Over a half of the diabetic individuals develop macrovascular complications that cause high mortality. Oxidative stress (OS) promotes endothelial dysfunction (ED) which is a critical early step toward diabetic macrovascular complications. Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular antioxidant defense system and combats diabetes-induced OS. Previously, we found that impaired NRF2 antioxidant signaling contributed to diabetes-induced endothelial OS and dysfunction in mice. The present study has investigated the effect of microRNA-200a (miR-200a) on NRF2 signaling and diabetic ED. In aortic endothelial cells (ECs) isolated from C57BL/6 wild-type (WT) mice, high glucose (HG) reduced miR-200a levels and increased the expression of kelch-like ECH-associated protein 1 (Keap1) – a target of miR-200a and a negative regulator of NRF2. This led to the inactivation of NRF2 signaling and exacerbation of OS and inflammation. miR-200a mimic (miR-200a-M) or inhibitor modulated KEAP1/NRF2 antioxidant signaling and manipulated OS and inflammation under HG conditions. These effects were completely abolished by knockdown of Keap1, indicating that Keap1 mRNA is a major target of miR-200a. Moreover, the protective effect of miR-200a-M was completely abrogated in aortic ECs isolated from C57BL/6 Nrf2 knockout (KO) mice, demonstrating that NRF2 is required for miR-200a’s actions. In vivo, miR-200a-M inhibited aortic Keap1 expression, activated NRF2 signaling, and attenuated hyperglycemia-induced OS, inflammation and ED in the WT, but not Nrf2 KO, mice. Therefore, the present study has uncovered miR-200a/KEAP1/NRF2 signaling that controls aortic endothelial antioxidant capacity, which protects against diabetic ED.
Ziping Jiang, Junduo Wu, Fuzhe Ma, Jun Jiang, Linlin Xu, Lei Du, Wenlin Huang, Zhaohui Wang, Ye Jia, Laijin Lu, and Hao Wu
Anneleen Segers, Louis Desmet, Shu Sun, Kristin Verbeke, Jan Tack, and Inge Depoortere
The known crosstalk between short-chain fatty acids (SCFAs) and the circadian clock is tightly intertwined with feeding time. We aimed to investigate the role of the core clock gene Bmal1 and feeding time in the diurnal rhythms in plasma and caecal SCFA levels and in their effect on the release of the hunger hormone ghrelin in the stomach and colon. WT, Bmal1 -/- (ad libitum fed) and night-time-restricted-fed (RF)-Bmal1 -/- littermates were killed at zeitgeber time (ZT) 4 and 16. SCFA concentrations were measured by gas chromatography. To investigate the effect of SCFAs on ghrelin release, stomach and colonic full-thickness strips were incubated with Krebs or a SCFA mix mimicking plasma or caecal concentrations, after which octanoyl ghrelin release was measured by RIA. Diurnal rhythms in caecal and plasma SCFAs oscillated in phase but rhythmic changes were abolished in Bmal1 -/- mice. RF of Bmal1 -/- mice restored fluctuations in caecal SCFAs. Plasma SCFA concentrations failed to affect gastric ghrelin release. The effect of caecal SCFA concentrations on colonic ghrelin release was rhythmic (inhibition at ZT 4, no effect at ZT 16). In Bmal1 -/- mice, the inhibitory effect of SCFAs at ZT 4 was abolished. RF Bmal1 -/- mice restored the inhibitory effect and increased colonic Clock expression. To conclude, diurnal fluctuations in caecal SCFAs and the effect of SCFAs on colonic ghrelin release are regulated by feeding time, independent of the core clock gene Bmal1. However, local entrainment of other clock genes might contribute to the observed effects.
Romain Fontaine, Eirill Ager-Wick, Kjetil Hodne, and Finn-Arne Weltzien
Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of the fshb and lhb promotors respectively. We found that Fsh cells appear in the pituitary at 8 dpf, while Lh cells were previously shown to appear at 14 dpf. Similar to Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juveniles and adults but not in larvae where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh cells start to produce Lhβ, a phenomenon enhanced by gonadotropin-releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol-sensitive proliferation and Gnrh promoted phenotypic conversion, and moreover, show that gonadotropes lose part of their identity when kept in cell culture.
Koichiro Taguchi, Kazuo Kajita, Yoshihiko Kitada, Masayuki Fuwa, Motochika Asano, Takahide Ikeda, Toshiko Kajita, Tatsuo Ishizuka, Itaru Kojima, and Hiroyuki Morita
Despite extensive investigation, the mechanisms underlying adipogenesis are not fully understood. We previously identified proliferative cells in adipose tissue expressing adipocyte-specific genes, which were named small proliferative adipocytes (SPA). In this study, we investigated the characteristics and roles of SPA in adipose tissue. Epididymal and inguinal fat was digested by collagenase, and then SPA were separated by centrifugation from stromal vascular cells (SVC) and mature white adipocytes. To clarify the feature of gene expression in SPA, microarray and real-time PCR were performed. The expression of adipocyte-specific genes and several neuronal genes was increased in the order of SVC < SPA < mature white adipocytes. In addition, proliferin was detected only in SPA. SPA differentiated more effectively into lipid-laden cells than SVC. Moreover, differentiated SPA expressed uncoupling protein 1 and mitochondria-related genes more than differentiated SVC. Treatment of SPA with pioglitazone and CL316243, a specific β3-adrenergic receptor agonist, differentiated SPA into beige-like cells. Therefore, SPA are able to differentiate into beige cells. SPA isolated from epididymal fat (epididymal SPA), but not SPA from inguinal fat (inguinal SPA), expressed a marker of visceral adipocyte precursor, WT1. However, no significant differences were detected in the expression levels of adipocyte-specific genes or neuronal genes between epididymal and inguinal SPA. The ability to differentiate into lipid-laden cells in epididymal SPA was a little superior to that in inguinal SPA, whereas the ability to differentiate into beige-like cells was greater in inguinal SPA than epididymal SPA. In conclusion, SPA may be progenitors of beige cells.
Wonsuk Choi, Joon Ho Moon, and Hail Kim
Serotonin is a biogenic amine synthesized from the essential amino acid tryptophan. Because serotonin cannot cross the blood-brain barrier, it functions differently in neuronal and non-neuronal tissues. In the CNS, serotonin regulates mood, behavior, appetite, and energy expenditure. Although most serotonin in the body is synthesized at the periphery, its biological roles have not been well elucidated. Older studies using chemical agonists and antagonists yielded conflicting results, because the complexity of serotonin receptors and the low selectivity of agonists and antagonists were not known. Several recent studies using specific knock-out of serotonin receptors have been performed to assess the role of peripheral serotonin in regulating energy metabolism. This review discusses (1) the tissue-specific roles of peripheral serotonin in regulating energy metabolism, (2) the mechanism by which dysfunctional peripheral serotonin signaling can progress to metabolic diseases, and (3) how peripheral serotonin signaling could be a therapeutic target for metabolic diseases.
Federico Gatto, Richard A Feelders, Rob van der Pas, Peter van Koetsveld, Eleonora Bruzzone, Marica Arvigo, Fadime Dogan, Steven Lamberts, Diego Ferone, and Leo Hofland
Pituitary-directed medical treatment for Cushing’s disease (CD) is currently represented by membrane receptor targeting drugs (somatostatin analogs and dopamine agonists). Somatostatin and dopamine receptors are regulated by β-arrestins, which have been shown to be differentially regulated by glucocorticoids in non-neuroendocrine cells. In this study we investigated the effects of glucocorticoids on β-arrestin expression in corticotroph tumor cells. First, AtT20 cells, a mouse model of CD, were exposed to dexamethasone (Dex) at different time points and β-arrestin expression was evaluated at mRNA and protein levels. Futhermore, β-arrestin mRNA expression was evaluated in 17 human corticotroph adenoma samples and correlated to patients’ pre-operative cortisol levels. We observed that Dex treatment induced a time-dependent increase in β-arrestin 1 mRNA expression and a decrease in β-arrestin 2. The same modulation pattern was observed at protein level. Dex-mediated modulation of β-arrestins was abolished by co-treatment with mifepristone, and Dex withdrawal restored β-arrestin expression to basal levels after 72 h. The evaluation of β-arrestin mRNA in corticotroph adenomas from CD patients with variable disease activity showed a significant positive correlation between β-arrestin 1 mRNA and urinary cortisol levels. The effect of glucocorticoids on β-arrestin levels was confirmed by the analysis of two samples from a single patient, which underwent adenomectomy twice, with different pre-operative cortisol levels. In conclusion, glucocorticoids induce an inverse modulation of the two β-arrestin isofoms in corticotroph tumor cells. Since β-arrestins regulate membrane receptor functions, this finding may help to better understand the variable response to pituitary-targeting drugs in patients with Cushing’s disease.
Yang Chen, Mingyue Zhao, Chenhao Wang, Huaizhen Wen, Yuntao Zhang, Mingxu Lu, Salah Adlat, Tingting Zheng, Mingjiao Zhang, Dan Li, Xiaodan Lu, Mengwei Guo, Hongyu Chen, Luqing Zhang, Xuechao Feng, and Yaowu Zheng
Excessive fat accumulation causes obesity and many diseases. Previous study demonstrates VEGFB universal knockout induces obese phenotypes including expansion of white adipose tissue, whitening of brown adipose tissue, increase of fat accumulation and reduction in energy consumption. However, roles of VEGFB in adipose tissues are not clear. In this study, we have generated a mouse model with adipose-specific VEGFB repression using CRISPR/dCas9 system (Vegfb AdipoDown) and investigated the roles of VEGFB in adipose development and energy metabolism. VEGFB repression induced significant changes in adipose tissue structure and function. Vegfb AdipoDown mice have larger body sizes, larger volume of white adipose tissues than its wild type littermates. Adipose-specific VEGFB repression induced morphological and functional transformation of adipose tissues toward white adipose for energy storage. Metabolic processes are broadly changed in Vegfb AdipoDown adipose tissues including carbohydrate metabolism, lipid metabolism, nucleotide metabolism and amino acid metabolism. We have demonstrated that adipose VEGFB repression can recapitulate most of the phenotypes of the whole body VEGFB knockout mouse. Intriguingly, approximately 50% VEGFB repression in adipose tissues can almost completely mimic the effects of universal Vegfb deletion, suggesting adipose VEGFB is a major regulator of energy metabolism and may be important in prevention and treatment of obesity.
Miguel del Campo, Néstor Lagos, and Hernán Lara
A high sympathetic tone is observed in the development and maintenance of the polycystic ovary (PCO) phenotype in rats. Neosaxitoxin (NeoSTX) specifically blocks neuronal voltage-dependent Na+ channels, and we studied the capacity of NeoSTX administered into the ovary to block sympathetic nerves and PCO phenotype that is induced by estradiol valerate (EV). The toxin was administered with a minipump inserted into the bursal cavity using two protocols: (1) the same day as EV administration and (2) 30 days after EV to block the final step of cyst development and maintenance of the condition. We studied the estrous cycling activity, follicular morphology, steroid plasma levels, and norepinephrine concentration. NeoSTX administered together with EV decreased NA intraovarian levels that were induced by EV, increased the number of corpora lutea, decreased the number of follicular cyst found after EV administration, and decreased the previously increased testosterone plasma levels induced by the PCO phenotype. Estrous cycling activity also recovered. NeoSTX applied after 30 days of EV administration showed near recovery of ovary function, suggesting that there is a specific window in which follicular development could be protected from cystic development. In addition, plasma testosterone levels decreased while those of progesterone increased. Our data strongly suggest that chronic inhibition of sympathetic nerves by a locally applied long-lasting toxin is a new tool to manage the polycystic phenotype in the rat and could be applied to other mammals depending on sympathetic nerve activity.
Hui-Fang Wang, Qing-Qing Yu, Rui-Fang Zheng, and Ming Xu
Cardiovascular complications of type 2 diabetes mellitus (T2DM) are associated with vascular remodeling in the arteries. Perivascular sympathetic neurons release an abundance of trophic factors to regulate vascular function via a paracrine signaling. Netrin-1, a diffusible protein that can be secreted outside the cell, is one of common signals of ‘conversation’ between nerve and vessel. The present study investigated whether netrin-1 is a novel modulator of sympathetic neurons paracrine signaling and played a critical role in vascular adventitial remodeling under T2DM. Vascular adventitial remodeling was observed in adventitial fibroblasts (AFs) responding to netrin-1 deficiency in the supernatant from primary rat superior cervical ganglia (SCG) neurons, shown as AFs proliferation, migration, and collagen deposition. Conditioned medium from the high glucose (HG)-treated SCG neurons contributed to AFs remodeling, which was effectively alleviated by exogenous netrin-1 supplementation. Further, it was found that uncoordinated-5-B (Unc5b) was mainly expressed in AFs among netrin-1 specific receptors. Treatment of netrin-1 inhibited H2O2 production derived from NADPH oxidase 4 (NOX4) through the UNC5b/CAMP/PKA signal pathway in AFs remodeling. In vivo, aorta adventitial remodeling was accompanied with the downregulation of netrin-1 in the perivascular sympathetic nerve in T2DM rats. Such abnormalities were restored by netrin-1 intervention, which was associated with the inhibition of NOX4 expression in the aorta adventitia. In conclusion, netrin-1 is a novel modulator of sympathetic neurons paracrine signaling to maintain AFs function. Vascular adventitial remodeling was aggravated by sympathetic neurons paracrine signaling under hyperglycemia, which was ameliorated by netrin-1 treatment through the UNC5b/CAMP/PKA/NOX4 pathway.
Qiaoyuan Zheng, Hesheng Xiao, Hongjuan Shi, Tingru Wang, Lina Sun, Wenjing Tao, Thomas D Kocher, Minghui Li, and Deshou Wang
The impacts of androgens and glucocorticoids on spermatogenesis have intrigued scientists for decades. 11β-hydroxylase, encoded by cyp11c1, is the key enzyme involved in the synthesis of 11-ketotestosterone and cortisol, the major androgen and glucocorticoid in fish, respectively. In the present study, a Cyp11c1 antibody was produced. Western blot and immunohistochemistry showed that Cyp11c1 was predominantly expressed in the testicular Leydig cells and head kidney interrenal cells. A mutant line of cyp11c1 was established by CRISPR/Cas9. Homozygous mutation of cyp11c1 caused a sharp decrease of serum cortisol and 11-ketotestosterone, and a delay in spermatogenesis which could be rescued by exogenous 11-ketotestosterone or testosterone, but not cortisol treatment. Intriguingly, this spermatogenesis restored spontaneously, indicating compensatory effects of other androgenic steroids. In addition, loss of Cyp11c1 led to undersized testes with a smaller efferent duct and disordered spermatogenic cysts in adult males. However, a small amount of viable sperm was produced. Taken together, our results demonstrate that cyp11c1 is important for testicular development, especially for the initiation and proper progression of spermatogenesis. 11-ketotestosterone is the most efficient androgen in tilapia.