Genomic Characterization of Metformin Hepatic Response

Abstract

Metformin is used as a first-line therapy for type 2 diabetes (T2D) and prescribed for numerous other diseases. However, its mechanism of action in the liver has yet to be characterized in a systematic manner. To comprehensively identify genes and regulatory elements associated with metformin treatment, we carried out RNA-seq and ChIP-seq (H3K27ac, H3K27me3) on primary human hepatocytes from the same donor treated with vehicle control, metformin or metformin and compound C, an AMP-activated protein kinase (AMPK) inhibitor (allowing to identify AMPK-independent pathways). We identified thousands of metformin responsive AMPK-dependent and AMPK-independent differentially expressed genes and regulatory elements. We functionally validated several elements for metformin-induced promoter and enhancer activity. These include an enhancer in an ataxia telangiectasia mutated (ATM) intron that has SNPs in linkage disequilibrium with a metformin treatment response GWAS lead SNP (rs11212617) that showed increased enhancer activity for the associated haplotype. Expression quantitative trait locus (eQTL) liver analysis and CRISPR activation suggest that this enhancer could be regulating ATM, which has a known role in AMPK activation, and potentially also EXPH5 and DDX10, its neighboring genes. Using ChIP-seq and siRNA knockdown, we further show that activating transcription factor 3 (ATF3), our top metformin upregulated AMPK-dependent gene, could have an important role in gluconeogenesis repression. Our findings provide a genome-wide representation of metformin hepatic response, highlight important sequences that could be associated with interindividual variability in glycemic response to metformin and identify novel T2D treatment candidates.

Pharmacogenetics of type 2 diabetes mellitus, the route toward tailored medicine

“…It is possible that the latter population studies failed to replicate the original findings because of inadequate statistical power or pharmacogenetic MET‐response interaction with rs11212617 may diverge at different stages of impaired glucose metabolism. In addition to this, more recently, enhancer assays of MET‐activated epigenetic sites showed increased enhancer activity in the ATM intron containing SNPs in LD with rs11212617 . Interestingly, the LD block encompasses several genes including EXPH5 (Exophilin 5, involved in exosome secretion and intracellular vesicle trafficking) and DDX10 (DEAD‐box helicase 10), which resulted upregulated by MET in vitro, while ATM expression was unchanged …”

“…In addition to this, more recently, enhancer assays of MET-activated epigenetic sites showed increased enhancer activity in the ATM intron containing SNPs in LD with rs11212617. 9 Interestingly, the LD block encompasses several genes including EXPH5 (Exophilin 5, involved in exosome secretion and intracellular vesicle trafficking) and DDX10 (DEAD-box helicase 10), which resulted upregulated by MET in vitro, while ATM expression was unchanged. 9 In a meta-analysis performed by the Metformin Genetics (MetGen) Consortium comprising 10 557 participants of European ancestry, a genome-wide statistically significant association was found for the intronic SNP rs8192675, located within SLC2A2, which encodes the GLUT2 glucose transporter.…”

“…9 Interestingly, the LD block encompasses several genes including EXPH5 (Exophilin 5, involved in exosome secretion and intracellular vesicle trafficking) and DDX10 (DEAD-box helicase 10), which resulted upregulated by MET in vitro, while ATM expression was unchanged. 9 In a meta-analysis performed by the Metformin Genetics (MetGen) Consortium comprising 10 557 participants of European ancestry, a genome-wide statistically significant association was found for the intronic SNP rs8192675, located within SLC2A2, which encodes the GLUT2 glucose transporter. 10 Each copy of rs8192675 C allele was associated with a greater MET-induced HbA1c reduction of 0·17% (P = 6·6 × 10 −14 ), which was attenuated after adjusting for baseline HbA1c (reduction of 0·07%; P = 2 × 10 −8 ).…”

Precision medicine in diabetes: an opportunity for clinical translation

“…Expression quantitative trait locus analysis and CRISPR-Cas9 activation suggest that this enhancer haplotype could be regulating ATM in the liver and activating transcription factor 3, leading to gluconeogenesis repression. 110 A noncoding variant in SLC2A2 (encoding the facilitated glucose transporter GLUT2) represents another identified genome-wide signal for metformin response, and it was associated with reduction in hemoglobin A1c in 10,577 participants of European ancestry. 111 This regulatory variant influences GLUT2 expression in the human liver, identifying hepatic GLUT2 as an effector of metformin action.…”

CRISPR/Cas9: at the cutting edge of hepatology

“…This technology has been used to characterise the metformin response in the liver. In one study, metformin responsive enhancers were identified, and targeted with a dCas9-VP64 fusion protein to measure the transcriptional response in the Huh7 cell line 57. Activation of genes in this region via the CRISPR-guided VP64 transactivator showed significantly increased levels of ataxia telangiectasia mutated ( ATM ), exophilin 5, DEAD-Box Helicase 10 mRNA, suggesting a possible role for these proteins in the mechanism of action of metformin.…”