Kyoung-Han Kim

BACKGROUND: Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS: We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. RESULTS: Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS: Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in children and young adults. Abnormalities in several signaling pathways are implicated in the pathogenesis of HCM, but the role of the RAS-RAF-MEK-ERK MAPK pathway has been controversial. Noonan syndrome (NS) is one of several autosomal-dominant conditions known as RASopathies, which are caused by mutations in different components of this pathway. Germline mutations in RAF1 (which encodes the serine-threonine kinase RAF1) account for approximately 3%-5% of cases of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. To explore the pathogenesis of such mutations, we generated knockin mice expressing the NS-associated Raf1(L613V) mutation. Like NS patients, mice heterozygous for this mutation (referred to herein as L613V/+ mice) had short stature, craniofacial dysmorphia, and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. Agonist-evoked MEK-ERK activation was enhanced in multiple cell types, and postnatal MEK inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice. These data show that different NS genes have intrinsically distinct pathological effects, demonstrate that enhanced MEK-ERK activity is critical for causing HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to the treatment of RASopathies.

The widely expressed dipeptidyl peptidase-4 enzyme rapidly cleaves the gut hormone glucagon-like peptide-1 [GLP-1(7-36)amide] at the N terminus to generate GLP-1(9-36)amide. Both intact GLP-1(7-36)amide and GLP-1(9-36)amide exert cardioprotective actions in rodent hearts; however, the mechanisms underlying the actions of GLP-1(9-36)amide remain poorly understood. We used mass spectrometry of coronary effluents to demonstrate that isolated mouse hearts rapidly convert infused GLP-1(7-36)amide to GLP-1(9-36)amide. After ischemia-reperfusion (I/R) injury of isolated mouse hearts, administration of GLP-1(9-36)amide or exendin-4 improved functional recovery and reduced infarct size. The direct actions of these peptides were studied in cultured neonatal mouse cardiomyocytes. Both GLP-1(9-36)amide and exendin-4 increased levels of cAMP and phosphorylation of ERK1/2 and the phosphoinositide 3-kinase target protein kinase B/Akt. In I/R injury models in vitro, both peptides improved mouse cardiomyocyte viability and reduced lactate dehydrogenase release and caspase-3 activation. These effects were attenuated by inhibitors of ERK1/2 and phosphoinositide 3-kinase. Unexpectedly, the cardioprotective actions of GLP-1(9-36)amide were blocked by exendin(9-39) yet preserved in Glp1r(-/-) cardiomyocytes. Furthermore, GLP-1(9-36)amide, but not exendin-4, improved the survival of human aortic endothelial cells undergoing I/R injury, actions sensitive to the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME). In summary, our findings demonstrate separate actions for GLP-1(9-36)amide vs. the GLP-1R agonist exendin-4 and reveal the existence of a GLP-1(9-36)amide-responsive, exendin(9-39)-sensitive, cardioprotective signaling pathway distinct from that associated with the classical GLP-1 receptor.