Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D 1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Ppar γ, Cebp α, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22–76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Ppar γ and Cebp α expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes.
Hui Juan Zhu, Hui Pan, Xu Zhe Zhang, Nai Shi Li, Lin Jie Wang, Hong Bo Yang and Feng Ying Gong
Ping Li, Yan Hao, Feng-Hui Pan, Min Zhang, Jian-Qiang Ma and Da-Long Zhu
This study investigates the effectiveness and mechanisms of a serum- and glucocorticoid-inducible kinase 1 (SGK1) inhibitor in counteracting hyperglycemia. In an in vivo experiment, we demonstrated that after an 8-week treatment with an SGK1 inhibitor, the fasting blood glucose and HbA1c level significantly decreased in db/db mice. RT-PCR and western blot analyses revealed that intestinal SGK1 and sodium glucose co-transporter 1 (SGLT1) expression were enhanced in db/db mice. Treatment with an SGK1 inhibitor decreased excessive SGLT1 expression in the intestine of db/db mice. In vitro experiments with intestinal IEC-6 cells showed that the co-administration of an SGK1 inhibitor partly reversed the SGLT1 expression and glucose absorption that were induced by dexamethasone. In conclusion, this study revealed that the favorable effect of an SGK1 inhibitor on hyperglycemia is partly due to decreased glucose absorption through SGLT1 in the small intestine. These data collectively suggest that SGK1 may be a potent target for the treatment of diabetes and other metabolic disorders.
Peng Zhang, Sheng Wang, Liang Wang, Bing Chen Shan, Hui Zhang, Fan Yang, Zhi Qiang Zhou, Xiao Wang, Ye Yuan and You Jia Xu
Postmenopausal osteoporosis is a global health issue. Although a lack of estrogen is considered the major reason for postmenopausal osteoporosis, other factors might also contribute the etiology of the disease. In previous reports, we and others proposed that iron accumulation after menopause accelerates osteoporosis, and here, we genetically modified the expression of an endogenous hormone, hepcidin, to modulate iron status in a mouse model. Our results show that hepcidin levels negatively correlate with bone loss in both knockout and overexpression (with ovariectomy) murine models. In addition, iron overload enhances reactive oxygen species (ROS) activity and attenuates the functions of primary osteoblasts, while iron depletion could reverse this phenomenon through inhibiting the functions of primary osteoclasts. Therefore, our results provide more evidence of the ‘iron accumulation’ hypothesis, which suggests that high iron levels are risk factors for osteoporosis, and the ‘Huang’s hypothesis’ that hepcidin is a potential drug target for the prevention of postmenopausal osteoporosis.
Yuxiao Tang, Xingjian Cai, Hongwei Zhang, Hui Shen, Wanyin Wang, Zhilei Shen, Wei Gu, Changquan Ling and Min Li
The mechanisms of hypothalamic–pituitary–adrenal (HPA) axis regulation have been studied persistently but still are not elucidated. Considering the emerging roles of microRNA in stress response, we conducted a microRNA microarray in mice hypothalamus to identify the potential role of microRNAs in regulating the HPA axis. In total, 41 microRNAs changed during heat stress in which we found that miR-212 contains a binding sequence with corticotropin-releasing hormone (Crh) 3′UTR according to a sequence analysis. We observed that miR-212 expression in the hypothalamus was escalated by repeated heat and restraint stress. By overexpression or inhibition of miR-212 and the dual-luciferase reporter assay, we proved that miR-212 could bind with Crh 3′UTR to regulate its expression in mice hypothalamus primary cells and in the hippocampus neuron cell line HT-22. In addition, we injected miR-212 agomir or antagomir in mice hypothalamus to overexpress or inhibit miR-212, which leads to alterations of CRH expression and HPA axis activity in vivo. Furthermore, miR-212 and CRH were both transcribed by the cAMP response element-binding protein (CREB). Overexpression and inhibition of miR-212 affect CREB-dependent CRH expression. Taken together, our results suggest an inhibitory role of miR-212 on the HPA axis, which acts in a counter-regulatory manner.
Yan-hui Bai, Yong Lv, Wei-qun Wang, Guang-li Sun and Hao-hao Zhang
Human corneal fibroblasts (HCFs) are implicated in corneal neovascularization (CRNV). The mechanisms underlying the inflammatory response in HCFs and the development of CRNV were explored in this study. Alkali burns were applied to the corneas of rats to establish a CRNV model. The expression of long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) and mRNA and protein levels of nuclear factor kappa B (NF-κB)- activating protein (NKAP) were examined by quantitative real-time (qRT-PCR) and Western blot methods, respectively. Lipopolysaccharide (LPS) is used to stimulate HCFs for inflammatory response. The level of inflammation factors in HCF supernatant was detected using an enzyme-linked immunosorbent assay (ELISA). Binding and interactions between NEAT1 and miRNA 1246 (miR-1246) were determined by RNA immunoprecipitation (RIP) and RNA pull-down assays in HCFs. Compared with the control group (n = 6), NEAT1 was upregulated in the corneas of the CRNV rat model (n = 6). The expression of NEAT1 in HCFs was upregulated by LPS. Downregulation of NEAT1 suppressed the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). NEAT1 could bind and interact with miR-1246. LPS regulated the expression of NKAP and NF-κB signaling via the NEAT1/miR-1246 pathway. Downregulation of NEAT1 in vivo inhibited CRNV progression in the CRNV rat model. The lncRNA NEAT1 induced secretion of inflammatory factors, mediated by NF-κB, by targeting miR-1246, thereby promoting CRNV progression.
Sarah X Zhang, Joshua J Wang, Azar Dashti, Kenneth Wilson, Ming-Hui Zou, Luke Szweda, Jian-Xing Ma and Timothy J Lyons
Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 μg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10–160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-κB (NF-κB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO−) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-κB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-κB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO− formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.