of T-cadherin was decreased in the muscle of fasted pigs, suggesting that the expression of T-cadherin can be regulated by feeding status. In summary, in young pigs, adiponectin mRNA was up-regulated by fasting in visceral, but not s.c., adipose tissue, whereas AdipoR1 and AdipoR2 mRNA were increased in s.c., but not visceral, adipose tissue. The adiponectin receptor, T-cadherin, was expressed in s.c. and visceral adipose tissue and in muscle, but only muscle mRNA expression was decreased by fasting.

Expressed transcripts associated with high rates of egg production in chicken ovarian follicles.

The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P<0.05, log(2)> or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

Ectopic expression of porcine peroxisome proliferator-activated receptor delta regulates adipogenesis in mouse myoblasts.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical role in regulating adipogenesis. The expression of peroxisome proliferator-activated receptor delta (PPARdelta) precedes that of PPARgamma during adipocyte differentiation in rodents. The current experiment was designed to study the function of porcine PPARdelta and the interaction of PPARdelta and PPARgamma in adipocyte differentiation. Inhibition of myogenesis was observed in mouse myoblasts expressing porcine PPARdelta, similar to myoblasts expressing PPARgamma. Treatment of myoblasts expressing PPARdelta with ligands for both PPARdelta and PPARgamma enhanced lipogenesis and adipogenesis to a greater extent than treatment with a PPARgamma ligand alone, suggesting that both genes were involved in regulating lipogenesis and adipogenesis. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts coexpressing PPARdelta with either wild type or mutated PPARgamma (Ser 112 was mutated to Ala; the mutated PPARgamma is more active than the wild type) compared with myoblasts expressing PPARgamma alone. Adipocyte differentiation in myoblasts coexpressing PPARdelta and mutated PPARgamma was greater than in myoblasts coexpressing PPARdelta and wild type PPARgamma, confirming that Ser 112 is important for the function of PPARgamma. Taken together, our results demonstrate that overexpression of PPARdelta inhibits myotube formation and also enhances adipocyte differentiation. However, the complexity and interaction of PPARdelta and PPARgamma in adipogenesis are not clearly understood.

The differential expression of hepatic genes between prelaying and laying geese.

Suppression subtractive hybridization was used to detect differential expression of genes in the livers of laying and prelaying geese. Liver tissues from prelaying and laying geese were dissected for mRNA extraction. The cDNA, reverse transcribed from liver mRNA of prelaying geese, was subtracted from the cDNA generated from the laying geese (forward subtraction). Five hundred seventy-six clones with possible differentially expressed gene fragments were observed by forward subtraction hybridization. After differential screening using the reverse and forward subtraction cDNA, 164 clones were subjected to gene sequence determination and further analysis. Using Northern analysis, 5 known and 8 unknown genes were shown to be highly expressed in the livers of laying geese compared with prelaying geese. Vitellogenin I, apoVLDL-II, ethanolamine kinase, G-protein gamma-5 subunit, and leucyl-tRNA synthase were highly expressed in the livers of laying geese compared with that from the prelaying geese (P<0.05). The expression of these known genes suggests that their function in the liver of laying geese is primarily involved in lipid and lipoprotein metabolism. Several of these differentially expressed genes were found to be responsive to estrogen stimulation, confirming the involvement of these genes in the egg-laying function of the goose.

The expression of pituitary gland genes in laying geese.

The purpose of this study was to detect differential expression of genes in the pituitary gland in laying geese by suppression subtractive hybridization (SSH). Pituitary glands from prelaying and laying geese were dissected for mRNA extraction. The cDNA from pituitary glands of prelaying geese was subtracted from the cDNA from the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. We screened 384 clones with possible differentially expressed gene fragments by differential screening. Sixty-five clones from the differential screening results were subjected to gene sequencing and further analysis. We found that at least 19 genes were highly expressed in the pituitary glands of laying geese compared with prelaying geese. Among these, 6 genes (including 4 novel genes) were confirmed by virtual Northern analysis. We found that prolactin and visinin-like protein were highly expressed in the pituitary glands of laying geese compared with prelaying geese (P < 0.05). Further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

Porcine peroxisome proliferator-activated receptor gamma induces transdifferentiation of myocytes into adipocytes.

Peroxisome proliferator-activated receptor gamma2 (PPARgamma) is a nuclear transcription factor that regulates adipocyte differentiation and lipogenic genes during adipogenesis. The activity of rodent PPARgamma is regulated by phosphorylation of serine 112. The current experiment was designed to study the ability of porcine PPARgamma to stimulate transdifferentiation of myoblasts to adipocytes by overexpressing wild-type PPARgamma or mutated PPARgamma (serine 112 was mutated to alanine) in mouse myoblast cells. The expression of adipogenic marker genes (adipocyte fatty acid binding protein, lipoprotein lipase, and glycerol-3 phosphate dehydrogenase) in cells stably expressing mutated porcine PPARgamma was greater than in cells with wild-type PPARgamma, indicating that the mutated PPARgamma has greater adipogenic capability than the wild-type PPARgamma. Under treatment with a ligand, both wild-type and mutant porcine PPARgamma-expressing C2C12 myoblasts differentiated into adipocytes in 10 d. The expression of myogenic marker genes (myogenin, myogenic regulatory factor-4) was suppressed in cells transfected with the mutated PPARgamma or wild-type PPARgamma. Moreover, wild-type and mutant PPARgamma were able to inhibit myogenesis without addition of a ligand. Our results suggest that porcine wild-type PPARgamma and mutated PPARgamma can both convert myoblast cells into adipocytes, and also that the ability to transdifferentiate was greater in cells containing the mutated PPARgamma than in cells containing the wild-type PPARgamma. Therefore, the existence of serine 112 in PPARgamma may have a role in regulating adipocyte differentiation.

Abundantly expressed genes in pig adipose tissue: an expressed sequence tag approach.

Adipose tissue plays a critical role in metabolism, storage, and release of fatty acids in mammals. Construction of a full-length cDNA library is an effective way to understand the functional expression of genes in adipose tissue, and in addition, novel genes for further research can be found in the library. In this study, adipose tissue RNA was extracted from three 18-mo-old Lee-Sung pigs. The mRNA was isolated, reverse transcribed, and used to construct a cDNA library. After transformation, 2,880 clones were selected and sequenced. Cluster analysis was performed, and the assembled contig of each cluster was subjected to search against DNA sequences in the nucleotide databases (NCBINR/TIGRGI). These sequences were clustered into 1,527 unique sequences; 80% of the sequences were categorized as known genes, and 20% of the sequences were categorized as unknown genes. In this adipose tissue cDNA library, approximately 16% of the genes contained full-length sequences with start and stop codons. Gene ontology analysis was performed to indicate the possible functions of these genes. Genes associated with mitochondrial function were abundant and represented 10% of the total. Several fatty acid transport genes and stearoyl coenzyme A desaturase were among the most abundant genes expressed. Tissue distribution of several abundant genes was analyzed by northern analysis, and many of these genes were transcribed in porcine adipose tissue in high copy number. Our full-length sequence data and tissue distribution data can be used to decipher the functional roles exhibited by the adipocyte under various perturbations via endocrine, environmental, genetic, nutritional, pharmacological, or physiological manipulations.

The expression of genes related to adipocyte differentiation in pigs.

The purpose of this study was to detect differential expression of genes related to adipocyte differentiation in pigs by suppression subtractive hybridization. Adipocytes and stromal vascular cells (a fraction containing preadipocytes) from pig adipose tissue were isolated for mRNA extraction. The cDNA from preadipocytes was subtracted from the cDNA from adipocytes. The subtracted gene fragments were cloned into pGEM-T Easy TA cloning vector. We selected 384 clones for gene sequence determination and for further analysis. These genes were subjected to a differential screening procedure to confirm the differential expression of genes between the 2 cell types. We found that at least 36 genes were highly expressed in the adipocytes compared with preadipocytes. Among these, 6 genes including 2 novel genes with the greatest differences were selected and confirmed by Northern analysis. We found that angiotensin I-converting enzyme (ACE), ataxia-telangiectasia mutated protein (ATM), calpain 1, and stearoyl coenzyme A desaturase 1 (SCD1) were highly expressed in adipocytes compared with preadipocytes (P < 0.05). The relative mRNA abundance of ACE, ATM, calpain 1, SCD1, and 2 novel genes discovered in the current study was increased at the later stages of adipocyte differentiation (P < 0.05). The results confirmed that the genes involved in lipid metabolism and adipocyte differentiation were highly expressed in porcine adipocytes. However, further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

Effect of docosahexaenoic acid and arachidonic acid on the expression of adipocyte determination and differentiation-dependent factor 1 in differentiating porcine adipocytes.

Adipocyte determination and differentiation-dependent factor 1 (ADD1) drives the expression of several lipogenic genes in mammals. Polyunsaturated fatty acids decrease ADD1 mRNA abundance in differentiating porcine adipocytes. The current study was designed to explore the mechanisms by which PUFA inhibit the expression of ADD1 in porcine adipocytes. Porcine preadipocytes were differentiated for 24 h with 0 or 100 microM of docosahexaenoic acid (DHA) and mixtures of different concentrations of antioxidants to investigate the effect of DHA and antioxidants on the ADD1 mRNA abundance. We found the relative mRNA abundance was decreased by the addition of 100 microM DHA to the medium for porcine differentiating adipocytes, and adding an antioxidant mixture to the medium prevented part of the decrease in ADD1 mRNA abundance. These data suggest that DHA decreased the steady-state transcription factor ADD1 mRNA through a mechanism related to fatty acid peroxidation. Indeed, adding 7.5 microM vitamin E (a natural antioxidant) also restored the concentrations of ADD1 and fatty acid synthase mRNA, which were decreased by DHA treatment; however, the DHA or the antioxidant treatment did not change the expression of antioxidation genes (superoxide dismutase 1 and glutathione peroxidase 1) in porcine stromal vascular cells. When supplemented with the eicosanoid synthesis pathway inhibitors, the inhibition of the expression of ADD1 by arachidonic acid was partially recovered. These results suggest that the mechanism by which PUFA decrease ADD1 mRNA is due to the metabolic product of eicosanoids and peroxidation of these PUFA.