Enhanced tumor specific drug release by hypoxia sensitive dual-prodrugs based on 2-nitroimidazole.

Hypoxia in cancer is important in the development of cancer-selective medicines. Here, a novel hypoxia-responsible dual-prodrug is described. We designed and synthesized 2-nitroimidazole derivatives which spontaneously release both a PYG inhibitor and gemcitabine under hypoxic conditions. One such derivative, a prodrug 9 was found to be stable against chemical and enzymatic hydrolysis, and upon chemical reduction of the nitro group on imidazole, successfully releases both drugs. In an in vitro proliferation assay using human pancreatic cells, compound 9 exhibited significant anti-proliferative effects in hypoxia but fewer effects in normoxia. Consequently, prodrug 9 should be useful for cancer treatment due to its improved cancer selectivity and potential to overcome drug resistance.

TJ-17 (Goreisan) mitigates renal fibrosis in a mouse model of folic acid-induced chronic kidney disease.

BACKGROUND AND PURPOSE: TJ-17 (Goreisan), a traditional Japanese Kampo medicine, has been generally used to treat edema, such as heart failure, due to its diuretic effect. In the present study, we investigate the effects of TJ-17 on chronic kidney disease (CKD). METHODS: We the preventive action of TJ-17 against acute kidney injury (AKI) transition to CKD in vivo using a folic acid (FA)-induced mouse model. Mice were treated with food containing TJ-17 at 48 h after FA intraperitoneal injection (AKI phase). RESULTS: Histological analysis, as well as renal function and renal injury markers, deteriorated in mice with FA-induced CKD and were ameliorated by TJ-17 treatment. Increased levels of inflammatory cytokines and macrophage infiltration were also alleviated in mice treated with TJ-17. Renal fibrosis, a crucial factor in CKD, was induced by FA administration and inhibited by TJ-17 treatment. Pretreatment with TJ-17 did not exert an inhibitory effect on FA-induced AKI. The increase in urinary volume in FA-induced CKD mice was ameliorated by TJ-17 treatment, with a concurrent correction of reduced aquaporins expression in the kidney. CONCLUSION: TJ-17 may have a novel preventive effect against inflammation, oxidative stress, and fibrosis, contributing to innovation in the treatment of CKD.

CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses.

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.

Antifungal action of the combination of ferulic acid and ultraviolet-A irradiation against Saccharomyces cerevisiae.

AIMS: To examine the antifungal action of photocombination treatment with ferulic acid (FA) and ultraviolet-A (UV-A) light (wavelength, 365 nm) by investigating associated changes in cellular functions of Saccharomyces cerevisiae. METHODS AND RESULTS: When pre-incubation of yeast cells with FA was extended from 0.5 to 10 min, its photofungicidal activity increased. Flow cytometry analysis of stained live and dead cells revealed that 10-min UV-A exposure combined with FA (1 mg ml-1 ) induced a ~99.9% decrease in cell viability although maintaining cell membrane integrity when compared with pre-exposure samples. When morphological and biochemical analysis were performed, treated cells exhibited an intact cell surface and oxidative DNA damage similar to control cells. Photocombination treatment induced cellular proteins oxidation, as shown by 2.3-fold increasing in immunostaining levels of ~49-kDa carbonylated proteins compared with pre-irradiation samples. Pyruvate kinase 1 (PK1) was identified by proteomics analysis as a candidate protein whose levels was affected by photocombination treatment. Moreover, intracellular ATP levels decreased following FA treatment both in darkness and with UV-A irradiation, thus suggesting a possible FA-induced delay in cell growth. CONCLUSIONS: FA functions within the cytoplasmic membrane; addition of UV-A exposure induces increased oxidative modifications of cytosolic proteins such as PK1, which functions in ATP generation, without causing detectable genotoxicity, thus triggering inactivation of yeast cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial contamination is a serious problem that diminishes the quality of fruits and vegetables. Combining light exposure with food-grade phenolic acids such as FA is a promising disinfection technology for applications in agriculture and food processing. However, the mode of photofungicidal action of FA with UV-A light remains unclear. This study is the first to elucidate the mechanism using S. cerevisiae. Moreover, proteomics analyses identified a specific cytosolic protein, PK1, which is oxidatively modified by photocombination treatment.

Diphenhydramine may be a preventive medicine against cisplatin-induced kidney toxicity.

Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes kidney toxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced kidney toxicity is currently available. Here, based on a repositioning analysis of the Food and Drug Administration Adverse Events Reporting System, we found that a previously developed drug, diphenhydramine, may provide a novel treatment for cisplatin-induced kidney toxicity. To confirm this, the actual efficacy of diphenhydramine was evaluated in in vitro and in vivo experiments. Diphenhydramine inhibited cisplatin-induced cell death in kidney proximal tubular cells. Mice administered cisplatin developed kidney injury with significant dysfunction (mean plasma creatinine: 0.43 vs 0.15 mg/dl) and showed augmented oxidative stress, increased apoptosis, elevated inflammatory cytokines, and MAPKs activation. However, most of these symptoms were suppressed by treatment with diphenhydramine. Furthermore, the concentration of cisplatin in the kidney was significantly attenuated in diphenhydramine-treated mice (mean platinum content: 70.0 vs 53.4 µg/g dry kidney weight). Importantly, diphenhydramine did not influence or interfere with the anti-tumor effect of cisplatin in any of the in vitro or in vivo experiments. In a selected cohort of 98 1:1 matched patients from a retrospective database of 1467 patients showed that patients with malignant cancer who had used diphenhydramine before cisplatin treatment exhibited significantly less acute kidney injury compared to ones who did not (6.1 % vs 22.4 %, respectively). Thus, diphenhydramine demonstrated efficacy as a novel preventive medicine against cisplatin-induced kidney toxicity.

Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice.

AIMS/HYPOTHESIS: Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron-storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. METHODS: Conditional macrophage-specific H-ferritin (Fth, also known as Fth1) knockout (LysM-Cre Fth KO) mice were used and divided into four groups: wild-type (WT) and LysM-Cre Fth KO mice with normal diet (ND), and WT and LysM-Cre Fth KO mice with high-fat diet (HFD). These mice were analysed for characteristics of obesity and diabetes, tissue iron content, inflammation, oxidative stress, insulin sensitivity and metabolic measurements. RAW264.7 macrophage cells were used for in vitro experiments. RESULTS: Iron concentration reduced, and mRNA expression of ferroportin increased, in macrophages from LysM-Cre Fth KO mice. HFD-induced obesity was lower in LysM-Cre Fth KO mice than in WT mice at 12 weeks (body weight: KO 34.6 ± 5.6 g vs WT 40.1 ± 5.2 g). mRNA expression of inflammatory cytokines and infiltrated macrophages and oxidative stress increased in the adipose tissue of HFD-fed WT mice, but was not elevated in HFD-fed LysM-Cre Fth KO mice. However, WT mice fed an HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre Fth KO mice fed an HFD (adipose tissue [µmol Fe/g protein]: KO 1496 ± 479 vs WT 2316 ± 866; spleen [µmol Fe/g protein]: KO 218 ± 54 vs WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre Fth KO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and lipopolysaccharide-induced Tnf-α (also known as Tnf) mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. CONCLUSIONS/INTERPRETATION: Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes.

Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mouse myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of MAPK signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis.-Ikeda, Y., Satoh, A., Horinouchi, Y., Hamano, H., Watanabe, H., Imao, M., Imanishi, M., Zamami, Y., Takechi, K., Izawa-Ishizawa, Y., Miyamoto, L., Hirayama, T., Nagasawa, H., Ishizawa, K., Aihara, K.-I., Tsuchiya, K., Tamaki, T. Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress.

Novel Hydrophilic Camptothecin Derivatives Conjugated to Branched Glycerol Trimer Suppress Tumor Growth without Causing Diarrhea in Murine Xenograft Models of Human Lung Cancer.

Camptothecin possesses broad antitumor spectra on various cancers. In spite of its marked tumor-suppressing potency, camptothecin is too hydrophobic to be solved in water and therefore not currently in clinical use. CPT-11 (irinotecan) is one of the hydrophilic analogues of camptothecin and widely prescribed. However, its water solubility is still low and furthermore evokes severe diarrhea. Therefore, we designed and synthesized novel highly hydrophilic camptothecin derivatives by conjugating SN38 with branched glycerol trimer (SN38-BGL), which we have been developing as a unique strategy to endow hydrophobic molecule with much hydrophilicity, to maximize the benefit of CPT-11 and minimize the adverse effects. The SN38-BGLs exhibited equivalent or slightly stronger tumor-suppressing effects in murine xenograft human lung cancer models compared to CPT-11. However, neither early- nor late-onset diarrhea was observed when SN38-BGL was administered. Heights of villi in jejunum and ileum were bigger than those from CPT-11-treated mice, indicating that SN38-BGL is less harmful than CPT-11. Ex vivo digestion by liver microsome did not yield SN38 but a couple of other molecules against our expectations, which suggests the involvement of other active metabolites than SN38 and may explain the differences. Hence, SN38-BGLs can be a novel hydrophilic camptothecin derivative without causing severe diarrhea.

The uremic toxin indoxyl sulfate interferes with iron metabolism by regulating hepcidin in chronic kidney disease.

Background: Hepcidin secreted by hepatocytes is a key regulator of iron metabolism throughout the body. Hepcidin concentrations are increased in chronic kidney disease (CKD), contributing to abnormalities in iron metabolism. Levels of indoxyl sulfate (IS), a uremic toxin, are also elevated in CKD. However, the effect of IS accumulation on iron metabolism remains unclear. Methods: We used HepG2 cells to determine the mechanism by which IS regulates hepcidin concentrations. We also used a mouse model of adenine-induced CKD. The CKD mice were divided into two groups: one was treated using AST-120 and the other received no treatment. We examined control mice, CKD mice, CKD mice treated using AST-120 and mice treated with IS via drinking water. Results: In the in vitro experiments using HepG2 cells, IS increased hepcidin expression in a dose-dependent manner. Silencing of the aryl hydrocarbon receptor (AhR) inhibited IS-induced hepcidin expression. Furthermore, IS induced oxidative stress and antioxidant drugs diminished IS-induced hepcidin expression. Adenine-induced CKD mice demonstrated an increase in hepcidin concentrations; this increase was reduced by AST-120, an oral adsorbent of the uremic toxin. CKD mice showed renal anemia, decreased plasma iron concentration, increased plasma ferritin and increased iron content in the spleen. Ferroportin was decreased in the duodenum and increased in the spleen. These changes were ameliorated by AST-120 treatment. Mice treated by direct IS administration showed hepatic hepcidin upregulation. Conclusions: IS affects iron metabolism in CKD by participating in hepcidin regulation via pathways that depend on AhR and oxidative stress.

Mechanisms of the pH- and Oxygen-Dependent Oxidation Activities of Artesunate.

Artemisinin was discovered in 1971 as a constituent of the wormwood genus plant (Artemisia annua). This plant has been used as an herbal medicine to treat malaria since ancient times. The compound artemisinin has a sesquiterpene lactone bearing a peroxide group that offers its biological activity. In addition to anti-malarial activity, artemisinin derivatives have been reported to exert antitumor activity in cancer cells, and have attracted attention as potential anti-cancer drugs. Mechanisms that might explain the antitumor activities of artemisinin derivatives reportedly induction of apoptosis, angiogenesis inhibitory effects, inhibition of hypoxia-inducible factor-1α (HIF-1α) activation, and direct DNA injury. Reactive oxygen species (ROS) generation is involved in many cases. However, little is known about the mechanism of ROS formation from artemisinin derivatives and what types of ROS are produced. Therefore, we investigated the iron-induced ROS formation mechanism by using artesunate, a water-soluble artemisinin derivative, which is thought to be the underlying mechanism involved in artesunate-mediated cell death. The ROS generated by the coexistence of iron(II), artesunate, and molecular oxygen was a hydroxyl radical or hydroxyl radical-like ROS. Artesunate can reduce iron(III) to iron(II), which enables generation of ROS irrespective of the iron valence. We found that reduction from iron(III) to iron(II) was activated in the acidic rather than the neutral region and was proportional to the hydrogen ion concentration.

Nitrosonifedipine, a Photodegradation Product of Nifedipine, Suppresses Pharmacologically Induced Aortic Aneurysm Formation.

BACKGROUND/AIMS: We have reported that nitrosonifedipine (NO-NIF), a photodegradation product of nifedipine, has strong antioxidant and endothelial protective effects, and can suppress several cardiovascular diseases in animal models. The objective of the present study was to investigate the effects of NO-NIF on aortic aneurysm formation. METHODS: The mice were infused with ß-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. The oxidative stress was measured by dihydroethidium staining and nitrotyrosine staining. The expressions of inflammation-related genes were assessed by quantitative real-time PCR and immunohistochemical staining. To clarify the mechanisms of how NO-NIF suppresses vascular cell adhesion molecule (VCAM)-1, endothelial cells were used in in vitro system. RESULTS: NO-NIF suppressed pharmacologically induced the aortic aneurysm formation and aortic expansion without blood pressure changes. NO-NIF suppressed elastin degradation and matrix metalloproteinase-2 mRNA expression. NO-NIF suppressed the reactive oxygen species-cyclophilin A positive feedback loop. Upregulated mRNA expressions of inflammation-related genes and endothelial VCAM-1 were suppressed by NO-NIF co-treatment in aortae. CONCLUSION: NO-NIF has the potential to be a new, nifedipine-derived therapeutic drug for suppressing aortic aneurysm formation by directly improving aortic structure with its strong ability to reduce oxidative stress and inflammation.

Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation.

Renal anemia is a major complication in chronic kidney disease (CKD). Iron supplementation, as well as erythropoiesis-stimulating agents, are widely used for treatment of renal anemia. However, excess iron causes oxidative stress via the Fenton reaction, and iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CKD. EPO gene expression was suppressed in mice following direct iron treatment. Hypoxia-inducible factor-2 alpha (HIF-2α), a positive regulator of the EPO gene, was also diminished in the kidney of mice following iron treatment. Anemia-induced increase in renal EPO and HIF-2α expression was inhibited by iron treatment. In in vitro experiments using EPO-producing HepG2 cells, iron stimulation reduced the expression of the EPO gene, as well as HIF-2α. Moreover, iron treatment augmented oxidative stress, and iron-induced reduction of EPO and HIF-2α expression was restored by tempol, an antioxidant compound. HIF-2α interaction with the Epo promoter was inhibited by iron treatment, and was restored by tempol. These findings suggested that iron supplementation reduced EPO gene expression via an oxidative stress-HIF-2α-dependent signaling pathway.

Topical application of nitrosonifedipine, a novel radical scavenger, ameliorates ischemic skin flap necrosis in a mouse model.

Ischemic skin flap necrosis can occur in random pattern flaps. An excess amount of reactive oxygen species is generated and causes necrosis in the ischemic tissue. Nitrosonifedipine (NO-NIF) has been demonstrated to possess potent radical scavenging ability. However, there has been no study on the effects of NO-NIF on ischemic skin flap necrosis. Therefore, they evaluated the potential of NO-NIF in ameliorating ischemic skin flap necrosis in a mouse model. A random pattern skin flap (1.0 × 3.0 cm) was elevated on the dorsum of C57BL/6 mice. NO-NIF was administered by topical injection immediately after surgery and every 24 hours thereafter. Flap survival was evaluated on postoperative day 7. Tissue samples from the skin flaps were harvested on postoperative days 1 and 3 to analyze oxidative stress, apoptosis and endothelial dysfunction. The viable area of the flap in the NO-NIF group was significantly increased (78.30 ± 7.041%) compared with that of the control group (47.77 ± 6.549%, p < 0.01). NO-NIF reduced oxidative stress, apoptosis and endothelial dysfunction, which were evidenced by the decrease of malondialdehyde, p22phox protein expression, number of apoptotic cells, phosphorylated p38 MAPK protein expression, and vascular cell adhesion molecule-1 protein expression while endothelial nitric oxide synthase protein expression was increased. In conclusion, they demonstrated that NO-NIF ameliorated ischemic skin flap necrosis by reducing oxidative stress, apoptosis, and endothelial dysfunction. NO-NIF is considered to be a candidate for the treatment of ischemic flap necrosis.

Nitrite Activates 5'AMP-Activated Protein Kinase-Endothelial Nitric Oxide Synthase Pathway in Human Glomerular Endothelial Cells.

Recent studies have shown that orally supplied nitrates, which substantially exist in our daily diets, are reduced into nitrites and become significant sources of nitric oxide (NO) especially in hypoxic tissues. However, physiological significance of nitrites in normal tissues has not been elucidated though our serum concentrations of nitrites reach as high as micromolar levels. We investigated effects of nitrite on endothelial NO synthase (eNOS) using human glomerular endothelial cells to reveal potential glomerular-protective actions of nitrites with its underlying molecular mechanism. Here we demonstrate that nitrite stimulation evokes eNOS activation which is dependent on 5'AMP-activated protein kinase (AMPK) activation in accordance with ATP reduction. Thus, nitrites should facilitate AMPK-eNOS pathway in an energy level-dependent manner in endothelial cells. The activation of AMPK-eNOS signals is suggested to be involved in vascular and renal protective effects of nitrites and nitrates. Nitrites may harbor beneficial effects on metabolic regulations as AMPK activators.

Spontaneously hyperactive MEK-Erk pathway mediates paradoxical facilitation of cell proliferation in mild hypoxia.

BACKGROUND: Oxygen is important for common eukaryotic cells to generate ATP. Pathophysiological conditions such as ischemic diseases cause tissue hypoxia. In addition, oxygen availability in deep tissues is supposed to be far lower than surrounding atmosphere even in healthy animals, and the oxygen partial pressures in most normal tissues are estimated to be around 40-50mmHg, so-called mild hypoxia. Recent studies have demonstrated that mild hypoxia has distinct effects on living cells from severe hypoxia. For instance, mild hypoxia was reported to promote cell reprogramming. Although severe hypoxia is known to inhibit cell proliferation, mild hypoxia has been paradoxically demonstrated to increase cell proliferation. However, it has not been clarified by which molecular mechanisms mild hypoxia evokes the discontinuous increment of cell proliferation. METHODS: We established experimental conditions showing the opposite influences of mild and severe hypoxia on cell proliferation using undifferentiated Caco2 human colon carcinoma cells in order to clarify the underlying molecular mechanism. RESULTS: The basal activity of Erk, which is a typical mediator of mitogenic signals, is spontaneously increased specifically in cells exposed to mild hypoxia, and inhibition of MEK, an upstream kinase of the Erk, completely inhibited the mild hypoxia-induced enhancement of cell proliferation. CONCLUSIONS: Spontaneous hyperactivation of the MEK-Erk pathway by mild hypoxia should be the plausible molecular mechanism of the paradoxical promotion of cell proliferation. GENERAL SIGNIFICANCE: Our findings will provide clues to the molecular basis of mild hypoxia-evoked phenomena such as cell reprogramming.

Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM.

HIF-2α/ARNT complex regulates hair development via induction of p21(Waf1/Cip1) and p27(Kip1).

The hypoxia-inducible factors HIF-1α or HIF-2α form heterodimeric complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1α/ARNT and HIF-2α/ARNT complexes activate hypoxia-inducible genes that play critical roles in angiogenesis, anaerobic metabolism, and other processes in response to O2 deprivation. HIF-2α is known to regulate the function and/or differentiation of stem cells by activating the POU domain transcription factor Oct4; however, the precise underlying mechanism is unknown. This study examined the role of HIF-2α/ARNT in hair development using conditional-knockout mice, in which Arnt was specifically deleted in keratinocytes. In wild-type mice, HIF-2α and ARNT were highly expressed in the precortex above the hair matrix, an area containing differentiating stem cells. An analysis of hair size and type in these mice showed that loss of ARNT decreased the production of zigzag hairs, corresponding to reduced expression of HIF-2α and induction of the mammalian cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27 (Kip1). The results suggest that the HIF-2α/ARNT complex regulates hair follicle differentiation via induction of p21(Waf1/Cip1) and possibly p27(Kip1), as p27(Kip1) expression was not altered in ARNT knockout mice. The findings provide insight into a possible mechanism underlying hair growth disorders and can be useful for future studies on hair follicle response to insults, such as chemotherapy and ionizing radiation.

Angiotensin II alters the expression of duodenal iron transporters, hepatic hepcidin, and body iron distribution in mice.

PURPOSE: Angiotensin II (ANG II) has been shown to affect iron metabolism through alteration of iron transporters, leading to increased cellular and tissue iron contents. Serum ferritin, a marker of body iron storage, is elevated in various cardiovascular diseases, including hypertension. However, the associated changes in iron absorption and the mechanism underlying increased iron content in a hypertensive state remain unclear. METHODS: The C57BL6/J mice were treated with ANG II to generate a model of hypertension. Mice were divided into three groups: (1) control, (2) ANG II-treated, and (3) ANG II-treated and ANG II receptor blocker (ARB)-administered (ANG II-ARB) groups. RESULTS: Mice treated with ANG II showed increased serum ferritin levels compared to vehicle-treated control mice. In ANG II-treated mice, duodenal divalent metal transporter-1 and ferroportin (FPN) expression levels were increased and hepatic hepcidin mRNA expression and serum hepcidin concentration were reduced. The mRNA expression of bone morphogenetic protein 6 and CCAAT/enhancer-binding protein alpha, which are regulators of hepcidin, was also down-regulated in the livers of ANG II-treated mice. In terms of tissue iron content, macrophage iron content and renal iron content were increased by ANG II treatment, and these increases were associated with reduced expression of transferrin receptor 1 and FPN and increased expression of ferritin. These changes induced by ANG II treatment were ameliorated by the administration of an ARB. CONCLUSIONS: Angiotensin II (ANG II) altered the expression of duodenal iron transporters and reduced hepcidin levels, contributing to the alteration of body iron distribution.

Hypoxia decreases glucagon-like peptide-1 secretion from the GLUTag cell line.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.

Changes in intestinal microbiota and biochemical parameters in patients with inflammatory bowel disease and irritable bowel syndrome induced by the prolonged addition of soluble fibers to usual drug therapy.

OBJECTIVES: Partially hydrolyzed guar gum (PHGG) is a soluble dietary fiber;in addition to improving bowel movements, it maintains intestinal health by producing short-chain fatty acids. However, majority of clinical studies on PHGG have been concluded within a month and excluded usual drug therapy. Hence, this study aimed to determine the effects of long-term consumption of PHGG, in combination with drug therapy, on gut bacteria ratios, laboratory values for inflammatory response, and fecal characteristics. METHODS AND RESULTS: The study was performed in patients with irritable bowel syndrome (IBS), Crohn's disease (CD), and ulcerative colitis (UC), by the administration of PHGG for six months while they continued their usual treatment. PHGG treatment caused significant changes in patients with IBS, including an increase in the abundance of short-chain fatty acid-producing bacteria, a significant decrease in Bacteroides abundance, and normalization of the Bristol scale of stool. In patients with UC, non-significant normalization of soft stools and decrease in fecal calprotectin were observed. Adverse events were not observed in any of the groups. CONCLUSION: Thus, it would be beneficial to include PHGG in the usual drug therapies of patients with IBS. J. Med. Invest. 71 : 121-128, February, 2024.