Effect of pioglitazone on the expression of ubiquitin proteasome system and autophagic proteins in rat pancreas with metabolic syndrome

The metabolic syndrome (MetS) and pathologies associated with metabolic dysregulations a worldwide growing problem. Our previous study demonstrated that pioglitazone (PGZ) has beneficial effects on metabolic syndrome associated disturbances in the heart. However, mechanism mediating the molecular alterations of Ubiquitin proteasome system (UPS) and autophagy has not been investigated in rat pancreas with metabolic syndrome. For this reason, we first aimed to detect whether MetS effects on the expression of UPS (p97/VCP, SVIP, Ubiquitin) and autophagic (p62, LC3) proteins in rat pancreas. The second aim of the study was to find impact of pioglitazone on the expression of UPS and autophagic proteins in MetS rat pancreas. To answer these questions, metabolic syndrome induced rats were used as a model and treated with pioglitazone for 2 weeks. Pancreatic tissue injuries, fibrosis and lipid accumulation were evaluated histopathologically in control, MetS and MetS-PGZ groups. Apoptosis and cell proliferation of pancreatic islet cells were assessed in all groups. UPS and autophagic protein expressions of pancreas in all groups were detected by using immunohistochemistry, double-immunfluorescence and Western blotting. Compared with the controls, the rat fed with high sucrose exhibited signs of metabolic syndrome, such as higher body weight, insulin resistance, higher triglyceride level and hyperglycaemia. MetS rats showed pancreatic tissue degeneration, fibrosis and lipid accumulation when their pancreas were examined with Hematoxilen-eozin and Mallory trichrome staining. Metabolic, histopathologic parameters and cell proliferation showed greater improvement in MetS-PGZ rats and pioglitazone decreased apoptosis of islet cells. Moreover, SVIP, ubiquitin, LC3 and p62 expressions were significantly increased while only p97/VCP expression was significantly decreased in MetS-rat pancreas compared to control. PGZ treatment significantly decreased the MetS-induced increases in autophagy markers. Additionally, UPS and autophagy markers were found to colocalizated with insulin and glucagon. Colocalization ratio of UPS markers with insulin showed significant decrease in MetS rats and PGZ increased this ratio, whereas LC3-insulin colocalization displayed significant increase in MetS rats and PGZ reversed this effect. In conclusion, PGZ improved the pancreatic tissue degeneration by increasing the level of p97/VCP and decreasing autophagic proteins, SVIP and ubiquitin expressions in MetS-rats. Moreover, PGZ has an effect on the colocalization ratio of UPS and autophagy markers with insulin.

     

The determination of the relationship between p97/VCP,small VCP-interacting protein,two ERAD proteins and steroidogenesis in Leydig cell lines

Background

In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.

Methods and results

HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.

Conclusion

We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

     

Expression of p97/VCP and ubiquitin during postnatal development of the degenerating rat retina

In this study, we aimed to investigate the distribution pattern of ubiquitin and p97/VCP in the rat retina during postnatal development. Eyeballs from 1-, 4-, 10-, 36- and 72-week-old rats were examined by immunohistochemistry, and protein colocalization was determined by immunofluorescence microscopy. In the 1-week-old rat retina, p97/VCP was strongly expressed in the neuroblast layer, however no ubiquitin immunoreactivity was observed. p97/VCP immunoreactivity was present in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS) of the photoreceptor layer, and retinal pigment epithelium in the 4- and 10-week-old rat retinas. p97/VCP immunoreactivity increased significantly in the 10-week-old rat retinas. Ubiquitin was barely seen in the 4-week-old rat retinas, and ubiquitin expression was weak in the GCL and the IPL of the 10-week-old rat retinas. In the 36- and 72-week-old rats, the presence of ubiquitin was remarkable in the IS, INL, IPL and GCL, however, p97/VCP immunoreactivity was significantly decreased. Colocalization of ubiquitin and p97/VCP was also observed in the INL, IS, GCL and ONL of 36- and 72-week-old rat retinas. Our results indicate that p97/VCP immunoreactivity in the retina significantly decreases after rats reach 10 weeks of age, whereas ubiquitin immunoreactivity increases with aging. These results suggest that an altered expression pattern of p97/VCP and ubiquitin in the developing rat retina may associate with age-related retinal degeneration.     

Cell lines with reduced UDP-N-acetylhexosamine pool in the presence of ammonium.

The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium. Glucosamine-6-phosphate isomerase (GPI) has been proposed as the key enzyme responsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc) by accepting ammonium from the medium of cultured mammalian cells. As previously reported, the increased UDPGNAc pool then affects the N-glycan complexity in glycoproteins. To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK-21 cells and characterized. Thus, the complete pathway by which ammonium enters the cellular metabolism was elucidated. To reduce the negative effects of ammonium, GPI was inhibited using two different strategies. First, the addition of mannose to the culture media and, second, antisense RNA expression. In both cases, the cellular UDPGNAc pool was suppressed in the presence of high ammonium concentrations in the medium. However, constant suppression of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable. Further studies showed that the main reason for instability was the inducibility of GPI by its substrate ammonium. GPI was induced to a factor of two under ammonium-containing medium conditions. We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the presence of ammonium.     

Recombinant protein expression by targeting pre-selected chromosomal loci

Background  

Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Distribution of matrix metalloproteinases MMP-1, MMP-2, MMP-8 and tissue inhibitor of matrix metalloproteinases-2 in nasal polyposis and chronic rhinosinusitis

Nasal polyposis (NP), a chronic inflammatory disease of the upper airway, is a subgroup of chronic rhinosinusitis (CRS). Matrix metallo-proteinases (MMPs) and their tissue inhibitors (TIMPs) are considered to play important roles in the pathogenesis of nasal polyposis. The aim of the current study was to evaluate and compare the levels of MMP-1, MMP-2, MMP-8 and TIMP-2 in NP and CRS with normal nasal mucosa by using immunohistochemistry. Twenty-five patients with NP and fifteen patients with CRS underwent endoscopic sinus surgery. Diseased mucosal samples were obtained from ethmoidal sinuses. Control nasal mucosa (n=10) was obtained from inferior nasal turbinate. Immunohistochemistry for MMP-1, MMP-2, MMP-8 and TIMP-2 was performed. The expression of MMP-1, MMP-2 and MMP-8 significantly increased in NP and CRS compared with control (p<0.05). The distribution of TIMP-2 was higher in CRS than control and NP respectively (p<0.05). MMP-1 immunoreactivity was distributed in the extracellular matrix whereas MMP-2, MMP-8 and TIMP-2 immunostaining was present in the epithelium, submucosal glands, vascular endothelium and inflammatory cells in CRS and NP. We suggest that differences in histological features between CRS and NP might be related to the expression of MMP-1, MMP-2, MMP-8 and their tissue inhibitor-2.     

Neuroprotective effect of mexiletine in the central nervous system of diabetic rats

Both experimental and clinical studies suggests that oxidative stress plays an important role in the pathogenesis of diabetes mellitus type 1 and type 2. Hyperglycaemia leads to free radical generation and causes neural degeneration. In the present study we investigated the possible neuroprotective effect of mexiletine against streptozotocin-induced hyperglycaemia in the rat brain and spinal cord.30 adult male Wistar rats were divided into three groups: control, diabetic, and diabetic-mexiletine treated group. Diabetes mellitus was induced by a single injection of streptozotocin (60 mg/kg body weight). Mexiletine (50 mg/kg) was injected intraperitoneally every day for six weeks. After 6 weeks the brain, brain stem and cervical spinal cord of the rats were removed and the hippocampus, cortex, cerebellum, brain stem and spinal cord were dissected for biochemical analysis (the level of Malondialdehide [MDA], Nitric Oxide [NO], Reduced Glutathione [GSH], and Xanthine Oxidase [XO] activity). MDA, XO and NO levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the diabetic group increased significantly, when compared with control and mexiletine groups (P < 0.05). GSH levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the diabetic group decreased significantly when compared with control and mexiletine groups (P < 0.05).This study demonstrates that mexiletine protects the neuronal tissue against the diabetic oxidative damage.     

Disruption of p97/VCP induces autophagosome accumulation,cell cycle arrest and apoptosis in human choriocarcinoma cells

Molecular Biology Reports - Gestational choriocarcinoma is aggressive trophoblastic disease. The development, progression and the cure of this disease is not well-established. p97/Valosin...