Antihypertrophic Effects of Small Molecules that Maintain Mitochondrial ATP Levels Under Hypoxia

Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin β, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction

GPR40 full agonism exerts feeding suppression and weight loss through afferent vagal nerve.

GPR40/FFAR1 is a Gq protein-coupled receptor expressed in pancreatic β cells and enteroendocrine cells, and mediates insulin and incretin secretion to regulate feeding behavior. Several GPR40 full agonists have been reported to reduce food intake in rodents by regulating gut hormone secretion in addition to their potent glucose-lowering effects; however, detailed mechanisms of feeding suppression are still unknown. In the present study, we characterized T-3601386, a novel compound with potent full agonistic activity for GPR40, by using in vitro Ca2+ mobilization assay in Chinese hamster ovary (CHO) cells expressing FFAR1 and in vivo hormone secretion assay. We also evaluated feeding suppression and weight loss after the administration of T-3601386 and investigated the involvement of the vagal nerve in these effects. T-3601386, but not a partial agonist fasiglifam, increased intracellular Ca2+ levels in CHO cells with low FFAR1 expression, and single dosing of T-3601386 in diet-induced obese (DIO) rats elevated plasma incretin levels, suggesting full agonistic properties of T-3601386 against GPR40. Multiple doses of T-3601386, but not fasiglifam, in DIO rats showed dose-dependent weight loss accompanied by feeding suppression and durable glucagon-like peptide-1 elevation, all of which were completely abolished in Ffar1-/- mice. Immunohistochemical analysis in the nuclei of the solitary tract demonstrated that T-3601386 increased the number of c-Fos positive cells, which also disappeared in Ffar1-/- mice. Surgical vagotomy and drug-induced deafferentation counteracted the feeding suppression and weight loss induced by the administration of T-3601386. These results suggest that T-3601386 exerts incretin release and weight loss in a GPR40-dependent manner, and that afferent vagal nerves are important for the feeding suppression induced by GPR40 full agonism. Our novel findings raise the possibility that GPR40 full agonist can induce periphery-derived weight reduction, which may provide benefits such as less adverse effects in central nervous system compared to centrally-acting anti-obesity drugs

A Novel Antidiabetic Drug, Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1

<div><p>Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca²⁺ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs <i>in vivo.</i> Point mutations of FFAR1 differentially affected Ca²⁺ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.</p></div

Insulinotropic effects of fasiglifam are attenuated by pharmacological reduction of plasma FFA levels <i>in vivo</i>.

<p>(<i>A</i>) Effects of the lipolysis inhibitors acipimox (30 mg/kg) and fasiglifam (10 mg/kg) on plasma free fatty acids (FFAs) during the oral glucose tolerance test (OGTT) in N-STZ-1.5 rats. (<i>B</i>) Area under the curve (AUC) of plasma FFA during 0–120 min. Fas, fasiglifam. (<i>C</i>) Plasma glucose levels after coadministration of acipimox (30 mg/kg) and fasiglifam (10 mg/kg). (<i>D</i>) AUC of plasma glucose levels during 0–120 min. *<i>P</i><0.05, **<i>P</i><0.01 versus vehicle by Student’s t-test, ^$$<i>P</i><0.01 versus vehicle by Aspin–Welch test. (<i>E</i>) Plasma insulin concentrations after coadministration of acipimox and fasiglifam during OGTT. (<i>F</i>) Insulinotropic effects of fasiglifam (Fas) just before glucose load (time 0) shown in (<i>E</i>) in the absence and presence of acipimox. **<i>P</i><0.01 versus vehicle, ^$<i>P</i><0.05 versus acipimox alone by Student’s t-test, followed by Bonferroni’s correction for four time point comparisons. Data represent mean ± s.e.m. (n = 6).</p

Fasiglifam exhibits positive cooperativity with γ-LA in intracellular Ca²⁺ influx and insulin secretion.

<p>(<i>A</i> and <i>B</i>) Allosteric modulation of γ-linolenic acid (γ-LA) activity by fasiglifam (<i>A</i>) and fasiglifam activity by γ-LA (<i>B</i>) in the Ca²⁺ mobilization assay using hFFAR1/GPR40-expressing CHO cells (clone #2). (<i>C</i> and <i>D</i>) Positive cooperative effect of fasiglifam (Fas) on γ-LA activity (<i>C</i>) and of γ-LA on fasiglifam activity (<i>D</i>) in insulin secretion in mouse pancreatic β cell line, MIN6 cells. (<i>E</i> and <i>F</i>) Fasiglifam-induced insulin secretion in the absence and presence of γ-LA in pancreatic islets of wild-type (<i>E</i>) and FFAR1-knockout mice (<i>F</i>). All insulin secretion assays (<i>C</i>-<i>F</i>) were conducted in the presence of 16 mM glucose (G). All data are representative of at least two replicates. Error bars indicate s.e.m. (n = 3). *<i>P</i><0.025 by one-tailed Shirley-Williams test, ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus DMSO alone by Student’s t-test.</p

Point mutations of FFAR1/GPR40 differentially affect Ca²⁺ influx activities of fasiglifam and γ-LA.

<p>(<i>A</i>) Relative cell surface expression levels of FLAG-tagged FFAR1 wild<b>-</b>type and mutant receptors in transfected HEK293T cells were determined using flow cytometric analysis (FACS). (<i>B</i><b>-</b><i>J</i>) Effects of FFAR1 point mutations on the Ca²⁺ influx activities of FFAR1 agonists. HEK293T cells were transiently transfected with mock vector (<i>B</i>), wild-type (<i>C</i>), S8A (<i>D</i>), Y91A (<i>E</i>), H137A (<i>F</i>), R183A (<i>G</i>), L186F (<i>H</i>), N244A (<i>I</i>), and R258A (<i>J</i>) constructs. Data are representative of three independent experiments. Error bars indicate s.e.m. (n = 3); γ-LA, γ-linolenic acid.</p

Fasiglifam does not exacerbate FFA-induced apoptotic signaling in MIN6 cells.

<p>Caspase 3/7 activity in the mouse pancreatic β cell line MIN6 after 72-h exposure to 0.25–1 mM palmitic acid (<i>A</i>) or γ-linolenic acid (γ-LA) (<i>B</i>) in combination with fasiglifam (Fas, 0.1–10 µM). “PA+Fas” and “γ-LA+Fas” indicate “1 mM palmitic acid +10 µM fasiglifam” and “1 mM γ-LA +10 µM fasiglifam”, respectively. Data shown are mean ± s.e.m. (n = 3).</p

Partial agonist activity of fasiglifam is affected by FFAR1/GPR40 expression levels.

<p>(<i>A</i>) The chemical structure of fasiglifam. (<i>B</i> and <i>C</i>) FFAR1 agonist activities of fasiglifam and free fatty acids (FFAs) in the intracellular Ca²⁺ mobilization assay using CHO cell lines expressing hFFAR1 (clone #104) (<i>B</i>) or mFFAR1 (<i>C</i>). Data are representative of three experiments. (<i>D</i>) hFFAR1 mRNA levels of hFFAR1-expressing CHO clones were evaluated by qRT-PCR. (<i>E</i><b>-</b><i>H</i>) Relative Ca²⁺ influx activities of γ-LA and fasiglifam in CHO clones #104 (<i>E</i>), #19 (<i>F</i>), #2 (<i>G</i>), and #4 (<i>H</i>) with various hFFAR1 expression levels. Error bars indicate s.e.m. (n = 3).</p