BBA - Molecular Basis of Disease (v.1842, #11)
Editorial Board (i).
S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis by Mikael Brisslert; Li Bian; Mattias N.D. Svensson; Rita F. Santos; Ing-Marie Jonsson; Igor Barsukov; Malin Erlandsson; Karin Andersson; Alexandre M. Carmo; Maria I. Bokarewa (2049-2059).
Objectives: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. Methods: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. Results: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3+ Treg cells, while the number of RORγt+ and (pTyr705)STAT3+ cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70 kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. Conclusion: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.S100A4 coordinates a balance between proliferation and differentiation of antigen-exposed T-cells promoting CD5–Fyn–RhoA–STAT3 dependent effects.Scr-tyrosine kinases Lck and Fyn control T-cell receptor (TCR) signalling by the phosphorylation of an immunoreceptor tyrosine-based activation motif of the CD3 chains and of the TCR-inhibitor CD5. S100A4 binding to Lck and Fyn reciprocally regulates their kinase activity and gains control over T-cell proliferation. It limits the Lck-dependent phosphorylation of ZAP-70 (theta-chain associated protein kinase of 70 kDa) and facilitates Fyn-dependent phosphorylation of CD5. S100A4 promotes a link between the Fyn and RhoA (Ras homolog gene family, member A)-system, which coordinates phosphorylation of IRF4 (interferon regulatory factor 4) and STAT3 (signal transducer and activator of transcription 3), the transcription factors critical for the expression of RORγt (retinoic acid orphan receptor gamma t) and the formation of Th (T helper) 17 cells.Display Omitted
Keywords: S100A4; Th17 cell; TCR; Src-tyrosine kinase; Arthritis; CD5;
A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition by Karl A. Rodriguez; Pawel A. Osmulski; Anson Pierce; Susan T. Weintraub; Maria Gaczynska; Rochelle Buffenstein (2060-2072).
The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~ 31 years) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although Heat shock protein 72 kDa (HSP72) and Heat shock protein 40 kDa (Homolog of bacterial DNAJ1) (HSP40(Hdj1)) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging.Display Omitted
Keywords: Proteasome; Heat shock protein; Naked mole-rat; Protease inhibitor; Protein degradation; Aging;
Autophagy inhibition suppresses the tumorigenic potential of cancer stem cell enriched side population in bladder cancer by Rani Ojha; Vivekanand Jha; Shrawan Kumar Singh; Shalmoli Bhattacharyya (2073-2086).
The mechanisms that underlie tumor formation and progression have not been elucidated in detail in cancer biology. Recently, the identification of a tumor cell subset defined as cancer stem cells (CSCs), which is enriched for tumor initiating capacity, has engendered new perspectives towards selective targeting of tumors. In this study, we isolated the side population (SP) cells which share characteristics of CSCs from bladder cancer cell lines, T24 and UM-UC-3 by fluorescence activated cell sorting. The cells were cultured in serum free medium and expression profile of stem cell like markers (SOX-2, NANOG, KLF-4 and OCT-4), drug resistant genes (ABCG2 and MDR1) and spheroid forming capability were examined in SP, non-side population (NSP) and bulk T24 and UM-UC-3 cells. We observed that SP cells possessed a higher mRNA expression of SOX-2, NANOG, KLF-4, OCT-4, ABCG2, and MDR1 as well as a higher spheroid forming ability as compared to other bulk cells or NSP cells. The SP cells had low ROS levels and high GSH/GSSG ratio which may contribute to radio-resistance. The SP cells also showed substantial resistance to gemcitabine, mitomycin and cisplatin compared with the NSP counterpart. A high autophagic flux was observed in the SP cells. Both pharmacological and siRNA mediated inhibition of autophagy potentiated the chemotherapeutic effects of gemcitabine, mitomycin and cisplatin in these cells. We concluded that the ABCG2 expressing SP cells show autophagy associated cell survival and may be a potent target for developing more effective treatment in bladder carcinoma to enhance patient survival.Display Omitted
Keywords: Side population; Cancer stem cell; Autophagy; Bladder cancer; T24; UM-UC-3;
Overexpression of Oct4 suppresses the metastatic potential of breast cancer cells via Rnd1 downregulation by Long Shen; Kunhua Qin; Dekun Wang; Yan Zhang; Nan Bai; Shengyong Yang; Yunping Luo; Rong Xiang; Xiaoyue Tan (2087-2095).
Although Oct4 is known as a critical transcription factor involved in maintaining “stemness”, its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo. Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo. Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression.
Keywords: Oct4; Metastasis; Breast cancer; E-cadherin; Rnd1;
The arachidonic acid–LTB4–BLT2 pathway enhances human B-CLL aggressiveness by Nathalie Guriec; Catherine Le Jossic- Corcos; Brigitte Simon; Jean-Christophe Ianotto; Adrian Tempescul; Yvonne Dréano; Jean-Pierre Salaün; Christian Berthou; Laurent Corcos (2096-2105).
Deregulation of the oxidative cascade of poly-unsaturated fatty acids (PUFAs) has been associated with several cancers, including chronic lymphocytic leukemia (B-CLL). Leukotriene B4 (LTB4), a metabolite of arachidonic acid (AA), is produced by B-CLL and contributes to their survival. The aim of the present study was to analyze the activity of the oxidative cascade of PUFAs in B-CLL. Purified B cells from patients and normal B CD5 positive cells were subjected to flow cytometry, Western-blot and RT-qPCR analyses. LTB4 plasma and intracellular concentrations were determined by ELISA. Our results showed that aggressive B-CLL tumor cells, i.e. cells with an annual proliferation index above 2, over-expressed calcium-dependent and calcium-independent phospholipases A2 (cPLA2-alpha and iPLA2-beta, respectively), 5-lipoxygenase (5LOX) and leukotriene A4 hydroxylase (LTA4H). Intracellular LTB4 levels were lower in the most aggressive cells than in cells with a smaller proliferation index, despite equivalent plasma levels, and lower expression of cytochrome P450 4F3A (CYP4F3A), one major enzyme involved in LTB4 inactivation. Since BLT2, a LTB4 membrane receptor was also more often expressed on aggressive tumor cells, and since a BLT2 inhibitor significantly impaired B-CLL viability in vitro, we propose that LTB4 was efficiently trapped onto BLT2 present on aggressive tumors, thereby eliciting an autocrine response. Taken together our results demonstrate a major deregulation of the pathway leading to LTB4 synthesis and degradation in B-CLL cells, and provide a framework for understanding how these modifications promote cell survival and proliferation, especially in the most aggressive BCLL.Legend: 5LOX: 5-lipoxygenase, LTA4H: leukotriene A4 hydrolase, AA: arachidonic acid, PL: membrane phospholipids, cPLA2: calcium-dependent phospholipase A2alpha, iPLA2: calcium-independent phospholipase A2beta.Display Omitted
Keywords: B-CLL; LTB4; PPAR; BLT2; Disease progression;
Vascular wall extracellular matrix proteins and vascular diseases by Junyan Xu; Guo-Ping Shi (2106-2119).
Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension.
Keywords: Extracellular matrix protein; Atherosclerosis; Aortic aneurysm; Varicose vein; Hypertension;
Increased serum 2-oxoglutarate associated with high myocardial energy expenditure and poor prognosis in chronic heart failure patients by Ping-An Chen; Zhi-Hao Xu; Yu-Li Huang; Yi Luo; Ding-Ji Zhu; Peng Wang; Zhi-Yong Du; Yang Yang; Dai-Hong Wu; Wen-Yan Lai; Hao Ren; Ding-Li Xu (2120-2125).
Myocardial energy expenditure (MEE) and 2-oxoglutarate are elevated in chronic heart failure (CHF) patients compared with healthy controls. To explore whether 2-oxoglutarate could reflect the levels of MEE and predict the prognosis of CHF, 219 CHF patients and 66 healthy controls were enrolled. 2-Oxoglutarate was assayed with Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (LC/MS/MS). CHF patients were divided into 4 groups according to interquartile range of MEE and followed for death or recurrent hospital admission due to CHF for the mean follow-up time 6.64 ± 0.16 months. 2-Oxoglutarate was increased in CHF patients compared with controls (P < 0.01) and correlated with estimated glomerular filtration rate (r = 0.142, P = 0.036), age (r = − 0.269, P < 0.01) and MEE levels (r = 0.307, P < 0.01) in a multiple linear correlation analysis in CHF patients. Furthermore, 2-oxoglutarate (OR = 3.470, 95% CI = 1.557 to 7.730, P = 0.002), N-terminal pro-B-type natriuretic peptide (OR = 4.013, 95% CI = 1.553 to 10.365, P = 0.004), age (OR = 1.611, 95% CI = 1.136 to 2.283, P = 0.007) and left ventricular ejection fraction (OR = 7.272, 95% CI = 3.110 to 17.000, P < 0.001) were independently associated with MEE on multiple logistic regression analysis. Kaplan–Meier event curves showed that high 2-oxoglutarate levels were associated with adverse outcomes (Log Rank, Chi2 = 4.026, P = 0.045). This study showed that serum 2-oxoglutarate is associated with MEE levels, which can be used as potential biomarkers for MEE, and it can reflect the clinical severity and short-term outcome of CHF.
Keywords: 2-Oxoglutarate; Myocardial energy expenditure; Biomarker; Heart failure;
Long-term effects of combined neonatal and adolescent stress on brain-derived neurotrophic factor and dopamine receptor expression in the rat forebrain by Rachel A. Hill; Szerenke Kiss Von Soly; Udani Ratnayake; Maren Klug; Michele D. Binder; Anthony J. Hannan; Maarten van den Buuse (2126-2135).
Altered brain-derived neurotrophic factor (BDNF) signalling and dopaminergic neurotransmission have been shown in the forebrain in schizophrenia. The ‘two hit’ hypothesis proposes that two major disruptions during development are involved in the pathophysiology of this illness. We therefore used a ‘two hit’ rat model of combined neonatal and young-adult stress to assess effects on BDNF signalling and dopamine receptor expression. Wistar rats were exposed to neonatal maternal separation (MS) stress and/or adolescent/young-adult corticosterone (CORT) treatment. At adulthood the medial prefrontal cortex (mPFC), caudate putamen (CPu) and nucleus accumbens (NAc) were analysed by qPCR and Western blot. The ‘two hit’ combination of MS and CORT treatment caused significant increases in BDNF mRNA and protein levels in the mPFC of male, but not female rats. BDNF mRNA expression was unchanged in the CPu but was significantly reduced by CORT in the NAc. DR3 and DR2 mRNA were significantly up-regulated in the mPFC of two-hit rats and a positive correlation was found between BDNF and DR3 expression in male, but not female rats. DR2 and DR3 expression were significantly increased following CORT treatment in the NAc and a significant negative correlation between BDNF and DR3 and DR2 mRNA levels was found. Our data demonstrate male-specific two-hit effects of developmental stress on BDNF and DR3 expression in the mPFC. Furthermore, following chronic adolescent CORT treatment, the relationship between BDNF and dopamine receptor expression was significantly altered in the NAc. These results elucidate the long-term effects of ‘two hit’ developmental stress on behaviour.
Keywords: BDNF; Stress; Dopamine; Sex difference; Prefrontal cortex; Striatum;
Oligomerization of Cysteine String Protein alpha mutants causing adult neuronal ceroid lipofuscinosis by Yong-quan Zhang; Sreeganga S. Chandra (2136-2146).
Cysteine String Protein alpha (CSPα) is a palmitoylated, synaptic vesicle co-chaperone that is essential for neuroprotection. Two mutations in CSPα — L115R and L116Δ — cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly-inherited neurodegenerative disease. To elucidate the pathogenesis of ANCL, the intrinsic biochemical properties of human wildtype (WT) and disease mutant CSPα were examined. Mutant proteins purified from Escherichia coli exhibited high potency to oligomerize in a concentration, temperature, and time dependent manner, with L115R possessing the greatest potency. When freshly purified, ANCL mutant proteins displayed normal co-chaperone activity and substrate recognition similar to WT. However, co-chaperone activity was impaired for both CSPα mutants upon oligomerization. When WT and mutant CSPα were mixed together they co-oligomerized leading to an overall decrease of co-chaperone activity. The oligomerization properties of ANCL mutants were faithfully replicated in HEK 293T cells. Interestingly, the oligomers were covalently tagged by ubiquitination instead of palmitoylation. Taken together, ANCL mutations result in both a gain and partial loss-of-function.
Keywords: ATPase; Heat shock cognate; Lysosomal storage disease; Synapse maintenance;
A functional polymorphism affecting the APOA5 gene expression is causally associated with plasma triglyceride levels conferring coronary atherosclerosis risk in Han Chinese Population by Weihua Shou; Ying Wang; Fang Xie; Beilan Wang; Lin Yang; Hong Wu; Yi Wang; Zhimin Wang; Jinxiu Shi; Wei Huang (2147-2154).
Apolipoprotein A5 (APOA5) gene plays a key role in plasma triglyceride (TG) metabolism, and shows the involvement in coronary artery disease (CAD). A set of single nucleotide polymorphisms around the APOA5 gene was identified to be associated with plasma TG levels. It is of biological and clinical importance to discern the genuine genetic determinants. A polymorphism in 3′ untranslated region of the APOA5 gene, rs2266788, is deserving of investigation for suggestive clues from the association in multiple independent studies. In this study, rs2266788 was genotyped in 3222 unrelated subjects consisting of 2062 CAD cases and 1160 controls. The statistical analyses indicated that the minor C allele of rs2266788 was significantly associated with elevated plasma TG levels and higher CAD risk. In normal human liver tissues, comparison of global APOA5 mRNA levels among genotypes and allelic expression imbalance analysis showed the decreased gene expression for the C allele. Luciferase assays confirmed a concordant result that transcriptional activity was lowered for the C allele compared with the T allele in four cell lines. Multiple lines of evidence in our study supported that rs2266788 was causally associated with plasma TG levels conferring CAD risk in Han Chinese population owing to a cis-acting effect to the APOA5 gene expression.
Keywords: APOA5 gene; Triglyceride; Coronary artery disease; Single nucleotide polymorphism; Gene expression;
Role of extracellular and intracellular microRNAs in sepsis by Kobina Essandoh; Guo-Chang Fan (2155-2162).
Sepsis is the major cause of death in the intensive care unit (ICU). Numerous biomarkers have been studied to identify the cause and severity of sepsis but these factors cannot differentiate between infectious and non-infectious inflammatory response. MicroRNAs (miRNAs) are non-coding RNA transcripts that regulate the expression of genes by repressing translation or degrading mRNA. Importantly, miRNAs can be released outside cells and easily detectable in bodily fluids such as blood, sweat, urine and breast milk. Numerous studies have explored the idea of utilizing extracellular miRNAs as biomarkers for sepsis by profiling the dysregulation of miRNAs in blood samples of sepsis patients. So far, miR-223, miR-146a and miR-150 have been identified to have promising prognostic and diagnostic value to sepsis. In addition, various intracellular miRNAs have been implicated to play critical roles in regulating the TLR-NF-κB pathway, which is a well-known inflammatory signaling pathway involved in the process of sepsis. Here, we summarize the recent progress on the role of extracellular and intracellular miRNAs in sepsis. Specifically, we discuss the possible role of circulating miRNA biomarkers for the diagnosis of sepsis and how intracellular miRNAs regulate the inflammatory responses in sepsis.
Keywords: Sepsis; miRNAs; Biomarkers; Inflammatory response; NF-κB;
FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1 by Angel L. Pey; Clare F. Megarity; David J. Timson (2163-2173).
NAD(P)H quinone oxidoreductase 1 is involved in antioxidant defence and protection from cancer, stabilizing the apoptosis regulator p53 towards degradation. Here, we studied the enzymological, biochemical and biophysical properties of two cancer-associated variants (p.R139W and p.P187S). Both variants (especially p.187S) have lower thermal stability and greater susceptibility to proteolysis compared to the wild-type. p.P187S also has reduced activity due to a lower binding affinity for the FAD cofactor as assessed by activity measurements and direct titrations. Native gel electrophoresis and dynamic light scattering also suggest that p.P187S has a higher tendency to populate unfolded states under native conditions. Detailed thermal stability studies showed that all variants irreversibly denature causing dimer dissociation, while addition of FAD restores the stability of the polymorphic forms to wild-type levels. The kinetic destabilization induced by polymorphisms as well as the kinetic protection exerted by FAD was confirmed by measuring denaturation kinetics at temperatures close to physiological. Our data suggest that the main molecular mechanisms associated with these cancer-related variants are their low binding affinity for FAD and/or kinetic instability. Thus, pharmacological chaperones may be useful in the treatment of patients bearing these polymorphisms.Display Omitted
Keywords: NAD(P)H quinone oxidoreductase 1; Cancer-associated polymorphism; NQO1*2; NQO1*3; Kinetic instability;
Cathepsin G activity lowers plasma LDL and reduces atherosclerosis by Jing Wang; Sara Sjöberg; Ting-Ting Tang; Katariina Öörni; Wenxue Wu; Conglin Liu; Blandine Secco; Viviane Tia; Galina K. Sukhova; Cleverson Fernandes; Adam Lesner; Petri T. Kovanen; Peter Libby; Xiang Cheng; Guo-Ping Shi (2174-2183).
Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr–/– ) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3 months. When mice consume this diet for 6 months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r = –0.535, P < 0.0001) and LDL cholesterol (r = –0.559, P < 0.0001), but not with HDL cholesterol (P = 0.901) or triglycerides (P = 0.186). Such inverse correlations with total cholesterol (r = –0.504, P < 0.0001) and LDL cholesterol (r = –0.502, P < 0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.
Keywords: Cathepsin G; Atherosclerosis; Low-density lipoprotein; Elastin; Angiotensin-II;
Akt1 isoform modulates phenotypic conversion of vascular smooth muscle cells by Sung Ji Yun; Jung Min Ha; Eun Kyoung Kim; Young Whan Kim; Seo Yeon Jin; Dong Hyung Lee; Sang Heon Song; Chi Dae Kim; Hwa Kyoung Shin; Sun Sik Bae (2184-2192).
In this study, we investigated the role of Akt1 isoform in phenotypic change of vascular smooth muscle cells (VSMCs) and neointima formation. Laminin-induced conversion of synthetic VSMCs into contractile VSMCs was measured by expression of marker proteins for contractile VSMCs and collagen gel contraction assay. Culture of synthetic VSMCs on laminin-coated plates induced expression of marker proteins for contractile VSMCs and showed contraction in response to angiotensin II (AngII) stimulation. Silencing integrin-linked kinase attenuated activation of Akt and blocked phenotypic conversion of VSMCs resulting in the loss of AngII-dependent contraction. Laminin-induced phenotypic conversion of VSMCs was abrogated by phosphatidylinositol 3-kinase inhibitor or in cells silencing Akt1 but not Akt2. Proliferation of contractile VSMCs on laminin-coated plate was enhanced in cells silencing Akt1 whereas silencing Akt2 did not affect. Promoter activity of myocardin and SM22α was enhanced in contractile phenotype and overexpression of myocardin stimulated promoter activity of SM22α in synthetic phenotype. Promoter activity of myocardin and SM22α was reduced in cells silencing Akt1 and promoter activity of SM22α was restored by overexpression of myocardin in cells silencing Akt1. However, silencing of Akt2 affected neither promoter activity of myocardin nor SM22α. Finally, neointima formation in carotid artery ligation and high fat-diet-induced atherosclerosis was facilitated in mice lacking Akt1. This study demonstrates that Akt1 isoform stimulates laminin-induced phenotypic conversion of synthetic VSMCs by regulating the expression of myocardin. VSMCs become susceptible to shifting from contractile to synthetic phenotype by the loss of Akt1 in pathological conditions.Regulation of VSMC phenotype by extracellular matrix.Display Omitted
Keywords: Akt; Laminin; Integrin-linked kinase; Neointima; Phenotypic change;
Excessive microglial activation aggravates olfactory dysfunction by impeding the survival of newborn neurons in the olfactory bulb of Niemann–Pick disease type C1 mice by Yoojin Seo; Hyung-Sik Kim; Yooyoung Shin; Insung Kang; Soon Won Choi; Kyung-Rok Yu; Kwang-Won Seo; Kyung-Sun Kang (2193-2203).
Progressive olfactory impairment is one of the earliest markers of neurodegeneration. However, the underlying mechanism for this dysfunction remains unclear. The present study investigated the possible role of microgliosis in olfactory deficits using a mouse model of Niemann–Pick disease type C1 (NPC1), which is an incurable neurodegenerative disorder with disrupted lipid trafficking. At 7 weeks of age, NPC1 mutants showed a distinct olfactory impairment in an olfactory test compared with age-matched wild-type controls (WT). The marked loss of olfactory sensory neurons within the NPC1 affected olfactory bulb (NPC1-OB) suggests that NPC1 dysfunction impairs olfactory structure. Furthermore, the pool of neuroblasts in the OB was diminished in NPC1 mice despite the intact proliferative capacity of neural stem/progenitor cells in the subventricular zone. Instead, pro-inflammatory proliferating microglia accumulated extensively in the NPC1-OB as the disease progressed. To evaluate the impact of abnormal microglial activation on olfaction in NPC1 mice, a microglial inhibition study was performed using the anti-inflammatory agent Cyclosporin A (CsA). Importantly, long-term CsA treatment in NPC1 mice reduced reactive microgliosis, restored the survival of newly generated neurons in the OB and improved overall performance on the olfactory test. Therefore, our study highlights the possible role of microglia in the regulation of neuronal turnover in the OB and provides insight into the possible therapeutic applications of microglial inhibition in the attenuation or reversal of olfactory impairment.
Keywords: Niemann–Pick disease type C1; Olfaction; Neurodegeneration; Microglia; Cyclosporin A;
Inhibitory effects of long noncoding RNA MEG3 on hepatic stellate cells activation and liver fibrogenesis by Yong He; Yu-ting Wu; Cheng Huang; Xiao-Ming Meng; Tao-tao Ma; Bao-Ming Wu; Feng-yun Xu; Lei Zhang; Xiong-Wen Lv; Jun Li (2204-2215).
Long noncoding RNAs (lncRNAs) are being increasingly recognized as major players in governing fundamental biological processes through diverse mechanisms. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA correlated with several human cancers. Recently, the methylation-dependent downregulation of MEG3 has been described in liver cancers. However, its biological functional role in liver fibrosis remains unknown. In our study, MEG3 levels were remarkably decreased in CCl4-induced mouse liver fibrosis models and human fibrotic livers as demonstrated by real-time quantitative PCR. Moreover, the expression of MEG3 was downregulated in human hepatic stellate cell lines LX-2 cells in response to transforming growth factor-β1 (TGF-β1) stimulation in dose and time-dependent manner. Enforced expression of MEG3 in LX-2 cells inhibited TGF-β1-induced cell proliferation, while promoting cell apoptosis. In addition, hypermethylation of MEG3 promoter was identified by methylation-specific PCR and MEG3 expression was robustly increased by the inhibition of methylation with either 5-aza-2-deoxycytidine (5-azadC), or siRNA to DNA methyltransferase 1 (DNMT1) in TGF-β1-induced LX-2 cells. More importantly, overexpression of MEG3 could activate p53 and mediate cytochrome c release, subsequently leading to caspase-3-dependent apoptosis in TGF-β1-treated LX-2 cells. These findings suggested that MEG3 may play an important role in stellate cell activation and liver fibrosis progression and act as a novel potential therapeutic target for liver fibrosis.
Keywords: lncRNAs; MEG3; Liver fibrosis; Methylation; p53;
Cataract-causing mutation R233H affects the stabilities of βB1- and βA3/βB1-crystallins with different pH-dependence by Yi-Bo Xi; Wei-Jie Zhao; Xiao-Tong Zuo; Harry Christianto Tjondro; Jing Li; An-Bang Dai; Sha Wang; Yong-Bin Yan (2216-2229).
Disease-causing mutations can be stabilizing or destabilizing. Missense mutations of structural residues are generally destabilizing, while stabilizing mutations are usually linked to alterations in protein functions. Stabilizing mutations are rarely identified in mutations linked to congenital cataract, a disease caused by the opacification of the lens. In this research, we found that R233H mutation had little impact on βB1-crystallin structure, solubility and thermal stability under neutral solution pH conditions. The mutation increased βB1 stability against guanidine hydrochloride-induced denaturation, suggesting that Arg233 might be a functional residue. Further analysis indicated that the R233H mutation did not affect the formation of βA3/βB1 heteromer, but significantly reduced heteromer stability against heat- and guanidine hydrochloride-induced denaturation. The R233H mutation negatively affected the thermal stabilities and aggregatory propensities of βB1 and βA3/βB1 with different pH-dependence, implying that the protonation of His side chains during acidification played a regulatory role in crystallin stability and aggregation. Molecular dynamic simulations indicated that Arg233 is one of the residues forming an inter-subunit ion-pairing network with intrinsically dynamic nature. Based on these observations, we proposed that the highly dynamic ion-pairing network contributed to the tradeoff among βB1 solubility, stability, aggregatory propensity and function of protecting βA3.Display Omitted
Keywords: β-Crystallin; Cataract-causing mutation; Molecular dynamic simulation; Ion-paring network; Protein aggregation; Stabilizing mutation;
Effect of conjugated linoleic acid mixture supplemented daily after carcinogen application on linoleic and arachidonic acid metabolites in rat serum and induced tumours by Małgorzata Jelińska; Agnieszka Białek; Hanna Mojska; Iwona Gielecińska; Andrzej Tokarz (2230-2236).
Conjugated linoleic acid (CLA) is thought to exert anticarcinogenic, antiatherogenic, anti-inflammatory and weight loss effects. The impact on eicosanoid biosynthesis may be one of the mechanisms of its action. The aim of this study was to establish whether CLA mixture supplemented daily after administration of carcinogen (7, 12-dimethylbenz[a]anthracene, DMBA) influenced the concentration of linoleic and arachidonic acid metabolites: 13- or 9-hydroxyoctadecadienoic acids (13-, 9-HODE) and 15-, 12- or 5-hydroxyeicosatetraenoic acids (15-, 12- or 5-HETE) and prostaglandin E2 (PGE2) in rat serum and DMBA-induced tumours. The correlations between polyunsaturated fatty acids (PUFA) and HETE and HODE contents in serum were also investigated.Female Sprague–Dawley rats divided into three groups according to the diet (1% Bio-C.L.A., 2% Bio-C.L.A. and plant oil in the control group) were used in the study. On the 50th day of life some of the animals in every dietary group were administered DMBA to induce tumours. Since that day, the rats were fed one of the above-mentioned diets. After 15 weeks the animals were sacrificed and blood and tumours were collected. HETE and HODE were extracted using a solid-phase extraction (SPE) method on C18 columns and analysed with LC-MS/MS.The results of our study showed that CLA daily supplementation after carcinogen administration influence LA and AA metabolite levels in serum and tumours. However, the ratios of eicosanoids having opposite effects (e.g. 12-HETE/15-HETE), not concentrations of particular compounds, appear to be better indicators of pathological processes.
Keywords: Hydroxyeicosatetraenoic acid; Hydroxyoctadecadienoic acid; Conjugated linoleic acid; 15-, 12- or 5-HETE; 13-HODE; 9-HODE;
Stat3 signaling activation crosslinking of TGF-β1 in hepatic stellate cell exacerbates liver injury and fibrosis by Ming-Yi Xu; Jun-Jie Hu; Jie Shen; Mei-Ling Wang; Qing-Qing Zhang; Ying Qu; Lun-Gen Lu (2237-2245).
The role of signal transducer and activator of transcription 3 (Stat3) in liver fibrosis is still controversial. Since hepatic stellate cells (HSCs) and transforming growth factor-β1 (TGF-β1) are central to the fibrogenesis, our goal was to clarify the mechanism of Stat3 crosslinking of TGF-β1 signaling.Stat3, TGF-β1 mRNA and protein expressions were examined in liver tissues of chronic hepatitis B (CHB) patients and diethylinitrosamine (DEN)-induced rat fibrosis model. The effect of Stat3 activation or suppression on TGF-β1 signaling in HSCs was tested in vitro and in vivo.Stat3 expression as well as TGF-β1 was increased in CHB patients and DEN-induced fibrosis rat model. This was strongly correlated with increase in fibrosis staging. TGF-β1, a mediator of fibrosis, was enhanced by Stat3, but suppressed by siRNA-mediated RNA knockdown of Stat3 (siStat3) or Janus kinase 2 inhibitor (AG490) both in vivo and in vitro. Stat3 crosslinking TGF-β1 signaling plays an important role in HSC activation and increasing fibrosis related products. TGF-β1 could not achieve profibrogenic cytokine and anti-apoptosis characteristics without Stat3 activation in HSCs.We provide a novel role of Stat3 cooperating TGF-β1 in activation and anti-apoptotic effect of HSCs. Stat3 worsens liver fibrosis through the up-regulation of TGF-β1 and fibrotic product expression.
Keywords: Hepatic stellate cell; Signal transducer and activator of transcription 3; Transforming growth factor-β1; Liver fibrosis;
Novel role of silent information regulator 1 in acute endothelial cell oxidative stress injury by Yue Li; Kun Wang; Yingtong Feng; Chongxi Fan; Feng Wang; Juanjuan Yan; Jian Yang; Haifeng Pei; Zhenxing Liang; Shuai Jiang; Xiangjun Chen; Yang Yang; Yan Qu (2246-2256).
Silent information regulator 1 (SIRT1), a class III histone deacetylase, retards aging and plays roles in cellular oxidative stress injury (OSI). However, the biological context in which SIRT1 promotes oxidative injury is not fully understood. Here, we show that SIRT1 essentially mediates hydrogen peroxide (H2O2)-induced cytotoxicity in human umbilical vein endothelial cell (HUVEC). In HUVECs, SIRT1 protein expression was significantly increased in a dose-dependent manner after H2O2 treatment, whereas the acetylation levels of the NF-κB p65 subunit and p53 were decreased. EX527 (a specific SIRT1 inhibitor) conferred protection to the HUVECs against H2O2, as indicated by an improved cell viability, adhesion, an enhanced migratory ability, a decreased apoptotic index, decreased reactive oxygen species (ROS) production and reductions in several biochemical parameters. Immunofluorescence and Western blot analyses demonstrated that H2O2 treatment up-regulated SIRT1, phosphorylated-JNK (p-JNK), p-p38MAPK, and p-ERK expression. EX527 pretreatment reversed these effects on SIRT1, p-JNK, and p-p38MAPK but further increased the p-ERK levels. Similar results were confirmed in SIRT1 siRNA experiments. In summary, SIRT1 signaling pathway inhibition imparts protection against acute endothelial OSI, and modulation of MAPKs (JNK, p38MAPK, and ERK) may be involved in the protective effect of SIRT1 inhibition.
Keywords: Silent information regulator 1; Acute oxidative stress injury; EX527; Mitogen activated protein kinases;
A cell-type-specific role for murine Commd1 in liver inflammation by Paulina Bartuzi; Tobias Wijshake; Daphne C. Dekker; Alina Fedoseienko; Niels J. Kloosterhuis; Sameh A. Youssef; Haiying Li; Ronit Shiri-Sverdlov; Jan-Albert Kuivenhoven; Alain de Bruin; Ezra Burstein; Marten H. Hofker; Bart van de Sluis (2257-2265).
The transcription factor NF-κB plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-κB activation may protect tissues from stress, a prolonged NF-κB activation can have a detrimental effect on tissue homeostasis and therefore accurate termination is crucial. Copper Metabolism MURR1 Domain-containing 1 (COMMD1), a protein with functions in multiple pathways, has been shown to suppress NF-κB activity. However, its action in controlling liver inflammation has not yet been investigated. To determine the cell-type-specific contribution of Commd1 to liver inflammation, we used hepatocyte and myeloid-specific Commd1-deficient mice. We also used a mouse model of NAFLD to study low-grade chronic liver inflammation: we fed the mice a high fat, high cholesterol (HFC) diet, which results in hepatic lipid accumulation accompanied by liver inflammation. Depletion of hepatocyte Commd1 resulted in elevated levels of the NF-κB transactivation subunit p65 (RelA) but, surprisingly, the level of liver inflammation was not aggravated. In contrast, deficiency of myeloid Commd1 exacerbated diet-induced liver inflammation. Unexpectedly we observed that hepatic and myeloid Commd1 deficiency in the mice both augmented hepatic lipid accumulation. The elevated levels of proinflammatory cytokines in myeloid Commd1-deficient mice might be responsible for the increased level of steatosis. This increase was not seen in hepatocyte Commd1-deficient mice, in which increased lipid accumulation appeared to be independent of inflammation. Our mouse models demonstrate a cell-type-specific role for Commd1 in suppressing liver inflammation and in the progression of NAFLD.
Keywords: COMMD1; Inflammation; NAFLD; Macrophages; NF-κB inhibitor;
High glucose-induced hyperosmolarity impacts proliferation, cytoskeleton remodeling and migration of human induced pluripotent stem cells via aquaporin-1 by Rosalinda Madonna; Yong-Jian Geng; Harnath Shelat; Peter Ferdinandy; Raffaele De Caterina (2266-2275).
Background and objective: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5 mmol/L glucose, high glucose (HG) at 30.5 mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with β-catenin. HG and HM induced the expression of β-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. Conclusions: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1.
Keywords: Induced pluripotent stem cell; Aquaporin-1; Cytoskeleton remodeling; Hyperosmolarity; Diabetes; Hyperglycemia;
Amyloid-β(1-42) protofibrils stimulate a quantum of secreted IL-1β despite significant intracellular IL-1β accumulation in microglia by Shana E. Terrill-Usery; Michael J. Mohan; Michael R. Nichols (2276-2285).
Neuroinflammation is a characteristic feature of the Alzheimer’s disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-β protein (Aβ). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aβ plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aβ(1-42) protofibrils. Aβ(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) mRNA, and intracellular pro and mature forms of IL-1β protein. The accumulation of both IL-1β forms indicated that Aβ(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aβ-induced accumulation of intracellular mature IL-1β did not translate into greater IL-1β secretion. Instead, we found that Aβ elicited a quantized burst of secreted IL-1β and this process occurred even prior to Aβ priming of the microglia suggesting a basal level of either pro or mature IL-1β in the cultured primary microglia. The IL-1β secretion burst was rapid but not sustained, yet could be re-evoked with additional Aβ stimulation. The findings from this study demonstrated multiple sites of IL-1β regulation by Aβ(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1β secretory process. These results underscore the wide-ranging effects of Aβ on the innate immune response.
Keywords: Amyloid-beta protein; Protofibril; Microglia; Toll-like receptors; NLRP3 inflammasome; Interleukin 1-beta;
Advanced diffusion MRI fiber tracking in neurosurgical and neurodegenerative disorders and neuroanatomical studies: A review by Kumar Abhinav; Fang-Cheng Yeh; Sudhir Pathak; Valerie Suski; David Lacomis; Robert M. Friedlander; Juan C. Fernandez-Miranda (2286-2297).
Diffusion MRI enabled in vivo microstructural imaging of the fiber tracts in the brain resulting in its application in a wide range of settings, including in neurological and neurosurgical disorders. Conventional approaches such as diffusion tensor imaging (DTI) have been shown to have limited applications due to the crossing fiber problem and the susceptibility of their quantitative indices to partial volume effects. To overcome these limitations, the recent focus has shifted to the advanced acquisition methods and their related analytical approaches. Advanced white matter imaging techniques provide superior qualitative data in terms of demonstration of multiple crossing fibers in their spatial orientation in a three dimensional manner in the brain. In this review paper, we discuss the advancements in diffusion MRI and introduce their roles. Using examples, we demonstrate the role of advanced diffusion MRI-based fiber tracking in neuroanatomical studies. Results from its preliminary application in the evaluation of intracranial space occupying lesions, including with respect to future directions for prognostication, are also presented. Building upon the previous DTI studies assessing white matter disease in Huntington's disease and Amyotrophic lateral sclerosis; we also discuss approaches which have led to encouraging preliminary results towards developing an imaging biomarker for these conditions.
Keywords: Fiber tracking; Diffusion spectrum imaging (DSI); Diffusion tensor imaging (DTI); High angular resolution diffusion imaging (HARDI); Neurodegeneration; Intracranial mass lesion;
The soluble form of HFE protein regulates hephaestin mRNA expression in the duodenum through an endocytosis-dependent mechanism by Bruno Silva; Joana Ferreira; Vera Santos; Cilénia Baldaia; Fátima Serejo; Paula Faustino (2298-2305).
Dietary iron absorption regulation is one of the key steps for the maintenance of the body iron homeostasis. HFE gene expression undergoes a complex post-transcriptional alternative splicing mechanism through which two alternative transcripts are originated and translated to a soluble HFE protein isoform (sHFE). The first purpose of this study was to determine if sHFE transcript levels respond to different iron conditions in duodenal and macrophage cell models. In addition, we aimed to determine the functional effect of the sHFE protein on the expression of iron metabolism-related genes in a duodenal cell model as well as, in vivo, in duodenum biopsy samples.Levels of sHFE transcripts were measured in HuTu-80, Caco-2, HT-29 and activated THP1 cells, after holo-Tf stimulus, and in total RNA from duodenum biopsies of functional dyspepsia patients. Also, the expression of several iron metabolism-related genes was determined after endogenous and exogenous overexpression of sHFE protein in a duodenal cell model. sHFE endocytosis mechanism was studied using endocytosis inhibitors.Our results showed that sHFE transcript expression was up-regulated after holo-Tf stimuli. Hephaestin and duodenal cytochrome b expressions were down-regulated by both endogenous HFE and sHFE proteins in a duodenal cell model. Exogenous sHFE was able to down-regulate hephaestin mRNA levels by a clathrin-independent, dynamin-mediated, and RhoA-regulated endocytosis mechanism. Moreover, HEPH levels negatively correlated with sHFE levels in the duodenum of functional dyspepsia patients. Thus, sHFE seems to be an important iron metabolism regulator playing a role in the control of dietary iron absorption in the duodenum.
Keywords: Alternative splicing; Iron metabolism; Enterocyte; Iron absorption; Macrophage; Endocytosis;
A novel proteolysis-resistant cyclic helix B peptide ameliorates kidney ischemia reperfusion injury by Cheng Yang; Zhongliang Xu; Zitong Zhao; Long Li; Tian Zhao; Dian Peng; Ming Xu; Ruiming Rong; Ya-Qiu Long; Tongyu Zhu (2306-2317).
Helix B surface peptide (HBSP), derived from erythropoietin, displays powerful tissue protection during kidney ischemia reperfusion (IR) injury without erythropoietic side effects. We employed cyclization strategy for the first time, and synthesized thioether-cyclized helix B peptide (CHBP) to improve metabolic stability and renoprotective effect. LC–MS/MS analysis was adopted to examine the stability of CHBP in vitro and in vivo. The renoprotective effect of CHBP in terms of renal function, apoptosis, inflammation, extracellular matrix deposition, and histological injury was also detected in vivo and in vitro. Antibody array and western blot were performed to analyze the signal pathway of involvement by CHBP in the IR model and renal tubular epithelial cells. In this study, thioether-cyclized peptide was significantly stable in vivo and in vitro. One dose of 8 nmol/kg CHBP administered intraperitoneally at the onset of reperfusion improved renal protection compared with three doses of 8 nmol/kg linear HBSP in a 48 h murine IR model. In a one-week model, the one dose CHBP-treated group exhibited remarkably improved renal function over the IR group, and attenuated kidney injury, including reduced inflammation and apoptosis. Interestingly, we found that the phosphorylation of autophagy protein mTORC1 was dramatically reduced upon CHBP treatment. We also demonstrated that CHBP induced autophagy via inhibition of mTORC1 and activation of mTORC2, leading to renoprotective effects on IR. Our results indicate that the novel metabolically stable CHBP is a promising therapeutic medicine for kidney IR injury treatment.
Keywords: Cyclic helix B peptide; Metabolic stability; Kidney ischemia reperfusion injury; Autophagy; Apoptosis; Inflammation;
Glycogen storage disease type III: A novel Agl knockout mouse model by Serena Pagliarani; Sabrina Lucchiari; Gianna Ulzi; Raffaella Violano; Michela Ripolone; Andreina Bordoni; Monica Nizzardo; Stefano Gatti; Stefania Corti; Maurizio Moggio; Nereo Bresolin; Giacomo P. Comi (2318-2328).
Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease.
Keywords: Glycogen storage disease type III; Metabolic disease; Glycogenosis; Glycogen debranching enzyme; Mouse model;
Nonalcoholic fatty liver disease: A main driver of insulin resistance or a dangerous liaison? by Nanda Gruben; Ronit Shiri-Sverdlov; Debby P.Y. Koonen; Marten H. Hofker (2329-2343).
Insulin resistance is one of the key components of the metabolic syndrome and it eventually leads to the development of type 2 diabetes, making it one of the biggest medical problems of modern society. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are tightly associated with insulin resistance. While it is fairly clear that insulin resistance causes hepatic steatosis, it is not known if NAFLD causes insulin resistance. Hepatic inflammation and lipid accumulation are believed to be the main drivers of hepatic insulin resistance in NAFLD. Here we give an overview of the evidence linking hepatic lipid accumulation to the development of insulin resistance, including the accumulation of triacylglycerol and lipid metabolites, such as diacylglycerol and ceramides. In particular, we discuss the role of obesity in this relation by reviewing the current evidence in terms of the reported changes in body weight and/or adipose tissue mass. We further discuss whether the activation or inhibition of inflammatory pathways, Kupffer cells and other immune cells influences the development of insulin resistance. We show that, in contrast to what is commonly believed, neither hepatic steatosis nor hepatic inflammation is sufficient to cause insulin resistance. Many studies show that obesity cannot be ignored as an underlying factor in this relationship and NAFLD is therefore less likely to be one of the main drivers of insulin resistance.
Keywords: Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis; Insulin resistance; Inflammation; Kupffer cell; Lipid;