Inhibition of MGAT2 modulates fat-induced gut peptide release and fat intake in normal mice and ameliorates obesity and diabetes in ob/ob mice fed on a high-fat diet.

Monoacylglycerol O-acyltransferase 2 (MGAT2) is one of the key enzymes responsible for triglyceride (TG) re-synthesis in the small intestine. We have previously demonstrated that pharmacological inhibition of MGAT2 has beneficial effects on obesity and metabolic disorders in mice. Here, we further investigate the effects of MGAT2 inhibition on (a) fat-induced gut peptide release and fat intake in normal mice and (b) metabolic disorders in high-fat diet (HFD)-fed ob/ob mice, a model of severe obesity and type 2 diabetes mellitus, using an orally bioavailable MGAT2 inhibitor Compound B (CpdB). CpdB inhibited elevation of plasma TG in mice challenged with an oil-supplemented liquid meal. Oil challenge stimulated the secretion of two gut anorectic hormones (peptide tyrosine-tyrosine and glucagon-like peptide-1) into the bloodstream, and these responses were augmented in mice pretreated with CpdB. In a two-choice test using an HFD and a low-fat diet, CpdB selectively inhibited intake of the HFD in normal mice. Administration of CpdB to HFD-fed ob/ob mice for 5 weeks suppressed food intake and body weight gain and inhibited elevation of glycated hemoglobin. These results indicate that pharmacological MGAT2 inhibition modulates fat-induced gut peptide release and fat intake in normal mice and improves obesity and diabetes in HFD-fed ob/ob mice and thus may have potential for development into a treatment of obesity and its related metabolic diseases.

CDK5 Regulatory Subunit-Associated Protein 1-like 1 Negatively Regulates Adipocyte Differentiation through Activation of Wnt Signaling Pathway.

CDK5 Regulatory Subunit-Associated Protein 1-like 1 (CDKAL1) was identified as a susceptibility gene for type 2 diabetes and body mass index in genome-wide association studies. Although it was reported that CDKAL1 is a methylthiotransferase essential for tRNALys(UUU) and faithful translation of proinsulin generated in pancreatic ß cells, the role of CDKAL1 in adipocytes has not been understood well. In this study, we found that CDKAL1 is expressed in adipose tissue and its expression is increased during differentiation. Stable overexpression of CDKAL1, however, inhibited adipocyte differentiation of 3T3-L1 cells, whereas knockdown of CDKAL1 promoted differentiation. CDKAL1 increased protein levels of ß-catenin and its active unphosphorylated form in the nucleus, thereby promoting Wnt target gene expression, suggesting that CDKAL1 activated the Wnt/ß-catenin pathway-a well-characterized inhibitory regulator of adipocyte differentiation. Mutant experiments show that conserved cysteine residues of Fe-S clusters of CDKAL1 are essential for its anti-adipogenic action. Our results identify CDKAL1 as novel negative regulator of adipocyte differentiation and provide insights into the link between CDKAL1 and metabolic diseases such as type 2 diabetes and obesity.

Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia, Obesity, and Diabetes by Change in Intestinal Fat Utilization.

Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.

Optimization of a novel series of N-phenylindoline-5-sulfonamide-based acyl CoA:monoacylglycerol acyltransferase-2 inhibitors: Mitigation of CYP3A4 time-dependent inhibition and phototoxic liabilities.

Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8 nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts.

The AP-1 complex regulates intracellular localization of insulin receptor substrate 1, which is required for insulin-like growth factor I-dependent cell proliferation.

The activation of the insulin/insulin-like growth factor I (IGF-I) receptor and the subsequent tyrosine phosphorylation of insulin receptor substrates (IRSs) are key initial events in a variety of insulin/IGF bioactivities, including mitogenesis. It has been reported that IRS-1 associates with intracellular membrane compartments, and this localization is believed to be important for insulin/IGF signal transduction. However, the molecular mechanisms underlying IRS-1 localization remain unclear. Here we show that in L6 myoblasts, IRS-1 associates with µ1A of the ubiquitously expressed AP-1 complex, which packages cargo proteins into clathrin-coated vesicles derived from intracellular membranes. While wild-type IRS-1 was predominantly localized to vesicular structures, IRS-1 mutants lacking three YXXΦ motifs responsible for binding to µ1A were mislocalized to the mannose-6-phosphate receptor-positive structures, suggesting that AP-1-dependent transport to peripheral vesicles is inhibited in these mutants. Furthermore, deletion of AP-1 binding sites in IRS-1 impaired IGF-I-induced cell proliferation, accompanied by reduced tyrosine phosphorylation of IRS-1 and its association with phosphoinositide (PI) 3-kinase. These data demonstrate the importance of AP-1-dependent localization of IRS-1 in mediating IGF-I-stimulated signaling and maximum mitogenic response.

Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation.

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type-specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA-mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type-specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation.

Differential subcellular localization of insulin receptor substrates depends on C-terminal regions and importin beta.

Insulin receptor substrates (IRSs) play essential roles in signal transduction of insulin and insulin-like growth factors. Previously, we showed that IRS-3 is localized to the nucleus as well as the cytosol, while IRS-1 and 2 are mainly localized to the cytoplasm. In the present study, we found that importin beta directly interacts with IRS-3 and is able to mediate nuclear transport of IRS-3. Importin beta interacted with the pleckstrin homology domain, the phosphotyrosine binding domain and the C-terminal region of IRS-3; indeed all of these fragments exhibited predominant nuclear localization. By contrast, almost no interaction of importin beta with IRS-1 and -2 was observed, and their C-terminal regions displayed discrete spotty images in the cytosol. In addition, using chimeric proteins between IRS-1 and IRS-3, we revealed that the C-terminal regions are the main determinants of the differing subcellular localizations of IRS-1 and IRS-3.