Fibroblast growth factor 21 reverses high‐fat diet‐induced impairment of vascular function via the anti‐oxidative pathway in ApoE knockout mice

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver‐secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the β‐klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H₂O₂)‐induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H₂O₂. Administration of N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)‐deficient mice that were fed a high‐fat diet (HFD). Endothelial function, as reflected by acetylcholine‐stimulated aortic relaxation, was improved after FGF21 treatment in ApoE‐deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE‐deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD‐induced vascular injury in ApoE‐deficient mice.     

不同活动、饥饿和饮食成分状态下FGF21的动力学表达

成纤维细胞生长因子21（fibroblast growth factor,FGF21）是FGF家族中的新成员．目前研究显示,FGF21是一个新的糖脂代谢调节因子,有望成为治疗糖尿病的新型药物．为探讨FGF21的生理功能,利用real-time PCR和Western印迹,检测FGF21在不同生理或病理状态下基因水平和蛋白水平的表达量变化规律．实验结果显示,在全天24 h中,小鼠肝脏中FGF21在晚18点至21点,表达量显著升高,这可能与啮齿类动物傍晚活动加强及进食习性有关|FGF21在饥饿后表达量显著升高,在饥饿后喂食FGF21的表达量下降,并且随着饥饿时间的延长,FGF21的表达量升高,说明FGF21与饥饿程度呈正相关|灌注葡萄糖后20 min内,FGF21的表达量下降,而灌注脂肪乳20 min内,FGF21的表达量上升,说明葡萄糖是FGF21的负调节因子,而脂肪乳是FGF21的正调节因子|利用谷氨酸钠造模的肥胖小鼠,肝脏中FGF21的表达量显著高于同龄对照组,说明肥胖可诱导FGF21高表达．综上所述,FGF21的表达量变化与小鼠夜间活动取食、饥饿程度、饮食中不同的成分以及肥胖有关.     

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9× higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.     

Identification of the cyclic AMP responsive element (CRE) that mediates transcriptional regulation of the pyruvate carboxylase gene in HepG2 cells

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis. Here we investigated the effect of various hormones including cAMP, dexamethasone and insulin on the abundance of PC mRNA in the human hepatocyte cell line, HepG2. Treatment of HepG2 cells with 1 μM of glucagon increased the expression of PC mRNA threefold within 72 h. Treatment with 1 mM 8-Br-cAMP caused the abundance of PC mRNA to increase by 2-3-fold by 48 h, peak at fourfold at 72 h, and remain unchanged to 96 h. This is in contrast to phosphoenolpyruvate carboxykinase (PEPCK) for which expression was decreased after 72 h, suggesting a distinct difference in the control of these two enzymes in the long term. Dexamethasone or insulin alone did not affect the abundance of PC mRNA whereas treatment of HepG2 cells with the combination of 1 mM 8-Br-cAMP and 0.5 μM dexamethasone further increased the abundance of PC mRNA, suggesting the predominant role of 8-Br-cAMP over dexamethasone. Transient transfection of the luciferase reporter construct driven by a 1.95 kbp 5′-flanking sequence of the mouse PC gene and a plasmid encoding the human cAMP-responsive element binding protein increased luciferase reporter activity to 7-fold similar to that observed with a PEPCK promoter-luciferase reporter construct. Deletion of the 5′-flanking region of the PC gene to 781 bp resulted in the complete loss of CREB-mediated induction of reporter gene, suggesting the presence of the cAMP-responsive unit is located between 1.95 kbp and 781 bp upstream of the mouse PC gene. Electrophoretic mobility shifted and chromatin immunoprecipitation assays demonstrated that CREB bind to −1639/−1631 CRE of mouse PC gene in vitro and in vivo, respectively.     

Characterization,expression and localization of valosin-containing protein in ovaries of the giant tiger shrimp Penaeus monodon

Valosin-containing protein (VCP), a member of the ATPase-associated with diverse cellular activity (AAA) family, was identified from the giant tiger shrimp (Penaeus monodon). The full-length cDNA of the PmVCP mRNA consisted of 2724 bp containing an ORF of 2367 bp corresponding to a deduced polypeptide of 788 amino acids. The deduced PmVCP protein contained two putative Cdc48 domains (positions 17–103, E-value = 2.00e− 36 and 120–186, E-value = 3.60e− 11) and two putative AAA domains (positions 232–368, E-value = 3.67e− 24 and 505–644, E-value = 3.73e− 25). PmVCP mRNA expression in ovaries was greater than that in testes in both juveniles and broodstock. PmVCP was significantly up-regulated in stages II and IV ovaries in intact wild broodstock (P < 0.05) but it was not differentially expressed during ovarian development in eyestalk-ablated broodstock (P > 0.05). The expression level of PmVCP mRNA in ovaries of 14-month-old shrimp was not affected by progesterone injection (0.1 μg/g body weight, P > 0.05). In contrast, exogenous 5-HT administration (50 μg/g body weight) resulted in an increase of PmVCP mRNA in ovaries of 18-month-old shrimp at 6 and 24 h post-injection (hpi) (P < 0.05). The rPmCdc48-VCP protein and its polyclonal antibody were successfully produced. Cellular localization revealed that PmVCP was localized in the ooplasm of previtellogenic oocytes. Subsequently, it was translocated into the germinal vesicle of vitellogenic oocytes. Interestingly, PmVCP was found in nucleo-cytoplasmic compartments, in the cytoskeletal architecture and in the plasma membrane of mature oocytes in both intact and eyestalk-ablated broodstock.     

Effect of RFRP-3 on reproduction is sex- and developmental status-dependent in the striped hamster (Cricetulus barabensis)

RFamide-related peptides (RFRPs) are orthologous to gonadotropin-inhibitory hormone (GnIH) inhibiting gonadotropin release. There are only two RFRP sequences (RFRP-1 and RFRP-3) encoded in rodents. RFRP-3, which was considered as a hypothetical inhibitor on GnRH, shows a stimulatory effect on the male Syrian and male Siberian hamster in short days. As a dominant rodent pest in northern China farmland, the striped hamster (Cricetulus barabensis) has higher reproductive activities and could act as a model to study the mechanism of reproduction. However, the effect of RFRP-3 on the reproductive activity for the striped hamster is less understood. In the study, we cloned 643 bp RFRP cDNA from the striped hamster hypothalamus, which contained an ORF of 570 bp encoding two RFamide-related peptide (RFRP) sequences: SPAPANKVPHSAANLPLRF-NH₂ (C. barabensis RFRP-1) and TLSRVPSLPQRF-NH₂ (C. barabensis RFRP-3). We also investigated the expression variation of RFRP mRNA and GnRH mRNA in the hypothalamus from hamsters with different developmental statuses (7-week-, 13-week- and 1.5-year-olds) using FQ-PCR, in which the 13-week-old female individuals were in estrous. The striped hamsters that are 7 weeks and 1.5 years old are non-breeding individuals, and those that are 13-week hamsters have breeding phenomena. The highest hypothalamus RFRP mRNA level was found in breeding males as compared to non-breeding males. Conversely, the lowest RFRP mRNA level in the hypothalamus was observed in breeding females, with no significant level when the breeding females were compared to the 7-week-old individuals. Additionally, the investigation of GnRH expression level showed a declining expression trend across the developmental stages (7-week-, 13-week- and 1.5-year-olds) in both sexes. Significant negative and positive relationships were detected in the 13-week estrous female (r = − 0.997, P = 0.035) and the 13-week male (r = 0.998, P = 0.029) striped hamsters respectively, which suggest that RFRP-3 has inhibitory and stimulatory effects on female and male adults respectively. Our results suggest that the effects of RFRP-3 on reproduction are sex- and developmental status-dependent in the striped hamster.     

The extrapituitary prolactin promoter polymorphism is associated with rheumatoid arthritis and anti-CCP antibodies in Mexican population

Prolactin (PRL) is a hormone–cytokine that has been involved in autoimmunity due to its immunoregulatory and lymphoproliferative effects. It is produced by various extrapituitary sites including immune cells, under control of a superdistal promoter that contains a single nucleotide polymorphism − 1149 G/T previously associated with rheumatoid arthritis (RA) susceptibility in European population. The aim of this study was to investigate the association of the extrapituitary PRL − 1149 G/T promoter polymorphism with clinical parameters, clinical activity and disability indices in RA patients from Western Mexico and to analyze the PRL mRNA expression according to the PRL − 1149 G/T promoter polymorphism in total leucocytes from RA patients and controls. We conducted a case–control study that included 258 RA patients and 333 control subjects (CS). The DNA samples were genotyped using the PCR–RFLP method and the PRL mRNA expression was determined by quantitative real time PCR. PRL serum levels and antibodies to cyclic citrullinated peptides (anti-CCP) were measured with ELISA. We found significant differences in the genotype (p = 0.022) and allelic (p = 0.046) distribution of the polymorphism between RA patients and control subjects. According to the dominant genetic model, there is an association between the T allele (GT + TT genotypes) and decreased RA susceptibility in comparison to the G allele carriers (GG genotype) (OR 0.64, 95% CI 0.45–0.92; p = 0.011). The T allele carriers (GT + TT genotypes) had lower titers of anti-CCP antibodies in comparison to the G allele carriers (GG genotype) (median, 66 U/mL vs. 125 U/mL; p = 0.03). Furthermore, the GG homozygotes had higher PRL mRNA expression in comparison to the GT heterozygotes, and this latter with respect to the TT homozygotes, in both groups (RA: 1 > 0.72 > 0.19; CS: 1 > 0.54 > 0.28). However, PRL serum levels were similar in both groups. Our results suggest that the PRL − 1149 T allele is a genetic marker for decreased RA susceptibility and is associated with lower titers of anti-CCP antibodies in Mexican population. We also suggest influence of genotype upon PRL mRNA expression.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.