Matching Items (4)

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Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

Description

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase,

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m[superscript 2]) and lean (BMI, 22±1 kg/m[superscript 2]) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h[superscript -1]) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

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Agent

Created

Date Created
  • 2016-08-17

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Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery

Description

Background
Obesity is a disease that is caused by genetic and environmental factors. However, epigenetic mechanisms of obesity are less well known. DNA methylation provides a mechanism whereby environmental factors

Background
Obesity is a disease that is caused by genetic and environmental factors. However, epigenetic mechanisms of obesity are less well known. DNA methylation provides a mechanism whereby environmental factors can influence gene transcription. The aim of our study was to investigate skeletal muscle DNA methylation of sorbin and SH3 domain containing 3 (SORBS3) with weight loss induced by Roux-en-Y gastric bypass (RYGB).
Results
Previously, we had shown increased methylation (5.0 to 24.4%) and decreased gene expression (fold change − 1.9) of SORBS3 with obesity (BMI > 30 kg/m[superscript 2]) compared to lean controls. In the present study, basal muscle biopsies were obtained from seven morbidly obese (BMI > 40 kg/m[superscript 2]) female subjects pre- and 3 months post-RYGB surgery, in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We identified 30 significantly altered promoter and untranslated region methylation sites in SORBS3 using reduced representation bisulfite sequencing (RRBS). Twenty-nine of these sites were decreased (− 5.6 to − 24.2%) post-RYGB compared to pre-RYGB. We confirmed the methylation in 2 (Chr.8:22,423,690 and Chr.8:22,423,702) of the 29 decreased SORBS3 sites using pyrosequencing. This decreased methylation was associated with an increase in SORBS3 gene expression (fold change + 1.7) post-surgery. In addition, we demonstrated that SORBS3 promoter methylation in vitro significantly alters reporter gene expression (P < 0.0001). Two of the SORBS3 methylation sites (Chr.8:22,423,111 and Chr.8:22,423,205) were strongly correlated with fasting plasma glucose levels (r = 0.9, P = 0.00009 and r = 0.8, P = 0.0010). Changes in SORBS3 gene expression post-surgery were correlated with obesity measures and fasting insulin levels (r = 0.5 to 0.8; P < 0.05).
Conclusions
These results demonstrate that SORBS3 methylation and gene expression are altered in obesity and restored to normal levels through weight loss induced by RYGB surgery.

Contributors

Created

Date Created
  • 2017-09-02

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Next Generation Sequencing Methylation Profiling in Skeletal Muscle Pre- and Post-Roux-en-Y Gastric Bypass Surgery

Description

Epigenetic mechanisms such as DNA methylation have been found to affect metabolic processes, which leads to conditions like type 2 diabetes and obesity. The aim of this project was to

Epigenetic mechanisms such as DNA methylation have been found to affect metabolic processes, which leads to conditions like type 2 diabetes and obesity. The aim of this project was to validate differentially methylated cytosines (DMCs) identified in skeletal muscle from seven obese, non-diabetic women pre- and 3 months post- Roux-en-Y gastric bypass surgery. DNA samples extracted from skeletal muscle were sent to the Mayo Genotyping Core to undergo reduced representation bisulfite sequencing (RRBS). Differentially methylated cytosines at chr14.105353824 of the gene CEP170B, chr19.16437949 of the KLF2 gene, chr7.130126082 of MEST, and chr15.62457572 of C2CD4B were captured from the RRBS analysis using MethylSig. Bisulfite sequencing PCR (BSP) was performed on all DMCs listed above which resulted in no significant changes in methylation post-surgery. It was concluded that an alternate, more precise method should be used for validation of the RRBS, such as pyrosequencing.

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Agent

Created

Date Created
  • 2016-05

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Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes

Description

Background
Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the

Background
Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity.
Results
Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m[superscript 2]) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m[superscript 2]) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change −1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530–22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity.
Conclusions
These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.

Contributors

Agent

Created

Date Created
  • 2016-07-18