Two Novel InDels within the 5’UTR of SIRT1 are Associated with Growth Traits in Chickens

Abstract Background: SIRT1, a NAD+ dependent histone deacetylase, is involved in lipid metabolism, glucose metabolism, apoptosis, and insulin secretion. However, the function of the SIRT1 gene in chickens has not been elucidated. Results: In our study, we identified two novel InDels (c.-1552_-1553insCG and c.-450_-451delCG) in the 5’UTR of the chicken SIRT1 gene. After genotyping 1,141 chickens from 7 breeds, we found that the wild type genotypes for both sites were the most common. An association study using 860 chickens from a Gushi ×Anka F2 resource population showed that c.-1552_-1553insCG was significantly correlated with growth traits and serum lipid indicators. The insertion genotype was most highly associated with body weight in 0-, 2-, and 4-week old chickens, and with shank length and shank circumference in 4-week and 8-week old chickens. The wild type genotype at this site was most highly associated with serum lipid indicators. In contrast, c.-450_-451delCG was significantly correlated with muscle fiber diameter. We also analyzed SIRT1 gene expression in chickens with different InDel genotypes and found that SIRT1 expression in muscle and fat tissue was significantly higher with heterozygous genotypes at both sites, relative to expression in chickens with the corresponding homozygous genotypes. Finally, we analyzed the effects of different haplotypes on SIRT1 promoter activity. The results showed that promoter activity depends on haplotype, with haplotype HapII exhibiting the highest activity. Conclusion: We conclude that the SIRT1 gene is associated with chicken growth traits and that the two InDels influence SIRT1 promoter activity in chickens.

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Lipid Metabolism Gene Expression And Cellularity of Intramuscular Adipocytes Within The Longissimus Muscle of Angus- And Wagyu-Sired Cattle When Raised To A Similar Age or Body Weight

10.21203/rs.3.rs-764272/v1 ◽

2021 ◽

Author(s):

Jerad Jaborek ◽

Francis Fluharty ◽

Kichoon Lee ◽

Henry Zerby ◽

Alejandro Relling

Keyword(s):

Gene Expression ◽

Body Weight ◽

Fatty Acid ◽

Lipid Metabolism ◽

Binding Protein ◽

Peroxisome Proliferator ◽

Longissimus Muscle ◽

Peroxisome Proliferator Activated Receptor ◽

Lipid Metabolism Gene ◽

Metabolism Gene

Abstract Background: This study investigates intramuscular (IM) adipocyte development and growth in the Longissimus muscle (LM) between Wagyu- and Angus-sired steers compared at a similar age and days on feed (DOF) endpoint or similar body weight (BW) endpoint by measuring IM adipocyte cell area and lipid metabolism gene expression. Methods: Angus-sired steers (AN, n=6) were compared with steers from two different Wagyu sires, selected for either growth or marbling, to be compared at a similar DOF (WA-GD, n=5 and WA-MD, n=5) in experiment 1 or BW (WA-GB, n=4 and WA-MB, n=5) in experiment 2, respectively. Results: In experiment 1, WA-MD steers had a greater percentage of IM fat in the LM compared with AN and WA-GD steers. In experiment 2, WA-MB steers had a greater percentage of IM fat in the LM compared with AN and WA-GB steers. The distribution of IM adipocyte area was unimodal at all biopsy collections, with IM adipocyte area becoming progressively larger as cattle age and BW increased (P≤0.01). Peroxisome proliferator activated receptor delta (PPARd) was upregulated earlier for WA-MD and WA-MB cattle compared with other steers at a similar age and BW (P≤0.02; treatment×biopsy interaction). An earlier upregulation of PPARd is believed to have then upregulated peroxisome proliferator activated receptor gamma (PPARg) at a lesser BW for WA-MB steers (P=0.09; treatment×biopsy interaction), while WA-MD steers had a greater (P≤0.04) overall mean PPARg expression compared with other steers. Glycerol-3-phosphate acyltransferase, lipin 1, and hormone sensitive lipase demonstrated expression patterns similar to PPARg and PPARd or CCAAT enhancer binding protein beta, which emphasizes their importance in marbling development and growth. Additionally, WA-MD and WA-MB steers often had a greater early expression of fatty acid transporters (fatty acid transport protein 1; P<0.02; treatment×biopsy interaction) and binding proteins (fatty acid binding protein 4) compared with other steers. With many lipolytic genes upregulated at harvest, acetyl-CoA carboxylase beta may be inhibiting fatty acid oxidation in the LM to allow greater IM fat accumulation.Conclusions: Cattle with a greater marbling propensity appear to upregulate adipogenesis at a lesser maturity through PPARd, PPARg, and possibly adipogenic regulating compounds in lysophosphatidic acid and diacylglycerol.

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Transcriptomic analysis of PPARα-dependent alterations during cardiac hypertrophy

Physiological Genomics ◽

10.1152/physiolgenomics.90296.2008 ◽

2008 ◽

Vol 36 (1) ◽

pp. 15-23 ◽

Cited By ~ 26

Author(s):

Pascal J. H. Smeets ◽

Heleen M. de Vogel-van den Bosch ◽

Peter H. M. Willemsen ◽

Alphons P. Stassen ◽

Torik Ayoubi ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Lipid Metabolism ◽

Cardiac Hypertrophy ◽

Signaling Pathways ◽

Differentially Expressed Genes ◽

Left Ventricular ◽

Transcriptomic Analysis ◽

Differentially Expressed ◽

Wild Type

Peroxisome proliferator-activated receptor (PPAR)α regulates lipid metabolism at the transcriptional level and modulates the expression of genes involved in inflammation, cell proliferation, and differentiation. Although PPARα has been shown to mitigate cardiac hypertrophy, knowledge about underlying mechanisms and the nature of signaling pathways involved is fragmentary and incomplete. The aim of this study was to identify the processes and signaling pathways regulated by PPARα in hearts challenged by a chronic pressure overload by means of whole genome transcriptomic analysis. PPARα−/− and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days, and left ventricular gene expression profile was determined with Affymetrix GeneChip Mouse Genome 430 2.0 arrays containing >45,000 probe sets. In unchallenged hearts, the mere lack of PPARα resulted in 821 differentially expressed genes, many of which are related to lipid metabolism and immune response. TAC resulted in a more pronounced cardiac hypertrophy and more extensive changes in gene expression (1,910 and 312 differentially expressed genes, respectively) in PPARα−/− mice than in wild-type mice. Many of the hypertrophy-related genes were related to development, signal transduction, actin filament organization, and collagen synthesis. Compared with wild-type hypertrophied hearts, PPARα−/− hypertrophied hearts revealed enrichment of gene clusters related to extracellular matrix remodeling, immune response, oxidative stress, and inflammatory signaling pathways. The present study therefore demonstrates that, in addition to lipid metabolism, PPARα is an important modulator of immune and inflammatory response in cardiac muscle.

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SpoIIE Regulates Sporulation but Does Not Directly Affect Solventogenesis in Clostridium acetobutylicum ATCC 824

Journal of Bacteriology ◽

10.1128/jb.187.6.1930-1936.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 1930-1936 ◽

Cited By ~ 37

Author(s):

Miles C. Scotcher ◽

George N. Bennett

Keyword(s):

Gene Expression ◽

Antisense Rna ◽

Clostridium Acetobutylicum ◽

Promoter Activity ◽

Normal Growth ◽

Wild Type ◽

Solvent Production ◽

Intact Copy ◽

Clostridium Acetobutylicum Atcc 824 ◽

Production Strain

ABSTRACT Using gene expression reporter vectors, we examined the activity of the spoIIE promoter in wild-type and spo0A-deleted strains of Clostridium acetobutylicum ATCC 824. In wild-type cells, the spoIIE promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator spo0A is disrupted, no spoIIE promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress spoIIE and to decrease spoIIE expression via antisense RNA targeted against spoIIE, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of spoIIE. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in spoIIE-downregulated cells are mediated by effects on the cell during sporulation.

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Investigating the Role of Farnesoid X Receptor in Heme Biosynthesis and Ductular Reaction

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1650 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A810-A811

Author(s):

Angela E Dean ◽

Emilian Jungwirth ◽

Katrin Panzitt ◽

Martin Wagner ◽

Sayeepriyadarshini Anakk

Keyword(s):

Gene Expression ◽

Body Weight ◽

Weight Ratio ◽

Heme Biosynthesis ◽

Farnesoid X Receptor ◽

Whole Body ◽

Wild Type ◽

Body Weight Ratio ◽

Ductular Reaction ◽

Male And Female

Abstract Bile acids (BAs) have gained traction not just as emulsifiers of fat, but also as hormones. Nuclear receptor Farnesoid X receptor (FXR) is the master regulator of BAs and can also control glucose and lipid metabolism. We examined if FXR contributed towards heme biosynthesis and induction of a ductular reaction. Male and female whole body Fxr knockout (FxrKO) mice, as well as liver- and intestine-specific knockouts (LFxrKO and IFxrKO, respectively) were treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, a ferrochelatase inhibitor) for two weeks. At the end of the two weeks, mice were fasted for four hours and euthanized. All groups of mice had lost a similar percentage of body weight when fed the DDC diet. However, female FxrKO mice had significantly increased liver to body weight ratio, while male FxrKO mice had significantly decreased liver to body weight ratio when fed the DDC diet compared with their wild type counterparts. Serum liver injury markers were analyzed and liver histology and changes in genes involved in the heme biosynthesis pathway were examined. Both male and female whole body FxrKO livers had decreased ductular reaction with minimal bile plugs (porphyrin accumulation) compared with their wild type counterparts. LFxrKO mice mimicked diminished ductular reaction, while IFxrKO mice exhibited severe ductular reaction similar to that of wild type mice, indicating that the ductular reaction is dependent on hepatic FXR. ChIP-Seq for FXR revealed binding peaks in the heme biosynthesis genes, Alas1, Alad, Uros, and Fech, suggesting that FXR may act as a transcription factor for these genes. Further investigation revealed that Pbgd gene expression was increased, while Fech gene expression was decreased in female FxrKO mice compared to wild type mice. In male mice, Pbgd, Uros, Urod, and Cpox gene expression was increased in the absence of Fxr. In conclusion, Fxr is necessary to mount a ductular reaction and plays a key role in heme biosynthesis in the liver.

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HCK Transcription Is Regulated By AP1, NF-Kb and STAT3 Transcription Factors in MYD88 Mutated WM and ABC-DLBCL Cells

Blood ◽

10.1182/blood.v128.22.2931.2931 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2931-2931

Author(s):

Xia Liu ◽

Jiaji G Chen ◽

Jie Chen ◽

Lian Xu ◽

Nicholas Tsakmaklis ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Cell Lines ◽

Binding Sites ◽

Research Funding ◽

Promoter Activity ◽

Promoter Sequence ◽

Wild Type ◽

Mutation Status ◽

Bcr Signaling

Abstract Hematopoietic cell kinase (HCK) is a member of the SRC family of tyrosine kinases (SFKs). HCK transcription is aberrantly upregulated in Waldenström's Macroglobulinemia (WM) and Activated B-cell (ABC) subtype Diffuse Large B-cell Lymphoma (DLBCL) in response to activating mutations in MYD88 (Yang et al, Blood 2016). To clarify the mechanism responsible for the aberrant upregulation of HCK transcription inMYD88 mutated cells, we analyzed the promoter sequence of HCK using PROMO and identified consensus binding sites for transcription factors (AP1, NF-kB, STAT3, and IRF1) that are regulated by mutated MYD88 (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013; Juilland et al, Blood 2016; Yang et al, Blood 2016). We performed Chromatin Immuno-precipitation (ChIP) assays using ChIP grade antibodies to JunB, c-Jun, NF-kB-p65, STAT3 and IRF1 in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1, OCI-Ly3) cells that highly express HCK transcripts, as well as wild type MYD88 expressing GCB DLBCL (OCI-Ly7, OCI-Ly19) cells that show low HCK transcription. Following ChIP, a HCK promoter specific quantitative PCR assay was used to detect HCK promoter sequences. These studies showed that JunB, NF-kB-p65 and STAT3 bound more robustly to the HCK promoter in MYD88 mutated WM and ABC-DLBCL cells versus MYD88 wild type GCB DLBCL cell lines, while c-Jun bound more abundantly to the HCK promoter sequence in all DLBCL cell lines, regardless of MYD88 mutation status. In contrast c-Jun binding was low in MYD88 mutated WM cells. IRF1 binding to the HCK promoter was similar in all cell lines, regardless of the MYD88 mutation status. To further investigate HCK regulation, we developed an HCK promoter driven luciferase reporter vector (WT) with mutated AP-1 binding (AP1-mu-1~6), NF-kB binding (NF-kB-mu-1~5), and STAT3 binding (STAT3-mu) sites and investigated their impact on HCK promoter activity in MYD88 mutated BCWM.1 cells. We observed that mutation of AP1-mu-1,4,5,6; NF-kB-mu-1,4,5, as well as STAT3-mu binding sites greatly reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 transcription factors in HCK gene expression in MYD88 mutated cells. To further clarify the importance of these transcription factors in aberrant HCK gene expression in MYD88 mutated cells, we treated BCWM.1, MWCL-1, TMD-8 and HBL-1 cells with the AP-1 inhibitor SR 11302; NF-kB inhibitor QNZ; and the STAT3 inhibitor STA-21. Treatment of cells for 2 hours with SR 11302, QNZ, and STA-21 at sub-EC50 concentrations resulted in decreased HCK expression in MYD88 mutated all cell lines. Lastly, we investigated the contribution of BCR signaling to HCK transcription. BCWM.1, MWCL-1, TMD-8, and HBL-1 cells were treated with the Syk kinase inhibitor R406, and HCK transcription levels were then assessed. Differences in HCK expression were observed between MYD88 mutated WM and ABC DLBCL cells following R406, supporting a contributing role for BCR signaling in ABC DLBCL but not WM cells to HCK expression. Our data provide critical new insights into HCK regulation, and a framework for targeting pro-survival HCK signaling in WM and ABC DLBCL cells dependent on activating MYD88 mutations. Disclosures Castillo: Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding; Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.

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Exploration of Genetic Variants within the Goat A-Kinase Anchoring Protein 12 (AKAP12) Gene and Their Effects on Growth Traits

Animals ◽

10.3390/ani11072090 ◽

2021 ◽

Vol 11 (7) ◽

pp. 2090

Author(s):

Yangyang Bai ◽

Rongrong Yuan ◽

Yunyun Luo ◽

Zihong Kang ◽

Haijing Zhu ◽

...

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Body Weight ◽

Genetic Variants ◽

Growth And Development ◽

Growth Traits ◽

Indel Mutation ◽

Anchoring Protein ◽

Cashmere Goats ◽

Regulation Of Growth

The A-kinase anchoring protein 12 gene (AKAP12) is a scaffold protein, which can target multiple signal transduction effectors, can promote mitosis and cytokinesis and plays an important role in the regulation of growth and development. In our previous study, P1–7 bp (intron 3) and P2–13 bp (3′UTR) indels within the AKAP12 gene significantly influenced AKAP12 gene expression. Therefore, this study aimed to identify the association between these two genetic variations and growth-related traits in Shaanbei white cashmere goats (SBWC) (n = 1405). Herein, we identified two non-linkage insertions/deletions (indels). Notably, we found that the P1–7 bp indel mutation was related to the height at hip cross (HHC; p < 0.05) and the P2–13 bp indel was associated with body weight, body length, chest depth, chest width, hip width, chest circumference and cannon (bone) circumference in SBWC goats (p < 0.05). Overall, the two indels’ mutations of AKAP12 affected growth traits in goats. Compared to the P1–7 bp indel, the P2–13 bp indel is more suitable for the breeding of goat growth traits.

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PSXI-27 Association of an increased marbling score by dietary glycerol supplementation with lipid metabolism gene expression in Korean cattle steers

Journal of Animal Science ◽

10.1093/jas/skz258.764 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 383-384

Author(s):

Seon Pil Yoo ◽

Dilla Fassah ◽

Myunggi Baik ◽

sang Weon Na ◽

Inhyuk Jeong ◽

...

Keyword(s):

Gene Expression ◽

Body Weight ◽

Lipid Metabolism ◽

Fatty Acid Synthase ◽

Mrna Levels ◽

Lipid Uptake ◽

Marbling Score ◽

Adipose Tissues ◽

Korean Cattle ◽

Subcutaneous Adipose

Abstract This study investigated effects of dietary glycerol supplementation on liver, muscle, and adipose gene expression related with gluconeogenesis and lipid metabolism and association of gene expression levels with marbling score in Korean cattle steers. Fourteen Korean cattle steers (average age 28.4 months; average body weight 733 kg) were equally assigned to two groups (0 and 5% glycerol supplementation). Glycerol was provided with glycerol (63%)-adsorbed ground wheat bran (37%, DM) by top dressing during roughage feeding. A concentrate (1.2% of body weight) and 1.0 kg of ryegrass were individually fed twice daily. After four months of study, steers were slaughtered, and marbling score was evaluated. Longissimus thoracis (LT) and subcutaneous adipose tissue at the 13th thoracic vertebra area and liver were collected and analyzed for mRNA levels by quantitative real-time PCR. Statistical significance was analyzed by analysis of variance. Correlations were analyzed using Pearson’s correlation analysis. Glycerol supplementation increased (P = 0.01) marbling score. In the LT, glycerol supplementation tended to increase (0.05 < P ≤ 0.10) lipid uptake CD36 and lipoprotein lipase (LPL) mRNA levels. In subcutaneous adipose tissues, glycerol supplementation increased (P ≤ 0.05) LPL, adipogenic sterol regulatory element binding protein 1 (SREBP1), and lipogenic acetyl CoA carboxylase (ACC) mRNA levels and tended to increase (0.05 < P < 0.10) CD36, adipogenic peroxisome proliferator-activated gamma (PPARG), and lipogenic fatty acid synthase (FASN) expression. It did not affect (P > 0.05) mRNA levels of hepatic gluconeogenesis genes. Marbling score showed significant positive correlations (0.57 < r < 0.68; P < 0.05) with mRNA levels of several genes including LPL, PPARG, SREBP1, and ACC in adipose tissues, but not with any genes examined in the LT. Our study demonstrates that lipid uptake, adipogenesis and lipogenesis may mainly contribute to the increased marbling score by glycerol supplementation.

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Differential body weight and feeding responses to high-fat diets in rats and mice lacking cholecystokinin 1 receptors

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00002.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. R55-R63 ◽

Cited By ~ 48

Author(s):

Sheng Bi ◽

Jie Chen ◽

R. Ryan Behles ◽

Jayson Hyun ◽

Alan S. Kopin ◽

...

Keyword(s):

Gene Expression ◽

Body Weight ◽

Food Intake ◽

Wild Type ◽

High Fat ◽

Oletf Rats ◽

Equal Degree ◽

Cck1 Receptor ◽

Long Evans ◽

Rats And Mice

Prior data demonstrated differential roles for cholecystokinin (CCK)1 receptors in maintaining energy balance in rats and mice. CCK1 receptor deficiency results in hyperphagia and obesity of Otsuka Long-Evans Tokushima Fatty (OLETF) rats but not in mice. To ascertain the role of CCK1 receptors in high-fat-diet (HFD)-induced obesity, we compared alterations in food intake, body weight, fat mass, plasma glucose, and leptin levels, and patterns of hypothalamic gene expression in OLETF rats and mice lacking CCK1 receptors in response to a 10-wk exposure to HFD. Compared with Long-Evans Tokushima Otsuka (LETO) control rats, OLETF rats on HFD had sustained overconsumption over the 10-wk period. High fat feeding resulted in greater increases in body weight and plasma leptin levels in OLETF than in LETO rats. In situ hybridization determinations revealed that, while HFD reduced neuropeptide Y (NPY) mRNA expression in both the arcuate nucleus (Arc) and the dorsomedial hypothalamus (DMH) of LETO rats, HFD resulted in decreased NPY expression in the Arc but not in the DMH of OLETF rats. In contrast to these results in OLETF rats, HFD increased food intake and induced obesity to an equal degree in both wild-type and CCK1 receptor−/− mice. NPY gene expression was decreased in the Arc in response to HFD, but was not detectable in the DMH in both wild-type and CCK1 receptor−/− mice. Together, these data provide further evidence for differential roles of CCK1 receptors in the controls of food intake and body weight in rats and mice.

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Dietary isoflavone supplementation modulates lipid metabolism via PPARα-dependent and -independent mechanisms

Physiological Genomics ◽

10.1152/physiolgenomics.00155.2005 ◽

2006 ◽

Vol 26 (1) ◽

pp. 8-14 ◽

Cited By ~ 49

Author(s):

Orsolya Mezei ◽

Yilan Li ◽

Eimear Mullen ◽

Jennifer S. Ross-Viola ◽

Neil F. Shay

Keyword(s):

Gene Expression ◽

Lipid Metabolism ◽

Soy Protein ◽

Knockout Mice ◽

Estrogenic Activity ◽

Serum Triglyceride ◽

Peroxisome Proliferator ◽

Wild Type ◽

Serum Triglycerides ◽

Regulated Gene Expression

Intake of soy protein has been associated with improvements in lipid metabolism, with much attention being focused on the serum cholesterol-lowering property of soy. The component or components of soy that are responsible for improvements in lipid metabolism have been investigated and their specific actions debated. One component, the isoflavones, has been shown to have weak estrogenic activity, and recently, several research groups have suggested that isoflavones are activating peroxisome proliferator-activated receptors (PPARs). The three different isoforms of PPARs (α, γ, and δ) have overlapping tissue distributions and functions associated with lipid metabolism. The goal of the present study was to investigate the hypothesis that the effect of isoflavones is mediated through the PPARα receptor. Male and female 129/Sv mice were obtained, including both wild-type and genetically altered PPARα knockout mice. Groups of mice were fed high-fat atherogenic diets containing soy protein +/- isoflavones and PPARα agonist fenofibrate for 6 wk. At the end of 6 wk, serum and tissue lipid levels were measured along with hepatic gene expression. Most notably, serum triglycerides were reduced by isoflavone consumption. Compared with intake of a low-isoflavone basal diet, isoflavone intake reduced serum triglyceride levels by 36 and 52% in female and male wild-type mice, respectively, compared with 55 and 52% in fenofibrate-treated mice. Isoflavones also improved serum triglyceride levels in knockout mice, whereas fenofibrate did not, suggesting that two different regulatory mechanisms may be affected by isoflavone intake. Isoflavone intake resembled action of fenofibrate on PPARα-regulated gene expression, although less robustly compared with fenofibrate. We suggest that, at the levels consumed in this study, isoflavone intake is altering lipid metabolism in a manner consistent with activation of PPARα and also via a PPARα-independent mechanism as well.

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Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy

Skeletal Muscle ◽

10.1186/s13395-019-0218-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Cynthia Shu ◽

Ariana N. Kaxon-Rupp ◽

Judd R. Collado ◽

Robert Damoiseaux ◽

Rachelle H. Crosbie

Keyword(s):

Gene Expression ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Calcium Channel ◽

Muscle Cell ◽

High Throughput ◽

Promoter Activity ◽

Muscle Cells ◽

Wild Type ◽

Protein Levels

Abstract Background Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Methods Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. Results We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. Conclusions We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

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