Atsuhiko Ichimura

Dietary Gut Microbial Metabolites, Short-chain Fatty Acids, and Host Metabolic Regulation

During feeding, the gut microbiota contributes to the host energy acquisition and metabolic regulation thereby influencing the development of metabolic disorders such as obesity and diabetes. Short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate, which are produced by gut microbial fermentation of dietary fiber, are recognized as essential host energy sources and act as signal transduction molecules via G-protein coupled receptors (FFAR2, FFAR3, OLFR78, GPR109A) and as epigenetic regulators of gene expression by the inhibition of histone deacetylase (HDAC). Recent evidence suggests that dietary fiber and the gut microbial-derived SCFAs exert multiple beneficial effects on the host energy metabolism not only by improving the intestinal environment, but also by directly affecting various host peripheral tissues. In this review, we summarize the roles of gut microbial SCFAs in the host energy regulation and present an overview of the current understanding of its physiological functions.

Free fatty acid receptors act as nutrient sensors to regulate energy homeostasis

Free fatty acids (FFAs) have been demonstrated to act as ligands of several G-protein-coupled receptors (GPCRs) (FFAR1, FFAR2, FFAR3, GPR84, and GPR120). These fatty acid receptors are proposed to play critical roles in a variety of types of physiological homeostasis. FFAR1 and GPR120 are activated by medium- and long-chain FFAs. GPR84 is activated by medium-chain, but not long-chain, FFAs. In contrast, FFAR2 and FFAR3 are activated by short-chain FFAs. FFAR1 is expressed mainly in pancreatic beta-cells and mediates insulin secretion, whereas GPR120 is expressed abundantly in the intestine and promotes the secretion of glucagon-like peptide-1 (GLP-1). FFAR3 is expressed in enteroendocrine cells and regulates host energy balance through effects that are dependent upon the gut microbiota. In this review, we summarize the identification, structure, and pharmacology of these receptors and present an essential overview of the current understanding of their physiological roles.

Sustained activation of ERK1/2 by NGF induces microRNA‐221 and 222 in PC12 cells

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate gene expression by inhibiting translation and/or inducing degradation of target mRNAs, and they play important roles in a wide variety of biological functions including cell differentiation, tumorigenesis, apoptosis and metabolism. However, there is a paucity of information concerning the regulatory mechanism of miRNA expression. Here we report identification of growth factor‐regulated miRNAs using the PC12 cell line, an established model of neuronal growth and differentiation. We found that expression of miR‐221 and miR‐222 expression were induced by nerve growth factor (NGF) stimulation in PC12 cells, and that this induction was dependent on sustained activation of the extracellular signal‐regulated kinase 1 and 2 (ERK1/2) pathway. Using a target prediction program, we also identified a pro‐apototic factor, the BH3‐only protein Bim, as a potential target of miR‐221/222. Overexpression of miR‐221 or miR‐222 suppressed the activity of a luciferase reporter activity fused to the 3′ UTR of Bim mRNA. Furthermore, overexpression of miR‐221/222 decreased endogenous Bim mRNA expression. These results reveal that the ERK signal regulates miR‐221/222 expression, and that these miRNAs might contribute to NGF‐dependent cell survival in PC12 cells.

MicroRNA-34a inhibits cell proliferation by repressing mitogen-activated protein kinase kinase 1 during megakaryocytic differentiation of K562 cells.

Phorbol 12-myristate 13-acetate (PMA) induces megakaryocytic differentiation of the human chronic myelocytic leukemia cell line K562. We examined the potential regulatory role of microRNAs (miRNAs) in this process. Genome-wide expression profiling identified 21 miRNAs (miRs) that were induced by the treatment of K562 cells with PMA. Among them, the expression of miR-34a, miR-221, and miR-222 was induced in the early stages and maintained throughout the late stages of differentiation. Cell signaling analysis showed that the activation of extracellular signal-regulated protein kinase (ERK) in response to PMA strongly induced miR-34a expression by transactivation via the activator protein-1 binding site in the upstream region of the miR-34a gene. Reporter gene assays identified mitogen-activated protein kinase kinase 1 (MEK1) as a direct target of miR-34a and c-fos as a direct target of miR-221/222. Although overexpression of the three miRNAs had little effect on cell differentiation, overexpression of miR-34a significantly repressed the proliferation of K562 cells with a concomitant reduction in MEK1 protein expression. Conversely, a locked nucleic acid probe against miR-34a significantly enhanced the proliferation of PMA-treated K562 cells. Taken together, the results show that PMA activates the MEK-ERK pathway and strongly induces miRNA-34a expression, which in turn inhibits cell proliferation by repressing the expression of MEK1. Thus, the results highlight an important regulatory role for miR-34a in the process of megakaryocytic differentiation, especially in the arrest of cell growth, which is a prerequisite for cells to enter differentiation.

Global correlation analysis for micro-RNA and mRNA expression profiles in human cell lines

AbstractMicroribonucleic acids (miRNAs) are small noncoding RNAs that negatively regulate gene expression at the posttranscriptional level. Although considerable progress has been made in studying the function of miRNAs, they still remain largely unclear, mainly because of the difficulty in identifying target genes for miRNA. We performed a global analysis of both miRNAs and mRNAs expression across 16 human cell lines and extracted negatively correlated pairs of miRNA and mRNA which indicate miRNA-target relationship. The many of known-target of miR-124a showed negative correlation, suggesting our analysis were valid. We further extracted physically relevant miRNA-target gene pairs, applying computational target prediction algorithm with inverse correlations of miRNA and messenger RNA (mRNA) expression. Furthermore, gene-ontology-based annotation and functional enrichment analysis of the extracted miRNA-target gene pairs made it possible to indicate putative functions of miRNAs. The data collected here will be of value for further studies into the function of miRNA.

Plasminogen Activator Inhibitor-1 Antagonist TM5441 Attenuates Nω-Nitro-l-Arginine Methyl Ester–Induced Hypertension and Vascular Senescence

Background Long-term inhibition of nitric oxide synthase (NOS) by L-arginine analogues such as Nω-nitro-L-arginine methyl ester (L-NAME) has been shown to induce senescence in vitro and systemic hypertension and arteriosclerosis in vivo. We previously reported that PAI-1-deficient mice (PAI-1−/−) are protected against L-NAME-induced pathologies. In this study, we investigated whether a novel, orally active PAI-1 antagonist (TM5441) has a similar protective effect against L-NAME treatment. Additionally, we studied whether L-NAME can induce vascular senescence in vivo and investigated the role of PAI-1 in this process.Background— Long-term inhibition of nitric oxide synthase by L-arginine analogues such as N&ohgr;-nitro-L-arginine methyl ester (L-NAME) has been shown to induce senescence in vitro and systemic hypertension and arteriosclerosis in vivo. We previously reported that plasminogen activator inhibitor-1 (PAI-1)–deficient mice (PAI-1−/−) are protected against L-NAME-induced pathologies. In this study, we investigated whether a novel, orally active PAI-1 antagonist (TM5441) has a similar protective effect against L-NAME treatment. Additionally, we studied whether L-NAME can induce vascular senescence in vivo and investigated the role of PAI-1 in this process. Methods and Results— Wild-type mice received either L-NAME or L-NAME and TM5441 for 8 weeks. Systolic blood pressure was measured every 2 weeks. We found that TM5441 attenuated the development of hypertension and cardiac hypertrophy compared with animals that had received L-NAME alone. Additionally, TM5441-treated mice had a 34% reduction in periaortic fibrosis relative to animals on L-NAME alone. Finally, we investigated the development of vascular senescence by measuring p16Ink4a expression and telomere length in aortic tissue. We found that L-NAME increased p16Ink4a expression levels and decreased telomere length, both of which were prevented with TM5441 cotreatment. Conclusions— Pharmacological inhibition of PAI-1 is protective against the development of hypertension, cardiac hypertrophy, and periaortic fibrosis in mice treated with L-NAME. Furthermore, PAI-1 inhibition attenuates the arterial expression of p16Ink4a and maintains telomere length. PAI-1 appears to play a pivotal role in vascular senescence, and these findings suggest that PAI-1 antagonists may provide a novel approach in preventing vascular aging and hypertension.

Nutritional Signaling via Free Fatty Acid Receptors

Excess energy is stored primarily as triglycerides, which are mobilized when demand for energy arises. Dysfunction of energy balance by excess food intake leads to metabolic diseases, such as obesity and diabetes. Free fatty acids (FFAs) provided by dietary fat are not only important nutrients, but also contribute key physiological functions via FFA receptor (FFAR)-mediated signaling molecules, which depend on FFAs’ carbon chain length and the ligand specificity of the receptors. Functional analyses have revealed that FFARs are critical for metabolic functions, such as peptide hormone secretion and inflammation, and contribute to energy homeostasis. In particular, recent studies have shown that the administration of selective agonists of G protein-coupled receptor (GPR) 40 and GPR120 improved glucose metabolism and systemic metabolic disorders. Furthermore, the anti-inflammation and energy metabolism effects of short chain FAs have been linked to the activation of GPR41 and GPR43. In this review, we summarize recent progress in research on FFAs and their physiological roles in the regulation of energy metabolism.

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

Tric-b knockout mice serve as a model for studying the bone defects of osteogenesis imperfecta. The TRIC to building strong bones During bone development, osteoblasts secrete a collagen-rich matrix that is necessary for bone mineralization. Defects in collagen deposition cause osteogenesis imperfecta (OI), a disease characterized by brittle bones. Zhao et al. found that mice lacking Tric-b, which encodes a trimeric intracellular cation channel that localizes to the endoplasmic reticulum (ER), had bone defects similar to those of OI patients. Although osteoblasts in Tric-b knockout mice synthesized collagen, it accumulated inside the cells instead of being secreted. The accumulation of intracellular collagen deposits was associated with morphological and biochemical markers of ER stress, including severe dilation, excess Ca2+ in the ER, and impaired Ca2+ release from the ER. These findings suggest that TRIC-B is necessary to maintain ER homeostasis, thus enabling osteoblasts to secrete the large amounts of collagen required to build strong bones. The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca2+ release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca2+ release from ER and increased ER Ca2+ content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.

Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis

Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.

Small Molecule Inhibitors of Plasminogen Activator Inhibitor-1 Elicit Anti-Tumorigenic and Anti-Angiogenic Activity.

Numerous studies have shown a paradoxical positive correlation between elevated levels of plasminogen activator inhibitior-1 (PAI-1) in tumors and blood of cancer patients with poor clinical outcome, suggesting that PAI-1 could be a therapeutic target. Here we tested two orally bioavailable small molecule inhibitors of PAI-1 (TM5275 and TM5441) for their efficacy in pre-clinical models of cancer. We demonstrated that these inhibitors decreased cell viability in several human cancer cell lines with an IC50 in the 9.7 to 60.3 μM range and induced intrinsic apoptosis at concentrations of 50 μM. In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant. Pharmacokinetics studies indicated an average peak plasma concentration of 11.4 μM one hour after oral administration and undetectable levels 23 hours after administration. The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis. These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer.