Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/βKlotho complex from random peptide T7 phage display

AbstractFibroblast growth factor receptor-1c (FGFR1c)/βKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist

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

<div><p>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.</p></div

Effect of compound A (compA) on postprandial triglyceride (TG) excursion.

<p>Fasted C57BL/6J mice were given a liquid meal orally with intraperitoneal injection of Pluronic F-127 to inhibit plasma TG lipolysis. (A) Structure of compA. Plasma samples were collected at 0, 2, and 4 h after oral gavage of a liquid meal. (B) Time course of changes in plasma chylomicron TG (CM/TG) levels and (C) postprandial TG excursion of 3 or 10 mg/kg compA at 6 h after dosing. (D) Time course of changes in plasma TG levels and (E) postprandial TG excursion of 30 mg/kg compA at 16 h after dosing. n = 6 (B, C), and n = 7 (D, E). #: <i>P</i> < 0.025 vs. vehicle group by one-tailed Williams’ test. **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 vs. vehicle group by Student’s t-test.</p

Acute anorectic effect of compA under high-fat diet (HFD)-feeding conditions.

<p>Fasted mice were orally administrated vehicle or compA and were fed either HFD or normal chow (NC) for 2 h. (A) Amount of HFD intake by C57BL/6J mice. (B) Amount of HFD intake by vehicle or 10 mg/kg compA-treated MGAT2 KO mice and WT littermates. (C) Amount of NC or HFD intake by vehicle or 10 mg/kg compA-treated C57BL/6J mice. n = 5 (A, B) or n = 7 (C). #: <i>P</i> < 0.025 vs. vehicle group by one-tailed Williams’ test. %%%: <i>P</i> < 0.001 vs. vehicle-administrated WT mice by Student’s t-test. *: <i>P</i> < 0.05 vs. HFD-fed vehicle group by Student's t-test. N.S.: not significant.</p

Lipid metabolic flux analysis of compA in intestinal mucosa.

<p>Fasted C57BL/6J mice were orally administered 30 mg/kg compA. Two hours after dosing, they were orally administered a liquid meal containing the ²H-labeled oleoylglycerol. Glycerol-labeled monoacylglycerol (MG) was used in panels 2A and 2C, and fatty acid-labeled MG was used in panel 2B as a tracer. (A) Intestinal ²H-labeled MG, diacylglycerol (DG), and triglyceride (TG) levels. (B) Intestinal ²H-labeled fatty acid and acylcarnitine levels. (C) Intestinal ²H-labeled phospholipids levels. n = 5. **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 vs. vehicle group by Student's t-test. &: <i>P</i> < 0.05, &&: <i>P</i> < 0.01, &&&: <i>P</i> < 0.001 vs. vehicle group by Aspin–Welch test. (D) Scheme of MGAT2 inhibitor-mediated intestinal fat metabolism. (1) Most of the dietary MG and free fatty acids (FFAs) is used as a source of TG resynthesis in enterocytes. Some of the synthesized DGs are pooled as phospholipids. (2) MGAT2 inhibitor suppresses DG/TG resynthesis. Transiently stored MG is hydrolyzed into glycerol and FFAs. The activated FFA (acylcarnitine) is transported into mitochondria as a substrate of β-oxidation.</p

Intestinal remodeling by compA in high-fat diet-streptozotocin (HFD-STZ) mice.

<p>The small intestine of HFD-STZ mice was collected after 6-week administration of 30 mg/kg compA. Mean (A) tissue weight of the upper and (B) lower halves of the small intestine. Mean levels of gene expression for select enzymes involved in cholesterol metabolism in the (C) upper and (D) lower halves of the small intestine. n = 7 (for the weight of the upper intestine) or n = 8. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 vs. the vehicle-treated group by Student’s t-test. &: <i>P</i> < 0.05 vs. the vehicle-treated group by Aspin–Welch test.</p

Anti-obesity effects of compA in C57BL/6J mice fed a high-fat diet (HFD).

<p>Male C57BL/6J mice were fed HFD and orally administered vehicle, 30 mg/kg compA, or 10 mg/kg sibutramine for 5 weeks. (A) Time course of changes in body weight (BW). (B) Food intake. Mean (C) fat mass and (D) lean mass. n = 6. *: <i>P</i> < 0.05, ***: <i>P</i> < 0.001 vs. WT mice by two-tailed Dunnett’s test. The results of Fig 4D were analyzed using two-tailed Steel’s test. (E) GLUTag cells were treated with assay buffer containing free fatty acid (oleic acid), monoacylglycerol (2-monoacylglycerol), diacylglycerol (diolein), and triglyceride (triolein) for 2h. Secreted GLP-1 levels in the assay buffer was measured by GLP-1 ELISA. n = 4. #: P < 0.025 vs. control by one-tailed Williams test.</p