Intramuscular Fat Accumulation Research Articles

Salvia hispanica L. (chia) seed improves redox state and reverts extracellular matrix collagen deposition in skeletal muscle of sucrose-rich diet-fed rats.

Skeletal muscle (SkM) is a plastic and dynamic tissue, essential in energy metabolism. Growing evidence suggests a close relationship between intramuscular fat accumulation, oxidative stress (OS), extracellular matrix (ECM) remodeling, and metabolic deregulation in SkM. Nowadays natural products emerge as promising alternatives for the treatment of metabolic disorders. We have previously shown that chia seed administration reverts SkM lipotoxicity and whole-body insulin resistant (IR) in sucrose-rich diet (SRD) fed rats. The purpose of the present study was to assess the involvement of OS and fibrosis in SkM metabolic impairment of insulin-resistant rats fed a long-term SRD and the effects of chia seed upon these mechanisms as therapeutic strategy. Results showed that insulin-resistant SRD-fed rats exhibited sarcopenia, increase in lipid peroxidation, altered redox state, and ECM remodeling-increased collagen deposition and lower activity of the metalloproteinase 2 (MMP-2) in SkM. Chia seed increased ferric ion reducing antioxidant power and glutathione reduced form levels, and the activities of glutathione peroxidase and glutathione reductase enzymes. Moreover, chia seed reversed fibrosis and restored the MMP-2 activity. This work reveals a participation of the OS and ECM remodeling in the metabolic alterations of SkM in our experimental model. Moreover, current data show novel properties of chia seed with the potential to attenuate SkM OS and fibrosis, hallmark of insulin-resistant muscle.

Intrasynovial autograft for reconstruction of chronic large rotator cuff tears in a rabbit model: biomechanical, computed tomography, and histological results

BackgroundRotator cuff (RC) tears are a common cause of shoulder dysfunction and pain, posing significant challenges for orthopedic surgeons. Grafts have been proposed as a solution to augment or bridge torn tendons, but optimal clinical outcomes are not always achieved due to poor graft integration, suboptimal mechanical properties, and immunological reactions. The aim of this study was to investigate the biomechanical, CT and histological results of RC reconstruction using an intrasynovial tendon autograft, in a chronic large tear subscapularis rabbit model.MethodsTwenty-six adult male Zealand white rabbits were used in this study. Large defects in the subscapularis tendons were produced bilaterally in 20 rabbits. After 6 weeks, secondary procedures were performed to the right shoulder of the rabbits, which were reconstructed with an intrasynovial interposition autograft (graft group). The left shoulder did not undergo any further treatment (defect group). The specimens were randomly divided into two equal time groups and underwent biomechanical testing, CT analysis, and histological evaluation at 6, and 12 weeks after reconstruction. In addition, 6 rabbits that were not operated, were used as a control group.ResultsAt 12 weeks post-repair, the graft group exhibited a significant increase in ultimate failure load compared to the defect group (p < 0.05). Furthermore, the 12-week graft group demonstrated comparable stiffness to that of the control group. CT analysis indicated no significant progression of intramuscular fat accumulation in both graft groups, in contrast to the 12-week defect group when compared to the control group. Finally, histological evaluation revealed a gradual integration of the graft with the host tissue at 12 weeks.ConclusionOur study suggests that intrasynovial flexor tendon autografts hold promise as an effective interposition graft for the reconstruction of chronic large RC tears, as they improve the biomechanical and biological properties of the repaired tendon. Nonetheless, further investigations in preclinical large animal models are warranted to validate and extrapolate these findings to human studies.

Subtherapeutic Kitasamycin Promoted Fat Accumulation in the Longissimus Dorsi Muscle in Growing-Finishing Pigs.

Kitasamycin (KM), a broad-spectrum macrolide antibiotic, has implications for growth performance and residue in animals and humans. This study aimed to explore the effects of different KM doses on intramuscular fat accumulation, cecal microflora, and short-chain fatty acids (SCFAs) using a growing-finishing pig model. Forty-two pigs were divided into three groups: control, subtherapeutic KM (50 mg/kg, KM50), and therapeutic KM (200 mg/kg, KM200) diets over 8 weeks. KM50 led to increased back fat thickness, fat content in the longissimus dorsi muscle (LM), and elevated plasma total cholesterol (TC) levels (p < 0.05), supported by upregulated lipid synthesis gene expression (Acc1, Fas, Scd1) (p < 0.05) in the LM. KM50 altered cecal microflora, reducing Lactobacillus spp. and Bifidobacterium spp. abundance, while increasing SCFA concentrations (acetic acid, propionic acid, total SCFAs) (p < 0.05). KM200 had minimal effects on intestinal weight and density, with increased apparent digestibility of nutrients. These findings highlight the dose-dependent impact of KM on intramuscular fat deposition. Subtherapeutic KM induced ectopic fat deposition, emphasizing potential risks in disease treatment for humans and animals.

Lower limb muscle MRI fat fraction is a responsive outcome measure in CMT X1, 1B and 2A.

With potential therapies for many forms of Charcot-Marie-Tooth disease (CMT), responsive outcome measures are urgently needed for clinical trials. Quantitative lower limb MRI demonstrated progressive calf intramuscular fat accumulation in the commonest form, CMT1A with large responsiveness. In this study, we evaluated the responsiveness and validity in the three other common forms, due to variants in GJB1 (CMTX1), MPZ (CMT1B) and MFN2 (CMT2A). 22 CMTX1, 21 CMT1B and 21 CMT2A patients and matched controls were assessed at a 1-year interval. Intramuscular fat fraction (FF) was evaluated using three-point Dixon MRI at thigh and calf level along with clinical measures including CMT examination score, clinical strength assessment, CMT-HI and plasma neurofilament light chain. All patient groups had elevated muscle fat fraction at thigh and calf levels, with highest thigh FF and atrophy in CMT2A. There was moderate correlation between calf muscle FF and clinical measures (CMTESv2 rho = 0.405; p = 0.001, ankle MRC strength rho = -0.481; p < 0.001). Significant annualised progression in calf muscle FF was seen in all patient groups (CMTX1 2.0 ± 2.0%, p < 0.001, CMT1B 1.6 ± 2.1% p = 0.004 and CMT2A 1.6 ± 2.1% p = 0.002). Greatest increase was seen in patients with 10-70% FF at baseline (calf 2.7 ± 2.3%, p < 0.0001 and thigh 1.7 ± 2.1%, p = 0.01). Our results confirm that calf muscle FF is highly responsive over 12 months in three additional common forms of CMT which together with CMT1A account for 90% of genetically confirmed cases. Calf muscle MRI FF should be a valuable outcome measure in upcoming CMT clinical trials.

Recent Advances in Nutraceuticals for the Treatment of Sarcopenic Obesity.

Sarcopenic obesity, low muscle mass, and high body fat are growing health concerns in the aging population. This review highlights the need for standardized criteria and explores nutraceuticals as potential therapeutic agents. Sarcopenic obesity is associated with insulin resistance, inflammation, hormonal changes, and reduced physical activity. These factors lead to impaired muscle activity, intramuscular fat accumulation, and reduced protein synthesis, resulting in muscle catabolism and increased fat mass. Myostatin and irisin are myokines that regulate muscle synthesis and energy expenditure, respectively. Nutritional supplementation with vitamin D and calcium is recommended for increasing muscle mass and reducing body fat content. Testosterone therapy decreases fat mass and improves muscle strength. Vitamin K, specifically menaquinone-4 (MK-4), improves mitochondrial function and reduces muscle damage. Irisin is a hormone secreted during exercise that enhances oxidative metabolism, prevents insulin resistance and obesity, and improves bone quality. Low-glycemic-index diets and green cardamom are potential methods for managing sarcopenic obesity. In conclusion, along with exercise and dietary support, nutraceuticals, such as vitamin D, calcium, vitamin K, and natural agonists of irisin or testosterone, can serve as promising future therapeutic alternatives.

Muscle proteome of crossbred cattle that received vitamin A at birth: impacts on meat quality traits

This study aimed to evaluate the skeletal muscle proteome of calves that received an intramuscular application of vitamin A at birth and its impacts on meat quality. Forty male crossbred calves were used from birth to feedlot finishing. On the day of birth, 20 calves were injected intramuscularly with a single dose of 300,000 IU of vitamin A and the other 20 animals received placebo (control). After weaning at 210 days, the animals of the two treatments were feedlot finished for 180 days. Longissimus muscle samples were collected for proteome analysis and meat quality assessment. There was a trend (P = 0.07) towards more tender meat in animals that received vitamin A compared to control (WBSF = 3.92 ± 0.16 kg vs. 4.23 ± 0.15). Meat color, pH and cooking loss were not affected (P > 0.05). Vitamin A treatment resulted in a more than 50% increase in intramuscular fat (IMF) content (P < 0.05) compared to control (4.10 ± 0.35% vs. 2.57 ± 0.27%). The intramuscular injection of vitamin A affected the post-mortem muscle proteome and the protein-protein interactions. A greater abundance of proteins involved in three main pathways were observed these being energy metabolism (GAPDH, ENO3, TPI1, CKM, and COX5A), muscle contraction (ACTB, ACTC1, ACTG1, ACTG2, ACTA1, ACTA2, ACTN1, ACTN2, ACTN3, TPM1, TPM2, TPM3, MYH1, PDLIM3, and TNNT3), protein binding, transport, and signaling (TUBA4A, VIM, TBA1B, and EEF1A2). A greater abundance of oxidative stress and cellular defense proteins (HSPA8 and DNAJC18) was specifically observed in the control treatment. Vitamin A upregulated key biological processes related to energy metabolism, which favored IMF accumulation during the finishing phase.

Muscle MRI in periodic paralysis shows myopathy is common and correlates with intramuscular fat accumulation.

The periodic paralyses are muscle channelopathies: hypokalemic periodic paralysis (CACNA1S and SCN4A variants), hyperkalemic periodic paralysis (SCN4A variants), and Andersen-Tawil syndrome (KCNJ2). Both episodic weakness and disabling fixed weakness can occur. Little literature exists on magnetic resonance imaging (MRI) in muscle channelopathies. We undertake muscle MRI across all subsets of periodic paralysis and correlate with clinical features. A total of 45 participants and eight healthy controls were enrolled and underwent T1-weighted and short-tau-inversion-recovery (STIR) MRI imaging of leg muscles. Muscles were scored using the modified Mercuri Scale. A total of 17 patients had CACNA1S variants, 16 SCN4A, and 12 KCNJ2. Thirty-one (69%) had weakness, and 9 (20%) required a gait-aid/wheelchair. A total of 78% of patients had intramuscular fat accumulation on MRI. Patients with SCN4A variants were most severely affected. In SCN4A, the anterior thigh and posterior calf were more affected, in contrast to the posterior thigh and posterior calf in KCNJ2. We identified a pattern of peri-tendinous STIR hyperintensity in nine patients. There were moderate correlations between Mercuri, STIR scores, and age. Intramuscular fat accumulation was seen in seven patients with no fixed weakness. We demonstrate a significant burden of disease in patients with periodic paralyses. MRI intramuscular fat accumulation may be helpful in detecting early muscle involvement, particularly in those without fixed weakness. Longitudinal studies are needed to assess the role of muscle MRI in quantifying disease progression over time and as a potential biomarker in clinical trials.

A single-cell atlas of bovine skeletal muscle reveals mechanisms regulating intramuscular adipogenesis and fibrogenesis.

Intramuscular fat (IMF) and intramuscular connective tissue (IMC) are often seen in human myopathies and are central to beef quality. The mechanisms regulating their accumulation remain poorly understood. Here, we explored the possibility of using beef cattle as a novel model for mechanistic studies of intramuscular adipogenesis and fibrogenesis. Skeletal muscle single-cell RNAseq was performed on three cattle breeds, including Wagyu (high IMF), Brahman (abundant IMC but scarce IMF), and Wagyu/Brahman cross. Sophisticated bioinformatics analyses, including clustering analysis, gene set enrichment analyses, gene regulatory network construction, RNA velocity, pseudotime analysis, and cell-cell communication analysis, were performed to elucidate heterogeneities and differentiation processes of individual cell types and differences between cattle breeds. Experiments were conducted to validate the function and specificity of identified key regulatory and marker genes. Integrated analysis with multiple published human and non-human primate datasets was performed to identify common mechanisms. A total of 32708 cells and 21 clusters were identified, including fibro/adipogenic progenitor (FAP) and other resident and infiltrating cell types. We identified an endomysial adipogenic FAP subpopulation enriched for COL4A1 and CFD (log2FC=3.19 and 1.92, respectively; P<0.0001) and a perimysial fibrogenic FAP subpopulation enriched for COL1A1 and POSTN (log2FC=1.83 and 0.87, respectively; P<0.0001), both of which were likely derived from an unspecified subpopulation. Further analysis revealed more progressed adipogenic programming of Wagyu FAPs and more advanced fibrogenic programming of Brahman FAPs. Mechanistically, NAB2 drives CFD expression, which in turn promotes adipogenesis. CFD expression in FAPs of young cattle before the onset of intramuscular adipogenesis was predictive of IMF contents in adulthood (R2 =0.885, P<0.01). Similar adipogenic and fibrogenic FAPs were identified in humans and monkeys. In aged humans with metabolic syndrome and progressed Duchenne muscular dystrophy (DMD) patients, increased CFD expression was observed (P<0.05 and P<0.0001, respectively), which was positively correlated with adipogenic marker expression, including ADIPOQ (R2 =0.303, P<0.01; and R2 =0.348, P<0.01, respectively). The specificity of Postn/POSTN as a fibrogenic FAP marker was validated using a lineage-tracing mouse line. POSTN expression was elevated in Brahman FAPs (P<0.0001) and DMD patients (P<0.01) but not in aged humans. Strong interactions between vascular cells and FAPs were also identified. Our study demonstrates the feasibility of beef cattle as a model for studying IMF and IMC. We illustrate the FAP programming during intramuscular adipogenesis and fibrogenesis and reveal the reliability of CFD as a predictor and biomarker of IMF accumulation in cattle and humans.

Distinct human skeletal muscle-derived CD90 progenitor subsets for myo-fibro-adipogenic disease modeling and treatment in multiplexed conditions.

Chronic muscle injuries, such as massive rotator cuff tears, are associated with progressive muscle wasting, fibrotic scarring, and intramuscular fat accumulation. While progenitor cell subsets are usually studied in culture conditions that drive either myogenic, fibrogenic, or adipogenic differentiation, it is still unknown how combined myo-fibro-adipogenic signals, which are expected to occur in vivo, modulate progenitor differentiation. We therefore evaluated the differentiation potential of retrospectively generated subsets of primary human muscle mesenchymal progenitors in multiplexed conditions in the presence or absence of 423F drug, a modulator of gp130 signaling. We identified a novel CD90+CD56- non-adipogenic progenitor subset that maintained a lack of adipogenic potential in single and multiplexed myo-fibro-adipogenic culture conditions. CD90-CD56- demarcated fibro-adipogenic progenitors (FAP) and CD56+CD90+ progenitors were typified as myogenic. These human muscle subsets exhibited varying degrees of intrinsically regulated differentiation in single and mixed induction cultures. Modulation of gp130 signaling via 423F drug mediated muscle progenitor differentiation in a dose-, induction-, and cell subset-dependent manner and markedly decreased fibro-adipogenesis of CD90-CD56- FAP. Conversely, 423F promoted myogenesis of CD56+CD90+ myogenic subset, indicated by increased myotube diameter and number of nuclei per myotube. 423F treatment eliminated FAP-derived mature adipocytes from mixed adipocytes-FAP cultures but did not modify the growth of non-differentiated FAP in these cultures. Collectively, these data demonstrate that capability of myogenic, fibrogenic, or adipogenic differentiation is largely dependent on the intrinsic features of cultured subsets, and that the degree of lineage differentiation varies when signals are multiplexed. Moreover, our tests performed in primary human muscle cultures reveal and confirm the potential triple-therapeutic effects of 423F drug which simultaneously attenuates degenerative fibrosis, fat accumulation and promotes myo-regeneration.

Long Non-Coding RNA BNIP3 Inhibited the Proliferation of Bovine Intramuscular Preadipocytes via Cell Cycle

The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5' RACE and 3' RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5'RACE, and 464 bp of 3'RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2'- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality.

Effect of electromyostimulation training on intramuscular fat accumulation determined by ultrasonography in older adults.

PurposeElectromyostimulation (EMS) induces a short-term change in muscle metabolism, and EMS training induces long-term improvements of muscle atrophy and function. However, the effects of EMS training on intramuscular fat in older adults are still poorly known. The purpose of this study was to examine whether the intramuscular fat index and biochemical parameters change with EMS training of the quadriceps femoris muscles in older adults.MethodsNineteen non-obese older men and women performed EMS training of the quadriceps femoris for 12 weeks (3 times/week; single session for 30 min). The intramuscular fat content index was estimated by echo intensity of the vastus lateralis and rectus femoris muscles on ultrasonography, and muscle thickness was also measured. Muscle strength was assessed as the maximal voluntary contraction during isometric knee extension. Echo intensity, muscle thickness, and muscle strength were measured before and after EMS training. A rested/fasting blood samples were collected before and after EMS training for measuring plasma glucose, insulin, free fatty acid, triglyceride, and interleukin-6 concentrations. To examine the acute effect of a single-EMS session on biochemical parameters, blood samples were taken before and after the EMS session.ResultsEMS training did not significantly change echo intensity in muscles, muscle thickness, muscle strength, or biochemical parameters. Regarding the acute effect on blood lipid concentrations, a single-EMS session increased free fatty acid and glucose concentrations.ConclusionEMS sessions had an acute effect of increasing free fatty acid and glucose concentrations, but EMS training intervention did not improve intramuscular fat content.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00421-022-05074-3.

MRI correlates of motoneuron loss in SMA

BackgroundMagnetic resonance imaging (MRI) is currently explored as supplemental tool to monitor disease progression and treatment response in various neuromuscular disorders. We here assessed the utility of a multi-parametric magnetic resonance imaging (MRI) protocol including quantitative water T2 mapping, Dixon-based proton density fat fraction (PDFF) estimation and diffusion tensor imaging (DTI) to detect loss of spinal motor neurons and subsequent muscle damage in adult SMA patients.MethodsSixteen SMA patients and 13 age-matched controls were enrolled in this prospective, longitudinal study. All participants underwent MRI imaging including measurements of Dixon-based PDFF and DTI of the sciatic nerve. SMA patients furthermore underwent measurements of muscle water T2 (T2w) of the biceps femoris muscle (BFM) and quadriceps femoris muscle (QFM). Ten participants returned for a second scan six months later. MRI parameter were correlated with clinical data. All patients were on nusinersen treatment.ResultsThere were significantly higher intramuscular fat fractions in the BFM and QFM of SMA patients compared to healthy controls at baseline and after 6 months. Furthermore, T2 values significantly correlated positively with intramuscular fat fractions. The Hammersmith functional motor scale significantly correlated with the QFM’s intramuscular fat fractions. DTI scans of the sciatic nerve were not significantly different between the two groups.ConclusionThis study demonstrates that, water T2 mapping and Dixon-based PDFF estimation may distinguish between adult SMA patients and controls, due to massive intramuscular fat accumulation in SMA. More extensive long-term studies are warranted to further evaluate these two modalities as surrogate markers in SMA patients during treatment.

Effect Of Electromyostimulation Training On Intramuscular Fat Accumulation And Blood Lipids In Older Adults

PURPOSE: Aging induces muscle atrophy, decline of muscular metabolism, and excessive intramuscular fat accumulation. Aerobic and anerobic training enhance glycolytic or oxidative enzymes, and improve muscle size in older adults. However, these trainings are assumed difficult for older adult with disfunction of lower leg. Electromyostimulation (EMS) is commonly used in clinical settings to improve the muscular functions in older adults. The purpose of this study was to examine the effects of EMS training on intramuscular fat content and blood lipids of quadriceps femoris muscles in older adults. METHODS: Nineteen physically active, non-obese older men and women (mean age 71.5 ± 5.4 years) were performed EMS training of the quadriceps femoris muscle for 12 weeks (3 times/week). Single EMS session consisted of 7 s contraction/relaxation cycle for 30 min. Intramuscular fat content was estimated by echo intensity of vastus lateralis using ultrasonography. Free fatty acids (FFA) were measured from blood samples. Echo intensity and FFA were measured before and after EMS training. To determine acute effect of single EMS session, FFA was measured before and after the EMS session. RESULTS: EMS training did not significant change echo intensity (pre 63.7 ± 11.1 a.u.; post 62.4 ± 9.0 a.u.) and FFA (pre 523.3 ± 187.0 μEq/L; post 503.5 ± 223.0 μEq/L). Regarding acute effect on blood lipid, single EMS session increased FFA (pre 523.3 ± 187.0 μEq/L; post 606.9 ± 185.2μEq/L, p < 0.01). CONCLUSIONS: Acute effect of EMS session on blood lipids was obtained, but EMS training intervention for 12 weeks did not improve intramuscular fat content of quadriceps femoris muscle and blood lipids. Supported by a Grant in Aid (KAKENHI 18 K17764).

Accelerate Clinical Trials in Charcot-Marie-Tooth Disease (ACT-CMT): A Protocol to Address Clinical Trial Readiness in CMT1A.

With therapeutic trials on the horizon for Charcot-Marie-Tooth type 1A (CMT1A), reliable, valid, and responsive clinical outcome assessments and biomarkers are essential. Accelerate Clinical Trials in CMT (ACT-CMT) is an international study designed to address important gaps in CMT1A clinical trial readiness including the lack of a validated, responsive functional outcome measure for adults, and a lack of validated biomarkers for multicenter application in clinical trials in CMT1A. The primary aims of ACT-CMT include validation of the Charcot-Marie-Tooth Functional Outcome Measure, magnetic resonance imaging of intramuscular fat accumulation as a lower limb motor biomarker, and in-vivo reflectance confocal microscopy of Meissner corpuscle sensory receptor density, a sensory biomarker. Initial studies have indicated that these measures are feasible, reliable and valid. A large prospective, multi-site study is necessary to fully validate and examine the responsiveness of these outcome measures in relation to existing outcomes for use in future clinical trials involving individuals with CMT1A. Two hundred 15 adults with CMT1A are being recruited to participate in this prospective, international, multi-center study. Serial assessments, up to 3 years, are performed and include the CMT-FOM, CMT Exam Score-Rasch, Overall Neuropathy Limitations Scale, CMT-Health Index, as well as nerve conduction studies, and magnetic resonance imaging and Meissner corpuscle biomarkers. Correlations using baseline data will be examined for validity. Longitudinal analyses will document the changes in function, intramuscular fat accumulation, Meissner corpuscle sensory receptor density. Lastly, we will use anchor-based and other statistical methods to determine the minimally clinically important change for these clinical outcome assessments and biomarkers in CMT1A. Reliable, and responsive clinical outcome assessments of function and disease progression biomarkers are urgently needed for application in early and late phase clinical trials in CMT1A. The ACT-CMT study protocol will address this need through the prospective, longitudinal, multicenter examination in unprecedented detail of novel and existing clinical outcome assessments and motor and sensory biomarkers, and enhance international clinical trial infrastructure, training and preparedness for future therapeutic trials in CMT and related neuropathies.

The Effects of Maternal Intake of EPA and DHA Enriched Diet During Pregnancy and Lactation on Offspring's Muscle Development and Energy Homeostasis.

EPA and DHA are n-3 long-chain polyunsaturated fatty acids with a diversity of health benefits on offspring. The objective of this study was to test the in vivo effect of maternal ingestion of EPA and DHA on fetal and offspring muscle development and energy balance. Two groups of female C57BL/6 mice were fed EPA and DHA enriched diet (FA) and diet devoid of EPA and DHA (CON) respectively throughout the entire period of gestation and lactation. Embryos at E13 and offspring at age of D1 and D21 were selected for sample collection and processing. No change in birth number and body weight were observed between groups at D1 and D21. Transient increase in the expression levels of myogenesis regulating genes was detected at D1 (p < 0.05) in FA group. Most of the expression of muscle protein synthesis regulating genes were comparable (p > 0.05) between FA and CON groups at D1 and D21. The significant increase in MHC4, and IGF-1 was not linked to increased muscle mass. A persistent increase in ISR expression (p < 0.05) but not in GLUT-4 (p > 0.05) was detected in offspring. Up-regulation of adipogenesis regulating genes was accompanied by increasing intramuscular fat accumulation in the offspring of FA group. Considerable increase in transcripts of genes regulating lipid catabolism and thermogenesis in liver (p < 0.05) was noticed in FA group at D21; whereas, only the levels of carnitine palmitoyl transferase 1A (Cpt1α) and Enoyl-CoA Hydratase And 3-Hydroxyacyl CoA Dehydrogenase (Ehhadh) increased at D1. Similarly, genes regulating lipolysis were highly expressed at D21 in FA group. EPA and DHA treatment promoted BAT development and activity by increasing the expression of BAT signature genes (p < 0.05). Also, maternal intake of EPA and DHA enriched diet enhanced browning of sWAT. Taken together, maternal ingestion of EPA/DHA may be suggested as a therapeutic option to improve body composition and counteract childhood obesity- related metabolic disorders and confer lifelong positive metabolic impact on offspring.

Growth and Meat Quality of Grass Carp (Ctenopharyngodon idellus) Responded to Dietary Protein (Soybean Meal) Level Through the Muscle Metabolism and Gene Expression of Myosin Heavy Chains.

The aim of this study was to investigate the effect of dietary protein level (soybean meal) on growth performance, flesh quality of grass carp, and the related molecular mechanisms. The results showed that appropriate dietary protein levels improved the growth performance, hardness, and pH of muscle while decreasing muscle crude lipid content and cooking loss and altering the antioxidant capacity and metabolic enzymes activities. In addition, appropriate dietary protein promoted the gene expression of myhc-1, myhc-4, myf5, myod, myog, and fgf6a, whereas inhibited that of myhc-7, myhc-2, mrf4, and mstn. Transcriptome profiling of muscle revealed that the flesh quality-specific differences were related to tight junctions and intramuscular fat (IMF) accumulation. GSEA showed that fatty acid metabolism and oxidative phosphorylation were downregulated in SM5 compared with SM1. To conclude, appropriate protein levels improved the growth and flesh quality by regulating muscle antioxidant capacity and gene expression of myhcs and fat metabolism-related signaling molecules.

Mfsd2a Promotes the Proliferation, Migration, Differentiation and Adipogenesis of Chicken Intramuscular Preadipocytes

Intramuscular fat (IMF) content is a crucial parameter for estimating meat quality. Growing evidence indicates that gene regulation plays an important role in IMF deposition. This study aimed to determine the function of Mfsd2a in chicken intramuscular preadipocytes. In the present study, high Mfsd2a mRNA levels were observed in the liver and adipose tissues of broilers. Subsequently, we synthesized small interfering RNAs to silence the expression of Mfsd2a in chicken intramuscular preadipocytes. The following results suggested that CDK2, PCNA, CCND1, CCND2 and MKI67 were inhibited, with CCK-8 and EdU assays revealing that cell proliferation was inhibited. Scratch test showed that cell migration ratios were declined. We also found that Mfsd2a silencing decreased the mRNA levels of PPARγ, RXRG and their target genes. The similar results were found in some key genes that contribute to lipid synthesis, including C/EBPα, C/EBPβ, FABP4, FASN, ACACA and ACSL1. Finally, Oil red O staining showed that IMF accumulation was blocked after Mfsd2a silencing. In conclusion, our results implied that Mfsd2a promotes the proliferation and migration of chicken intramuscular preadipocytes, as well as the differentiation and adipogenesis through PPARγ signaling pathway, which may provide a potential target to improve chicken meat quality.

PSIV-6 Differential gene expression of fibro/adipogenic progenitors between Wagyu and Brahman cattle: A possible contribution to their different meat quality

Abstract The amount of intramuscular fat directly influences the meat quality. However, significant differences in the ability to accumulate intramuscular fat are present among different beef cattle breeds. While Wagyu, a cattle breed that originated from Japan, is renowned for abundant intramuscular fat, Brahman cattle generally have very little intramuscular fat accumulation and produce tougher meat. We identified that bovine intramuscular fat is derived from a group of bipotent progenitor cells named fibro/adipogenic progenitors (FAPs) which also give rise to fibroblasts. Thus, the variation in intramuscular fat development between Wagyu and Brahman is likely attributed to the difference in FAPs between these two breeds. In order to understand the gene expression difference between FAPs of the two breeds, single-cell RNA-seq was performed using total single-nucleated cells isolated from the longissimus muscle of young purebred Wagyu, purebred Brahman, and Wagyu-Brahman cross cattle. FAPs constitute the largest single-nucleated cell population in both Wagyu and Brahman skeletal muscle. Multiple subpopulations of FAPs with different gene expression profiles were identified, suggesting that FAP is a heterogeneous population. A unique FAP cluster expressing lower levels of fibrillar collagen and extracellular remodeling enzyme genes but higher levels of select proadipogenic genes was identified exclusively in Wagyu skeletal muscle, which likely contributes to the robust intramuscular adipogenic efficiency of Wagyu FAPs. In conclusion, the difference in the cellular composition and gene expression of FAPs between Wagyu and Brahman cattle likely contribute to their distinct meat quality.

Dietary calcium supplementation promotes the accumulation of intramuscular fat

BackgroundIn the livestock industry, intramuscular fat content is a key factor affecting meat quality. Many studies have shown that dietary calcium supplementation is closely related to lipid metabolism. However, few studies have examined the relationship between dietary calcium supplementation and intramuscular fat accumulation.MethodsHere, we used C2C12 cells, C57BL/6 mice (n = 8) and three-way cross-breeding pigs (Duroc×Landrace×Large white) (n = 10) to study the effect of calcium addition on intramuscular fat accumulation. In vitro, we used calcium chloride to adjust the calcium levels in the medium (2 mmol/L or 3 mmol/L). Then we measured various indicators. In vivo, calcium carbonate was used to regulate calcium levels in feeds (Mice: 0.5% calcium or 1.2% calcium) (Pigs: 0.9% calcium or 1.5% calcium). Then we tested the mice gastrocnemius muscle triglyceride content, pig longissimus dorsi muscle meat quality and lipidomics.ResultsIn vitro, calcium addition (3 mmol/L) had no significant effect on cell proliferation, but promoted the differentiation of C2C12 cells into slow-twitch fibers. Calcium supplementation increased triglyceride accumulation in C2C12 cells. Calcium addition increased the number of mitochondria and also increased the calcium level in the mitochondria and reduced the of key enzymes activity involved in β-oxidation such as acyl-coenzyme A dehydrogenase. Decreasing mitochondrial calcium level can alleviate lipid accumulation induced by calcium addition. In addition, calcium addition also reduced the glycolytic capacity and glycolytic conversion rate of C2C12 cells. In vivo, dietary calcium supplementation (1.2%) promoted the accumulation of triglycerides in the gastrocnemius muscle of mice. Dietary calcium supplementation (1.5%) had no effect on pig weight, but significantly improved the flesh color of the longissimus dorsi muscle, reduced the backfat thickness and increased intramuscular fat content in pigs. Besides, calcium addition had no effect on longissimus dorsi pH, electrical conductivity and shear force.ConclusionsThese results suggest that calcium addition promotes intramuscular fat accumulation by inhibiting the oxidation of fatty acids. These findings provide a new tool for increasing intramuscular fat content and an economical strategy for improving meat quality.

Effects of Moringa oleifera and Brosimum alicastrum partial feed substitution in intramuscular fat and adipose tissues and on the expression of lipogenic genes of Mexican hairless pigs

The present study evaluated the effects of the inclusión of ground Moringa oleifera and Brosimum alicastrum leaf meal in the diet of Mexican hairless pigs (MHP) on the amount of intramuscular fat, subcutaneous fat, leg muscle (Biceps femoris) fat, loin muscle (Longissimus dorsi) fat, leg and back fat, and the expression of lipid metabolism genes. Hairless pigs are reared in the Mexican tropics and are characterised by their body and intramuscular fat accumulation. Eighteen male pigs fed for 82 d were randomly allotted to three experimental isoenergetic and isoproteic diets, where M. oleifera or B. alicastrum (six pigs per diet) replaced wheat bran. The diets used were a control diet, a diet with 10% M. oleifera leaf meal, and a diet with 10% ground B. alicastrum leaf meal. The M. oleifera diet decreased (P<0.05) the fat ratio in the Longissimus dorsi muscles, back fat, rib fat, total carcass fat, and the carcass fat: meat ratio. The B. alicastrum diet only decreased fat in the Biceps femoris muscle, back fat, and rib fat. Moringa oleifera and B. alicastrum diets also promoted the overexpression of mRNA from the stearoyl-CoA desaturase (SCD), fatty acid synthase (FASN), acetyl-CoA carboxylase alpha (ACACA), sterol regulatory element-binding protein 1 (SREBP1) and acyl carrier protein (ACP) lipogenic genes in the Biceps femoris muscle and leg fat (P<0.001). In addition, lower ACACA and SREBP1 mRNA expression in the Longissimus dorsi muscle and back fat (P<0.001) were related to the lower amount of fat in pigs fed M. oleifera and B. alicastrum. The inclusion of Moringa oleifera and Brosimum alicastrum meals 10% in the diet of the pig MHP reduces fat, this is an important finding because fat is abundant in this type of pig.