Browsing by Author "Jiang, Honglin"
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- Acute and chronic heat stress alters the metabolic profile of skeletal muscle in growing swineWon, Samantha Gwai Lan (Virginia Tech, 2012-08-02)Heat stress (HS) causes significant losses to the U.S. swine industry in several production and health areas including efficient lean tissue accretion. Perturbations in skeletal muscle metabolism may participate in this defect. The study objectives were to examine the cellular bioenergetic profile in skeletal muscle of piglets subjected to thermal stress in utero and/or during postnatal life. To accomplish this, 96 offspring from 14 sows were prenatally exposed to 1 of 4 environmental treatments involving thermal neutral (TN, 25°C) or HS conditions (cyclical 28-34°C). Sows exposed to TN or HS throughout gestation are denoted TNTN and HSHS, respectively whereas sows heat-stressed for the first or second half of gestation are denoted HSTN and TNHS, respectively. At 14 weeks of age, offspring were exposed to one of two postnatal thermal environments, constant TN (21°C) or HS (35°C) for 24 hrs (acute study) or 5 weeks (chronic study). Pigs were sacrificed after treatment and longissimus dorsi skeletal muscle samples collected for molecular analyses. Differences (p<0.05) were observed in protein abundance of p-4eBP1 and total Rs6 and gene expression of Cox5B, CytB, EEF2, HK2, MURF, ND1, PGC-1α, SDHA, and TFAM during the acute heat stress study. Differences (p<0.05) were observed in protein abundance of 4eBP1, total Akt, and p-Rs6 and gene expression of CytB, MURF, and PGC-1α during the chronic heat stress study. These data indicate that acute postnatal HS alters skeletal muscle metabolism, which may favor a reduction in mitochondrial respiration and protein synthesis potentially via the mTOR pathway.
- Additive effects among uterine paracrine factors in promoting bovine trophoblast cell proliferationXie, Ming (Virginia Tech, 2014-06-10)Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes.
- The anti-diabetic mechanisms by isoflavone genisteinFu, Zhuo (Virginia Tech, 2011-05-10)Diabetes is growing public health problem in the United States. Both in Type 1 and Type 2 diabetes, the deterioration of glycemic control over time is largely due to insulin secretory dysfunction and significant loss of functional β-cells. As such, the search for novel agents that promote β-cell survival and preserve functional β-cell mass are one of the essential strategies to prevent and treat the onset of diabetes. Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. It was recently shown that dietary intake of foods containing genistein improves diabetes in both experimental animals and humans. However, the potential anti-diabetic mechanisms of genistein are unclear. In the present study, we first investigated the effect of genistein on β-cell insulin secretion and proliferation and cellular signaling related to these effects in vitro and in vivo. We then determined its anti-diabetic potential in insulin-deficient and obese diabetic mouse models. The results in our study showed that exposure of clonal insulin secreting (INS1E) cells or isolated pancreatic islets to genistein at physiologically relevant concentrations (1-10 μM) enhanced glucose-stimulated insulin secretion (GSIS), whereas insulin content was not altered, suggesting that genistein-enhanced GSIS is not due to a modulation of insulin synthesis. This genistein's effect is protein tyrosine kinase- and KATP channel-independent. In addition, genistein had no effect on glucose transporter-2 expression or cellular ATP production, but similarly augmented pyruvate-stimulated insulin secretion in INS1E cells, indicating that genistein improvement of insulin secretion in β-cells is not related to an alternation in glucose uptake or the glycolytic pathway. Further, genistein (1-10 μM) induced both INS1 and human islet β-cell proliferation following 24 h of incubation, with 5 μM genistein inducing a maximal 27% increase. The effect of genistein on β-cell proliferation was neither dependent on estrogen receptors, nor shared by 17β-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of PKA or ERK1/2 completely abolished genistein-stimulated β-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for β-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in both insulin deficient type 1 and obese type 2 diabetic mice, concomitant with improved islet β-cell proliferation, survival, and mass. These changes were not due to alternations in animal body weight gain, food intake, fat deposit, plasma lipid profile, or peripheral insulin sensitivity. Collectively, these findings provide better understanding of the mechanism underlying the anti-diabetic effects of genistein. Loss of functional β-cell mass through apoptosis is central to the development of both T1D and T2D and islet β-cell preservation and regeneration are very important components of β-cell adaptation to increased apoptosis and insulin resistance and therefore holds promise as a treatment for this disease. In this context, these findings may potentially lead to the development of novel low-cost natural agents for prevention and treatment of diabetes.
- Assessing Hepatic Gene Expression in Response to Xenobiotic Exposure in MiceBoorgula, Smitha (Virginia Tech, 2007-04-12)Xenobiotics are plant derived compounds metabolized by phase I and II liver enzymes. Phase I enzymes increase, and phase II enzymes decrease, xenobiotic toxicity. Xenobiotics considered were ergotamine, associated with fescue toxicosis, and sulforaphane, a phase II inducer. Hypothesized responses in liver gene expression and enzyme activity due to exposure to these xenobiotics were tested. Polymorphic mice were gavaged with sulforaphane, ergotamine or control over four daily dosing periods (2, 5, 8 and 11 d), with at least 5 mice per treatment. Mice were killed and livers collected 24 h after last dosing. With ergotamine, expression of phase II genes catecholâ Oâ amine methyltransferase 1 (P = 0.009) on d 8, and glutathioneâ Sâ transferase (Gst) mu1 (Gstm1; P = 0.049) on d 11 was increased, and sulfotransferase 5a1 on d 11 decreased (P = 0.02). Sulforaphane increased expression of cytochrome P450 1a2 on d 5 (P = 0.02) and flavin containing monooxygenases 1 on d 11 (P = 0.002), both phase I genes. It also increased expression of a phase II gene transcription factor (P = 0.03) and quinone reductase 02 (P = 0.007) on d 5, and Gstm1 on d 8 (P = 0.04) and d 11 (P = 0.01). Moreover, sulforaphane treated mice had higher (P < 0.05) Gstm1 expression across days. Among enzymes, only sufloraphane treated mice had higher (P < 0.05) Gst activity. The increase in both Gstm1 expression and Gst activity indicate a consistent benefit of sufloraphane on phase II enzyme activity.
- An Assessment of the Molecular Basis of Toxin-induced Dilated Cardiomyopathy in an Avian Animal ModelTian, Xi (Virginia Tech, 2008-12-09)Dilated cardiomyopathy (DCM), a disease of the myocardium, causes morbidity and premature death in humans and other domestic animals including turkeys. Though DCM results from many different factors including those that are unknown or idiopathic, genetic factor is a major cause of idiopathic DCM. In this study, I assessed the molecular basis of toxin-induced DCM in turkeys by evaluating the association and effect of mutations in candidate genes in the nucleus and mitochondria on the incidence and severity of this disease. Echocardiographic measurements at 3 weeks of age showed that birds on furazolidone-containing diet exhibited a significant DCM phenotype (increased left ventricular end diastolic dimension and left ventricular end systolic dimension) with a marked decrease in the left ventricular shortening fraction. Pathological phenotype confirmed the dilated heart with extended cell necrosis. Two mutations, both in NADH dehydrogenase genes, were found to be associated with DCM. Real-time RT-PCR quantification indicated that mRNA expression of alpha cardiac actin gene (ACTC) were significantly different between control and treatment birds. While ACTC expression increased, though moderately, in control birds from week 1 to 3, it decreased significantly in treatment birds. These findings suggest that the mitochondrial DNA variation and ACTC expression may be associated with the turkey's response to toxin. Therefore, further research is needed to investigate the molecular mechanism of toxin-induced DCM in the turkey.
- Association of growth hormone deficiency with an increased number of preadipocytes in subcutaneous fatZhao, Lidan; Jia, Dan; Tan, Zhendong; Jiang, Honglin (Frontiers, 2023-05)The inhibitory effect of growth hormone (GH) on adipose tissue growth is well known, but the underlying mechanism is not fully understood. In this study, we determined the possibility that GH inhibits adipose tissue growth by inhibiting adipogenesis, the process of formation of adipocytes from stem cells, in the lit/lit mice. The lit/lit mice are GH deficient because of a spontaneous mutation to the GH releasing hormone receptor (ghrhr) gene, and they have more subcutaneous fat despite being smaller than the lit/+ mice at the same age. We found that cells of the stromal vascular fraction (SVF) of subcutaneous fat from the lit/lit mice had greater adipogenic potential than those from the lit/+ mice, as evidenced by forming greater numbers of lipid droplets-containing adipocytes and having greater expression of adipocyte marker genes during induced adipocyte differentiation in culture. However, addition of GH to the culture did not reverse the superior adipogenic potential of subcutaneous SVF from the lit/lit mice. Through florescence-activated cell sorting and quantification of mRNAs of preadipocyte markers, including CD34, CD29, Sca-1, CD24, Pref-1, and PPAR gamma, we found that subcutaneous SVF from the lit/lit mice contained more preadipocytes than that from the lit/+ mice. These results support the notion that GH inhibits adipose tissue growth in mice at least in part by inhibiting adipogenesis. Furthermore, these results suggest that GH inhibits adipogenesis in mice not by inhibiting the terminal differentiation of preadipocytes into adipocytes, rather by inhibiting the formation of preadipocytes from stem cells or the recruitment of stem cells to the fat depot.
- Characterization of seasonal reproduction in Virginia Tech Selection Line, St. Croix, and Suffolk ewesJordan, Katherine Mead (Virginia Tech, 2008-08-04)This dissertation research contained three studies. The first two studies were conducted to investigate the ability of ewes to rebreed while lactating during seasonal anestrus. Breeds studied included the Virginia Tech Out-of-season (OOS) Line, which is a wool line genetically selected to lamb in the fall, and the St. Croix, a hair breed of tropical origin thought to be lowly seasonal. When January-lambing ewes were exposed to rams while lactating in April, significantly more OOS than St. Croix ewes were marked by rams in the first 21 d and total 39 d of ram exposure (58.3 vs. 8.7%, P = 0.0003 and 95.8 vs. 43.5%, P < 0.0001). Percentages of ewes diagnosed pregnant (53.2%) and percentages of ewes lambing (41.3%) were not different between breeds. When March-lambing OOS ewes were exposed to rams while lactating in May, 52.9% of ewes were marked though only 20% of ewes exposed to rams gave birth to viable lambs. Both OOS and St. Croix ewes appear to be well suited to accelerated production systems involving 7 to 8 mo lambing intervals. However, reduction of lambing intervals to 6 to 7 mo appeared to have detrimental effects on fetal survival in OOS ewes. In a third study, alterations in endocrine profiles associated with differing degrees of hypothalamic sensitivity to estradiol-negative feedback and changing daylength in OOS, St. Croix, and Suffolk ewes in the absence of rams were investigated for 1 yr. The results show for the first time that based on progesterone profiles from intact ewes, St. Croix ewes do not have shorter anestrous periods than ewes of wool breeds, as previously thought. Based on luteinizing hormone profiles from ovariectomized ewes treated with estradiol implants, the duration of luteinizing hormone inhibition was shorter in OOS than Suffolk ewes (68 vs. 170.2 d, P = 0.02), but was not different from that found in St. Croix ewes (124.8 d). Specific roles for thyroxine and prolactin in timing the breeding season could not be assigned. This study was the first known use of the ovariectomized, estradiol-implanted ewe model to compare degree of reproductive seasonality in different breeds.
- Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiationLyu, Pengcheng; Jiang, Honglin (2024-03-12)Background: Satellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation. Results: Using chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers. Conclusions: Satellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.
- Cloning, Expression, and Developmental and Dietary Regulations of a Chicken Intestinal Peptide Transporter and Characterization and Regulation of an Ovine Gastrointestinal Peptide Transporter Expressed in a Mammalian Cell LineChen, Hong (Virginia Tech, 2001-09-28)To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken cDNA library. The cDNA was 2,914-bp and encoded a protein of 714 amino acid residues. Twenty-three di-, tri-, and tetra-peptides were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary (CHO) cells. For most di- and tripeptides tested, the Kt was in the micromolar range, except Lys-Lys and Lys-Trp-Lys. Northern analysis demonstrated that cPepT1 is expressed strongly in the small intestine, and at lower levels in kidney and cecum. These results demonstrated the presence and functions of a peptide transporter in chickens. cPepT1 mRNA abundance was evaluated in response to developmental and dietary regulations. In Experiment 1, eggs at incubation day 18 (E18) and Cobb chicks after hatch (d 0) were sampled before treatments. Three groups of chicks were fed diets containing 12, 18, or 24% crude protein (CP). Feed intake of chicks fed the 18 or 24% CP diets was restricted to that of chicks fed the 12% CP diet. In Experiment 2, a fourth group with free access to the 24% CP diet was added. cPepT1 mRNA abundance was quantified from northern blots. By d 0, there was a 50-fold increase in cPepT1 mRNA abundance compared with E 18. In chicks fed the 12% CP diet, cPepT1 mRNA abundance decreased throughout the 35 d. Chicks fed 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance with time. In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35. Our results indicate that cPepT1 mRNA is regulated by both dietary protein and developmental stage. To investigate the kinetics of an ovine peptide transporter (oPepT1), CHO cells were transfected with oPepT1 cDNA. Uptake of Gly-Sar by transfected cells was pH-dependent, concentration-dependent, and saturable. Competition studies showed that all di-, tri-, and tetra-peptides inhibited uptake of Gly-Sar. Pretreatment of the cells with staurosporine resulted in an increase in peptide transport. This increase was blocked by pretreatment with PMA. The results indicate that protein kinase plays a role in oPepT1 function.
- Cloning, Sequencing and Expression of a Porcine Intestinal Peptide Transporter in a Mammalian Cell LineKlang, Judith Elisa (Virginia Tech, 2002-12-10)Absorption of dietary proteins can be met through the uptake of free amino acids or as small peptides. A peptide transport protein, PepT1, is responsible for the absorption of intact peptides arising from digestion of dietary proteins. PepT1 is driven by a H+-coupled transport system that allows for the absorption of small peptides through the intestinal brush border membrane. Screening of a porcine intestinal cDNA library with a sheep PepT1 cDNA probe resulted in the identification of three porcine PepT1 (pPepT1) cDNAs of varying sizes and sequences. Each variant cDNA isolated was cloned into a mammalian expression vector, sequenced, and expressed in Chinese hamster ovary (CHO) cells. Peptide transport was assessed by uptake studies using the radiolabeled dipeptide [3H]-Gly-Sar. Only one of the three cDNAs encoding for a protein of 708 amino acids induced H+-dependent peptide transport activity. Through computer analysis, a putative protein structure for pPepT1 was developed. The transporter has an unusual 13 transmembrane structure with the N-terminus located extracellularly and the C-terminus located intracellularly. Seven glycosylation sites and three protein kinase C phosphorylation sites are located throughout the protein. Expression of pPepT1 activity in CHO cells had a optimal peptide uptake at 18-24 hours. The transporter showed optimal uptake at a pH of 5.5-6.0. Eighteen different unlabeled dipeptides and tripeptides were found to inhibit the uptake of [3H] -Gly-Sar in competition studies. The IC50 of 13 of the dipeptides and two tripeptides ranged between 0.015 to 0.4 mmol/L. The exceptions were Lys-Lys, Arg-Lys, and Lys-Trp-Lys, which showed IC50 values greater than 1.37 mmol/L and appear to be poor substrates for pPepT1. All three of the tetrapeptides examined showed very high IC50 values and inhibition of the uptake of Gly-Sar was too small to measure even at a 10mM concentration. Dipeptides and tripeptides appear to be substrates for the porcine intestinal peptide transporter while tetrapeptides do not appear to be transported.
- Defective excitation-contraction coupling is partially responsible for impaired contractility in hindlimb muscles of Stac3 knockout miceCong, Xiaofei; Doering, Jonathan; Grange, Robert W.; Jiang, Honglin (Nature, 2016-05-17)The Stac3 gene is exclusively expressed in skeletal muscle, and Stac3 knockout is perinatal lethal in mice. Previous data from Stac3-deleted diaphragms indicated that Stac3-deleted skeletal muscle could not contract because of defective excitation-contraction (EC) coupling. In this study, we determined the contractility of Stac3-deleted hindlimb muscle. In response to frequent electrostimulation, Stac3- deleted hindlimb muscle contracted but the maximal tension generated was only 20% of that in control (wild type or heterozygous) muscle (P < 0.05). In response to high [K⁺], caffeine, and 4-chloro-m-cresol (4-CMC), the maximal tensions generated in Stac3-deleted muscle were 29% (P < 0.05), 58% (P = 0.08), and 55% (P < 0.05) of those in control muscle, respectively. In response to 4-CMC or caffeine, over 90% of myotubes formed from control myoblasts contracted, but only 60% of myotubes formed from Stac3- deleted myoblasts contracted (P = 0.05). However, in response to 4-CMC or caffeine, similar increases in intracellular calcium concentration were observed in Stac3-deleted and control myotubes. Gene expression and histological analyses revealed that Stac3-deleted hindlimb muscle contained more slow type-like fibers than control muscle. These data together confirm a critical role of STAC3 in EC coupling but also suggest that STAC3 may have additional functions in skeletal muscle, at least in the hindlimb muscle.
- Deoxynivalenol interferes with intestinal motility via injuring the contractility of enteric smooth muscle cells: A novel hazard to the gastrointestinal tract by environmental toxinsJi, Xu; Qiao, Yu; Zheng, Weijiang; Jiang, Honglin; Yao, Wen (2021-11)Deoxynivalenol (DON) is a prevalent Fusarium mycotoxin, occurs predominantly in the global environment, especially in cereals, animal feed and food commodities. The widespread contamination causes a serious risk to human and animal health. DON usually impairs weight gain, which is presumably from its capacity to reduce feed intake by interfering with intestinal motility. To clarify the role of smooth muscle cells (SMCs) contractility intestinal motility and growth inhibition caused by DON, twelve weaned piglets were firstly divided into two groups to feed control or Fusarium mycotoxin-contaminated (MC) diet. Results showed that the final body weight, average daily gain and average daily feed intake were significantly reduced in piglets fed the MC diet. Exposure to the MC diet also significantly decreased the thickness of smooth muscle layer and SMCs contractile markers expression (myosin heavy chain 11, smooth muscle actin gamma 2, transgelin, calponin 1) in jejunum and ileum of piglets. Furthermore, oral DON supplementation (3 mg/kg body weight) to mice in six consecutive days could significantly inhibit the upper intestinal transit, impede normal defecation and downregulate SMCs contractile markers expression in small intestine. Finally, we generated a porcine enteric smooth muscle cell line (PISMC), and found that DON could depress its contractility by decreasing PISMC proliferation, migration and contractile markers expression. In conclusion, these findings in vivo and in vitro suggest that DON, as a common environmental toxin, can not only reduce proliferative and motile phenotype, but also decrease contractile apparatus components (contractile markers expression) in SMCs, which in turn influences SMCs contractility and then interferes with intestinal motility and growth performance.
- Developmental and Growth Hormone Regulation of the Expression of Liver-Enriched Transcription Factors in Bovine LiverEleswarapu, Satyanarayana Venkata (Virginia Tech, 2004-05-11)Liver gene expression changes during development and is affected by growth hormone (GH). These changes in gene expression may be due to the differential expression of the liver-enriched transcription factors (LETFs). To study the potential involvement of LETFs in the regulation of gene expression in the bovine liver, we cloned the cDNA fragments of nine bovine LETFs, including hepatocyte nuclear factor (HNF)-1Æ Ã , 1Æ Ã , 3Æ Ã , 3Æ Ã , 3Æ Ã , 6, albumin D-element binding protein (DBP), and CCAAT/enhancer-binding proteins (C/EBP) -Æ Ã and Æ Ã , and compared the expression levels of them between adult and fetal bovine liver and between GH-treated and untreated adult bovine liver. The mRNA abundance of the LETFs was determined by ribonuclease protection assay (RPA). The cloned bovine LETF cDNA sequences showed high degrees of similarity (79 % to 99 %) to the LETF sequences of other species. The mRNA levels of HNF-1Æ Ã , HNF-3Æ Ã , and HNF-6 were significantly higher (P < 0.05) in the fetal liver (n=3) than in the adult liver (n=7). There were significant increases (P < 0.05) in the mRNA expression of HNF-3Æ Ã and HNF-6 in the liver of cows 24 h (n=6) and 1w (n=6) after GH administration. The results of this study suggest that HNF-1Æ Ã , HNF-3Æ Ã , and HNF-6 may play a role in differential regulation of gene expression between the fetal and adult bovine liver and that HNF-3Æ Ã and HNF-6 may be also involved in GH regulation of gene expression in the bovine liver.
- Developmental Regulation of the Expression of Nutrient Transporter and BrushBorder Membrane Hydrolase Genes in the Small Intestine of PigletsXiao, Xunjun (Virginia Tech, 2005-12-14)The objective of this study was to evaluate developmental regulation of the expression of nutrient transporter and brushborder hydrolase genes in the small intestine of piglets. Seventy piglets from seven sows were killed at birth (d 0), during suckling (d 1, 3, 7, 14, 21) and postweaning (d 22, 24, 28, 35), and intestinal segments (duodenum, jejunum and ileum) were collected. The mRNA abundance was determined by Northern blot using specific cDNA probes for three disaccharidases (lactase-phlorizin hydrolase, LPH, sucrase-isomaltase, SI, and maltase-glucoamylase, MGA), three peptide hydrolases (aminopeptidase A, APA, aminopeptidase N, APN, and dipeptidyl peptidase IV, DPP IV), two sugar transporters (Na+-dependent glucose transporter 1, SGLT1, and facilitated glucose transporter 5, GLUT5), a peptide transporter (H+-dependent peptide transporter 1, PepT1), four amino acid transporters (excitatory amino acid carrier 1, EAAC1, Na+-dependent neutral amino acid transporter, ATB0, the light chain of a heterodimeric transport system b0,+ involved in the heteroexchange of cationic and neutral amino acids, b0,+AT, and Na+-independent large branched and aromatic neutral amino acid transporter 2, LAT2), and two iron transporters (divalent metal ion transporter 1, DMT1, and iron-regulated transporter 1, IREG1). Protein expression was quantified by Western blot using specific antibodies for LPH, SI, SGLT1, and PepT1. During suckling, the abundance of LPH, APA, APN, DPP IV, b0,+AT mRNA increased quadratically (P < 0.001) with age from birth to d 7 or 14 then remained unchanged or slightly declined with age to d 21. The mRNA abundance of SI increased and LAT2 decreased linearly (P < 0.001) with age, and the abundance of MGA and GLUT5 mRNA remained unchanged with age. There was an age x intestinal segment interaction (P < 0.001) for the abundance of EAAC1 and ATB0 mRNA. The abundance of EAAC1 mRNA increased from d 0 through 14 and remained stable to d 21 in the ileum, and it was low and slightly increased with age through d 21 in the duodenum and jejunum. The abundance of ATB0 mRNA generally increased from d 0 to 21 in the duodenum and ileum, and increased from d 0 to 7 and then decreased to d 21 in the jejunum. The abundance of SGLT1 and PepT1 mRNA was substantial at birth and transiently declined to d 1. The abundance of SGLT1 mRNA generally increased from d 1 to 21, and PepT1 mRNA abundance increased to d 3 and then plateaued through d 21. Postweaning, the mRNA abundance of all of these carbohydrate and protein assimilation related genes increased during the first day (3 d for ATB0) after weaning then declined to the levels at weaning in the jejunum and ileum, followed by a subsequent change pattern that varied among genes. During suckling, the mRNA abundance of LPH, SGLT1, and APA was greater in the duodenum and jejunum than the ileum (P < 0.001). The PepT1 and APN mRNA was evenly distributed among intestinal segments, and the expression of MGA, DPP IV, EAAC1, b0,+AT, ATB0, and LAT2 mRNA was generally greater in the jejunum and ileum than the duodenum or greatest in the ileum. Postweaning, the mRNA abundance of all of these carbohydrate and protein assimilation related genes examined was generally greater in the jejunum and ileum than the duodenum or highest in the ileum. From d 0 through 35, DMT1 and IREG1 mRNA was predominantly (P < 0.05) distributed in the duodenum, where the abundance of DMT1 and IREG1 mRNA increased with age during suckling, and then rapidly decreased after weaning. The protein expression of LPH and SI exhibited a similar developmental pattern as that for the mRNA abundance. Unlike the developmental regulation of their respective mRNA abundance, the protein expression of SGLT1 exhibited a general decline from suckling to postweaning. The protein expression of PepT1 gradually decreased with age from birth to d 35 in the duodenum, and initially declined from birth to the lowest value then slightly increased with age through d 21, followed by an increase to d 35 in the jejunum and ileum. In conclusion, the gene expression of these brushborder hydrolases and nutrient transporters was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages of life. These differences in ontogenetic regulation and the distribution may be related to the luminal substrate concentration as well as the nutrient categories, and the developmental regulation of these genes may occur not only at the transcriptional level but also at the posttranscriptional level.
- Dietary and Developmental Regulation of Nutrient Transporter Gene Expression in the Small Intestine of Two Lines of BroilersGilbert, Elizabeth R. (Virginia Tech, 2008-08-12)To better understand the digestive and absorptive capacities of the chick intestine so that we may feed diets that better meet the nutritional needs of the chick, it is important to understand how expression of nutrient transporter genes changes in response to various factors. A series of feeding trials were conducted to evaluate the dietary and developmental regulation of nutrient transporter mRNA abundance in the small intestine of two lines of broilers selected on corn-based (Line A) or wheat-based (Line B) diets. Abundance of mRNA was quantified in all experiments using real time PCR and the absolute quantification method. The objective of the first study was to investigate intestinal nutrient transporter and enzyme mRNA in Line A and B broilers at embryo day 18 and 20, day of hatch, and d 1, 3, 7, and 14 posthatch. Genes evaluated included the peptide transporter, PepT1, 10 AA transporters (rBAT, bo,+AT, ATBo,+, CAT1, CAT2, LAT1, y+LAT1, y+LAT2, BoAT and EAAT3), four sugar transporters (SGLT1, SGLT5, GLUT5, and GLUT2), and a digestive enzyme, APN. For PepT1, Line B had greater quantities of mRNA compared with Line A (P = 0.001), suggesting a greater capacity for absorption of AA as peptides. Levels of PepT1 mRNA were greatest in the duodenum (P < 0.05), whereas the abundances of SGLT1, GLUT5 and GLUT2 mRNA were greatest in the jejunum (P < 0.05). Abundances of EAAT3, bo,+AT, rBAT, BoAT, LAT1, CAT2, SGLT5 and APN mRNA were greatest in the ileum (P < 0.05). Quantities of PepT1, EAAT3, BoAT, SGLT1, GLUT5, and GLUT2 mRNA increased linearly (P < 0.01), while CAT1, CAT2, y+LAT1, and LAT1 mRNA decreased linearly (P < 0.05) with age. The objective of the second study was to evaluate the effect of dietary protein quality on intestinal peptide, AA, and glucose transporter, and digestive enzyme mRNA abundance in Line A and B broilers. At day of hatch (doh), chicks from both lines were randomly assigned to corn-based diets containing 24% crude protein (CP) with either soybean meal (SBM) or corn gluten meal (CGM) as the supplemental protein source, ad libitum. Groups of chicks from both lines were also assigned to the SBM diet at a quantity restricted to that consumed by the CGM group (SBM-RT). Abundance of PepT1, EAAT3, and GLUT2 mRNA was greater in Line B (P < 0.03), while APN and SGLT1 were greater in Line A (P < 0.04). When feed intake was equal (CGM vs restricted SBM), a greater abundance of PepT1 and bo,+AT mRNA was associated with the higher quality SBM (P < 0.04), while a greater abundance of EAAT3 and GLUT2 mRNA was associated with the lower quality CGM (P < 0.01). When feed intake was restricted (SBM vs SBM-RT), a greater abundance of PepT1 mRNA was associated with the restricted intake (P < 0.04). The objective of the third study was to determine the effect of dietary protein composition on mRNA abundance of peptide and AA transporters, and a digestive enzyme. From day 8 to day 15 posthatch, Line A and B broilers were fed equal amounts of 1 of 3 diets (24% CP). Dietary protein sources included whey protein concentrate (whey), a partial whey hydrolysate (hydro), or a mixture of free amino acids (AA) similar to the composition of whey. Intestine was collected at days 8, 9, 11, 13, and 15. Expression of all genes except LAT1 was greater (P < 0.05) in Line B compared with A. Abundance of PepT1, EAAT3, y+LAT2, CAT1, bo,+AT, and APN mRNA varied little across diets in Line A but for CAT1 mRNA was greatest (P = 0.005) in Line A birds that consumed the AA diet. Expression of these genes was greatest (P < 0.006) in Line B birds consuming the hydro diet. A greater (P < 0.05) age response of bo,+AT, EAAT3, CAT1, and APN mRNA was observed in birds consuming the hydro or AA diets relative to the whey diet. Results from these studies collectively demonstrate that nutrient transporter gene expression is responsive to a variety of factors, including developmental stage, dietary manipulation, and genetic selection. Information from these studies can be used to improve dietary formulation so that nutrient utilization is enhanced, resulting in improved growth of the broiler.
- Dietary Fat and Sugar Induce Obesity and Impair Glucose Tolerance in Prepubertal Pigsvan Eyk, Gregory Ryan (Virginia Tech, 2012-04-30)A pig model of childhood obesity was used to study the effects of dietary energy on body adiposity, and blood parameters associated with impaired glucose clearance. Prepubertal female pigs weaned at 21 d of age were fed control (CON), refined sugar (SUG), fat (FAT), and sugar-fat (SUGFAT) diets in a completely randomized arrangement for 16 wk. Calories from fat were 8.9% for CON, 5.6% for SUG, 35.5% for FAT and 32.3% for SUGFAT. Calories from sugar were 36.0% for SUG and 30.7% for SUGFAT. Adding fat, sugar or both to diets increased (P < 0.003) calorie intake. Percentage body fat was higher (P < 0.0001) in all treatments compared to CON, and in SUGFAT and FAT compared to SUG. Ultrasound back fat depth was positively correlated (r2 = 0.909; P < 0.001) with percentage body fat and negatively (r = 0.912; P-value ) with percentage body protein. Area under the curve (AUC) in response to oral glucose tolerance at 14 wk was higher (P < 0.03) in FAT (+14.6%) and SUGFAT (+25.5%) pigs compared to CON. Glucose AUC from sugar-fed pigs was not different (P = 0.2) from fat alone-fed pigs. Adding sugar, fat, or their combination to diets increased (P < 0.008) blood glucose and decreased (P < 0.0009) plasma insulin AUC. These data show that inclusion of fat and refined sugar in pig diets increases body adiposity and impairs glucose homeostasis and suggests that the composition of calories consumed may have different effects than simply consumption of excess of calories.
- Dietary manipulation causes childhood obesity-like characteristics in pigsFisher, Kimberly Denise (Virginia Tech, 2011-12-01)An animal model to study complications resulting from childhood obesity is lacking. Our objective was to develop a porcine model for studying mechanisms underlying diet-induced childhood obesity. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing tallow and refined sugars, or a control corn-based diet (n = 11) for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but, by wk 5, consumed more (P < 0.001) energy per kg body weight. At wk 15 and 22, pigs were subjected to an oral glucose tolerance test (OGTT); blood glucose increased (P < 0.05) in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01), even 3 h post-challenge. During OGTT, glucose area under the curve was higher and insulin area under the curve was lower in HED pigs compared to controls (P = 0.001). Pigs given 6 wk of dietary intervention, consuming a control diet, marginally improved glucose area under the curve and LDL-cholesterol although insulin area under the curve was unaffected. Chronic HED intake increased (P < 0.05) subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia; however, a 6 wk dietary intervention partially recovered a normal physiology. These data suggest pre-pubertal pigs fed HED are a viable animal model for studying childhood obesity.
- Effects of beta-hydroxy beta-methylbutyrate (HMB) supplementation on gluteus medius muscle fiber composition and muscle performance in adult Thoroughbred horses exercising to fatigue on a high-speed treadmillBusse Esser, Nicolas Ignacio (Virginia Tech, 2021-09-16)Consumption of β-hydroxy β-methylbutyrate (HBM), a leucine metabolite, alters muscle composition and metabolism leading to strength and agility improvements in human athletes. To determine if HMB affects athletic performance and muscle function in horses, Thoroughbred geldings were fed a control (CON; n=5) or HMB (n=6) supplement (30 mg/kg/day) for 6 weeks prior to completing a standardized exercise test (SET). Gluteus medius (GM) muscle samples were obtained before the SET for fiber-typing and venous blood was collected before and immediately upon completion of the SET for lactate measurements. Heart rate (HR), biceps femoris (BF) and semitendinosus (ST) surface electromyograms, and fore- and hindlimb metacarpophalangeal joint angles were captured for the duration of the SET. Results demonstrate that HMB supplementation increased (P < 0.05) the percentage of type IIA muscle fibers in the GM with a corresponding decrease (P < 0.05) in type IIX fibers. The percentage of type I fibers was unaffected by diet. Supplementation with HMB did not result in any significant effects on performance, muscle function or biomechanical properties by comparison to CON. Increasing treadmill speed resulted in an increase (P < 0.05) in stride length and maximal extension angle of the fore fetlock, and a shortening (P < 0.05) of the stance phase of the gait cycle. Integrated EMG (iEMG) increased (P < 0.05) with increasing treadmill speeds for both the BF and ST, with the BF exhibiting greater iEMG values than the ST. In summary, HMB increased the percentage of type IIA fibers which did not translate into immediate, improved athletic performance
- Effects of Growth Hormone and Insulin-Like Growth Factor-I on Milk Protein Gene Expression and Nutrient Uptake and Cell Proliferation in Clonal Bovine Mammary Epithelial CellsZhou, Yinli (Virginia Tech, 2007-08-23)The overall objective of this research was to further understand the mechanism by which growth hormone (GH) stimulates milk production in cattle. Three studies were conducted toward this objective. In the first study, the effects of GH and insulin-like growth factor-I (IGF-I), a major mediator of GH action in vivo, on cell proliferation, nutrient transport, and milk protein gene expression in bovine mammary epithelial cell line MAC-T cells were determined. GH increased (P < 0.01) expression of four major milk protein genes in MAC-T cells transfected with GHR expression plasmid. Cotransfection analyses indicated that GH also stimulated (P < 0.01) luciferase reporter gene expression from the promoters of the four milk protein genes in MAC-T cells. These findings together with the fact that GHR mRNA and protein are expressed in the epithelial cells of the bovine mammary gland suggest that GH may directly stimulate milk protein gene expression in the mammary gland. This study also showed that IGF-I increased the proliferation (P < 0.01) and amino acid transport (P < 0.05) in MAC-T cells. Because GH is known to stimulate IGF-I production in animals, IGF-I-mediated mammary epithelial cell proliferation and amino acid uptake may be additional mechanisms by which GH increases milk production in cattle. In the second study, the role of connective tissue growth factor (CTGF) on IGF-I-stimulated proliferation of MAC-T cells was investigated. A microarray analysis revealed that IGF-I decreased CTGF mRNA expression in MAC-T cells (P < 0.01). This effect of IGF-I was further found to be mediated through the PI-3 kinase/Akt signaling pathway from the IGF-I receptor (IGF-IR). CTGF alone stimulated MAC-T cell proliferation (P < 0.01). However, together with IGF-I, CTGF attenuated the proliferating effect of IGF-I on MAC-T cells, and this attenuation was reversed by additional IGF-I. Therefore, IGF-I inhibition of CTGF expression may benefit IGF-I stimulation of MAC-T cell proliferation. CTGF had no effect on IGF-I-induced phosphorylation of IGF-IR or total IGF-IR expression in MAC-T cells, suggesting that CTGF may attenuate IGF-I stimulation of MAC-T cell proliferation through a postreceptor inhibition of the IGF-IR signaling pathway. In the third study, whether a milk yield-associated T/A polymorphism in exon 8 of the bovine GHR gene affected GHR signaling was determined. It was found that the two corresponding GHR variants did not differ in mediating GH induction of gene expression, suggesting that the two GHR variants are not functionally different and hence are unlikely to mediate different effects of GH on milk production. In summary, the results of this dissertation research suggest that GH may directly stimulate milk protein gene expression and indirectly stimulate mammary epithelial cell proliferation and amino acid uptake through IGF-I, thereby stimulating milk production in cattle. The results also suggest that IGF-I stimulation of mammary epithelia cell proliferation may involve an inhibition of CTGF expression in the cells.
- The Effects of Tamoxifen on Mammary Development in Prepubertal HeifersTucker, Hannah L. (Virginia Tech, 2013-08-28)Our purpose was to determine the effects on mammary gland development in prepubertal heifers given the anti-estrogen tamoxifen. Sixteen Holstein calves were randomly assigned to one of two treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were subcutaneously injected daily from 28 to 120 days of age with 0.3 mg/kg tamoxifen or carrier. At 120 days calves were euthanized and udders removed. Weight of trimmed parenchymal tissue (left rear quarter) was dramatically lower in TAM calves than in CON calves (p < 0.0003; 16.1 vs. 34.8 g). Parenchymal samples from three regions of the left rear quarter (lower, middle and outer regions) were processed for immunohistochemical staining for Estrogen Receptor α and Progesterone Receptor, myoepithelial cells, and label retaining cells. Overall, the proportion of neither ER nor PR labeled cells was impacted by TAM treatment. However, imaging analysis indicated a markedly higher intensity of ER expression in CON calves. TAM caused an increase in myoepithelial cell differentiation similar to what is seen in ovariectomy. We were able to effectively use a new technique of multispectral imaging to identify label retaining cells, which led to the discovery of an increase in the percentage of label retaining cells in TAM compared to CON. While treatment with the anti-estrogen tamoxifen reduced mammary parenchymal mass similarly to OVX, the mechanism(s) involved appear to differ. This suggests that the impacts of ovariectomy are only partially explained by the absence of estrogen.