Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3′splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexon

TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes

Introduction Non-coding genetic variation at TCF7L2 is the strongest genetic determinant of type 2 diabetes (T2D) risk in humans. TCF7L2 encodes a transcription factor mediating the nuclear effects of WNT signaling in adipose tissue (AT). In vivo studies in transgenic mice have highlighted important roles for TCF7L2 in adipose tissue biology and systemic metabolism. Objective To map the expression of TCF7L2 in human AT, examine its role in human adipose cell biology in vitro, and investigate the effects of the fine-mapped T2D-risk allele at rs7903146 on AT morphology and TCF7L2 expression. Methods Ex vivo gene expression studies of TCF7L2 in whole and fractionated human AT. In vitro TCF7L2 gain- and/or loss-of-function studies in primary and immortalized human adipose progenitor cells (APCs) and mature adipocytes (mADs). AT phenotyping of rs7903146 T2D-risk variant carriers and matched controls. Results Adipose progenitors (APs) exhibited the highest TCF7L2 mRNA abundance compared to mature adipocytes and adipose-derived endothelial cells. Obesity was associated with reduced TCF7L2 transcript levels in whole subcutaneous abdominal AT but paradoxically increased expression in APs. In functional studies, TCF7L2 knockdown (KD) in abdominal APs led to dose-dependent activation of WNT/β-catenin signaling, impaired proliferation and dose-dependent effects on adipogenesis. Whilst partial KD enhanced adipocyte differentiation, near-total KD impaired lipid accumulation and adipogenic gene expression. Over-expression of TCF7L2 accelerated adipogenesis. In contrast, TCF7L2-KD in gluteal APs dose-dependently enhanced lipid accumulation. Transcriptome-wide profiling revealed that TCF7L2 might modulate multiple aspects of AP biology including extracellular matrix secretion, immune signaling and apoptosis. The T2D-risk allele at rs7903146 was associated with reduced AP TCF7L2 expression and enhanced AT insulin sensitivity. Conclusions TCF7L2 plays a complex role in AP biology and has both dose- and depot-dependent effects on adipogenesis. In addition to regulating pancreatic insulin secretion, genetic variation at TCF7L2 might also influence T2D risk by modulating AP function

Dataset for: Intact regulation of muscle expression and circulating levels of myokines in response to exercise in patients with type 2 diabetes

Aim: Regular exercise plays an important role in the prevention and treatment of type 2 diabetes (T2D). The synthesis and secretion of myokines in response to contraction may contribute to the beneficial metabolic effects of exercise. However, some exercise-induced responses may be attenuated in T2D. Here, we investigated whether the effect of acute exercise on selected myokines are impaired in T2D. Methods: Skeletal muscle biopsies and blood samples were obtained from 13 men with T2D and 14 weight-matched, glucose-tolerant men before, immediately after and 3-h after acute exercise (60 min cycling) to examine muscle expression and plasma/serum levels of selected myokines. Results: One-hour of exercise increased muscle expression of IL6, FGF21, ANGPTL4, CHI3L1, CTGF and CYR61 of which FGF21, ANGPTL4 and CHI3L1 increased further 3-h into recovery, whereas expression of IL6, CYR61 and CTGF returned to baseline levels. There was no immediate effect of exercise on IL15 expression, but it decreased 3-h into recovery. Plasma IL-6 increased robustly, whereas circulating levels of FGF21, ANGPTL4, IL-15 and CHI3L1 increased only modestly in response to exercise. All returned toward baseline levels 3-h into recovery except for plasma ANGPTL4, which increased further. No significant differences in these responses to exercise were observed between the groups. Conclusion: Our results demonstrate that muscle expression and circulating levels of selected known and putative myokines were equally regulated by acute exercise in patients with T2D and weight-matched controls. This suggests that the potential beneficial metabolic effects of these myokines are not impaired in patients with T2D