Expression In Skeletal Muscle Research Articles

Magnetization of mesenchymal stem cells using magnetic liposomes enhances their retention and immunomodulatory efficacy in mouse inflamed skeletal muscle

Sarcopenia, an age-related reduction in skeletal muscle mass and strength, is mainly caused by chronic inflammation. Because mesenchymal stem cells (MSCs) have the capacity to both promote myogenic cell differentiation and suppress inflammation, they are a promising candidate for sarcopenia treatment. In this study, to achieve the long-term retention of MSCs in skeletal muscle, we prepared magnetized MSCs using magnetic anionic liposome/atelocollagen complexes that we had previously developed, and evaluated their retention efficiency and immunomodulatory effects in mouse inflamed skeletal muscle. Mouse MSCs were efficiently magnetized by incubation with magnetic anionic liposome/atelocollagen complexes for 30 min under a magnetic field. The magnetized MSCs differentiated normally into osteoblasts and adipocytes. Additionally, non-magnetized MSCs and magnetized MSCs increased IL-6 and inducible nitric oxide synthase mRNA expression and decreased TNF-α and IL-1β mRNA expression in C2C12 mouse skeletal muscle myotubes through paracrine effects. Moreover, magnetized MSCs were significantly retained in cell culture plates and mouse skeletal muscle after their local injection in the presence of a magnetic field. Furthermore, magnetized MSCs significantly increased IL-6 and IL-10 mRNA expression and decreased TNF-α and IL-1β mRNA expression in inflamed skeletal muscle. These results suggest that magnetized MSCs may be useful for effective sarcopenia treatment.

Physical performance level in sarcomeric mitochondria creatine kinase knockout mouse model throughout ageing.

The objective of the present study was to establish the role of sarcomeric mitochondrial creatine kinase (Mt-CK) in muscle energy output during exercise in a murine model of ageing (the Mt-CK knock-out mouse, Mt-CK-/-). Three age groups of Mt-CK-/- mice and control male mice (6, 9, and 18months of age) underwent incremental treadmill running tests. The maximum speed (Vpeak) and maximal oxygen consumption (VO2peak) values were recorded. Urine samples were analyzed using metabolomic techniques. The skeletal muscle (quadriceps) expression of proteins involved in mitochondria biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and dynamin-related GTPase mitofusin 2 (Mnf2) were quantified. The VO2 peak (normalized to heart weight: HW) of 18-month-old (mo) Mt-CK-/- mice was 27% (p<0.001) lower than in 18-mo control mice. The VO2peak/HW ratio was 29% (p<0.001) lower in 18-mo Mt-CK-/- mice than in 6-mo (p<0.001) and 32% (p<0.001) than 9-mo Mt-CK-/- mice. With a 0° slope, Vpeak was 10% (p<0.05) lower in 18-mo Mt-CK-/- mice than in 6-mo Mt-CK-/- mice but did not differ when comparing the 18-mo and 6-mo control groups. The skeletal muscles weight normalized on body weight in 6-mo Mt-CK-/- were 13 to 14% (p<0.001, p<0.05) lower versus the 6-mo control, in addition, the presence of branched-chain amino acids in the urine of 6-mo Mt-CK-/- mice suggests an imbalance in protein turnover (catabolism rather than anabolism) but we did not observe any age-related differences. The expression of PGC-1α and Mnf2 proteins in the quadriceps showed that age-related effects were more prominent than genotype effects. The present study showed ageing is potentialized by Mt-CK deficiency with regard to VO2peak, Vpeak and mitochondrial protein expression. Our results support that Mt-CK-/- mice undergo physiological adaptations, enabling them to survive and to perform as well as wild-type mice. Furthermore, it is possible that these adaptations in Mt-CK-/- mice have a high energy cost and might trigger premature ageing.

MOTS-c is an exercise-induced mitochondrial-encoded regulator of age-dependent physical decline and muscle homeostasis

Healthy aging can be promoted by enhanced metabolic fitness and physical capacity. Mitochondria are chief metabolic organelles with strong implications in aging that also coordinate broad physiological functions, in part, using peptides that are encoded within their independent genome. However, mitochondrial-encoded factors that actively regulate aging are unknown. Here, we report that mitochondrial-encoded MOTS-c can significantly enhance physical performance in young (2 mo.), middle-age (12 mo.), and old (22 mo.) mice. MOTS-c can regulate (i) nuclear genes, including those related to metabolism and proteostasis, (ii) skeletal muscle metabolism, and (iii) myoblast adaptation to metabolic stress. We provide evidence that late-life (23.5 mo.) initiated intermittent MOTS-c treatment (3x/week) can increase physical capacity and healthspan in mice. In humans, exercise induces endogenous MOTS-c expression in skeletal muscle and in circulation. Our data indicate that aging is regulated by genes encoded in both of our co-evolved mitochondrial and nuclear genomes.

Development of antisense-mediated myostatin knockdown for the treatment of insulin resistance

Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. However, there is currently no clinically effective myostatin inhibitor, and therefore novel methods are required. We evaluated the use of antisense phosphorodiamidate morpholino oligomers (PMO) to reduce myostatin expression in skeletal muscle and measured their effects on muscle mass and glucose uptake. C57/Bl6 mice received intramuscular or intravenous injections of anti-myostatin PMOs. Repeated intramuscular administration lead to a reduction in myostatin transcript levels (~ 20–40%), and an increase in muscle mass in chow and high-fat diet (HFD)-fed mice, but insulin-stimulated glucose uptake was reduced in PMO-treated muscles of HFD-fed mice. Five weekly intravenous administrations of 100 nmol PMO did not reduce myostatin expression, and therefore had no significant physiological effects. Unexpectedly, exon skipping levels were higher after intramuscular administration of PMO in HFD- than chow-fed mice. These results suggest that a modest PMO-induced reduction in myostatin transcript levels is sufficient to induce an increase in muscle mass, but that a greater degree of inhibition may be required to improve muscle glucose uptake.

Antidiabetic and antioxidant activity of phlorotannins extracted from the brown seaweed Cystoseira compressa in streptozotocin-induced diabetic rats.

Diabetes mellitus is considered a set of diseases that lead to high glucose level due to the absolute or relative absence of insulin. The study investigated the antioxidant activity and antidiabetic effect of phlorotannins extracted from brown seaweed Cystoseira compressa. Phlorotannins were extracted from C. compressa. It was confirmed by 2,4 dimethoxy benzaldehyde assay (DMBA), ultraviolet spectra, Fourier transform infrared spectroscopy, and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The free radical scavenging activity of phlorotannins was estimated by total antioxidant capacity, 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, and 2,2 azino-bis3-ethylbenthiazoline-6-sulfonic acid assays. Four groups of albino rats used in this study include control normal, control phlorotannins extract, diabetic by intraperitoneally administering of streptozotocin, and diabetic treatment with 60mg/kg of phlorotannin extract after 4weeks of diabetes induction. The main compound identified by UPLC-MS/MS in C. compressa extract belonged to the fuhalol. C. compressa extract showed high antioxidant properties. Phlorotannins significantly decreased serum glucose, liver malondialdehyde, and α-amylase, glucosidase activities. However, total antioxidant capacity, serum insulin, hepatic glutathione, and AMPKα2 expression in skeletal muscle were improved compared to the diabetic group. The histopathological examination showed that phlorotannins markedly reduced damage in β cells of pancreases. Phlorotannins from C. compressa have efficient antioxidant activity and the antidiabetic effect that may be utilized in human health.

Influence of irisin on diet-induced metabolic syndrome in experimental rat model.

To evaluate the influence of irisin on the experimental paradigm of non-alcoholic fatty liver (NAFL) as a part of MetS cluster. Forty male albino rats were divided into four groups; normal control, standard diet+irisin, high carbohydrate and fat diet (HCHF), and HCHF+irisin. After the experimental period, levels of fasting blood sugar (FBS), insulin, lipid profile, kidney functions, salusin-alpha (Sal-α), adropin, and retinol-binding protein-4 (RBP-4) were evaluated. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) expression in skeletal muscle was evaluated by quantitative real-time PCR. Aorta, liver, pancreas, and skeletal muscle tissue samples were prepared for histopathological examination. Rats administrated HCHF showed elevated levels of FBS, lipid profile, kidney functions, RBP-4, and downregulation of PGC-1α expression along with a decline in levels of insulin, Sal-α, and adropin while administration of irisin significantly attenuated these levels. Irisin as based therapy could emerge as a new line of treatment against MetS and its related diseases.

14-Deoxy-11,12-Didehydroandrographolide Ameliorates Glucose Intolerance Enhancing the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 Signaling Pathway and Inducing GLUT4 Expression in Myotubes and Skeletal Muscle of Obese Mice.

14-Deoxy-11,12-didehydroandrographolide (deAND), a bioactive component of Andrographis paniculata, has antidiabetic activity. AMP-activated protein kinase (AMPK) regulates glucose transport and ameliorates insulin resistance. The aim of the present study was to investigate whether activation of AMPK is involved in the mechanism by which deAND ameliorates insulin resistance in muscles. deAND amounts up to 40 [Formula: see text]M dose-dependently activated phosphorylation of AMPK[Formula: see text] and TBC1D1 in C2C12 myotubes. In addition, deAND significantly activated phosphorylation of LKB1 at 6 h after treatment, and this activation was maintained up to 48 h. deAND increased glucose uptake at 18 h after treatment, and this increase was time dependent up to 72 h. Compound C, an inhibitor of AMPK, suppressed deAND-induced phosphorylation of AMPK[Formula: see text] and TBC1D1 and reversed the effect on glucose uptake. In addition, the expression of GLUT4 mRNA and protein in C2C12 myotubes was up-regulated by deAND in a time-dependent manner. Promotion of GLUT4 gene transcription was verified by a pGL3-GLUT4 (837 bp) reporter assay. deAND also increased the nuclear translocation of MEF-2A and PPAR[Formula: see text]. After 16 weeks of feeding, the high-fat diet (HFD) inhibited phosphorylation of AMPK[Formula: see text] and TBC1D1 in skeletal muscle of obese C57BL/6JNarl mice, and deactivation of AMPK[Formula: see text] and TBC1D1 by the HFD was abolished by deAND supplementation. Supplementation with deAND significantly promoted membrane translocation of GLUT4 compared with the HFD group. Supplementation also significantly increased GLUT4 mRNA and protein expression in skeletal muscle compared with the HFD group. The hypoglycemic effects of deAND are likely associated with activation of the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 signaling pathway and stimulation of MEF-2A- and PPAR[Formula: see text]-dependent GLUT4 gene expression, which account for the glucose uptake into skeletal muscle and lower blood glucose levels.

NAD+-boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin.

Rationale: Nicotinamide adenine dinucleotide+ (NAD+)-boosting therapy has emerged as a promising strategy to treat various health disorders, while the underlying molecular mechanisms are not fully understood. Here, we investigated the involvement of fibronectin type III domain containing 5 (Fndc5) or irisin, which is a novel exercise-linked hormone, in the development and progression of nonalcoholic fatty liver disease (NAFLD).Methods: NAD+-boosting therapy was achieved by administrating of nicotinamide riboside (NR) in human and mice. The Fndc5/irisin levels in tissues and blood were measured in NR-treated mice or human volunteers. The therapeutic action of NR against NAFLD pathologies induced by high-fat diet (HFD) or methionine/choline-deficient diet (MCD) were compared between wild-type (WT) and Fndc5-/- mice. Recombinant Fndc5/irisin was infused to NALFD mice via osmotic minipump to test the therapeutic action of Fndc5/irisin. Various biomedical experiments were conducted in vivo and in vitro to know the molecular mechanisms underlying the stimulation of Fndc5/irisin by NR treatment.Results: NR treatment elevated plasma level of Fndc5/irisin in mice and human volunteers. NR treatment also increased Fndc5 expression in skeletal muscle, adipose and liver tissues in mice. In HFD-induced NAFLD mice model, NR displayed remarkable therapeutic effects on body weight gain, hepatic steatosis, steatohepatitis, insulin resistance, mitochondrial dysfunction, apoptosis and fibrosis; however, these actions of NR were compromised in Fndc5-/- mice. Chronic infusion of recombinant Fndc5/irisin alleviated the NAFLD pathological phenotypes in MCD-induced NAFLD mice model. Mechanistically, NR reduced the lipid stress-triggered ubiquitination of Fndc5, which increased Fndc5 protein stability and thus enhanced Fndc5 protein level. Using shRNA-mediated knockdown screening, we found that NAD+-dependent deacetylase SIRT2, rather than other sirtuins, interacts with Fndc5 to decrease Fndc5 acetylation, which reduces Fndc5 ubiquitination and stabilize it. Treatment of AGK2, a selective inhibitor of SIRT2, blocked the therapeutic action of NR against NAFLD pathologies and NR-induced Fndc5 deubiquitination/deacetylation. At last, we identified that the lysine sites K127/131 and K185/187/189 of Fndc5 may contribute to the SIRT2-dependent deacetylation and deubiquitination of Fndc5.Conclusions: The findings from this research for the first time demonstrate that NAD+-boosting therapy reverses NAFLD by regulating SIRT2-deppendent Fndc5 deacetylation and deubiquitination, which results in a stimulation of Fndc5/irisin, a novel exerkine. These results suggest that Fndc5/irisin may be a potential nexus between physical exercise and NAD+-boosting therapy in metabolic pathophysiology.

Effects of Rehmannia glutinosa polysaccharides on bone tissue structure and skeletal muscle atrophy in rats with disuse

ABSTRACTPurposeTo study effects of Rehmannia glutinosa polysaccharides (RGP) on bone tissue structure and skeletal muscle atrophy in rats with disuse.MethodsA rat model of disuse osteoporosis combined with muscle atrophy was established by removing the bilateral ovaries of rats and fixing their hind limbs for a long time. Forty SD rats were administered intragastrically for 12 weeks. The bone histomorphometry parameters and the level of oxidative stress were measured. In addition, the changes of muscle atrophy F-box (MAFbx), muscle RING-finger protein-1 (MuRF1), forkhead box O1 (FOXO1) mRNA expression in skeletal muscle of rats were observed.ResultsRGP significantly increased the percentage of fluorescence perimeter and bone mineralization deposition rate of the second lumbar vertebrae of rats. It also significantly increased the wet weight ratio and muscle fiber cross-sectional area of the gastrocnemius muscle of rats. At the same time, RGP significantly increased the levels of super oxide dismutase (SOD) and catalase (CAT) in the skeletal muscle of rats, and reduced the content of malondialdehyde (MDA). Rehmannia glutinosa polysaccharides also significantly reduced the expression levels of FOXO1, MAFbx and MuRF1 mRNA in rat skeletal muscle.ConclusionsRGP could improve the bone structure of osteoporotic rats. It could also improve muscle that atrophy may be related to the inhibition of FOXO1-mediated ubiquitin-proteasome pathway.

The effects of exercise on skeletal and heart muscle citrate synthase and carnitine palmitoyltransferase mRNA expressions in high-calorie fed rats.

In this study, the effect of high-calorie feeding and aerobic exercise on skeletal and cardiac muscle citrate synthase (CS), carnitine palmitoyltransferase-I (CPT-I), and -II (CPT-II) mRNA expressions were evaluated. Genetically non-obese rats were grouped as normal-high calorie and sedentary-exercising. Gastrocnemius-soleus and heart muscles' CS, CPT-I, and CPT-II expressions and skeletal muscles' histopathological characteristics were evaluated. High-fat diet had increased body weight by 10% and aerobic exercise did not make any difference. Skeletal muscle CS expression was increased significantly in normal-calorie exercising group. Exercise and high-fat diet did not change CPT-I and CPT-II expressions in both heart and skeletal muscle. Histopathological evaluations demonstrated increased cytoplasmic lipid droplets in high-calorie fed sedentary rats, and exercise had reduced lipid droplets in skeletal muscle. Also, both mitochondria and nuclei distribution were impaired in high-calorie groups. In conclusion, aerobic exercise without food restriction was not enough to make significant changes in fat transportation mechanism into skeletal and heart muscle.

Exercise Training of Secreted Protein Acidic and Rich in Cysteine (Sparc) KO Mice Suggests That Exercise-Induced Muscle Phenotype Changes Are SPARC-Dependent

We previously identified secreted protein acidic and rich in cysteine (Sparc) as an exercise-induced gene in young and elderly individuals. Via this animal experiment, we aim to identify selected implications of SPARC mainly within the muscle in the contexts of exercise. Mice were divided into eight groups based on three variables (age, genotype and exercise): Old (O) or young (Y) × Sparc knock-out (KO) or wild-type (WT) × sedentary (Sed) or exercise (Ex). The exercised groups were trained for 12 weeks at the lactate threshold (LT) speed (including 4 weeks of adaptation period) and all mice were sacrificed afterwards. Body and selected tissues were weighed, and lactate levels in different conditions measured. Expression of skeletal muscle (SM) collagen type I alpha 1 chain (COL1A1) and mitochondrially encoded cytochrome c oxidase I (MT-CO1) in addition to SM strength (grip power) were also measured. Ageing increased the body and white adipose tissue (WAT) weights but decreased SM weight percentage (to body weight) and MT-CO1 expression (in WT). Exercise increased SM COL1A1 in WT mice and MT-CO1 expression, as well as weight percentage of the tibialis anterior muscle, and decreased WAT weight (trend). Compared to WT mice, Sparc KO mice had lower body, muscle and WAT weights, with a decrease in SM MT-CO1 and COL1A1 expression with no genotype effect on lactate levels in all our blood lactate measures. Sparc KO effects on body composition, adiposity and metabolic patterns are toward a reduced WAT and body weight, but with a negative metabolic and functional phenotype of SM. Whereas such negative effects on SM are worsened with ageing, they are relatively improved by exercise. Importantly, our data suggest that the exercise-induced changes in the SM phenotype, in terms of increased performance (metabolic, strength and development), including lactate-induced changes, are SPARC-dependent.

Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles

Oligonucleotide therapeutics hold promise for the treatment of muscle- and heart-related diseases. However, oligonucleotide delivery across the continuous endothelium of muscle tissue is challenging. Here, we demonstrate that docosanoic acid (DCA) conjugation of small interfering RNAs (siRNAs) enables efficient (~5% of injected dose), sustainable (>1 month), and non-toxic (no cytokine induction at 100 mg/kg) gene silencing in both skeletal and cardiac muscles after systemic injection. When designed to target myostatin (muscle growth regulation gene), siRNAs induced ~55% silencing in various muscle tissues and 80% silencing in heart, translating into a ~50% increase in muscle volume within 1 week. Our study identifies compounds for RNAi-based modulation of gene expression in skeletal and cardiac muscles, paving the way for both functional genomics studies and therapeutic gene modulation in muscle and heart.

MuscleAtlasExplorer: a web service for studying gene expression in human skeletal muscle

MuscleAtlasExplorer is a freely available web application that allows for the exploration of gene expression data from human skeletal muscle. It draws from an extensive publicly available dataset of 1654 skeletal muscle expression microarray samples. Detailed, manually curated, patient phenotype data, with information such as age, sex, BMI and disease status, are combined with skeletal muscle gene expression to provide insights into gene function in skeletal muscle. It aims to facilitate easy exploration of the data using powerful data visualization functions, while allowing for sample selection, in-depth inspection and further analysis using external tools.AvailabilityMuscleAtlasExplorer is available at https://mae.crc.med.lu.se/mae2.

Benzyl Isothiocyanate Ameliorates High-Fat Diet-Induced Hyperglycemia by Enhancing Nrf2-Dependent Antioxidant Defense-Mediated IRS-1/AKT/TBC1D1 Signaling and GLUT4 Expression in Skeletal Muscle.

Obesity caused lipotoxicity, which results in insulin resistance. We studied whether benzyl isothiocyanate (BITC) improved insulin resistance in muscle. BITC was studied in vivo in mice fed a high-fat diet (HFD) and in vitro in C2C12 myotubes treated with palmitic acid (PA). In C2C12 cells, BITC mitigated PA inhibition of glucose uptake and phosphorylation of IRS-1, AKT, and TBC1D1 in response to insulin. BITC upregulated the expression of HO-1, GSTP, and GCLM mRNA and protein as well as GSH contents, which suppressed oxidative damage. Knockdown of Nrf2 abrogated BITC enhancement of antioxidant defense and subsequently reversed BITC protection against PA-induced insulin resistance. Moreover, BITC upregulated the expression of GLUT4, PPARγ, and C/EBPα. In HFD-fed mice, plasma total cholesterol, nonesterified fatty acid, and glucose levels and HOMA-IR were dose-dependently decreased with 0.05 or 0.1% BITC administration. In gastrocnemius muscle, compared with the HFD group, BITC increased the phosphorylation of AKT and TBC1D1, GSH contents, and the expression of antioxidant enzymes as well as GLUT4. These results indicate that BITC ameliorates obesity-induced hyperglycemia by enhancing insulin sensitivity in muscle. This is partly attributed to its inhibition of lipotoxicity-induced oxidative insult and upregulation of GLUT4 expression.

A Class I Histone Deacetylase Inhibitor Attenuates Insulin Resistance and Inflammation in Palmitate-Treated C2C12 Myotubes and Muscle of HF/HFr Diet Mice.

Histone deacetylase (HDAC) inhibitors, which regulate gene expression by inhibiting the deacetylation of histones and nonhistone proteins, have been shown to exert a wide array of biological effects; these include anti-cancer, anti-obesity, and anti-diabetes effects, as well as cardiovascular-protective activity. However, the effects of class I HDAC inhibition on lipotoxicity in C2C12 myotubes and skeletal muscle tissue remain poorly understood. In this study, we investigated the molecular mechanism underlying the protective effect of class I HDAC inhibition under lipotoxic conditions, i.e., in palmitate (PA)-treated C2C12 myotubes and skeletal muscle tissue in high fat (HF)/high fructose (HFr) diet mice. PA treatment of C2C12 myotubes increased HDAC3 protein expression and impaired mitochondrial oxidation, resulting in increased mitochondrial ROS generation and an accumulation of intracellular triglycerides (TG). Prolonged exposure led to increased inflammatory cytokine expression and insulin resistance. In contrast, MS-275, a class I HDAC inhibitor, dramatically attenuated lipotoxicity, preventing PA-induced insulin resistance and inflammatory cytokine expression. Similar beneficial effects were also seen following HDAC3 knockdown. In addition, MS-275 increased the mRNA expression of peroxisome proliferator activator receptor γ-coactivator 1α (PGC1α) and mitochondrial transcription factor A (TFAM), which serve as transcriptional coactivators in the context of mitochondrial metabolism and biogenesis, and restored expression of peroxisome proliferator-activated receptor alpha (PPARα), medium-chain acyl-coenzyme A dehydrogenase (MCAD), enoyl-CoA hydratase, and 3-hydroxyacyl CoA dehydrogenase (EHHADH). In vivo, treatment of HF/HFr-fed mice with MS-275 ameliorated hyperglycemia, insulin resistance, stress signals, and TNF-α expression in skeletal muscle. Taken together, these results suggest that HDAC3 inhibition rather than HDAC1/2 inhibition by MS-275 protects against lipotoxicity in C2C12 myotubes and skeletal muscle, and may be effective for the treatment of obesity and insulin resistance.

The effects of glucose and fatty acids on CXCL10 expression in skeletal muscle cells.

Skeletal muscles produce secretory factors termed as myokines, which alter physiological functions of target tissues. We recently identified C-X-C chemokine ligand 10 (CXCL10) as a novel myokine, which is downregulated in response to exercise. In the present study, we investigated whether the nutritional changes affect CXCL10 expression in mouse skeletal muscle. Expression of CXCL10 was evaluated in mice fed a normal diet or a high fat diet for 10weeks. In animals fed on HFD, Cxcl10 expression was significantly induced in fast-twitched muscles, and was accompanied by increased blood glucose and free fatty acid levels. In vitro experiments using C2C12 myotubes suggested that the increased levels of glucose and palmitic acids directly enhanced CXCL10 expression. Interestingly, the effect of palmitic acids was attenuated by palmitoleic acids. Considering its potent angiostatic activity, induction of CXCL10 by nutritional changes may contribute to the impairment of microvascular networks in skeletal muscles.

PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD)

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3′UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5′luc3′ reporter cell line in which the 5′- and 3′-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.

Follicle stimulating hormone receptors are expressed in ewe’s reproductive and nonreproductive tissues

Follicle stimulating hormone receptors (FSHR) are present in granulosa cells, Sertoli cells, and other sites within the reproductive tract (uterine tube, uterus, placenta, and cervix). They are also present in many nonreproductive tissues (skin and adipose tissue, bone and cartilage, thyroid tissue, bladder and urethra, monocytes and endothelial cells within colon, pancreas, kidney, lung, liver, and in stomach cancer tissues of various species). However, the expression of FSHR outside of the reproductive tract in sheep, has not been reported. Objective was to determine FSHR expression in ovine ovary, uterus, liver, kidney, heart, and skeletal muscle. Tissue samples were collected at slaughter and FSHR expression was determined via immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections. Within the reproductive tract, FSHR were expressed in granulosa cells, luteal cells, and endometrial glandular epithelial cells. In liver, FSHR were expressed within hepatocytes and vascular endothelial cells. Expression of FSHR in cardiac myocytes was limited to cell membranes. Kidneys had FSHR expression on the renal tubular epithelial cells and the capillary endothelial cells. Lastly, in the skeletal muscle, FSHR were expressed in the endomysium. In human cancers, FSHR activation in nonreproductive tissues is involved in vascular remodeling, angiogenesis, cell proliferation, migration, and invasion. Effects of FSHR activation on ovine nonreproductive tissues remains unknown and warrants investigation into risks of frequent use of FSH agonists in sheep for nonseasonal estrous cycle manipulation.

Gene modification strategies using AO-mediated exon skipping and CRISPR/Cas9 as potential therapies for Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease affecting 1 in 5000 young males worldwide annually. Patients experience muscle weakness and loss of ambulation at an early age, with ∼75% reduced life expectancy. Recently developed genetic editing strategies aim to convert severe DMD phenotypes to a milder disease course. Among these, the antisense oligonucleotide (AO)-mediated exon skipping and the adeno-associated viral-delivered clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (adeno-associated viral (AAV)-delivered CRISPR/Cas9) gene editing have shown promising results in restoring dystrophin protein expression and functionality in skeletal and heart muscle in both animals and human cells in vivo and in vitro. However, therapeutic benefits currently remain unclear. The aim of this review is to compare the potential therapeutic benefits, efficacy, safety, and clinical progress of AO-mediated exon skipping and CRISPR/Cas9 gene-editing strategies. Both techniques have demonstrated therapeutic benefit and long-term efficacy in clinical trials. AAV-delivery of CRISPR/Cas9 may potentially correct disease-causing mutations following a single treatment compared to the required continuous AO/PMO-delivery of exon skipping drugs. The latter has the potential to increase the dystrophin expression in skeletal/heart muscle with sustained effects. However, therapeutic challenges including the need for optimised delivery must be overcome in to advance current clinical data.

Antidiabetic effect of a flavonoid-rich extract from Sophora alopecuroides L. in HFD- and STZ- induced diabetic mice through PKC/GLUT4 pathway and regulating PPARα and PPARγ expression

Headings ethnopharmacological relevanceSophora alopecuroides L. is a traditional ethnopharmacological plant, which is widely used in traditional Chinese medicine and Mongolian and Uighur medicine to ameliorate “thirst disease”. Aim of the studyThis study aimed to investigate the antidiabetic activities and mechanisms of a flavonoid-rich extract from Sophora alopecuroides L. (SA-FRE) both in vivo and vitro. Materials and methodsThe main six chemical constituents of SA-FRE were elucidated based on an off-line semi-preparative liquid chromatography nuclear magnetic resonance (LC-NMR) protocol. Myc-GLUT4-mOrange-L6 cell models and mouse model with diabetes induced by high-fat diet combined with STZ injection were respectively adopted to investigate the antidiabetic effects of SA-FRE both in vitro and vivo. ResultsIn vivo, 4-week treatment of SA-FRE ameliorated hyperglycemia, dyslipidemia, and insulin resistance in diabetic mice. Mechanically, SA-FRE regulated PPARα and PPARγ expression in white adipose tissue (WAT) and liver, thereby ameliorating dyslipidemia. Moreover, SA-FRE increased the phosphorylation of PKC and further stimulated the GLUT4 expression in WAT and skeletal muscle, thus increasing the glucose utilization in vivo. In vitro, 50 μg/mL SA-FRE increased GLUT4 translocation to about 1.91-fold and glucose uptake to 1.82-fold in L6-myotubes. SA-FRE treatment increased the GLUT4 expression at both gene and protein levels. Furthermore, only Gö6983, a PKC inhibitor, reversed the SA-FRE-induced GLUT4 translocation and expression at the gene and protein levels. ConclusionsGenerally, SA-FRE ameliorated hyperglycemia, dyslipidemia, and insulin resistance partly through activating PKC/GLUT4 pathway and regulating PPARα and PPARγ expression.