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Aijaz A John, Ravi Prakash and Divya Singh

miRNAs have appeared as critical controllers of gene expression at post-transcriptional level either by degrading RNA transcripts or repressing translation. It is evident from the ever-growing scientific literature that miRNAs play a significant role in osteoblast commitment and differentiation. Here, we report that overexpression of miR-487b-3p leads to inhibition of osteoblastic differentiation. Using in silico approaches, Nrarp was found to be the direct target of miR-487b-3p, which was further validated by luciferase 3′ UTR reporter assay. Nrarp inhibits Notch-1 signaling and promotes Wnt signaling by stabilization of LEF-1. Role of miR-487b-3p in regulating canonical Wnt and Notch signaling was determined by western blotting. Protein levels of Nrarp, RUNX-2, Lef1 and β catenin were reduced in osteoblasts cells transfected with miR-487b-3p, whereas protein levels of Notch1, Hes1 and P-β catenin were upregulated when osteoblast cells were transfected with miR-487b-3p. These outcomes were reversed after treating cells with anti-miR-487b-3p. Further silencing of miR-487b-3p in neonatal Balb/c mice attenuated all the inhibitory actions of miR-487b-3p on osteoblast differentiation. Importantly, in vivo action of anti-miR-487b-3p to ovariectomized osteopenic BALB/c mice steered to significant enhancement in trabecular bone microarchitecture. Furthermore, the bio-mechanical properties of isolated femurs were enhanced in anti-miR-487b-3p-treated mice. Overall, miR-487b-3p negatively regulates osteogenesis by suppressing Nrarp expression, which in turn, suppresses Runx-2 and Wnt signaling, both of which play a pivotal action in osteoblast differentiation.

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Bo-Kyung Son, Taro Kojima, Sumito Ogawa and Masahiro Akishita

Abdominal aortic aneurysm (AAA), one of the pathological phenotypes of vascular aging, is characterized by aortic dilation with impaired arterial wall integrity. Recent epidemiologic studies have shown that men with AAA have lower serum testosterone compared to men without. However, the underlying mechanisms remain unclear. In this study, we investigated the effects of testosterone on AAA formation using a murine AAA model under the conditions of depletion and administration of testosterone. In wild-type male mice (C57BL/6J), AAA was induced by CaCl2 application and angiotensin II infusion at 5 weeks after castration. Exacerbated AAA formation was seen in castrated mice, compared with sham-operated mice. Histological analysis revealed marked infiltration of macrophages in the destroyed aorta and IL-6/pSTAT3 expression was significantly elevated, suggesting that AAA development by castration is attributable to pronounced inflammation. Conversely, both 4-week and 9-week administration of testosterone significantly prevented AAA formation, and improvement of histological findings was confirmed. Aortic F4/80, Il-1b and Il-6 expression were significantly inhibited both by testosterone administration. Indeed, mice with implanted flutamide exhibited exacerbated AAA formation and aortic F4/80, Il-1b and Il-6 expression were significantly increased. Taken together, these results demonstrate that testosterone depletion and AR blockade precede AAA formation, and conversely, testosterone administration could suppress AAA formation by regulating macrophage-mediated inflammatory responses. This anti-inflammatory action of testosterone/AR on AAA formation might provide a mechanistic insight into the vascular protective actions of testosterone and suggest that its proper administration or selective AR modulators might be novel therapeutic strategies for this aortic pathology.

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Shanqi Fu, Miho Kuwahara, Yoko Uchida, Sei Kondo, Daichi Hayashi, Yuji Shimomura, Asami Takagaki, Takashi Nishida, Yusuke Maruyama, Mika Ikegame, Atsuhiko Hattori, Satoshi Kubota and Takako Hattori

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary BALB/c mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan and Sox9, but inhibited Col10a1 expression. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2 and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1 and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

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Kira G Slepchenko, Kathryn L Corbin and Craig S Nunemaker

Glucose-stimulated insulin secretion (GSIS) is a well-accepted method to investigate the physiological and pathophysiological function of islets. However, there is little consensus about which method is best for normalizing and presenting GSIS data. In this study, we evaluated the sufficiency of islet area, total protein, total DNA and total insulin content as parameters to normalize GSIS data. First, we tested if there is a linear correlation between each parameter and the number of islets (10, 20, 30 and 40 islets). Islet area, total protein and insulin content produced excellent linear correlations with islet number (R 2 > 0.9 for each) from the same islet material. Insulin secretion in 11 mM glucose also correlated reasonably well for islet area (R 2 = 0.69), protein (R 2 = 0.49) and insulin content (R 2 = 0.58). DNA content was difficult to reliably measure and was excluded from additional comparisons. We next measured GSIS for 18 replicates of 20 islets each, measuring 3 mM and 11 mM glucose to calculate the stimulation index and to compare each normalization parameter. Using these similar islet masses for each replicate, none of the parameters produced linear correlations with GSIS (R 2 < 0.05), suggesting that inherent differences in GSIS dominate small differences in islet mass. We conclude that when comparing GSIS for islets of reasonably similar size (<50% variance), normalization does not improve the representation of GSIS data. Normalization may be beneficial when substantial differences in islet mass are involved. In such situations, we suggest that using islet cross-sectional area is superior to other commonly used techniques for normalizing GSIS data.

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Anne M Houbrechts, Jolien Van houcke and Veerle M Darras

Thyroid hormones are crucial mediators of many aspects of vertebrate life, including reproduction. The key player is the biologically active 3,5,3’-triiodothyronine (T3), whose local bio-availability is strictly regulated by deiodinase enzymes. Deiodinase type 2 (Dio2) is present in many tissues and is the main enzyme for local T3 production. To unravel its role in different physiological processes, we generated a mutant zebrafish line, completely lacking Dio2 activity. Here we focus on the reproductive phenotype studied at the level of offspring production, gametogenesis, functioning of the hypothalamic–pituitary–gonadal axis and sex steroid production. Homozygous Dio2-deficient zebrafish were hypothyroid, displayed a delay in sexual maturity and the duration of their reproductive period was substantially shortened. Fecundity and fertilization were also severely reduced. Gamete counts pointed to a delay in oogenesis at onset of sexual maturity and later on to an accumulation of oocytes in mutant ovaries due to inhibition of ovulation. Analysis of spermatogenesis showed a strongly decreased number of spermatogonia A at onset of sexual maturity. Investigation of the hypothalamic–pituitary–gonadal axis revealed that dysregulation was largely confined to the gonads with significant upregulation of igf3, and a strong decrease in sex steroid production concomitant with alterations in gene expression in steroidogenesis/steroid signaling pathways. Rescue of the phenotype by T3 supplementation starting at 4 weeks resulted in normalization of reproductive activity in both sexes. The combined results show that reproductive function in mutants is severely hampered in both sexes, thereby linking the loss of Dio2 activity and the resulting hypothyroidism to reproductive dysfunction.

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Folami Y Ideraabdullah, Anthony M Belenchia, Cheryl S Rosenfeld, Seth W Kullman, Megan Knuth, Debabrata Mahapatra, Michael Bereman, Edward D Levin and Catherine A Peterson

Vitamin D is an essential nutrient that is metabolized in the body to generate an active metabolite (1,25(OH)2D) with hormone-like activity and highly diverse roles in cellular function. Vitamin D deficiency (VDD) is a prevalent but easily preventable nutritional disturbance. Emerging evidence demonstrates the importance of sufficient vitamin D concentrations during fetal life with deficiencies leading to long-term effects into adulthood. Here, we provide a detailed review and perspective of evidence for the role of maternal VDD in offspring long-term health, particularly as it relates to developmental origins of health and disease (DOHaD). We focus on the roles in neurobehavioral and cardiometabolic disorders in humans and highlight recent findings from zebrafish and rodent models that probe potential mechanisms linking early life VDD to later life health outcomes. Moreover, we explore evidence implicating epigenetic mechanisms as a mediator of this link. Gaps in our current understanding of how maternal VDD might result in deleterious offspring outcomes later in life are also addressed.

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Shiyun Tong, Shumin Yang, Ting Li, Rufei Gao, Jinbo Hu, Ting Luo, Hua Qing, Qianna Zhen, Renzhi Hu, Xuan Li, Yi Yang, Chuan Peng and Qifu Li

Bisphenol-A (BPA) is a common environmental pollutant, and exposure to it is associated with proteinuria and may predict the progression of chronic kidney disease; however, the mechanism is not clear. Neutrophil extracellular traps (NETs) are a DNA skeleton coated with various proteases, and it is associated with various types of autoimmune nephritis. In this study, we examine whether NETs is involved in BPA-induced chronic kidney injury. In vivo, BPA exposure resulted in impaired renal function and altered renal morphology, including glomerular mesangial matrix expansion and increased renal interstitial fibroblast markers. Meanwhile, more dsDNA can be detected in the serum, and the NETs-associated proteins, MPO and citH3 were deposited in the renal system. In vitro, BPA and NETs treatment caused podocyte injury, a loss of marker proteins and disorder in the actin skeleton. After NETs inhibition via DNase administration, BPA-induced injuries were significantly relieved. In conclusion, the increase of NETosis in circulation and the renal system during BPA exposure suggests that NETs may be involved in BPA-induced chronic kidney injury.

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Peixin Li, Zhijian Rao, Brenton Thomas Laing, Wyatt Bunner, Taylor Landry, Amber Prete, Yuan Yuan, Zhong-Tao Zhang and Hu Huang

Vertical sleeve gastrectomy (VSG) is an effective surgery to treat obesity and diabetes. However, the direct effect of VSG on metabolic functions is not fully understood. We aimed to investigate if alterations in hypothalamic neurons were linked with perturbations in liver metabolism after VSG in an energy intake-controlled obese mouse model. C57BL/6 and hrNPY-GFP reporter mice received HFD for 12 weeks and were then divided into three groups: Sham (ad lib), Sham (pair-fed) with VSG and VSG. Food intake was measured daily, and blood glucose levels were measured before and after the study. Energy expenditure and body composition were determined. Serum parameters, liver lipid and glycogen contents were measured and gene/protein expression were analyzed. Hypothalamic POMC, AgRP/NPY and tyrosine hydroxylase-expressing neurons were counted. The following results were obtained. VSG reduced body weight gain and adiposity induced by HFD, increased energy expenditure independent of energy intake. Fed and fasted blood glucose levels were reduced in the VSG group. While serum active GLP-1 level was increased, the active ghrelin and triglycerides levels were decreased along with improved insulin resistance in VSG group. Liver lipid accumulation, glycogen content and gluconeogenic gene expression were reduced in the VSG group. In the hypothalamus, TH-expressing neuron population was decreased, and the POMC-expressing neuron population was increased in the VSG group. In conclusion, our data suggest that VSG improves metabolic symptoms by increasing energy expenditure and lowering lipid and glycogen contents in the liver. These physiological alterations are possibly related to changes in hypothalamic neuron populations.

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Elizabeth K Fletcher, Monica Kanki, James Morgan, David W Ray, Lea M Delbridge, Peter J Fuller, Colin D Clyne and Morag J Young

We previously identified a critical pathogenic role for mineralocorticoid receptor (MR) activation in cardiomyocytes that included a potential interaction between the MR and the molecular circadian clock. While glucocorticoid regulation of the circadian clock is undisputed, studies on MR interactions with circadian clock signalling are limited. We hypothesised that the MR influences cardiac circadian clock signalling, and vice versa. Aldosterone or corticosterone (10 nM) regulated Cry1, Per1, Per2 and ReverbA (Nr1d1) gene expression patterns in H9c2 cells over 24 h. MR-dependent regulation of circadian gene promoters containing GREs and E-box sequences was established for CLOCK, Bmal, CRY1 and CRY2, PER1 and PER2 and transcriptional activators CLOCK and Bmal modulated MR-dependent transcription of a subset of these promoters. We also demonstrated differential regulation of MR target gene expression in hearts of mice 4 h after administration of aldosterone at 08:00 h vs 20:00 h. Our data support MR regulation of a subset of circadian genes, with endogenous circadian transcription factors CLOCK and BMAL modulating the response. This unsuspected relationship links MR in the heart to circadian rhythmicity at the molecular level and has important implications for the biology of MR signalling in response to aldosterone as well as cortisol. These data are consistent with MR signalling in the brain where, like the heart, it preferentially responds to cortisol. Given the undisputed requirement for diurnal cortisol release in the entrainment of peripheral clocks, the present study highlights the MR as an important mechanism for transducing the circadian actions of cortisol in addition to glucocorticoid receptor (GR) in the heart.

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Jirapas Sripetchwandee, Hiranya Pintana, Piangkwan Sa-nguanmoo, Chiraphat Boonnag, Wasana Pratchayasakul, Nipon Chattipakorn and Siriporn C Chattipakorn

Obese-insulin resistance following chronic high-fat diet consumption led to cognitive decline through several mechanisms. Moreover, sex hormone deprivation, including estrogen and testosterone, could be a causative factor in inducing cognitive decline. However, comparative studies on the effects of hormone deprivation on the brain are still lacking. Adult Wistar rats from both genders were operated upon (sham operations or orchiectomies/ovariectomies) and given a normal diet or high-fat diet for 4, 8 and 12 weeks. Blood was collected to determine the metabolic parameters. At the end of the experiments, rats were decapitated and their brains were collected to determine brain mitochondrial function, brain oxidative stress, hippocampal plasticity, insulin-induced long-term depression, dendritic spine density and cognition. We found that male and female rats fed a high-fat diet developed obese-insulin resistance by week 8 and brain defects via elevated brain oxidative stress, brain mitochondrial dysfunction, impaired insulin-induced long-term depression, hippocampal dysplasticity, reduced dendritic spine density and cognitive decline by week 12. In normal diet-fed rats, estrogen deprivation, not testosterone deprivation, induced obese-insulin resistance, oxidative stress, brain mitochondrial dysfunction, impaired insulin-induced long-term depression, hippocampal dysplasticity and reduced dendritic spine density. In high-fat–diet-fed rats, estrogen deprivation, not testosterone deprivation, accelerated and aggravated obese-insulin resistance and brain defects at week 8. In conclusion, estrogen deprivation aggravates brain dysfunction more than testosterone deprivation through increased oxidative stress, brain mitochondrial dysfunction, impaired insulin-induced long-term depression and dendritic spine reduction. These findings may explain clinical reports which show more severe cognitive decline in aging females than males with obese-insulin resistance.