Leptins and leptin receptor expression in the goldfish (Carassius auratus). Regulation by food intake and fasting/overfeeding conditions

Leptin is a hormone involved in feeding and body weight regulation in vertebrates, but the relationship between energy status and leptin has not been clearly established in fish. The aim of this study was to investigate in a teleost, the goldfish (Carassius auratus), the tissue expression pattern of two leptins (gLep-aI and gLep-aII) and leptin receptor (gLepR); and the effect of feeding on expression of these genes. Leptin system expression in goldfish was firstly analyzed in fish under overfeeding (2 weeks) or fasting (1 week), and secondly, at different postfeeding times (0, 3, 6, 9 and 12 h). Goldfish has two Lep-a paralog genes, gLep-aI was widely expressed in central and peripheral tissues, whereas gLep-aII was preferentially expressed in brain. This different distribution pattern of leptins suggests that they can play different physiological roles in goldfish. The gLepR mRNA was ubiquitous expressed, with the highest expression in the telencephalon and hypothalamus. No significant differences in the leptin system expression were found among control, overfed and fasting groups, suggesting an apparent lack of correlation between nutritional status and leptin system in goldfish. Hepatic expression of gLep-aI significantly increased 9 h after feeding time, while hypothalamic leptin system expression did not change after feeding. In summary, leptin in goldfish could signal short-term changes in food intake, as postprandial satiety, but seems to be independent of fasting/overfeeding conditions in this teleost. The widespread distribution of leptins and leptin receptor in goldfish strongly supports that this hormone may have pleitropic actions in fish.     

黄芪甲苷抑制缺血再灌注诱导的大鼠心肌细胞凋亡

摘要 目的：本研究旨在探究黄芪甲苷（Astragaloside IV,Ast-IV）在缺血再灌注（ischemia-reperfusion,I/R）大鼠模型中的保护作用,并讨论黄芪甲苷在抑制I/R诱导心肌细胞凋亡过程中的作用。方法：通过左冠状动脉前降支结扎构建I/R大鼠模型;将40只SD大鼠分为4组：假手术组（Sham组）、模型组（I/R组）、黄芪甲苷干预组（Ast组）和黄芪甲苷+LY294002干预组（Ast+LY组）。使用试剂盒测定血清中心脏功能障碍标记物CPK、ALT、LDH和tropornin-T的表达水平;通过HE染色和TUNEL分别检测心肌组织病理学变化和心肌细胞凋亡情况;通过超氧化物荧光探针染色检测细胞内ROS水平;通过ELISA试剂盒测定心肌组织MDA、GSH和GSH-PX含量;免疫组织化学检测SOD2和HO-1蛋白表达水平,分析心肌氧化应激状态;通过Western blot检测PI3K、p-Akt、Akt、p-eNOS、eNOS、caspase-3、Bax和Bcl-2蛋白表达水平变化。结果：Ast组大鼠血浆CPK、ALT、LDH和tropornin-T酶活性均明显低于I/R组（P<0.05）。Ast组大鼠心肌纤维断裂,心肌细胞坏死和炎性细胞浸润等病变程度均低于I/R组。Ast组大鼠TUNEL阳性细胞数低于I/R组（P<0.05)。相较于I/R组,Ast组大鼠Caspase3和Bax表达水平均明显下调,Bcl-2和PI3K蛋白表达水平上调,p-Akt/Akt和p-eNOS/eNOS比值均显著上调（P<0.05）。Ast组大鼠DHE荧光强度显著低于Ast+LY组（P<0.05）。与I/R组相比,Ast组大鼠心肌组织中MDA含量降低,GSH、GSH-PX、SOD2和HO-1表达水平升高（P<0.05）;与Ast组相比,Ast+LY组大鼠心肌组织中MDA含量升高,GSH、GSH-PX、SOD2和HO-1表达水平降低（P<0.05）。结论：黄芪甲苷通过激活PI3K/Akt信号通路,抑制心肌细胞氧化应激反应,从而减少I/R诱导大鼠心肌细胞凋亡,缓解I/R后大鼠心肌损伤。     

Population-related molecular responses on the effect of pesticides in Carassius auratus gibelio

The aim of this study was to evaluate population-related peculiarities of the adaptive responses of Carassius auratus gibelio. In order to do this, male specimens from polluted (B) and clean (Z) sites were exposed to commercial pesticides thiocarbamate Tatoo (9.1 μg·L(-1)and 91 μg·L(-1)) or tetrazine Apollo (2 μg·L(-1) and 10 μg·L(-1)) during fourteen days. The control fish from site B was distinguished by weakness of antioxidant defence (measured from superoxide dismutase and catalase activities, redox index of glutathione (GSH), superoxide anion (O(2)) and lipid peroxidation levels), imbalance of the concentrations of protein metallothionein (MT-SH) and MT-related metals (MT-Me) and neurotoxicity. Differences in glutathione-S-transferase activity in the liver and vitellogenin-like proteins in the serum were also showed between B and Z control groups. Common effects of pesticides were related to a decrease in GSH, an increase in O(2) production, ethoxyresorufin O-deethylase activity and hepatosomatic index. Apollo provoked particular elevation of MT-SH/MT-Me ratio. Population-related difference in the response was the activation of antioxidant defence in fish from site B and its inhibition in fish from site Z. The genotoxic effect of exposures was more expressed in fish from site B. Principal component analysis combine all exposed groups from site Z and control group from site B in one set, and separated each exposed group from site B. The main distinguishing index of each population selected by classification and regression tree analysis was MT-SH.     

Angiotensin II and III suppress food intake via angiotensin AT(2) receptor and prostaglandin EP(4) receptor in mice

Intracerebroventricularly administered angiotensin (Ang) II and III dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by AT(2) receptor-selective antagonist. Ang II did not suppress food intake in AT(2) receptor-knockout mice, while it did significantly in wild-type and AT(1) receptor-knockout mice. The suppression of food intake in AT(1) receptor-knockout mice was smaller than that in wild-type. The anorexigenic activities of Ang II and III were also blocked by a selective antagonist for prostaglandin EP(4) receptor. Taken together, centrally administered Ang II and III may decrease food intake through AT(2) receptor with partial involvement of AT(1) receptor, followed by EP(4) receptor activation, which is a novel pathway regulating food intake.     

Role of Keap1-Nrf2/ARE signal transduction pathway in protection of dexmedetomidine preconditioning against myocardial ischemia/reperfusion injury

Objective: To explore the role and mechanism of the Kelch sample related protein-1-nuclear factor erythroid-2 related factor 2/antioxidant response element (Keap1-Nrf2/ARE) signaling pathway in protection of dexmedetomidine (DEX) preconditioning against myocardial ischemia/reperfusion injury (MIRI). Methods: A total of 70 male SD rats were randomly divided into seven equal groups (n=10): blank control (S group), ischemia/reperfusion injury (C group), DEX preconditioning (DEX group), tertiary butylhydroquinone (tBHQ) control (tBHQ group), combined tBHQ and DEX preconditioning (tBHQ+DEX group), all-trans retinoic acid (ATRA) control (ATRA group), and combined ATRA and DEX preconditioning (ATRA+DEX group). Serum creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) concentrations were measured by ELISA kits, and the infarct size (IS) was assessed by Evan’s blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Oxidative stress was assessed through Western blotting for expression of Keap1-Nrf2/ARE pathway members and oxidative stress markers. Results: Cardioprotection of DEX, tBHQ, and tBHQ+DEX preconditioning treatments were shown as lower concentrations of serum CK-MB and cTnI and a smaller IS following MIRI in rats compared with those of MIRI rats without pre-treatment. In addition, tBHQ+DEX preconditioning exhibited stronger myocardial protection compared with DEX preconditioning. Mechanistically, the cardioprotection offered by DEX, tBHQ, and tBHQ+DEX preconditioning treatments was mediated via exerting antioxidant stress through activation of the Keap1-Nrf2/ARE signal transduction pathway. Conversely, the protective effects of DEX were diminished by blocking the Keap1-Nrf2/ARE pathway with inhibitor ATRA. Conclusion: DEX preconditioning protects against MIRI by exerting antioxidant stress through activation of the Keap1-Nrf2/ARE signal transduction pathway, while inhibition of the Keap1-Nrf2/ARE signal transduction pathway reverses the protective effect of DEX preconditioning on MIRI.     

Different culture conditions used for arresting the G0/G1 phase of the cell cycle in goldfish (Carassius auratus) caudal fin-derived fibroblasts

One of the most important factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We investigated the effects of serum starvation, culturing to confluence and roscovitine treatment on the cell cycle synchronization of goldfish caudal fin-derived fibroblasts by flow cytometric analysis. The results show that culturing the cells to confluence (85.5%) and roscovitine treatment (82.71%) yield a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than serum starvation (62.85%). Different concentrations of roscovitine (5, 10, or 15 μM) induce cell cycle arrest at the G0/G1 phase.     

The hepatoprotective effects of Hypericum perforatum L. on hepatic ischemia/reperfusion injury in rats

Little is known about the effective role of Hypericum perforatum on hepatic ischemia–reperfusion (I/R) injury in rats. Hence, albino rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. Hypericum perforatum extract (HPE) at the dose of 50 mg/kg body weight (HPE₅₀) was intraperitonally injected as a single dose, 15 min prior to ischemia. Rats were sacrificed at the end of reperfusion period and then, biochemical investigations were made in serum and liver tissue. Liver tissue homogenates were used for the measurement of malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GPx) levels. At the same time alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were assayed in serum samples and compared statistically. While the ALT, AST, LDH activities and MDA levels were significantly increased, CAT and GPx activities significantly decreased in only I/R-induced control rats compared to normal control rats (p < 0.05). Treatment with HPE₅₀ significantly decreased the ALT, AST, LDH activities and MDA levels, and markedly increased activities of CAT and GPx in tissue homogenates compared to I/R-induced rats without treatment–control group (p < 0.05). In oxidative stress generated by hepatic ischemia–reperfusion, H. perforatum L. as an antioxidant agent contributes an alteration in the delicate balance between the scavenging capacity of antioxidant defence systems and free radicals in favour of the antioxidant defence systems in the body.     

Intermediate filaments reminiscent of immature cells expressed by goldfish (Carassius auratus) astrocytes and oligodendrocytes in vitro

The expression of intermediate filaments is developmentally regulated. In the mammalian embryo keratins are the first to appear, followed by vimentin, while the principal intermediate filament of the adult brain is glial fibrillary acidic protein. The intermediate filaments expressed by a cell thus reflect its state of differentiation. The differentiation state of cells, and especially of glial cells, in turn determines their ability to support axonal growth. In this study we used three new antibodies directed against three fish intermediate filaments (glial fibrillary acidic protein, keratin 8 and vimentin), in order to determine the identity and level of expression of intermediate filaments present in fish glial cells in culture. We found that fish astrocytes and oligodendrocytes are both able to express keratin 8 and vimentin. We further demonstrate that under proliferative conditions astrocytes express high keratin 8 levels and most oligodendrocytes also express keratin 8, whereas under nonproliferative conditions the astrocytes express only low keratin 8 levels and most oligodendrocytes do not express keratin 8 at all. These results suggest that the fish glial cells retain characteristics of immature cells. The findings are also discussed in relation to the fish glial lineage.     

1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte

The steroid hormone 1,25(OH)₂-vitamin D₃ [1,25D] has been shown to affect the growth and proliferation of primary cultures of ventricular myocytes isolated from neonatal rat hearts. The research presented here shows that the vitamin D receptor [VDR] is present in murine cardiac myocytes (HL-1 cells), and that 1,25D affects the growth, proliferation and morphology of these cells. In addition we show that 1,25D effects expression of ANP, myotrophin, and c-myc. Furthermore, 1,25D effects expression and localization of the VDR within the cell. Murine HL-1 cardiac myocytes were grown and treated with 1,25D in culture, and growth and morphology were assessed with microscopic analysis. Cells were counted and protein levels were evaluated through Western blot analysis. Subcellular localization of the VDR was determined using immunofluorescence and confocal microscopy. 1,25D was found to decrease proliferation and alter cellular morphology of the HL-1 cells. Treatment with 1,25D increased expression of myotrophin while decreasing expression of atrial natriuretic peptide [ANP] and c-myc. 1,25D treatment also increased expression and nuclear localization of the VDR in these cardiac myocytes. Thus 1,25D is an important hormone involved in modulating and maintaining heart cell structure and function.     

Rooibos (Aspalathus linearis) offers cardiac protection against ischaemia/reperfusion in the isolated perfused rat heart

Rooibos, a unique South African herbal tea, is known to be an important source of unique polyphenolic compounds. In the present study we have quantified the main polyphenolic compounds in both fermented/traditional and unfermented/"green" rooibos (Aspalathus linearis) and evaluated its cardioprotective effects against ischaemia/reperfusion injury. Male Wistar rats consumed aqueous rooibos and green tea (Camellia sinensis) extracts (2%, w/v) for 7 weeks before their hearts were rapidly excised and perfused in a working heart perfusion apparatus. The results showed that the rooibos supplemented hearts significantly improved aortic output recovery after reperfusion when compared to the green tea supplemented hearts. Additionally, we showed that the rooibos extracts, containing the highest amount of flavonols, significantly decreased the level of cleaved caspase-3 and PARP, both pro-apoptotic proteins, during reperfusion when compared to green tea. Green tea supplementation increased phosphorylation of total PKB/Akt, Akt (threonine 308) and Akt (serine 473). The rooibos extracts did not cause significant change in the levels of the pro-survival PKB/Akt (threonine 308 and serinet 473). The GSH/GSSG ratio in the hearts of the green tea supplemented group was significantly (p<0.05) lower when compared to RF (37.78±28.63), RU (33.20±4.13) and C (45.50±14.96). The results clearly demonstrate the cardio-protective properties of aqueous rooibos extracts via the inhibition of apoptosis which can possibly be related to the flavonol content of this unique South African herbal tea.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

The effects of blinding,pinealectomy and exposure to constant dark conditions on gonadal activity in the female goldfish,Carassius auratus

Synopsis The effects of pinealectomy, blinding, and exposure to constant darkness were examined in female goldfish during different seasons. Neither blinding nor pinealectomy under short or decreasing photoperiod conditions, nor exposure to constant darkness had an effect on ovarian activity in goldfish during fall and winter. In spring, constant darkness has an inhibitory effect on ovarian activity. Pinealectomy under increasing photoperiod conditions partially inhibits reproductive activity, but not to the extent of constant darkness. Blinding, under the same regime also inhibited to some extent the ovarian response to increasing photoperiod. Our data suggest that retinal pathways and the pineal organ are involved in the photosexual response to increasing day lengths.Address correspondence to: Dr. V. L. de Vlaming, Marquette University     

The effects of cytokines, platelet activating factor, and arachidonate metabolites on C3B receptor (CR1, CD35) expression and phagocytosis by neutrophils

The cytokines tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin 1 (IL 1) all caused an upregulation of C3b receptors (CR1) on neutrophils that ranged from around 76% (G-CSF and IL 1) to 93% (TNF alpha and GM-CSF) of the upregulation obtained by pretreatment of the neutrophils with the chemotactic peptide FMLP. However, only TNF alpha and G-CSF caused a significant increase in phagocytosis of opsonized microspheres. Platelet derived growth factor, interleukin 2, and transforming growth factor beta had no effect on either of these parameters. The mediators platelet activating factor (PAF) and leukotriene B4 (LTB4) both caused a large upregulation of CR1 (93% and 80%, respectively, of the FMLP-mediated value); however, only PAF caused a significant enhancement of phagocytosis by the neutrophils. Prostaglandin E2 and thromboxane B2 had no effect on these parameters. Considerable individual variation was observed among some of the untreated and mediator-treated neutrophil preparations regarding CR1 expression and phagocytosis. The upregulation of CR1 and associated increase in phagocytic capacity of neutrophils caused by certain cytokines and other mediators may be important in host defense. Also the lack of enhancement of phagocytosis accompanying an upregulation of CR1 is unusual and may have important implications regarding the cellular mechanisms of phagocytosis by neutrophils.     

Regulation of sperm motility by PIP2(4,5) and actin polymerization

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. In previous studies, we demonstrated that the increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP₂). Here, we showed for the first time the involvement of PIP₂/gelsolin in human sperm motility before and during capacitation. Activation of gelsolin by causing its release from PIP₂ inhibited sperm motility, which could be restored by adding PIP₂ to the cells. Reduction of PIP₂ synthesis inhibited actin polymerization and motility, and increasing PIP₂ synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP₂ and F-actin. During capacitation there was an increase in PIP₂ and F-actin levels in the sperm head and a decrease in the tail. In sperm with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends on its binding to PIP₂. Activation of phospholipase C by Ca²⁺-ionophore or by activating the epidermal-growth-factor-receptor inhibits tyrosine phosphorylation of gelsolin. In conclusion, the data indicate that the increase of PIP₂ and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result the decrease of gelsolin in the tail allows keeping high level of F-actin in the tail, which is essential for the development of HA motility.     

Changes in gene expression induced by H(2)O(2) in cardiac myocytes

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.