Growth hormone and prolactin are important growth factors for pancreatic β-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in β-cells are largely ...mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Suppressors of cytokine signaling (SOCS) proteins are specific inhibitors of the JAK/STAT pathway acting through a negative-feedback loop. To investigate in vivo effects of SOCS-3 in growth hormone (GH)/prolactin signaling in β-cells we generated transgenic mice with β-cell-specific overexpression of SOCS-3. The relative β-cell proliferation and volume in the mice were measured by morphometry. β-Cell volume of transgenic female mice was reduced by over 30% compared with β-cell volume in wild-type female mice. Stimulation of transgenic islets in vitro with GH showed a reduced tyrosine phosphorylation of STAT-5 when compared with wild-type islets. Transduction of primary islet cultures with adenoviruses expressing various SOCS proteins followed by stimulation with GH or glucagon-like peptide-1 (GLP-1) revealed that SOCS-3 inhibited GH- but not GLP-1-mediated islet cell proliferation, indicating that the decreased β-cell volume observed in female transgenic mice could be caused by an inhibition of GH-induced β-cell proliferation by SOCS-3. In spite of the reduced β-cell volume the transgenic female mice exhibited enhanced glucose tolerance compared with wild-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic β-cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.
Combination therapies are increasingly common in the clinical management of type 2 diabetes. We investigated to what extent combined treatment with the human glucagon-like peptide-1 (GLP-1) analogue ...liraglutide and the dual PPARα/γ agonist ragaglitazar would improve glycaemic control in overtly diabetic Zucker diabetic fatty (ZDF) rats. Ninety overtly diabetic male ZDF rats were stratified into groups with matched haemoglobin A1c (HbA₁c) (9.0 ± 0.1%). Liraglutide (15 and 50 μg/kg subcutaneously twice daily), ragaglitazar (1 and 3 mg/kg perorally once daily) and their vehicles were studied as monotherapy and in combination in a 3 x 3 factorial design. After 4-week treatment, synergistic effects on HbA₁c, non-fasting morning blood glucose (BG) and/or 24-h BG profiles were observed with three of the four combinations. The relationship between plasma insulin and BG in combination-treated animals approached that of historical lean ZDF rats representing normal glucose homeostasis, suggesting that insulin secretion and insulin sensitivity were markedly improved. Increased insulin immunostaining in islets further supports the improved beta-cell function and/or insulin sensitivity in combination-treated animals. The synergistic effect on glycaemic control was found without a similar synergistic increase in beta-cell mass in the combination groups. Our data demonstrate that combination treatment with a human GLP-1 analogue and a dual PPARα/γ agonist through distinct mechanism of actions synergistically improves glycaemic control in the ZDF rat.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Many methods have been devised for double immunocytochemical staining. We now describe that moderate microwaving does not elute antibodies, but prevents their reactions with subsequently applied ...reagents. Thus, microwaving performed in between the first and second staining cycles permits double indirect immunofluorescence staining with antibodies raised in the same species. Moreover, microwaving also inhibits reactions with endogenous immunoglobulins present in extracellular compartments. This substantially reduces background in indirect immunostaining of mouse tissues with mouse monoclonal antibodies.
Binding of factor VIIa (FVIIa) to its cellular receptor tissue factor (TF) was previously shown to induce various intracellular signaling events, which were thought to be responsible for TF-mediated ...biologic effects, including angiogenesis, tumor metastasis, and restenosis. To understand the mechanisms behind these processes, we have examined the effect of FVIIa on apoptosis. Serum deprivation–induced apoptosis of BHK(+TF) cells was characterized by apoptotic blebs, nuclei with chromatin-condensed bodies, DNA degradation, and activation of caspase 3. FVIIa markedly decreased the number of cells with apoptotic morphology and prevented the DNA degradation as measured by means of TdT-mediated dUTP nick end labeling (TUNEL). The antiapoptotic effect of FVIIa was confirmed by the observation that FVIIa attenuated caspase 3 activation. FVIIa-induced antiapoptotic effect was dependent on its proteolytic activity and TF but independent of factor Xa and thrombin. FVIIa-induced cell survival correlated with the activation of Akt and was inhibited markedly by the specific PI3-kinase inhibitor, LY294002. Blocking the activation of p44/42 mitogen-activated protein kinase (MAPK) by the specific mitogen-induced extracellular kinase (MEK) inhibitor, U0126, impaired modestly the ability of FVIIa to promote cell survival. In conclusion, FVIIa binding to TF provided protection against apoptosis induced by growth factor deprivation, primarily through activation of PI3-kinase/Akt pathway, and to a lesser extent, p44/42 MAPK pathway.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
GH and PRL have been shown to stimulate proliferation and insulin production in islets of Langerhans. To identify genes regulated by GH/PRL in islets, we performed differential screening of a ...complementary DNA library from neonatal rat islets cultured for 24 h with human GH (hGH). One hGH-induced clone had 96% identity with mouse preadipocyte factor-1 (Pref-1, or delta-like protein (Dlk). The size of Pref-1 messenger RNA (mRNA) in islets was 1.6 kilobases, with two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-1 mRNA content of islets from adult rats was only 1% of that in neonatal islets. Pref-1 mRNA was markedly up-regulated in islets from pregnant rats from day 12 to term compared with those from age-matched female rats. Two peaks in mRNA expression were observed during gestation, one on day 14 and the other at term, whereafter it decreased to nonpregnant levels. Pref-1 mRNA was up-regulated 3- to 4-fold in neonatal rat islets of Langerhans after 48-h culture with hGH, as found also with bovine GH or ovine PRL. During the development of pancreas from embryonic day 12 (E12) to postnatal day 4, we observed a 2-fold increase in Pref-1 mRNA on E17 and a 5-fold increase at birth, followed by a rapid decline on postnatal day 4. Pref-1 immunoreactivity was found in a subpopulation of insulin cells of neonatal islets of Langerhans. At an early embryonal stage (E13), most cells of the pancreatic anlage were Pref-1 positive, becoming predominantly restricted to the insulin-producing cells during development. In conclusion, these findings suggest that Pref-1 is involved in both differentiation and growth of beta-cells.
A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No ...contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable (14)Ctetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Monospecific antisera against two fetal antigens (FA-1 and FA-2), alphafetoprotein (AFP) and two endometrial proteins (PP12 and PP14) were used to examine the distribution of these proteins and ...antigens in human trophoblast and gestational endometrium in first and third trimesters of pregnancy, normal human ovary and fetal tissues by indirect immunoperoxidase histochemical localisation techniques. Fetal liver stained exclusively for FA-1 and AFP which was used as a reference protein. Staining for FA-2 was seen in fetal connective tissue, in particular the basement membrane. FA-1 and FA-2 did not stain positively in decidua, trophoblast or ovarian tissue. Gestational endometrium stained positively for PP14 exclusively in the glandular epithelium, whilst staining for PP12 was seen only in the stromal cells. Trophoblast, both early and late, and ovarian tissue did not stain positively for any of the four substances tested.
Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many ...monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded porcine tissue sections using the highly sensitive tyramide signal amplification system. Combining this method with different antigen retrieval techniques enabled us to detect CD2, CD3, CD4, CD8 and SWC3 antigen expressing cells in porcine lymphoid tissue. Thus, we describe herein methods for the detection of several major cell types of the porcine immune system in fixed tissue with optimal preservation of histological details.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
4-hydroxyphenylpyruvate dioxygenase (HPD) is an important enzyme involved in tyrosine catabolism. HPD was shown to be identical to a protein named the F-antigen, exploited by immunologists because of ...its unique immunological properties. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. Decreased expression of HPD is often observed in association with the severe type I tyrosinemia, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. In this study we present a comprehensive analysis of tissue specific expression and intracellular localization of HPD in the rat. By combined use of in situ hybridization and immunohistochemistry we confirm previously known sites of expression in liver and kidney. In addition, we show that HPD is abundantly expressed in neurons in the cortex, cerebellum and hippocampus. By using immunoelectron microscopy and confocal laser scanning microscopy, we provide evidence that HPD contrary to earlier assumptions specifically localizes to membranes of the endoplasmic reticulum and the Golgi apparatus. Detailed mass spectrometric analyses of HPD purified from rat liver revealed N-terminal and C-terminal processing of HPD, and expression of recombinant HPD suggested that C-terminal processing enhances the enzymatic activity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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