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horseradish peroxidase; HSC, hepatic stellate cells; IL6, interleukin-6; IMDM, Iscove's Modified Dulbecco's Medium; ITGA5, integrin α 5 ; ITGB1, integrin β 1 ; ITS, insulin-selenitetransferrin; LAMA2, laminin α2; LAMA4, laminin α4; LAMA5, laminin α5; LAMB1, laminin β1; LAMB2, laminin β2; LAMC1, laminin γ1; LN-211, laminin-211; LN-521, laminin-521; LRAT, lecithin-retinol acyltransferase; MMP13, metalloproteinase 13; NGF, nerve growth factor; NID, nidogen; PBS, phosphate buffered saline; PCA, principal component analysis; PEG, polyethylene glycol; PPARγ, peroxisome proliferator-activated receptor γ; qPCR, quantitative polymerase chain reaction; SASP, senescence-associated secretory phenotype; SEC, sinusoidal endothelial cells; SEM, standard error of the mean; SPARCL1, secreted protein acidic and rich in cysteines-like 1; TGFb, transforming growth factor β; TLN, talin; TNFR, tumor necrosis factor receptor; VCL, vinculin; α-SMA, α-smooth muscle actin. AbstractHepatic blood flow and sinusoidal endothelial fenestration decrease during aging.Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α 5 /β 1 is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α 5 and β 1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α 5 and β 1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α 5 and laminin gene expression whereas integrin β 1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α 5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α 5 /β 1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration. K E Y W O R D S aging, hepatic stellate cells, integrins, laminins, mechanobiology S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section.

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-monthold mice, but doubled the ammonia concentration required for halfmaximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12month-old TauT KO mice. glutamine | hyperammonemia | taurine | scavenger cells | oxidative stress

Aims/hypothesis Exercise-induced hyperinsulinism (EIHI) is a hypoglycaemic disorder characterised by inappropriate insulin secretion following anaerobic exercise or pyruvate load. Activating promoter mutations in the MCT1 gene (also known as SCLA16A1), coding for monocarboxylate transporter 1 (MCT1), were shown to associate with EIHI. Recently, transgenic Mct1 expression in pancreatic beta cells was shown to introduce EIHI symptoms in mice. To date, MCT1 has not been demonstrated in insulin-producing cells from an EIHI patient. Methods In vivo insulin secretion was studied during an exercise test before and after the resection of an insulinoma. The presence of MCT1 was analysed using immunohistochemistry followed by laser scanning microscopy, western blot analysis and real-time RT-PCR of MCT1. The presence of MCT1 protein was analysed in four additional insulinoma patients.J. Marquard and A. Welters contributed equally to this work. Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2750-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users. -012-2750-7 Results Clinical testing revealed massive insulin secretion induced by anaerobic exercise preoperatively, but not postoperatively. MCT1 protein was not detected in the patient's normal islets. In contrast, immunoreactivity was clearly observed in the insulinoma tissue. Western blot analysis and real-time RT-PCR showed a four-to fivefold increase in MCT1 in the insulinoma tissue of the EIHI patient compared with human pancreatic islets. MCT1 protein was detected in three of four additional insulinomas. Conclusions/interpretation We show for the first time that an MCT1-expressing insulinoma was associated with EIHI and that MCT1 might be present in most insulinomas. Our data suggest that MCT1 expression in human insulinproducing cells can lead to EIHI and warrant further studies on the role of MCT1 in human insulinoma patients.Diabetologia (2013) 56:31-35 DOI 10.1007/s00125

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.

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