Inhibitory Effects of Hydrolysable Tannins on Lipid Accumulation in 3T3-L1 Cells.

Obesity is currently the most common cause of metabolic diseases including type 2 diabetes and hyperlipidemia. Obesity results from excess lipid accumulation in adipose tissue. Several studies have investigated the inhibitory effects of natural plant-derived products on adipocyte differentiation and lipid accumulation. In this study, we examined the effect of hydrolysable tannins composed of gallic acid and glucose on adipocyte differentiation in 3T3-L1 cells. 1,2,3,4,6-Penta-O-galloyl-ß-D-glucose (PGG) (1), a representative gallotannin, inhibited lipid accumulation in 3T3-L1 cells, whereas ellagitannins (tellimagrandin I, eugeniin and casuarictin) did not. The expression of adipocyte differentiation-related genes, including peroxisome proliferator activator γ2 (Pparγ2), CCAAT/enhancer binding protein α (C/EBPα) and adipocyte fatty acid binding protein (aP2), was significantly suppressed in PGG (1)-treated 3T3-L1 cells beginning at day 2 after induction of differentiation. While PGG (1) did not directly reduce Pparγ2 expression, it reduced the expression of its target genes in mature adipocytes. In addition, PGG (1) treatment inhibited mitotic clonal expansion, one of earliest events of adipocyte differentiation. These findings indicate that PGG (1) has an inhibitory effect on adipocyte differentiation through the suppression of mitotic clonal expansion.

Synthesis and Evaluation of Gallotannin Derivatives as Antioxidants and α-Glucosidase Inhibitors.

Gallotannins are phenolic natural products containing galloyl moieties connected to polyhydric alcohol cores, e.g., D-glucose. Some gallotannins are reported to have antidiabetic properties, such as α-glucosidase inhibitory activity. In this study, fourteen unnatural gallotannin derivatives with 1,5-anhydroalditol and inositol as the cyclic polyol cores were synthesized to investigate how their structures affected antioxidative and α-glucosidase inhibitory activities. Tannic acid demonstrated the most potent antioxidative activity (EC50 = 2.84 µM), with potency increasing proportionally to the number of galloyl moieties. Synthetic inositol derivatives outperformed 1,5-anhydroalditol derivatives in rat α-glucosidase inhibitory activity. Pentagalloyl glucose, a natural compound, demonstrated the highest activity (IC50 = 0.336 µM).

Inhibitory Effects of Indirubin-3'-oxime Derivatives on Lipid Accumulation in 3T3-L1 Cells.

Obesity elevates the risk of cardiovascular disease and has been strongly associated with increases in the incidence of many metabolic diseases. Therefore, prevention of obesity leads to the prevention of metabolic diseases. In light of this, substances that exert anti-obesity effects are crucial for the prevention of obesity. Indirubin, a 3,2' bisindole isomer of indigo, is the active component of the traditional Chinese medicine used for the treatment of chronic myelocytic leukemia. In particular, indirubin-3'-oxime (1) was shown to inhibit the differentiation of adipocytes. In this study, we investigated the inhibitory effects of nine indirubin-3'-oxime derivatives against lipid accumulation during differentiation in 3T3-L1 cells. Among the compounds tested, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) at 5 µM exhibited significantly stronger inhibitory activity than indirubin-3'-oxime (1). Furthermore, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) markedly suppressed the expression of CCAAT/enhancer-binding protein α, peroxisome proliferator activator γ2, and adipocyte protein 2, both of which are key adipogenic regulators at the intermediate stage of adipocyte differentiation. Our results demonstrate that 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) significantly down-regulated lipid accumulation during differentiation of 3T3-L1 cells, suggesting their potential as novel therapeutic drugs against the development of obesity.

Severe chronic kidney disease environment reduced calcium-sensing receptor expression in parathyroid glands of adenine-induced rats even without high phosphorus diet.

BACKGROUND: Chronic kidney disease (CKD) disrupts mineral homeostasis and its main underlying cause is secondary hyperparathyroidism (SHPT). We previously reported that calcium-sensing receptor (CaSR) mRNA and protein expression in parathyroid glands (PTGs) significantly decreased in a CKD rat model induced by a 5/6 nephrectomy that were fed a high phosphorus diet. However, there was a significant difference in the severity of CKD between high phosphorus and adequate phosphorus diet groups. Thus, it was unclear whether CKD environment or the high phosphorus diet influenced CaSR expression, and the underlying mechanism remains largely unknown. METHODS: CKD was induced in rats with 0.75% adenine-containing diet. CKD and control rats were maintained for 5 days and 2 weeks on diets with 0.7% or 1.3% phosphorus. For gene expression analysis, quantitative real-time polymerase chain reaction was performed with TaqMan probes. Protein expression was analyzed by immunohistochemistry. RESULTS: PTG CaSR expression significantly decreased in the presence of a severe CKD environment, even without the high phosphate load. Ki67 expressing cells in PTGs were significantly higher only in the CKD rats fed a high phosphorus diet. Furthermore, among the many genes that could affect CaSR expression, only vitamin D receptor (VDR) and glial cells missing 2 (Gcm2) showed significant changes. Moreover, Gcm2 was significantly reduced at an early stage without significant changes in serum calcium, phosphorus and 1,25(OH)2 vitamin D, and there was no significant reduction in CaSR and VDR expressions. Then, significantly elevated Ki67-positive cell numbers were also only observed in the early CKD PTGs with high-phosphorus diets. CONCLUSIONS: Our data suggest that the cause of the decreased PTG CaSR expression is the reduction in VDR and Gcm2 expression; Gcm2 may play a role in the onset and progression of SHPT.

Asymmetric N-H Insertion Reaction with Chiral Aminoalcohol as Catalytic Core of Cinchona Alkaloids.

In order to develop an efficient organocatalyst for the enantioselective N-H insertion reaction via carbene/carbenoid, the catalytic core of the cinchona alkaloids was investigated. According to our working hypothesis of an eight-membered ring transition state in the N-H insertion reaction, two pairs of enantiomers related to 2-amino-1-phenylethanol were investigated for their chiral inducing potential. Since both (1R,2S)-isomers gave the N-phenyl-1-phenylglycine derivative enriched in the R-form, while their enantiomers gave the S-form, the 2-amino-1-phenylethanol structure is concluded to be the catalytic core of the cinchona alkaloid in the enantioselective N-H insertion reaction via rhodium(II) carbenoid.

Synthesis and Comparative Structure-Activity Study of Carbohydrate-Based Phenolic Compounds as α-Glucosidase Inhibitors and Antioxidants.

Twenty-one natural and unnatural phenolic compounds containing a carbohydrate moiety were synthesized and their structure-activity relationship (SAR) was evaluated for α-glucosidase inhibition and antioxidative activity. Varying the position of the galloyl unit on the 1,5-anhydro-d-glucitol (1,5-AG) core resulted in changes in the α-glucosidase inhibitory activity and notably, particularly strong activity was demonstrated when the galloyl unit was present at the C-2 position. Furthermore, increasing the number of the galloyl units significantly affected the α-glucosidase inhibition, and 2,3,4,6-tetra-galloyl-1,5-AG (54) and 2,3,4,6-tetra-galloyl-d-glucopyranose (61) exhibited excellent activities, which were more than 13-fold higher than the α-glucosidase inhibitory activity of acertannin (37). Moreover, a comparative structure-activity study suggested that a hemiacetal hydroxyl functionality in the carbohydrate core and a biaryl bond of the 4,6-O-hexahydroxydiphenoyl (HHDP) group, which are components of ellagitannins including tellimagrandin I, are not necessary for the α-glucosidase inhibitory activity. Lastly, the antioxidant activity increased proportionally with the number of galloyl units.

Optimal route of diphtheria toxin administration to eliminate native nephron progenitor cells in vivo for kidney regeneration.

To address the lack of organs for transplantation, we previously developed a method for organ regeneration in which nephron progenitor cell (NPC) replacement is performed via the diphtheria toxin receptor (DTR) system. In transgenic mice with NPC-specific expression of DTR, NPCs were eliminated by DT and replaced with NPCs lacking the DTR with the ability to differentiate into nephrons. However, this method has only been verified in vitro. For applications to natural models, such as animal fetuses, it is necessary to determine the optimal administration route and dose of DT. In this study, two DT administration routes (intra-peritoneal and intra-amniotic injection) were evaluated in fetal mice. The fetus was delivered by caesarean section at E18.5, and the fetal mouse kidney and RNA expression were evaluated. Additionally, the effect of the DT dose (25, 5, 0.5, and 0.05 ng/fetus-body) was studied. Intra-amniotic injection of DT led to a reduction in kidney volume, loss of glomeruli, and decreased differentiation marker expression. The intra-peritoneal route was not sufficient for NPC elimination. By establishing that intra-amniotic injection is the optimal administration route for DT, these results will facilitate studies of kidney regeneration in vivo. In addition, this method might be useful for analysis of kidney development at various time points by deleting NPCs during development.

Deletion of Bmal1 Prevents Diet-Induced Ectopic Fat Accumulation by Controlling Oxidative Capacity in the Skeletal Muscle.

Brain and muscle arnt-like protein 1 (BMAL1), is a transcription factor known to regulate circadian rhythm. BMAL1 was originally characterized by its high expression in the skeletal muscle. Since the skeletal muscle is the dominant organ system in energy metabolism, the possible functions of BMAL1 in the skeletal muscle include the control of metabolism. Here, we established that its involvement in the regulation of oxidative capacity in the skeletal muscle. Muscle-specific Bmal1 KO mice (MKO mice) displayed several physiological hallmarks for the increase of oxidative capacity. This included increased energy expenditure and oxygen consumption, high running endurance and resistance to obesity with improved metabolic profiles. Also, the phosphorylation status of AMP-activated protein kinase and its downstream signaling substrate acetyl-CoA carboxylase in the MKO mice were substantially higher than those in the Bmal1flox/flox mice. In addition, biochemical and histological studies confirmed the substantial activation of oxidative fibers in the skeletal muscle of the MKO mice. The mechanism includes the regulation of Cacna1s expression, followed by the activation of calcium-nuclear factor of activated T cells (NFAT) axis. We thus conclude that BMAL1 is a critical regulator of the muscular fatty acid level under nutrition overloading and that the mechanism involves the control of oxidative capacity.

[Fragility Fractures in Hemodialysis Patients. Abnormal bone material characteristics of dialysis patients.]

The relationship between renal dysfunction and bone fragility is well known. Bone fragility, which is associated with an increased risk of bone fracture, affects quality of life and prognosis in patients with chronic kidney disease(CKD). The bone of patients with CKD is more fragile than the risk estimated by measurement of bone mineral density. Bone quality, which is defined by many factors including bone turnover, microarchitecture, collagen crosslinking, and matrix composition, is considered to be major cause of bone fragility of the patients with CKD. In this article, we will describe the bone quality of dialysis patients with CKD as representatives of patients with CKD.

5-Bromoindirubin 3'-(O-oxiran-2-ylmethyl)oxime: A long-acting anticancer agent and a suicide inhibitor for epoxide hydrolase.

Indirubin 3'-oxime (Indox (1b)) suppresses cancer cell growth (IC50: 15µM towards HepG2 cells) and inhibits cell cycle-related kinases such as cyclin-dependent kinases and glycogen synthase kinase-3ß. We have previously reported that the conjugation of 1b with oxirane, a protein-reactive component, enhanced the cytotoxic activity of Indox as determined from the IC50 value (1.7µM) of indirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/Ind (1c)). Here we prepared Epox/Ind derivatives with one or two halogen atoms or a methoxy group on the aromatic ring(s) of an Indox moiety and studied the structure-activity relationships of the substituent(s). We found that bromine-substitution at the 5-position on 1c or any Epox/Ind derivative(s) having bromine on the aromatic ring except Epox/6'-Br-Ind was efficient to improving anticancer activity. Of the 22 Epox/Ind derivatives, 5-bromoindirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/5-Br-Ind (2c)) was the best anticancer agent in both short- (24h) (IC50: 0.67µM) and extended-duration (72h) cultures. The high anticancer activity of 2c was partly due to it being a poor substrate and a suicide inhibitor for epoxide hydrolase as epoxide hydrolase was identified as the enzyme primarily responsible for the metabolism of 2c.

Neuroprotective effect of S-allyl-l-cysteine derivatives against endoplasmic reticulum stress-induced cytotoxicity is independent of calpain inhibition.

S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on µ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC.

Indirubin 3'-(O-oxiran-2-ylmethyl)oxime: a novel anticancer agent.

Indirubin is a potent inhibitor of cell cycle-related protein kinases by binding to the ATP-binding site and thus is a promising compound for development as an antitumor drug. We prepared indirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/Ind), in which the ATP-binding site orientated part was attached by non-specific alkylating group. The IC50 value of Epox/Ind at 1.7 µM in HepG2 cells is comparable to that of cisplatin (4.0 µM). Furthermore, Epox/Ind was shown to be metabolized by a HepG2 cell lysate into indirubin 3'-(O-2,3-dihydroxypropyl)oxime (E804), the sole extractable metabolite. The lower toxicity of this metabolite may explain the lack of cytotoxicity of 1 µM Epox/Ind observed in HepG2 cells beyond an initial loss of viability in the first 24h of treatment.

Sesquiterpene lactones derived from Saussurea lappa induce apoptosis and inhibit invasion and migration in neuroblastoma cells.

Neuroblastoma is among the most fatal of solid tumors in the pediatric age group, even when treated aggressively. Therefore, a new effective therapeutic drug(s) for neuroblastoma is urgently needed. To clarify the anticancer effects of the sesquiterpene lactones dehydrocostus lactone and costunolide, derived from Saussurea lappa, we examined the cytotoxic and migration/invasion-inhibitory effects of these compounds against neuroblastoma cell lines. Both the compounds exerted significant cytotoxicity against the neuroblastoma cell lines IMR-32, NB-39, SK-N-SH, and LA-N-1. Evidence of cellular apoptosis, such as nuclear condensation and membrane inversion, were observed after treatment with these compounds. Both compounds induced caspase-7 activation and PARP cleavage as confirmed by Western blotting. Furthermore, the sesquiterpene lactones also suppressed invasion and migration of the neuroblastoma cells. These results suggest that dehydrocostus lactone and costunolide are promising candidates for being developed into novel anticancer drugs effective against neuroblastoma.

Adiponectin regulates the circadian rhythm of glucose and lipid metabolism.

Adiponectin is a cytokine secreted from adipocytes and regulates metabolism. Although serum adiponectin levels show diurnal variations, it is not clear if the effects of adiponectin are time-dependent. Therefore, this study conducted locomotor activity analyses and various metabolic studies using the adiponectin knockout (APN (-/-)) and the APN (+/+) mice to understand whether adiponectin regulates the circadian rhythm of glucose and lipid metabolism. We observed that the adiponectin gene deficiency does not affect the rhythmicity of core circadian clock genes expression in several peripheral tissues. In contrast, the adiponectin gene deficiency alters the circadian rhythms of liver and serum lipid levels and results in the loss of the time dependency of very-low-density lipoprotein-triglyceride secretion from the liver. In addition, the whole-body glucose tolerance of the APN (-/-) mice was normal at CT10 but reduced at CT22, compared to the APN (+/+) mice. The decreased glucose tolerance at CT22 was associated with insulin hyposecretion in vivo. In contrast, the gluconeogenesis activity was higher in the APN (-/-) mice than in the APN (+/+) mice throughout the day. These results indicate that adiponectin regulates part of the circadian rhythm of metabolism in the liver.

Hypercalcemia caused by comorbid parathyroid adenoma and pulmonary tuberculosis.

Hypercalcemia is usually secondary to one etiology, although two coexisting etiologies can rarely cause hypercalcemia. Here, we report a 47-year-old woman with hypercalcemia caused by comorbid parathyroid adenoma and pulmonary tuberculosis. Primary hyperparathyroidism is the most common cause of hypercalcemia. Tuberculosis is a rare cause of hypercalcemia, but Japan continues to have an intermediate tuberculosis burden. Therefore, tuberculosis should be considered as a cause of hypercalcemia in Japan. Patients with tuberculosis are often asymptomatic, making the diagnosis difficult. In the previous cases in which these diseases coexisted, one disease was diagnosed after treatment of the other. In our case, the very high 1,25-dihydroxyvitamin D level (162 pg/mL) helped us to diagnose asymptomatic tuberculosis and both diseases were diagnosed promptly. It is necessary to consider comorbidities, including tuberculosis in a case with a very high 1,25-dihydroxyvitamin D level. We report a valuable case in which the early diagnosis and treatment of tuberculosis and primary hyperparathyroidism prevented the spread of tuberculosis.

Chiral Induction by cinchona alkaloids in the rhodium(II) catalyzed O-H insertion reaction.

Cinchona alkaloids are effective additives for enantioselective O-H insertion of alpha-phenyldiazoacetate and water by rhodium(II) complexes. Addition of silica gel promotes O-H insertion in the reaction rate and the reaction proceeds smoothly at less than the freezing point of water, e.g., -10 degrees C, and provided mandelate in up to 50% ee. The results reported here are the highest asymmetric inductions obtained to date for O-H insertions via a Rh-carbenoid.

Induction of apoptosis in human leukemia cell (Jurkat) by neolignans isolated from seeds of Licaria puchury-major.

Ethanol extract of the seeds of Licaria puchury-major, a Brazilian herbal medicine, was found to inhibit cell proliferation in human leukemia cell line (Jurkat) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bioassay-guided fractionation of the active components led to the isolation of one phenylpropanoid (1) and ten neolignans (2-11). The apoptosis-inducing activity of the compounds showing cytotoxicity in Jurkat cells was assessed by flow cytometric analysis. Among the identified neolignans, compounds 3, 4, 6 and 7 which have similar molecular structures, showed apoptotic activity. To elucidate the mechanism of apoptosis induction by neolignans, intracellular caspase-3, -8 and -9 activity in Jurkat cells was evaluated. Compound 4 markedly elevated the activity of caspase-3 and -9.

Effects of synthetic para-nonylphenol isomers administered chronically throughout pregnancy and lactation on reproductive system of mouse pups.

The aim of this study was to analyze the effects of synthetic para-nonylphenol isomers administered chronically throughout pregnancy and lactation on the reproductive system of mouse pups. The two synthetic isomers used in this study showed higher (3E22NP) or lower (44NP) estrogen receptor (ER) binding activity on in vitro yeast assay than a commercial NP (NPmix). Female mice were implanted with a tube filled with one of three NPs and estradiol-17 ß (E2) before mating. The tube was kept in the mice throughout pregnancy and lactation period, until their pups had weaned at 28 days of age (PND 28). The data indicated that in males, NPs decreased body weight in some dose groups on PND28 and increased weights of testes and epididymides. However, the rate of seminiferous tubules having elongate spermatids (steps 10-16) decreased significantly in NPs and E2 treated groups, except for the 44NP groups. In female mice, NPs and E2 increased weights of ovaries, uterus and vagina in the pups in some dose groups on PND 28. However, there were no differences in the day of vaginal opening and numbers of corpus lutea. The present study demonstrates that the effects of these two isomers of NP on the pup reproduction at PND 28 are not quite different, despite them showing different levels of ER binding activity using in vitro yeast assay.

Gcm2 regulates the maintenance of parathyroid cells in adult mice.

Glial cells missing homolog 2 (GCM2), a zinc finger-type transcription factor, is essential for the development of parathyroid glands. It is considered to be a master regulator because the glands do not form when Gcm2 is deficient. Remarkably, Gcm2 expression is maintained throughout the fetal stage and after birth. Considering the Gcm2 function in embryonic stages, it is predicted that Gcm2 maintains parathyroid cell differentiation and survival in adults. However, there is a lack of research regarding the function of Gcm2 in adulthood. Therefore, we analyzed Gcm2 function in adult tamoxifen-inducible Gcm2 conditional knockout mice. One month after tamoxifen injection, Gcm2-knockout mice showed no significant difference in serum calcium, phosphate, and PTH levels and in the expressions of calcium-sensing receptor (Casr) and parathyroid hormone (Pth), whereas Ki-67 positive cells were decreased and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) positive cell number did not change, as compared with those of controls. Seven months after tamoxifen injection, Gcm2-knockout mice showed shrinkage of the parathyroid glands and fewer parathyroid cells. A significant decrease was noted in Casr- and Pth-expressing cells and serum PTH and Ca levels, whereas serum phosphate levels increased, as compared with those of controls. All our results concluded that a reduction of Gcm2 expression leads to a reduction of parathyroid cell proliferation, an increase in cell death, and an attenuation of parathyroid function. Therefore, we indicate that Gcm2 plays a prominent role in adult parathyroid cell proliferation and maintenance.

Syntheses and estrogenic activity of 4-nonylphenol isomers.

Eight branched 4-nonylphenol (NP) isomers, which were identified from commercially available NP reagent, 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP-C), 4-(1,1-dimethyl-3-ethylpentyl)phenol (NP-D), 4-(1,3-dimethyl-1-ethylpentyl)phenol (NP-E(G)), diastereomeric mixture), 4-(1,1,4-trimethylhexyl)phenol (NP-F), 4-(1-methyl-1-n-propylpentyl)phenol (NP-H), 4-(1,1-dimethyl-2-ethylpentyl)phenol (NP-I), 4-(1,1,2-trimethylhexyl)phenol (NP-M), and 4-(1-ethyl-1-methylhexyl)phenol (NP-N), were synthesized by two different synthetic methods starting from 4-benzyloxyacetophenone or phenol. The chemical structures of the synthesized compounds were confirmed by MS and NMR spectroscopy. The estrogenic activities of these synthetic NP isomers were tested and exhibited different activities on the recombinant yeast screen system. NP-I was found to exhibit three times greater estrogenic activity than the commercial NP mixture.