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BBA - Molecular Basis of Disease (v.1832, #6)
Insulin-like growth factor 1 enhances bile-duct proliferation and fibrosis in Abcb4−/− mice by Sokolovic Aleksandar Sokolović; Carlos M. Rodriguez-Ortigosa; Lysbeth ten Bloemendaal; Ronald P.J. Oude Elferink; Jesús Prieto; Piter J. Bosma (pp. 697-704).
Adamant progression of chronic cholangiopathies towards cirrhosis and limited therapeutic options leave a liver transplantation the only effective treatment. Insulin-like growth factor 1 (IGF1) effectively blocks fibrosis in acute models of liver damage in mice, and a phase I clinical trial suggested an improved liver function. IGF1 targets the biliary epithelium, but its potential benefit in chronic cholangiopathies has not been studied. To investigate the possible therapeutic effect of increased IGF1 expression, we crossed Abcb4−/− mice (a model for chronic cholangiopathy), with transgenic animals that overexpress IGF1. The effect on disease progression was studied in the resulting IGF1-overexpressing Abcb4−/− mice, and compared to that of Abcb4−/− littermates. The specificity of this effect was further studied in an acute model of fibrosis. The overexpression of IGF1 in transgenic Abcb4−/− mice resulted in stimulation of fibrogenic processes — as shown by increased expression of Tgfß, and collagens 1, 3 and 4, and confirmed by Sirius red staining and hydroxyproline measurements. Excessive extracellular matrix deposition was favored by raise in Timp1 and Timp2, while a reduction of tPA expression indicated lower tissue remodeling. These effects were accompanied by an increase in expression of inflammation markers like Tnfα, and higher presence of infiltrating macrophages. Finally, increased number of Ck19-expressing cells indicated proliferation of biliary epithelium. In contrast to liver fibrosis associated with hepatocellular damage, IGF1 overexpression does not inhibit liver fibrogenesis in chronic cholangiopathy.► Effect of IGF1 on biliary fibrosis was tested in a model of chronic cholangiopathies. ► IGF1 overexpression increased fibrogenesis and lowered fibrolysis in Abcb4−/− mice. ► IGF1 overexpression stimulated inflammation and recruitment of macrophages. ► The enhanced IGF1 signaling stimulated cholangiocyte and bile duct proliferation. ► IGF1 is not an option for treating fibrosis caused by chronic cholangiopathies.
Keywords: Abbreviations; Abcb4; −/−; ATP-binding cassette, sub-family B member 4; ECM; extracellular matrix; F4/80; EGF-like module containing, mucin-like, hormone receptor-like sequence 1; HSCs; hepatic stellate cells; α-SMA; actin, alpha 2, smooth muscle; tPA; tissue-type plasminogen activator; Ck19; keratin 19; ALT; alanine transaminase; AST; aspartate aminotransferase; ALP; alkaline phosphataseLiver fibrosis; Abcb4; −/−; mice; IGF1; Primary sclerosing cholangitis; Cholangiocytes proliferation
Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate by Barros-Minones Lucía Barros-Miñones; Martin-de-Saavedra Dolores Martín-de-Saavedra; Sergio Perez-Alvarez; Lourdes Orejana; Suquia Verónica Suquía; Goni-Allo Beatriz Goñi-Allo; Isabel Hervias; Lopez Manuela G. López; Joaquin Jordan; Norberto Aguirre; Elena Puerta (pp. 705-717).
Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia–reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5mg/kg by mouth (p.o.)), given 30min before malonate (1.5μmol/2μL), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h.► Sildenafil affords a significant protection against malonate-induced chemical hypoxia. ► The mechanism relies on the inhibition of malonate-induced calpain/p25/cdk5 signaling pathway activation. ► Sildenafil also increases the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xL. ► This neuroprotection was extended to an in vitro model of oxygen glucose deprivation. ► Therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h.
Keywords: Abbreviations; BSA; bovine serum albumin; cdk5; cyclin-dependent kinase 5; cGMP; cyclic guanosine monophosphate; Cyt c; cytochrome c; DG; dentate gyrus; LDH; lactate dehydrogenase; MCAo; middle cerebral artery occlusion; MDL28170; carbobenzoxy-valinyl-phenyl-alaninal; MEF2; myocyte enhancer factor 2; OGD; oxygen glucose deprivation; PBS; phosphate-buffered saline; PDE5; Phosphodiesterase 5; PDEi; Phosphodiesterase 5 inhibitor; PI; propidium iodide; ROS; reactive oxygen species; SDH; succinate dehydrogenaseApoptosis; Calpain; Cyclin-dependent kinase 5 (cdk5); Excitotoxicity; Malonate; Sildenafil
Hepatocyte-specific Dyrk1a gene transfer rescues plasma apolipoprotein A-I levels and aortic Akt/GSK3 pathways in hyperhomocysteinemic mice by Asma Tlili; Frank Jacobs; Leanne de Koning; Sirine Mohamed; Linh-Chi Bui; Julien Dairou; Nicole Belin; Véronique Ducros; Thierry Dubois; Jean-Louis Paul; Jean-Maurice Delabar; Bart De Geest; Nathalie Janel (pp. 718-728).
Hyperhomocysteinemia, characterized by high plasma homocysteine levels, is recognized as an independent risk factor for cardiovascular diseases. The increased synthesis of homocysteine, a product of methionine metabolism involving B vitamins, and its slower intracellular utilization cause increased flux into the blood. Plasma homocysteine level is an important reflection of hepatic methionine metabolism and the rate of processes modified by B vitamins as well as different enzyme activity. Lowering homocysteine might offer therapeutic benefits. However, approximately 50% of hyperhomocysteinemic patients due to cystathionine-beta-synthase deficiency are biochemically responsive to pharmacological doses of B vitamins. Therefore, effective treatments to reduce homocysteine levels are needed, and gene therapy could provide a novel approach. We recently showed that hepatic expression of DYRK1A, a serine/threonine kinase, is negatively correlated with plasma homocysteine levels in cystathionine-beta-synthase deficient mice, a mouse model of hyperhomocysteinemia. Therefore, Dyrk1a is a good candidate for gene therapy to normalize homocysteine levels. We then used an adenoviral construct designed to restrict expression of DYRK1A to hepatocytes, and found decreased plasma homocysteine levels after hepatocyte-specific Dyrk1a gene transfer in hyperhomocysteinemic mice. The elevation of pyridoxal phosphate was consistent with the increase in cystathionine-beta-synthase activity. Commensurate with the decreased plasma homocysteine levels, targeted hepatic expression of DYRK1A resulted in elevated plasma paraoxonase-1 activity and apolipoprotein A-I levels, and rescued the Akt/GSK3 signaling pathways in aorta of mice, which can prevent homocysteine-induced endothelial dysfunction. These results demonstrate that hepatocyte-restricted Dyrk1a gene transfer can offer a useful therapeutic targets for the development of new selective homocysteine lowering therapy.► Plasma homocysteine level is decreased after hepatocyte-specific Dyrk1a gene transfer. ► The elevation of pyridoxal phosphate is consistent with the increase in CBS activity. ► The effect on aortic Akt/GSK3 pathways is consistent with the plasma apo A-I level.
Keywords: Abbreviations; Apo A-I; apolipoprotein A-I; CBS; cystathionine-β-synthase; DS; Down syndrome; DYRK1A; dual-specificity tyrosine-(Y)-phosphorylation regulated kinase-1A; HDL; high-density lipoprotein; Hcy; homocysteine; NFAT; nuclear factor of activated T cell; NQO1; NAD(P)H quinone oxidoreductase-1; PLP; pyridoxal phosphate; PON1; paraoxonase-1; RPPA; reverse phase protein array; SAM; S-adenosylmethionine; SAH; S-adenosylhomocysteine; SAHH; S-adenosylhomocysteine hydrolase; VEGF; vascular endothelial growth factorApolipoprotein A-I; Dyrk1a gene transfer; Homocysteine; Pyridoxal phosphate; Akt/GSK3 pathway; Aorta
A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice by Wint Nandar; Elizabeth B. Neely; Erica Unger; James R. Connor (pp. 729-741).
Because of the increasing evidence thatH63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences ofH63D HFE in vivo using mice that carryH67D HFE (homologous to humanH63D). Although total brain iron concentration did not change significantly in theH67D mice, brain iron management proteins expressions were altered significantly. The 6-month-oldH67D mice had increased HFE and H-ferritin expression. At 12months,H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia inH67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-oldH67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in theH67D mice. These findings demonstrate that theH63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. TheH67D knock-in mouse can be used as a model to evaluate how theH63D HFE mutation contributes to neurodegenerative diseases.►H63D HFE impacts brain iron homeostasis. ►H63D HFE promotes an environment for oxidative stress and gliosis. ► TheH67D mice develop adaptive mechanisms to maintain brain iron homeostasis. ► TheH67D mouse can be used to study the role ofH63D HFE in neurodegeneration.
Keywords: H63D HFE; Iron; Oxidative stress; Gliosis; Neurodegenerative disease
Enhancement of brain-type creatine kinase activity ameliorates neuronal deficits in Huntington's disease by Yow-Sien Lin; Tzu-Hao Cheng; Chin-Pang Chang; Hui-Mei Chen; Yijuang Chern (pp. 742-753).
Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin ( HTT) gene. Brain-type creatine kinase (CKB) is an enzyme involved in energy homeostasis via the phosphocreatine–creatine kinase system. Although downregulation of CKB was previously reported in brains of HD mouse models and patients, such regulation and its functional consequence in HD are not fully understood. In the present study, we demonstrated that levels of CKB found in both the soma and processes were markedly reduced in primary neurons and brains of HD mice. We show for the first time that mutant HTT (mHTT) suppressed the activity of the promoter of the CKB gene, which contributes to the lowered CKB expression in HD. Exogenous expression of wild-type CKB, but not a dominant negative CKB mutant, rescued the ATP depletion, aggregate formation, impaired proteasome activity, and shortened neurites induced by mHTT. These findings suggest that negative regulation of CKB by mHTT is a key event in the pathogenesis of HD and contributes to the neuronal dysfunction associated with HD. In addition, besides dietary supplementation with the CKB substrate, strategies aimed at increasing CKB expression might lead to the development of therapeutic treatments for HD.► CKB exists in the soma and neuronal processes of neurons. ► Mutant Huntingtin suppresses the promoter of the CKB gene. ► Overexpression of a functional CKB rescues the proteasome activity and neuritogenesis impaired by mutant Huntingtin. ► Creatine supplementation increases CKB expression and ameliorates major symptoms of HD. ► Strategies aimed at increasing the expression of CKB might lead to the development of therapeutic treatments for HD.
Keywords: Huntington's disease; Brain-type creatine kinase; Neuritogenesis; Protein aggregate; Proteasome
TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways by Raúl Vivar; Claudio Humeres; Pedro Ayala; Ivonne Olmedo; Catalan Mabel Catalán; Garcia Lorena García; Sergio Lavandero; Diaz-Araya Guillermo Díaz-Araya (pp. 754-762).
Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia–reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.► Ischemia/reperfusion induces cardiac fibroblast apoptosis. ► TGF-β1 only in reperfusion prevents cardiac fibroblast apoptosis. ► TGF-β1 activates ERK1/2, Akt and Smad2/3 to prevent apoptosis induced by I/R.
Keywords: TGF-β1; Cardiac fibroblast; Cell death, apoptosis, ischemia/reperfusion
Alternative splicing of iodothyronine deiodinases in pituitary adenomas. Regulation by oncoprotein SF2/ASF by Agnieszka Piekielko-Witkowska; Hanna Kedzierska; Piotr Poplawski; Anna Wojcicka; Beata Rybicka; Maria Maksymowicz; Wieslawa Grajkowska; Ewa Matyja; Tomasz Mandat; Wieslaw Bonicki; Pawel Nauman (pp. 763-772).
Pituitary tumors belong to the group of most common neoplasms of the sellar region. Iodothyronine deiodinase types 1 (DIO1) and 2 (DIO2) are enzymes contributing to the levels of locally synthesized T3, a hormone regulating key physiological processes in the pituitary, including its development, cellular proliferation, and hormone secretion. Previous studies revealed that the expression of deiodinases in pituitary tumors is variable and, moreover, there is no correlation between mRNA and protein products of the particular gene, suggesting the potential role of posttranscriptional regulatory mechanisms. In this work we hypothesized that one of such mechanisms could be the alternative splicing. Therefore, we analyzed expression and sequences of DIO1 and DIO2 splicing variants in 30 pituitary adenomas and 9 non-tumorous pituitary samples. DIO2 mRNA was expressed as only two mRNA isoforms. In contrast, nine splice variants of DIO1 were identified. Among them, five were devoid of exon 3. In silico sequence analysis of DIO1 revealed multiple putative binding sites for splicing factor SF2/ASF, of which the top-ranked sites were located in exon 3. Silencing of SF2/ASF in pituitary tumor GH3 cells resulted in change of ratio between DIO1 isoforms with or without exon 3, favoring the expression of variants without exon 3. The expression of SF2/ASF mRNA in pituitary tumors was increased when compared with non-neoplastic control samples. In conclusion, we provide a new mechanism of posttranscriptional regulation of DIO1 and show deregulation of DIO1 expression in pituitary adenoma, possibly resulting from disturbed expression of SF2/ASF.► Iodothyronine deiodinases DIO1 and DIO2 regulate biosynthesis of thyroid hormone. ► Thyroid hormone regulates pituitary functions, including proliferation. ► We report different splicing patterns of DIO1 and DIO2 in pituitary adenomas. ► SF2/ASF oncogene is upregulated in pituitary adenomas and regulates DIO1 splicing.
Keywords: Abbreviations; DIO1; iodothyronine deiodinase type 1; DIO2; iodothyronine deiodinase type 2; T4; 3,5,3′,5′-tetraiodothyronine; T3; 3,5,3′-triiodothyronine; rT3; reverse T3; SF2/ASF; splicing factor 2/alternative splicing factor; NFPA; nonfunctioning pituitary adenomaDeiodinase; Thyroid hormone; Alternative splicing; SF2/ASF; Pituitary adenoma; GH3 cells
Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism by Sara Violante; Lodewijk IJlst; Jos Ruiter; Janet Koster; Henk van Lenthe; Marinus Duran; Isabel Tavares de Almeida; Ronald J.A. Wanders; Sander M. Houten; Fátima V. Ventura (pp. 773-779).
Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.► We determined the substrate specificity of human carnitine acetyltransferase, CrAT. ► CrAT is responsible for the synthesis of short- and branched-chain acylcarnitines. ► Trans-2-enoyl-CoAs and 2-methylacyl-CoAs are poor substrates for CrAT. ► Short-chain dicarboxylic acyl-CoAs are not substrates for CrAT. ► Origin of most acylcarnitines detected in inborn errors of metabolism is resolved.
Keywords: Abbreviations; BCAAO; branched-chain amino acid oxidation; CPT1; carnitine palmitoyltransferase 1; CPT2; carnitine palmitoyltransferase 2; CrAT; carnitine acetyltransferase; CrOT; carnitine octanoyltransferase; mCrAT; mitochondrial CrAT; mFAO; mitochondrial fatty acid beta-oxidation; pCrAT; peroxisomal CrATAcylcarnitine; Fatty acid oxidation; Branched-chain amino acid oxidation; Enoyl-CoA intermediate; Carnitine shuttle
Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart by Bénédicte Demeulder; Elham Zarrinpashneh; Audrey Ginion; Benoit Viollet; Louis Hue; Mark H. Rider; Jean-Louis Vanoverschelde; Christophe Beauloye; Sandrine Horman; Luc Bertrand (pp. 780-790).
Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)–p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR–p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR–p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR–p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR–p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR–p70S6K pathway during ischemia. This challenges the accepted notion that mTOR–p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism.•We genetically prove that cardiac AMPKα2 inhibits eEF2 during myocardial ischemia.•AMPKα2 is not required for p70S6K inhibition during ischemia.•This challenges the accepted notion that p70S6K inhibition by ischemia is due to AMPK.•In normoxia, cardiac AMPKα2 puts a brake on p70S6K via Raptor.
Keywords: Abbreviations; AMPK; AMP-activated protein kinase; 4E-BP1; eIF-4E binding protein-1; eEF2; Eukaryotic elongation factor 2; eEF2K; eEF2 kinase; eIF-4E; Eukaryotic initiation factor-4E; IGF-1; Insulin-like growth factor-1; KO; knockout; mTOR; Mammalian target of rapamycin; mTORC1; mTOR complex 1; p70S6K; p70 ribosomal S6 protein kinase; PKB/Akt; protein kinase B; Raptor; Regulatory associated protein of mTOR; S6; Ribosomal S6 protein; TSC2; tuberous sclerosis complex 2AMPK; Myocardial ischemia; Protein synthesis; p70S6K; eEF2; PKB/Akt
Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits by Agnieszka Piwkowska; Dorota Rogacka; Małgorzata Kasztan; Stefan Angielski; Maciej Jankowski (pp. 791-804).
The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300nM, 5min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.► Insulin induced an increase in albumin permeability through PKGIα activation. ► Insulin signaling requires activation of the NOX4 subunit of NAD(P)H oxidase. ► Albumin permeability increases in glomeruli isolated from Zucker rats ► Augmentation of PKGIα and NOX4 expression in Zucker rats
Keywords: Abbreviations; PKGIα; protein kinase G type Iα; MLC; myosin light chain; MLCP; myosin light chain phosphatase; MLCK; myosin light chain kinase; MYPT1; myosin phosphatase target subunit 1Filtration barrier permeability; Insulin; NAD(P)H oxidase; Protein kinase G type Iα; Podocyte; Hyperinsulinemia
Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy by Zhen Zhang; Bao-ying Li; Xiao-li Li; Mei Cheng; Fei Yu; Wei-da Lu; Qian Cai; Jun-fu Wang; Rui-hai Zhou; Hai-qing Gao; Lin Shen (pp. 805-816).
Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1⁄2), Akt and glycogen synthase kinase-3beta (GSK-3β) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3β in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.•GSPB2 improves renal dysfunction and histological lesions in db/db mice.•MFG-E8 is up-regulated in diabetic kidney and back-regulated to normal after GSPB2 treatment identified by iTRAQ.•MFG-E8 silencing improves renal histological lesions by inhibiting the phosphorylation of ERK1⁄2, Akt and GSK-3β.•In contrast, over-expression of MFG-E8 results in the opposite effects.•GSPB2 ameliorates diabetic nephropathy by down-regulation MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.
Keywords: Abbreviations; AGEs; advanced glycation end products; BUN; blood urea nitrogen; Cr; plasma creatinine concentration; DM; diabetes mellitus; DN; diabetic nephropathy; ERK1/2; extracellular signal-regulated kinase1/2; ESRD; end-stage renal disease; FBG; fasting blood glucose; GSK-3β; glycogen synthase kinase-3beta; GSPB2; grape seed procyanidin B2; GSPE; grape seed proanthocyanidin extracts; iTRAQ; isobaric tags for relative and absolute quantification; MFG-E8; milk fat globule EGF-8Grape seed procyanidin B2; Diabetic nephropathy; MFG-E8; db/db mice; iTRAQ
Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site by Hee-Seong Jang; Jee In Kim; Kyong-Jin Jung; Jinu Kim; Ki-Hwan Han; Kwon Moo Park (pp. 817-825).
Increase of interstitial cell population, resulting in the expansion of interstitium, excessive production of extracellular matrix, and reduction of functioning tubules, is critical in fibrotic progression in the kidney of patients suffering from chronic renal diseases. Here, we investigated the contribution of bone marrow-derived cells (BMDC) in kidney fibrosis caused by ureteral obstruction (UO) using eGFP bone marrow-reconstituted chimeric mice. UO caused dramatic increases in the numbers of interstitial cells and expansion of the interstitium. Most kidney interstitial cells expressed GFP. Twenty nine percent of interstitial cells were cells that had proliferated and approximately 89% among them were BMDCs. Proliferation of fibroblasts differentiated from BMDCs significantly occurred in the interstitium of UO-kidney. Removal of BMDCs by whole body irradiation after UO resulted in reduction of kidney fibrosis, while injection of RAW264.7 cells, monocytes/macrophages, into irradiated mice induced a reversal of this reduction. Treatment with apocynin, an inhibitor of NADPH oxidase, reduced infiltration of BMDCs into the UO-kidney, leading to reduction of kidney fibrosis. In addition, only a few slow-cycling cells were observed in the interstitium of normal kidney. Even after UO, no change in the number of those cells was observed. Our findings demonstrate that BMDCs are a major source for interstitial expansion during kidney fibrosis via infiltration into damaged sites, differentiation to fibroblasts, and subsequent proliferation, contributing kidney fibrosis. These data provide a clear therapeutic target for treatment of chronic kidney disease.•BMDCs occupy the majority of increased interstitial population in fibrotic kidney.•BMDCs differentiate into the cell that has characteristics of fibroblast and subsequently proliferate in fibrotic kidney.•Inhibition of BMDCs infiltration and production prevents the kidney fibrosis.•Interstitial slow-cycling cell may not participate in increased fibroblast population in fibrotic kidney.
Keywords: Fibrosis; Fibroblast; Bone marrow-derived cell; Differentiation; Proliferation; Reactive oxygen species
The PrPC C1 fragment derived from the ovine A136R154R171 PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrPC protein in vitro by Lauren Campbell; Andrew C. Gill; Gillian McGovern; Clara M.O. Jalland; John Hopkins; Michael A. Tranulis; Nora Hunter; Wilfred Goldmann (pp. 826-836).
Expression of the cellular prion protein (PrPC) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrPC comprised of 25% more C1 fragment than PrPC from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrPC variants. We propose that the increased α-cleavage of ovine ARR PrPC contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrPC β-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.•Cleavage of prion protein in the brain may influence susceptibility to prion disease.•Levels of truncated PrPC (C1) varied in sheep of different PRNP genotypes.•Only C1 from ARR variant inhibited the in-vitro fibril formation of prion protein.•C1 fragment formation may exert negative dominance over scrapie susceptibility.•The α-cleavage of PrPC may be a therapeutic target in prion disease.
Keywords: Abbreviations; PrP; C; cellular prion protein; CNS; central nervous system; CJD; Creutzfeldt–Jakob disease; CWD; chronic wasting disease; BSE; bovine spongiform encephalopathy; PrP; Sc; disease associated misfolded prion protein; NPU; Neuropathogenesis Unit; AHVLA; Animal Health and Veterinary Laboratories Agency; PMSF; phenylmethanesulfonylfluoride; NEM; N-ethylmaleimide; PNGase F; peptide:N-glycosidase F; TBS; Tris-buffered saline; TBST; TBS-Tween; ALP; alkaline phosphatase; IPTG; isopropylthio-β-galactoside; HPLC; high pressure liquid chromatography; MES; 2-(N-morpholino)ethanesulfonic acid sodium salt; ThT; Thioflavin TPrion; Transmissible spongiform encephalopathy; Fibrillization; Protein-cleavage
Early cognitive stimulation compensates for memory and pathological changes in Tg2576 mice by Gorka Gerenu; Marta Dobarro; Maria J. Ramirez; Francisco J. Gil-Bea (pp. 837-847).
Education and cognitive occupations are commonly associated to reduce risk of Alzheimer's disease (AD) or dementia. Animal studies have demonstrated that cognitive stimulation (CS) achieved by social/physical activities and/or enriched environments compensates for memory decline. We have elaborated a novel paradigm of CS that is devoid of physical/social activity and enriched environments. 4month-old Tg2576 mice were cognitively trained for 8weeks and, after a break of 8months, long-lasting effects of CS on cognitive abilities and AD-like pathology were measured. Morris Water Maze (MWM) and Novel Object Recognition (NOR) tests showed that deficits in spatial and recognition memories were compensated by CS. These outcomes were accompanied by increased levels of hippocampal post-synaptic markers (PSD95 and NR1) and proteins involved in synaptic formation (Arc, β-catenin). CS softened amyloid pathology in terms of reduced levels of Aβ1–42 and the dodecameric assembly, referred as Aβ*56. CS appeared to affect the APP processing since differences in levels of ADAM17, BACE1 and C99/C83 ratio were found. Tau hyper-phosphorylation and high activities of tau kinases were also reduced by CS. In contrast, CS did not induce any of these molecular changes in wild-type mice. The present findings suggest beneficial and long-lasting effects of CS early in life on cognitive decline and AD-like pathology.•A novel paradigm to induce cognitive stimulation in mice has been tested.•Early-in-life cognitive stimulation delayed cognitive decline in Tg2565 mice.•Early-in-life cognitive stimulation compensated for synaptic pathology in Tg2576 mice.•Early-in-life cognitive stimulation reduced amyloid pathology in Tg2565 mice.•Early-in-life cognitive stimulation reduced tau hyperphosphorylation in Tg2576 mice.
Keywords: Abbreviations; AD; Alzheimer's disease; CS; cognitive stimulation; MWM; Morris Water Maze; NOR; novel object recognition; WT; wild-typeCognitive training; hAPP transgenic mouse; Synaptic; Amyloid; Tau
Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress by Maolong Dong; Nan Hu; Yinan Hua; Xihui Xu; Machender R. Kandadi; Rui Guo; Shasha Jiang; Sreejayan Nair; Dahai Hu; Jun Ren (pp. 848-863).
Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca2+ properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca2+ release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca2+ properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca2+ anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.•We examine the effect of Akt activation in sepsis-induced cardiomyopathy.•Chronic Akt activation alleviates LPS-induced cardiac mechanical defect.•Akt activation inhibits apoptosis and ER stress in sepsis.
Keywords: Sepsis; Heart; Contractile function; Akt; ER stress; Apoptosis