Recently, the pleiotropic benefits of incretin-based therapy have been reported. We have previously reported that Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, attenuates prostate cancer growth. Metformin is known for its anti-cancer effect. Here, we examined the anti-cancer effect of Exendin-4 and metformin using a prostate cancer model.

Incretin therapy has emerged as one of the most popular medications for type 2 diabetes. We have previously reported that the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin attenuates neointima formation after vascular injury in non-diabetic mice. In the present study, we examined whether combined treatment with linagliptin and the sodium glucose cotransporter 2 (SGLT2) inhibitor empagliflozin attenuates neointima formation in diabetic mice after vascular injury. Diabetic db/db mice were treated with 3 mg/kg/day linagliptin and/or 30 mg/kg/day empagliflozin from 5 to 10 weeks of age. Body weight was significantly decreased by empagliflozin and the combined treatment. Blood glucose levels and glucose tolerance test results were significantly improved by empagliflozin and the combined treatment, but not by linagliptin. An insulin tolerance test suggested that linagliptin and empagliflozin did not improve insulin sensitivity. In a model of guidewire-induced femoral artery injury in diabetic mice, neointima formation was significantly decreased in mice subjected to combined treatment. In an in vitro assay using rat aortic smooth muscle cells (RASMC), 100, 500, or 1000 nM empagliflozin significantly decreased the RASMC number in a dose-dependent manner. A further significant reduction in RASMC proliferation was observed after combined treatment with 10 nM linagliptin and 100 nM empagliflozin. These data suggest that combined treatment with the DPP-4 inhibitor linagliptin and SGLT2 inhibitor empagliflozin attenuates neointima formation after vascular injury in diabetic mice in vivo and smooth muscle cell proliferation in vitro.

The hippocampus plays an important role in the formation of contextual memory between the environment and the rewarding effect of abused drugs. The dopaminergic neural transmission in the hippocampus seems to be critical for such memory. Using conditioned place preference in rats, we found that the protein level of the dopamine D(1) receptor and its prerequisite mRNA in the hippocampus increased in animals that showed a clear preference for the environment paired with cocaine. The increase was not a simple reflection of the repeated administration of cocaine. Instead, it is attributable to conditioning, because systematic contingency between drug administration and exposure to a particular environment was necessary for the increase. Furthermore, we found that the mRNA of the dopamine D(1) receptors increased in the granule cell layer of the dentate gyrus. These results suggest that the alteration of dopamine D(1) receptor in the hippocampus, especially in the dentate gyrus, is related to the induction of drug-induced contextual memory. The finding implicates the relevance of the dopaminergic signal transduction in the hippocampus to drug dependence.

We previously identified the selective androgen receptor (AR) modulator S42, which does not stimulate prostate growth but has a beneficial effect on lipid metabolism. In the prostate cancer (PC) cell line LNCaP, S42 did not induce AR transactivation but antagonized 5α-dihydrotestosterone (DHT)‒induced AR activation. Next, we investigated whether S42 suppresses the growth of PC cell lines. Basal growth of LNCaP cells was significantly suppressed by treatment with S42 compared with vehicle, as determined by cell counting and 5-bromo-2'-deoxyuridine assays. The suppressive effect of S42 on cell growth was evident in the AR-positive PC cells LNCaP and 22Rv1 and was slightly observed even in the AR-negative PC-3 cells. However, S42 did not induce apoptosis as determined by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. S42 had an even greater suppressive effect on DHT-dependent LNCaP cell proliferation than on basal proliferation (P < 0.05). DHT treatment increased the expression of phosphorylated extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK), a major signaling molecule for PC proliferation, and this was significantly inhibited by S42. DHT also significantly upregulated AR, insulinlike growth factor-1 receptor (IGF-1R), and insulin receptor (IR)-β protein levels, which were similarly reduced by S42 treatment. Importantly, S42 administration to mice attenuated the growth of LNCaP tumors and reduced tumor expression of the prostate-specific antigen, P504S, Ki67, and phosphorylated ERK-MAPK. These data suggest that S42 attenuates LNCaP tumor growth not by inducing apoptosis but by inhibiting the expression of proliferation-related receptors, including IGF-1R, IR, and AR, and by suppressing ERK-MAPK activation. S42 may thus be a feasible candidate for PC treatment.

Mesenchymal stroma/stem cells (MSCs) exist in adult tissues, such as adipose tissue and bone marrow, and differentiate into cells of multiple lineages. In previous studies, we found that MSCs differentiate into steroidogenic cells by forced expression of steroidogenic factor 1 (SF-1)/adrenal 4 binding protein (Ad4BP), the master regulator of steroidogenesis and differentiation of pituitary gonadotrophs, adrenal glands, and gonads. In this study, SF-1/Ad4BP-induced steroidogenic cells derived from mouse adipose tissue-derived MSCs (ADSCs) were implanted under the kidney capsule of bilateral adrenalectomized (bAdx) mice. bAdx mice did not survive after 7 days. However, 4 of 9 bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, 1 of 10 bAdx mice transplanted with control ADSCs, and bAdx mice transplanted with an adrenal gland survived for 30 days. Plasma corticosterone levels in bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells and control ADSCs were 5.41 ± 2.26 ng/mL (mean ± SEM) and undetectable at 7 days after implantation, respectively. After removal of the kidney bearing the graft from the surviving mice at 30 days after implantation, plasma corticosterone was not detected in any of the samples. Immunohistochemical staining revealed SF-1/Ad4BP-positive cells under the capsule of the kidney. Although we performed an adrenocorticotropin (ACTH) loading test on bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, ACTH responsiveness was not observed. Implantation of steroidogenic cells derived from ADSCs into bAdx mice increased the basal plasma corticosterone level and extended the survival of bAdx mice, suggesting the capability of restoring steroidogenic cells by cell transplantation therapy for adrenal insufficiency.

To investigate the behaviours of polyethylene glycol (PEG) and its interaction with biomass constituents in coniferous wood (Japanese cypress), variable temperature solid-state NMR spectra and relaxation times were measured from 20-80 °C. Signal intensities in the 1H and 13C PST-MAS NMR spectra changed depending on both the measurement temperature and the melting point of the impregnated PEG. In the 13C CP-MAS NMR spectra with increasing temperature, although the signal intensities of biomass constituents slightly decreased, signal intensities of PEG molecules in the cypress maximized at 80 °C. PEG impregnation into cypress decreased the T 1H values at 80 °C for short to medium chain PEG in the liquid phase while it decreased T 1H values at ambient temperature for long chain PEG in the solid phase because the interactions of PEG molecules and the biomass constituents of coniferous wood were different for different chain lengths of the PEG. These variable temperature measurements of both solid-state NMR spectra and relaxation time indicated that impregnation of longer chain PEG molecules produced higher hydrophobicity because of the increased steric hinderance of PEG attached to carbohydrates. The variable temperature measurements also showed that long chain PEG molecules were restricted to the lumen while short to medium chain length PEG molecules infiltrated into the intercellular region of the cell wall in addition to the lumen. These results obtained from the variable temperature NMR measurements were also supported by ATR-IR spectroscopy analyses.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is a major societal, scientific, and economic problem. Several early-life factors associated with an increased risk for the clinical diagnosis of AD have recently been identified. In the present study, we investigated the involvement of early-life stress in the pathogenesis of AD using heterozygous amyloid precursor protein (APP) mutant mice (AppNL-G-F/wt) and wild-type (Appwt/wt) mice. We found that maternal-separated Appwt/wt mice showed narrowing of vessels and decreased pericyte coverage of capillaries in the prefrontal cortex, while maternal-separated AppNL-G-F/wt mice additionally showed the impairment of cognitive function, earlier formation of Aβ plaques, increased vessel-associated microglia, and disruption of the blood-brain barrier. Substantial activation of microglia was detected in the maternal-separated AppNL-G-F/wt mice and maternal-separated Appwt/wt mice. At an early stage, morphological changes and inflammatory responses were observed in the microglia of the maternal-separated AppNL-G-F/wt mice and maternal-separated Appwt/wt mice, and morphological changes in the microglia were observed in the non-maternal-separated AppNL-G-F/wt mice. Microglia activation induced by maternal separation in combination with the APP mutation may impair the vascular system, leading to AD progression. These findings therefore suggest that maternal separation results in the early induction of AD-related pathology via angiopathy.

Regulatory T cells (Treg) play essential roles in maintaining immune homeostasis. Resident Treg in visceral adipose tissue (VAT-Treg) decrease in male obese mice, which leads to the development of obesity-associated chronic inflammations and insulin resistance. Although gender differences in immune responses have been reported, the effects of the difference in metabolic environment on VAT-Treg are unclear. We investigated the localization of VAT-Treg in female mice in comparison with that in male mice. On a high-fat diet (HFD), VAT-Treg decreased in male mice but increased in female mice. The increase was abolished in ovariectomized and HFD-fed mice, but was restored by estrogen supplementation. The IL33 receptor ST2, which is important for the localization and maturation of VAT-Treg in males, was reduced in CD4+CD25+ T cells isolated from gonadal fat of obese mice of both genders, suggesting that a different system exists for VAT-Treg localization in females. Extensive analysis of chemokine expression in gonadal fat and adipose CD4+CD25+T cells revealed several chemokine signals related to female-specific VAT-Treg accumulation such as CCL24, CCR6, and CXCR3. Taken together, the current study demonstrated sexual dimorphism in VAT-Treg localization in obese mice. Estrogen may attenuate obesity-associated chronic inflammation partly through altering chemokine-related VAT-Treg localization in females.

Understanding of the mechanism of adipogenesis is essential for the control of obesity, which predisposes toward numerous health problems. High-mobility group box protein 2 (HMGB2) is a non-histone chromosomal protein that facilitates DNA replication, transcription, recombination, and repair. Here, we studied the role of HMGB2 in adipogenic differentiation. The expression of HMGB2 was measured at the mRNA and protein levels in cultured 3T3-L1 pre-adipocyte cells and during the process of adipogenic differentiation induced bya cocktail of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone. This increased in the early phase and decreased in the late phase of differentiation. However, 3T3-L1 pre-adipocyte cells did not differentiate into adipocytes after the knockdown of HMGB2 expression by small interfering RNA (siRNA). Similarly, mesenchymal stem cells (MSCs) isolated from Hmgb2-/- mice did not express peroxisome proliferator-activated receptor gamma (PPARγ) in response to the adipocyte differentiation cocktail and did not differentiate. Wnt/β-catenin signaling is a negative regulator of adipogenic differentiation. We found that β-catenin expression was downregulated during 3T3-L1 adipogenic differentiation, as expected, but not when endogenous HMBG2 expression was knocked down using siRNA. These results indicate that HMGB2 plays an essential role in the early phase of the differentiation of pre-adipocytes and MSCs, and probably interacts with other regulators, such as PPARγ and Wnt/β-catenin signaling.

Myocardial lactate production in the coronary circulation during acetylcholine (ACh)-provocation test (abbreviated as lactate production) provides supporting evidence for coronary spasm-induced myocardial ischemia. The purpose of this study was to examine the clinical features, predictive factors, and prognosis of patients with coronary vasospastic angina (VSA) and lactate production.

Transcranial direct-current stimulation (tDCS) is a non-invasive procedure that achieves polarity-dependent modulation of neuronal membrane potentials. It has recently been used as a functional intervention technique for the treatment of psychiatric and neurological diseases; however, its neuronal mechanisms have not been fully investigated in vivo.

Eutrema salsugineum (also known as Thellungiella salsuginea and formerly Thellungiella halophila), a species closely related to Arabidopsis thaliana, shows tolerance not only to salt stress, but also to chilling, freezing, and high temperatures. To identify genes responsible for stress tolerance, we conducted Full-length cDNA Over-eXpressing gene (FOX) hunting among a collection of E. salsugineum cDNAs that were stress-induced according to gene ontology analysis or over-expressed in E. salsugineum compared with A. thaliana. We identified E. salsugineum CSP41b (chloroplast stem-loop-binding protein of 41 kDa; also known as CRB, chloroplast RNA binding; named here as EsCSP41b) as a gene that can confer heat and salinity stress tolerance on A. thaliana. A. thaliana CSP41b is reported to play an important role in the proper functioning of the chloroplast: the atcsp41b mutant is smaller and paler than wild-type plants and shows altered chloroplast morphology and photosynthetic performance. We observed that AtCSP41b-overexpressing transgenic A. thaliana lines also exhibited marked heat tolerance and significant salinity stress tolerance. The EsCSP41b-overexpressing transgenic A. thaliana lines showed significantly higher photosynthesis activity than wild-type plants not only under normal growth conditions but also under heat stress. In wild-type plants, the expression levels of both EsCSP41b and AtCSP41b were significantly reduced under heat or salinity stress. We conclude that maintenance of CSP41b expression under abiotic stresses may alleviate photoinhibition and improve survival under such stresses.

Reduction of low-density lipoprotein cholesterol (LDL-C) is important for patients with a high risk for atherosclerotic events, such as patients with diabetes and other risk factors. Anagliptin was reported to reduce LDL-C for 12 weeks in phase III trials regardless of the use of statins, but it is uncertain whether this effect is common to other dipeptidylpeptidase-4 (DPP-4) inhibitors.

In Theiler's murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelinating lesions, and the distribution of axonal damage present during the early phase of infection corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal damage recruits inflammatory cells to sites of Wallerian degeneration, leading to demyelination. Three weeks after TMEV infection, axonal degeneration was induced in the posterior funiculus of mice by injecting the toxic lectin Ricinus communis agglutinin (RCA) I into the sciatic nerve. Neuropathology was examined 1 week after lectin injection. Control mice, infected with TMEV but receiving no RCA I, had inflammatory demyelinating lesions in the anterior/lateral funiculi. Other control mice that received RCA I alone did not develop inflammatory lesions. In contrast, RCA I injection into TMEV-infected mice induced lesions in the posterior funiculus in addition to the anterior/lateral funiculi. We found no differences in lymphoproliferative responses or antibody titers against TMEV among the groups. This suggests that axonal degeneration contributes to the recruitment of inflammatory cells into the central nervous system by altering the local microenvironment. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).

Early rejection is a critical issue to be overcome to achieve successful islet transplantation. NLRP3 inflammasome is a protein complex that mediates the maturation of pro-interleukin (IL)-1β and pro-IL-18 to IL-1β and IL-18, respectively, which induce cellular death. Here, we investigated the impact of NLRP3 inflammasome and the effect of its inhibition by MCC950 in a rodent model of islet transplantation. We assessed the therapeutic effects of MCC950, a specific inhibitor of NLRP3 inflammasome, on gene expression, islet survival ratio and viability, and islet transplantation in mice. NLRP3 inflammasome-related gene (Nlrp3 and Il1b) expression was upregulated in islets stimulated with proinflammatory cytokines and suppressed when incubated with MCC950. Survival ratio and viability of incubated islets were reduced by cytokine stimulation and improved by MCC950. Regarding islet transplantation, the number of apoptotic cells in transplanted islets was reduced by MCC950. Furthermore, the expression of IL-1β in transplanted islets, migration of macrophages around islets, and fluctuation of blood glucose levels were suppressed by MCC950. Our study revealed that NLRP3 inflammasome worsened the therapeutic outcomes of islet transplantation and that MCC950 administration improved glycaemic control in syngeneic mice that underwent islet transplantation by inhibiting inflammation, which suppressed islet death.

Aryl hydrocarbon receptor interacting protein (AIP) is thought to be a tumor suppressor gene, as indicated by a mutational analysis of pituitary somatotroph adenomas. However, the physiological significance of AIP inactivation in somatotroph cells remains unclear. Using CRISPR/Cas9, we identified a GH3 cell clone (termed GH3-FTY) in which Aip was genetically disrupted, and subsequently investigated its character with respect to growth hormone (Gh) synthesis and proliferation. Compared with GH3, GH3-FTY cells showed remarkably increased Gh production and a slight increase in cell proliferation. Gh-induced Stat3 phosphorylation is known to be a mechanism of Gh oversecretion in GH3. Interestingly, phosphorylated-Stat3 expression in GH3-FTY cells was increased more compared with GH3 cells, suggesting a stronger drive for this mechanism in GH3-FTY. The phenotypes of GH3-FTY concerning Gh overproduction, cell proliferation, and increased Stat3 phosphorylation were significantly reversed by the exogenous expression of Aip. GH3-FTY cells were less sensitive to somatostatin than GH3 cells in the suppression of cell proliferation, which might be associated with the reduced expression of somatostatin receptor type 2. GH3-FTY xenografts in BALB/c nude mice (GH3-FTY mice) formed more mitotic somatotroph tumors than GH3 xenografts (GH3 mice), as also evidenced by increased Ki67 scores. GH3-FTY mice were also much larger and had significantly higher plasma Gh levels than GH3 mice. Furthermore, GH3-FTY mice showed relative insulin resistance compared with GH3 mice. In conclusion, we established a somatotroph cell line, GH3-FTY, which possessed prominent Gh secretion and mitotic features associated with the disruption of Aip.

We previously reported the in vitro and in vivo antitumor effects of trametinib, a MEK inhibitor, on neuroblastoma with MAPK pathway mutations. As we observed eventual resistance to trametinib in our previous study, we evaluated the combination therapy of CA3, a YAP inhibitor, with trametinib, based on a recent report suggesting the potential involvement of YAP in the mechanism underlying the resistance to trametinib in neuroblastoma.

We previously identified a novel selective androgen receptor modulator, S42, that does not stimulate prostate growth but has a beneficial effect on lipid metabolism. S42 also increased muscle weight of the levator ani in orchiectomized Sprague-Dawley rats. These findings prompted us to investigate whether S42 has a direct effect on cultured C2C12 myotubes. S42 significantly lowered expression levels of the skeletal muscle ubiquitin ligase (muscle atrophy-related gene), atrogin1 and Muscle RING-Finger Protein 1(MuRF1) in C2C12 myotubes, as determined by real time PCR. Phosphorylation of p70 S6 kinase (p70S6K), an essential factor for promoting protein synthesis in skeletal muscle, was significantly increased by S42 to almost the same extent as by insulin, but this was significantly prevented by treatment with rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). However, phosphorylation of Akt, upstream regulator of mTORC1, was not changed by S42. S42 did not increase insulin-like growth factor 1 (Igf1) mRNA levels in C2C12 myotubes. These results suggest that S42 may have an anabolic effect through activation of mTORC1-p70S6K signaling, independent of IGF-1-Akt signaling and may exert an anti-catabolic effect through inhibition of the degradation pathway in cultured C2C12 myotubes.

Recently, glucagon-like peptide-1 (GLP-1)-based therapy, including dipeptidyl peptidase-4 (DPP-4) inhibitors and GLP-1 receptor agonists, has emerged as one of the most popular anti-diabetic therapies. Furthermore, GLP-1-based therapy has attracted increased attention not only for its glucose-lowering ability, but also for its potential as a tissue-protective therapy. In this study, we investigated the vascular-protective effect of the DPP-4 inhibitor, linagliptin, using vascular smooth muscle cells (VSMCs).

The Conflict Tactics Scale 1 (CTS1) is a widely used self-report measure of abusive attitudes of parents towards children. The factor structure of the CTS1 still remains to be clarified. The aim of this study was to examine the factor structure of the Japanese version of the CTS1 for postpartum women in community settings.