Zoe Daniel

Dose-dependent effects of vitamin D on transdifferentiation of skeletal muscle cells to adipose cells

Fat infiltration within muscle is one of a number of features of vitamin D deficiency, which leads to a decline in muscle functionality. The origin of this fat is unclear, but one possibility is that it forms from myogenic precursor cells present in the muscle, which transdifferentiate into mature adipocytes. The current study examined the effect of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), on the capacity of the C2C12 muscle cell line to differentiate towards the myogenic and adipogenic lineages. Cells were cultured in myogenic or adipogenic differentiation media containing increasing concentrations (0, 10−13, 10−11, 10−9, 10−7 or 10−5 M) of 1,25(OH)2D3 for up to 6 days and markers of muscle and fat development were measured. Mature myofibres were formed in both adipogenic and myogenic media, but fat droplets were only observed in adipogenic media. Relative to controls, low physiological concentrations (10−13 and 10−11 M) of 1,25(OH)2D3 increased fat droplet accumulation, whereas high physiological (10−9 M) and supraphysiological concentrations (≥10−7 M) inhibited fat accumulation. This increased accumulation of fat with low physiological concentrations (10−13 and 10−11 M) was associated with a sequential up-regulation of Pparγ2 (Pparg) and Fabp4 mRNA, indicating formation of adipocytes, whereas higher concentrations (≥10−9 M) reduced all these effects, and the highest concentration (10−5 M) appeared to have toxic effects. This is the first study to demonstrate dose-dependent effects of 1,25(OH)2D3 on the transdifferentiation of muscle cells into adipose cells. Low physiological concentrations (possibly mimicking a deficient state) induced adipogenesis, whereas higher (physiological and supraphysiological) concentrations attenuated this effect.

Examination of myosin heavy chain isoform expression in ovine skeletal muscles.

The contractile and associated metabolic characteristics of muscles are determined by their myosin heavy chain (MHC) isoform expression. In large mammals, the level of MHCIIB expression, which is associated with fast glycolytic-type muscle fibers, has not been fully characterized. In this study, quantitative reverse transcription-PCR and SDS-PAGE methodologies were developed for the analyses of adult ovine MHC isoform expression and used to characterize MHC expression in 3 skeletal muscles [LM, semitendinosus, and supraspinatus) from 66-d-old lambs. Three MHC isoforms (MHCI, MHCIIA, and MHCIIX) were detected at both the protein and messenger RNA levels in all 3 muscles, with greater proportions of type II than type I MHC. The expression of MHCIIB could not be detected at the protein level in any of the muscles and was detectable (in semitendinosus muscle) only at the messenger RNA level by using semiquantitative reverse transcription-PCR, indicating that MHCIIX is the predominant fast glycolytic fiber type in the sheep muscles studied. The methodologies developed are suitable for studying fiber type transformations at the molecular level, as well as allowing analyses of very small samples, including biopsies, when histochemical analysis may not be possible.

In utero effects on livestock muscle development and body composition

This review will focus on the evidence for in utero effects on development of skeletal muscle in farm and laboratory animals, particularly sheep and pigs. We will describe genetic and environmental factors that have been shown to alter the numbers of muscle fibres formed and outline our working hypothesis for the mechanism involved and the critical window during pregnancy when these effects are seen. We will then discuss the long-term consequences in terms of body composition. Although this review concentrates on skeletal muscle development, the mechanism we suggest might be equally applicable to other tissues in the body (e.g. the brain, kidneys or sex organs) and, therefore, impact on their physiological functions.

Use of the Affymetrix Human GeneChip array and genomic DNA hybridisation probe selection to study ovine transcriptomes.

Affymetrix GeneChip microarrays are a powerful tool to study global gene expression profiles and have been used on many species. However, no sheep-specific Affymetrix GeneChip is currently available and the bovine array is fairly limited in its coverage and annotation. Previously, a probe-selection method based on hybridisation of genomic DNA (gDNA) was developed, which enables GeneChips to be used for species that they were not designed for. This approach can greatly increase the number of potential annotated genes that can be studied beyond that which is currently available, particularly for species that do not have comprehensively characterised genomes. In this study, we demonstrate that gDNA-based probe selection on the Affymetrix Human U133+2 GeneChip array can be used to study gene expression profiles in sheep tissues. More than 20 000 transcripts were detected in triplicate ovine skeletal muscle and liver samples, which is more than would be possible using the commercially available sheep-specific microarray. The majority of the top 15 differentially expressed genes for each tissue were known to either be expressed in a tissue-specific manner or relate to specific functions of that tissue. Gene ontology analysis of the differentially expressed genes revealed the expected differences in gene expression profiles between the two tissues. Besides demonstrating that the probe selection method can be used to study the ovine transcriptome, the benefits of this approach are that it can greatly increase the number of annotated and novel genes that can be studied beyond those currently possible using ovine- or bovine-specific microarrays. This same method also has the potential to allow the study of other species where species-specific microarrays are not available or whose genomes have not been comprehensively characterised.

Maternal age effects on myometrial expression of contractile proteins, uterine gene expression, and contractile activity during labor in the rat

Advanced maternal age of first time pregnant mothers is associated with prolonged and dysfunctional labor and significant risk of emergency cesarean section. We investigated the influence of maternal age on myometrial contractility, expression of contractile associated proteins (CAPs), and global gene expression in the parturient uterus. Female Wistar rats either 8 (YOUNG n = 10) or 24 (OLDER n = 10) weeks old were fed laboratory chow, mated, and killed during parturition. Myometrial strips were dissected to determine contractile activity, cholesterol (CHOL) and triglycerides (TAG) content, protein expression of connexin‐43 (GJA1), prostaglandin‐endoperoxide synthase 2 (PTGS2), and caveolin 1 (CAV‐1). Maternal plasma concentrations of prostaglandins PGE2, PGF2α, and progesterone were determined by RIA. Global gene expression in uterine samples was compared using Affymetrix Genechip Gene 2.0 ST arrays and Ingenuity Pathway analysis (IPA). Spontaneous contractility in myometrium exhibited by YOUNG rats was threefold greater than OLDER animals (P < 0.027) but maternal age had no significant effect on myometrial CAP expression, lipid profiles, or pregnancy‐related hormones. OLDER myometrium increased contractile activity in response to PGF2α, phenylephrine, and carbachol, a response absent in YOUNG rats (all P < 0.002). Microarray analysis identified that maternal age affected expression of genes related to immune and inflammatory responses, lipid transport and metabolism, steroid metabolism, tissue remodeling, and smooth muscle contraction. In conclusion YOUNG laboring rat myometrium seems primed to contract maximally, whereas activity is blunted in OLDER animals and requires stimulation to meet contractile potential. Further work investigating maternal age effects on myometrial function is required with focus on lipid metabolism and inflammatory pathways.

Protein restriction in the pregnant mouse modifies fetal growth and pulmonary development: role of fetal exposure to β‐hydroxybutyrate

Maternal undernutrition during sensitive periods of pregnancy results in offspring predisposed towards the development of a number of diseases of adulthood, including hypertension and diabetes. In order to determine the nature of any gross alterations in fetal growth during early organogenesis, we supplied timed‐mated pregnant mice with diets containing 6% protein (6%P), 9% protein (9%P) or 18% protein (18%P; control) from day 0 of pregnancy. At embryonic days 11 (E11), 12 (E12) and 13 (E13), females were killed and fetuses removed. Gross morphological analysis revealed that fetal limb growth was impaired between E11 and E12 in 6%P animals, but this recovered by E13. Likewise, fetal liver growth and lung branching morphogenesis were seen to exhibit an initial growth impairment at E12 followed by a rapid recovery by E13. Coincident with the observed changes in fetal growth, we noted an elevation in maternal hepatic triglyceride content, expression of the ketogenic 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (Hmgcs2) and circulating plasma β‐hydroxybutyrate (BOHB). In addition, fetal liver Hmgcs2 expression was switched on by E13 in both 6%P‐ and 9%P‐exposed animals. Exogenous BOHB did not influence branching morphogenesis in fetal lung explant cultures; however, we cannot rule out the possibility that this may occur in vivo. In conclusion, we find that disturbance of fetal growth by maternal dietary protein restriction is associated and therefore potentially indicated by changes in maternal and fetal ketone body metabolism.

The effect of maternal undernutrition on the rat placental transcriptome: protein restriction up-regulates cholesterol transport

BackgroundFetal exposure to a maternal low protein diet during rat pregnancy is associated with hypertension, renal dysfunction and metabolic disturbance in adult life. These effects are present when dietary manipulations target only the first half of pregnancy. It was hypothesised that early gestation protein restriction would impact upon placental gene expression and that this may give clues to the mechanism which links maternal diet to later consequences.MethodsPregnant rats were fed control or a low protein diet from conception to day 13 gestation. Placentas were collected and RNA sequencing performed using the Illumina platform.ResultsProtein restriction down-regulated 67 genes and up-regulated 24 genes in the placenta. Ingenuity pathway analysis showed significant enrichment in pathways related to cholesterol and lipoprotein transport and metabolism, including atherosclerosis signalling, clathrin-mediated endocytosis, LXR/RXR and FXR/RXR activation. Genes at the centre of these processes included the apolipoproteins ApoB, ApoA2 and ApoC2, microsomal triglyceride transfer protein (Mttp), the clathrin-endocytosis receptor cubilin, the transcription factor retinol binding protein 4 (Rbp4) and transerythrin (Ttr; a retinol and thyroid hormone transporter). Real-time PCR measurements largely confirmed the findings of RNASeq and indicated that the impact of protein restriction was often striking (cubilin up-regulated 32-fold, apoC2 up-regulated 17.6-fold). The findings show that gene expression in specific pathways is modulated by maternal protein restriction in the day-13 rat placenta.ConclusionsChanges in cholesterol transport may contribute to altered tissue development in the fetus and hence programme risk of disease in later life.

Differential effects of short-term β agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle

Growth hormone (GH) and β agonists increase muscle mass, but the mechanisms for this response are unclear and the magnitude of response is thought to vary with age of animal. To investigate the mechanisms driving the muscle response to these agents, we examined the effects of short-term (6 day) administration of GH or cimaterol (a β2-adrenergic agonist, BA) on skeletal muscle phenotype in both young (day 60) and mature (day 120) lambs. Expression of myosin heavy chain (MyHC) isoforms were measured in Longissimus dorsi (LD), Semitendinosus (ST) and Supraspinatus (SS) muscles as markers of fibre type and metabolic enzyme activities were measured in LD. To investigate potential mechanisms regulating the changes in fibre type/metabolism, expression or activity of a number of signalling molecules were examined in LD. There were no effects of GH administration on MyHC isoform expression at either the mRNA or protein level in any of the muscles. However, BA treatment induced a proportional change in MyHC mRNA expression at both ages, with the %MyHCI and/or IIA mRNA being significantly decreased in all three muscles and %MyHCIIX/IIB mRNA significantly increased in the LD and ST. BA treatment induced de novo expression of MyHCIIB mRNA in LD, the fastest isoform not normally expressed in sheep LD, as well as increasing expression in the other two muscles. In the LD, the increased expression of the fastest MyHC isoforms (IIX and IIB) was associated with a decrease in isocitrate dehydrogenase activity, but no change in lactate dehydrogenase activity, indicating a reduced capacity for oxidative metabolism. In both young and mature lambs, changes in expression of metabolic regulatory factors were observed that might induce these changes in muscle metabolism/fibre type. In particular, BA treatment decreased PPAR-γ coactivator-1β mRNA and increased receptor-interacting protein 140 mRNA. The results suggest that the two agents work via different mechanisms or over different timescales, with only BA inducing changes in muscle mass and transitions to a faster, less oxidative fibre type after a 6-day treatment.

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20 ppm in feed) or Reporcin (recombinant growth hormone; GH; 10 mg/48 hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p = 0.002) and muscle weights (Vastus Lateralis: p < 0.001; Semitendinosus: p = 0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p < 0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p < 0.05) and 7 (p < 0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p < 0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth.

The Beta-adrenergic agonist, Ractopamine, increases skeletal muscle expression of Asparagine Synthetase as part of an integrated stress response gene program

Synthetic beta-adrenergic agonists (BA) have broad biomedical and agricultural application for increasing lean body mass, yet a poor understanding of the biology underpinning these agents is limiting further drug discovery potential. Growing female pigs (77 ± 7 kg) were administered the BA, Ractopamine (20 ppm in feed), or the recombinant growth hormone (GH), Reporcin (10 mg/48 hrs injected) for 1, 3, 7, 13 (n = 10 per treatment, per time point) or 27 days (n = 15 per treatment). Using RNA-sequencing and inferred pathway analysis, we examined temporal changes to the Longissimus Dorsi skeletal muscle transcriptome (n = 3 per treatment, per time point) relative to a feed-only control cohort. Gene expression changes were affirmed by quantitative-PCR on all samples (n = 164). RNA-sequencing analysis revealed that BA treatment had greater effects than GH, and that asparagine synthetase (Asns) was the 5th most significantly increased gene by BA at day 3. ASNS protein expression was dramatically increased by BA treatment at day 7 (p < 0.05). The most significantly increased gene at day 3 was activating transcription factor 5 (Atf5), a transcription factor known to regulate ASNS gene expression. Gene and protein expression of Atf4, another known regulator of Asns expression, was not changed by BA treatment. Expression of more than 20 known Atf4 target genes were increased by BA treatment, suggesting that BA treatment induces an integrated stress response (ISR) in skeletal muscle of pigs. In support of this, mRNA expression of sestrin-2 (Sesn2) and cyclin-dependant kinase 1 alpha (Cdkn1a), two key stress-responsive genes and negative regulators of cellular growth, were also strongly increased from day 3 of BA treatment. Finally, tRNA charging was the most significantly enriched pathway induced by BA treatment, suggesting alterations to the translational capacity/efficiency of the muscle. BA-mediated changes to the skeletal muscle transcriptome are highly indicative of an integrated stress response (ISR), particularly genes relating to amino acid biosynthesis and protein translational capacity.