Endocrine Abstracts

Background: GH secretion and action change with age in adult human subjects, and this play an important role in substrate metabolism in aging subjects. Targeted disruption of the GH receptor in mice extends longevity, and this is associated with decreased expression of apoptosis-related genes including caspase-9 (CASP9) in skeletal muscle.

Aim: To study DNA methylation of putative GH target genes in skeletal muscle of adult male subjects in relation to body composition, physical fitness, serum IGF1 levels and in vivo GH signaling.

Subjects and methods: In this pilot study we included 12 healthy adult subjects (ten males and two females) subdivided into a ‘young’ group (n=5) and ‘old’ group (n=7) (mean±S.E.M. age: 24±2.7 vs 25.6±2.0). Total and phosphorylated STAT5b were measured by WB at t=30 min, and IGF1 and SOCS/CIS gene expression at t=120 min. by RT-PCR in skeletal muscle following an i.v. GH bolus. In the GH-unstimulated state DNA methylation in muscle tissue was measured by Infinium HumanMethylation450 BeadChip technology (Illumina, CA, USA). We did a hypothesis-free analysis of the methylation level of each CpG locus in the 450k array comparing ‘old’ (n=7) vs ‘young’ (n=5) We focused on CpG loci located within 1500 and 200 bases upstream transcription start sites (TSS200 and TSS1500) increasing the possibility that the CpG site is regulating the gene transcription.

Results: Following the GH bolus significant STAT5b phosphorylation and gene expression of IGF1 and SOCS/CISH were recorded in all subjects with no significant difference between the two groups. Taking the false discovery rate into account the CASP9 gene methylation was statistically significant with an adjusted P value <0.05, while the mean beta-difference was only 0.011 being fully unmethylated in the ‘young’ group.

Conclusion: i) Significant activation of GH signaling in terms of pSTAT5b and expression of canonical target genes are detectable in vivo in skeletal muscle of adult human male subjects without a distinct impact of age, ii) methylation of the promotor region of CASP9 were increased in older subjects indicative of epigenetic modification; and iii) this in vivo model holds promises to disclose hitherto unrecognized regulatory mechanism of GH activity.