Publications by authors named "Asllan Gjinovci"

26 Publications

  • Page 1 of 1

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

Nat Commun 2020 12 11;11(1):6372. Epub 2020 Dec 11.

Epithelial Stem Cell Biology & Regenerative Medicine laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.
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http://dx.doi.org/10.1038/s41467-020-20082-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732825PMC
December 2020

An Whole-Organ Liver Engineering for Testing of Genetic Therapies.

iScience 2020 Dec 13;23(12):101808. Epub 2020 Nov 13.

MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.
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http://dx.doi.org/10.1016/j.isci.2020.101808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708813PMC
December 2020

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

Nat Commun 2018 10 16;9(1):4286. Epub 2018 Oct 16.

Stem Cell and Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College of London, London, WC1N 1EH, UK.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.
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http://dx.doi.org/10.1038/s41467-018-06385-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191423PMC
October 2018

Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.

Sci Rep 2018 05 30;8(1):8398. Epub 2018 May 30.

Stem Cells and Regenerative Medicine Section, Great Ormond Street Institute of Child Health, University College London, London, WC1N 1EH, UK.

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.
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http://dx.doi.org/10.1038/s41598-018-26371-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976677PMC
May 2018

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells.

PLoS One 2017 20;12(12):e0189586. Epub 2017 Dec 20.

MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738056PMC
January 2018

Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis.

Nat Commun 2016 07 20;7:12111. Epub 2016 Jul 20.

MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK.

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.
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http://dx.doi.org/10.1038/ncomms12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961739PMC
July 2016

Loss of prohibitin induces mitochondrial damages altering β-cell function and survival and is responsible for gradual diabetes development.

Diabetes 2013 Oct 17;62(10):3488-99. Epub 2013 Jul 17.

Department of Cell Physiology and Metabolism, University of Geneva Medical Centre, Geneva, Switzerland.

Prohibitins are highly conserved proteins mainly implicated in the maintenance of mitochondrial function and architecture. Their dysfunctions are associated with aging, cancer, obesity, and inflammation. However, their possible role in pancreatic β-cells remains unknown. The current study documents the expression of prohibitins in human and rodent islets and their key role for β-cell function and survival. Ablation of Phb2 in mouse β-cells sequentially resulted in impairment of mitochondrial function and insulin secretion, loss of β-cells, progressive alteration of glucose homeostasis, and, ultimately, severe diabetes. Remarkably, these events progressed over a 3-week period of time after weaning. Defective insulin supply in β-Phb2(-/-) mice was contributed by both β-cell dysfunction and apoptosis, temporarily compensated by increased β-cell proliferation. At the molecular level, we observed that deletion of Phb2 caused mitochondrial abnormalities, including reduction of mitochondrial DNA copy number and respiratory chain complex IV levels, altered mitochondrial activity, cleavage of L-optic atrophy 1, and mitochondrial fragmentation. Overall, our data demonstrate that Phb2 is essential for metabolic activation of mitochondria and, as a consequence, for function and survival of β-cells.
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http://dx.doi.org/10.2337/db13-0152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781460PMC
October 2013

Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

PLoS One 2012 20;7(9):e45844. Epub 2012 Sep 20.

Service of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045844PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447792PMC
February 2013

High-resolution magnetic resonance imaging quantitatively detects individual pancreatic islets.

Diabetes 2011 Nov 16;60(11):2853-60. Epub 2011 Sep 16.

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.

Objective: We studied whether manganese-enhanced high-field magnetic resonance (MR) imaging (MEHFMRI) could quantitatively detect individual islets in situ and in vivo and evaluate changes in a model of experimental diabetes.

Research Design And Methods: Whole pancreata from untreated (n = 3), MnCl(2) and glucose-injected mice (n = 6), and mice injected with either streptozotocin (STZ; n = 4) or citrate buffer (n = 4) were imaged ex vivo for unambiguous evaluation of islets. Exteriorized pancreata of MnCl(2) and glucose-injected mice (n = 6) were imaged in vivo to directly visualize the gland and minimize movements. In all cases, MR images were acquired in a 14.1 Tesla scanner and correlated with the corresponding (immuno)histological sections.

Results: In ex vivo experiments, MEHFMRI distinguished different pancreatic tissues and evaluated the relative abundance of islets in the pancreata of normoglycemic mice. MEHFMRI also detected a significant decrease in the numerical and volume density of islets in STZ-injected mice. However, in the latter measurements the loss of β-cells was undervalued under the conditions tested. The experiments on the externalized pancreata confirmed that MEHFMRI could visualize native individual islets in living, anesthetized mice.

Conclusions: Data show that MEHFMRI quantitatively visualizes individual islets in the intact mouse pancreas, both ex vivo and in vivo.
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http://dx.doi.org/10.2337/db11-0726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198086PMC
November 2011

In vivo conditional Pax4 overexpression in mature islet β-cells prevents stress-induced hyperglycemia in mice.

Diabetes 2011 Jun 26;60(6):1705-15. Epub 2011 Apr 26.

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.

Objective: To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in β-cells.

Research Design And Methods: Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR.

Results: Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content.

Conclusions: Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.
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http://dx.doi.org/10.2337/db10-1102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3114382PMC
June 2011

Reduction of plasma membrane glutamate transport potentiates insulin but not glucagon secretion in pancreatic islet cells.

Mol Cell Endocrinol 2011 May 1;338(1-2):46-57. Epub 2011 Mar 1.

Department of Cell Physiology and Metabolism, University Medical Centre, Geneva, Switzerland.

Glutamate is generated during nutrient stimulation of pancreatic islets and has been proposed to act both as an intra- and extra-cellular messenger molecule. We demonstrate that glutamate is not co-secreted with the hormones from intact islets or purified α- and β-cells. Fractional glutamate release was 5-50 times higher than hormone secretion. Furthermore, various hormone secretagogues did not elicit glutamate efflux. Interestingly, epinephrine even decreased glutamate release while increasing glucagon secretion. Rather than being co-secreted with hormones, we show that glutamate is mainly released via plasma membrane excitatory amino acid transporters (EAAT) by uptake reversal. Transcripts for EAAT1, 2 and 3 were present in both rat α- and β-cells. Inhibition of EAATs by L-trans-pyrrolidine-2,4-dicarboxylate augmented intra-cellular glutamate and α-ketoglutarate contents and potentiated glucose-stimulated insulin secretion from islets and purified β-cells without affecting glucagon secretion from α-cells. In conclusion, intra-cellular glutamate-derived metabolite pools are linked to glucose-stimulated insulin but not glucagon secretion.
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http://dx.doi.org/10.1016/j.mce.2011.02.019DOI Listing
May 2011

A role for pancreatic beta-cell secretory hyperresponsiveness in catch-up growth hyperinsulinemia: Relevance to thrifty catch-up fat phenotype and risks for type 2 diabetes.

Nutr Metab (Lond) 2011 Jan 18;8(1). Epub 2011 Jan 18.

Department of Medicine / Physiology, University of Fribourg, Switzerland.

Current notions about mechanisms by which catch-up growth predisposes to later type 2 diabetes center upon those that link hyperinsulinemia with an accelerated rate of fat deposition (catch-up fat). Using a rat model of semistarvation-refeeding in which catch-up fat is driven solely by elevated metabolic efficiency associated with hyperinsulinemia, we previously reported that insulin-stimulated glucose utilization is diminished in skeletal muscle but increased in white adipose tissue. Here, we investigated the possibility that hyperinsulinemia during catch-up fat can be contributed by changes in the secretory response of pancreatic beta-cells to glucose. Using the rat model of semistarvation-refeeding showing catch-up fat and hyperinsulinemia, we compared isocalorically refed and control groups for potential differences in pancreatic morphology and in glucose-stimulated insulin secretion during in situ pancreas perfusions as well as ex vivo isolated islet perifusions. Between refed and control animals, no differences were found in islet morphology, insulin content, and the secretory responses of perifused isolated islets upon glucose stimulation. By contrast, the rates of insulin secretion from in situ perfused pancreas showed that raising glucose from 2.8 to 16.7 mmol/l produced a much more pronounced increase in insulin release in refed than in control groups (p < 0.01). These results indicate a role for islet secretory hyperresponsiveness to glucose in the thrifty mechanisms that drive catch-up fat through glucose redistribution between skeletal muscle and adipose tissue. Such beta-cell hyperresponsiveness to glucose may be a key event in the link between catch-up growth, hyperinsulinemia and risks for later type 2 diabetes.
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http://dx.doi.org/10.1186/1743-7075-8-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033236PMC
January 2011

Comparison of tigecycline and vancomycin for treatment of experimental foreign-body infection due to methicillin-resistant Staphylococcus aureus.

Antimicrob Agents Chemother 2009 Jul 13;53(7):3150-2. Epub 2009 Apr 13.

Service of Infectious Diseases, University Hospitals of Geneva, 4 rue Gabrielle-Perret-Gentil, Geneva 14, Switzerland.

Twice-daily 7-day regimens of tigecycline (7 mg/kg) and vancomycin (50 mg/kg) were compared in a rat tissue cage model of chronic foreign-body infection due to methicillin (meticillin)-resistant Staphylococcus aureus strain MRGR3. Subcutaneously administered tigecycline reached levels in tissue cage fluid that were nearly equivalent or slightly superior to the antibiotic MIC (0.5 microg/ml) for strain MRGR3. After 7 days, equivalent, significant reductions in bacterial counts were recorded for tigecycline-treated and vancomycin-treated rats, compared with those for untreated animals.
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http://dx.doi.org/10.1128/AAC.01612-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704643PMC
July 2009

The Lou/C rat: a model of spontaneous food restriction associated with improved insulin sensitivity and decreased lipid storage in adipose tissue.

Am J Physiol Endocrinol Metab 2009 May 10;296(5):E1120-32. Epub 2009 Feb 10.

Department of Internal Medicine, Centre Médical Universitaire, 1211 Geneva 4, Switzerland.

The inbred Lou/C rat, originating from the Wistar strain, has been described as a model of resistance to diet-induced obesity, but little is known about its metabolism. Since this knowledge could provide some clues about the etiology of obesity/insulin resistance, this study aimed at characterizing glucose and lipid metabolism in Lou/C vs. Wistar rats. This was achieved by performing glucose and insulin tolerance tests, euglycemic hyperinsulinemic clamps, and characterization of intracellular insulin signaling in skeletal muscle. Substrate-induced insulin secretion was evaluated using perfused pancreas and isolated islets. Finally, body fat composition and the expression of various factors involved in lipid metabolism were determined. Body weight and caloric intake were lower in Lou/C than in Wistar rats, whereas food efficiency was similar. Improved glucose tolerance of Lou/C rats was not related to increased insulin output but was related to improved insulin sensitivity/responsiveness in the liver and in skeletal muscles. In the latter tissue, this was accompanied by improved insulin signaling, as suggested by higher activation of the insulin receptor and of the Akt/protein kinase B pathway. Fat deposition was markedly lower in Lou/C than in Wistar rats, especially in visceral adipose tissue. In the inguinal adipose depot, expression of uncoupling protein-1 was detected in Lou/C but not in Wistar rats, in keeping with a higher expression of peroxisome proliferator-activated receptor-gamma coactivator-1 in these animals. The Lou/C rat is a valuable model of spontaneous food restriction with associated improved insulin sensitivity. Independently from its reduced caloric intake, it also exhibits a preferential channeling of nutrients toward utilization rather than storage.
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http://dx.doi.org/10.1152/ajpendo.90592.2008DOI Listing
May 2009

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis.

J Biol Chem 2009 Jan 17;284(2):921-9. Epub 2008 Nov 17.

Department of Cell Physiology and Metabolism, Geneva, Switzerland.

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In betaGlud1(-/-) mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.
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http://dx.doi.org/10.1074/jbc.M806295200DOI Listing
January 2009

Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism.

Diabetes 2008 May 16;57(5):1176-85. Epub 2008 Apr 16.

Department of Genetic Medicine and Development, National Centre of Competence in Research-Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland.

Objective: Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents.

Research Design And Methods: Male CD-1 mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion.

Results: In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism.

Conclusions: Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.
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http://dx.doi.org/10.2337/db07-0630DOI Listing
May 2008

Cx36-mediated coupling reduces beta-cell heterogeneity, confines the stimulating glucose concentration range, and affects insulin release kinetics.

Diabetes 2007 Apr;56(4):1078-86

The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital L1, Stockholm, Sweden.

We studied the effect of gap junctional coupling on the excitability of beta-cells in slices of pancreas, which provide a normal environment for islet cells. The electrophysiological properties of beta-cells from mice (C57Bl/6 background) lacking the gap junction protein connexin36 (Cx36(-/-)) were compared with heterozygous (Cx36(+/-)) and wild-type littermates (Cx36(+/+)) and with frequently used wild-type NMRI mice. Most electrophysiological characteristics of beta-cells were found to be unchanged after the knockout of Cx36, except the density of Ca(2+) channels, which was increased in uncoupled cells. With closed ATP-sensitive K(+) (K(ATP)) channels, the electrically coupled beta-cells of Cx36(+/+) and Cx36(+/-) mice were hyperpolarized by the membrane potential of adjacent, inactive cells. Additionally, the hyperpolarization of one beta-cell could attenuate or even stop the electrical activity of nearby coupled cells. In contrast, beta-cells of Cx36(-/-) littermates with blocked K(ATP) channels rapidly depolarized and exhibited a continuous electrical activity. Absence of electrical coupling modified the electrophysiological properties of beta-cells consistent with the reported increase in basal insulin release and altered the switch on/off response of beta-cells during an acute drop of the glucose concentration. Our data indicate an important role for Cx36-gap junctions in modulating stimulation threshold and kinetics of insulin release.
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http://dx.doi.org/10.2337/db06-0232DOI Listing
April 2007

The Fas pathway is involved in pancreatic beta cell secretory function.

Proc Natl Acad Sci U S A 2007 Feb 13;104(8):2861-6. Epub 2007 Feb 13.

Clinic of Endocrinology and Diabetes, and Department of Pathology, University Hospital Zurich, CH-8091 Zurich, Switzerland.

Pancreatic beta cell mass and function increase in conditions of enhanced insulin demand such as obesity. Failure to adapt leads to diabetes. The molecular mechanisms controlling this adaptive process are unclear. Fas is a death receptor involved in beta cell apoptosis or proliferation, depending on the activity of the caspase-8 inhibitor FLIP. Here we show that the Fas pathway also regulates beta cell secretory function. We observed impaired glucose tolerance in Fas-deficient mice due to a delayed and decreased insulin secretory pattern. Expression of PDX-1, a beta cell-specific transcription factor regulating insulin gene expression and mitochondrial metabolism, was decreased in Fas-deficient beta cells. As a consequence, insulin and ATP production were severely reduced and only partly compensated for by increased beta cell mass. Up-regulation of FLIP enhanced NF-kappaB activity via NF-kappaB-inducing kinase and RelB. This led to increased PDX-1 and insulin production independent of changes in cell turnover. The results support a previously undescribed role for the Fas pathway in regulating insulin production and release.
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http://dx.doi.org/10.1073/pnas.0611487104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1815272PMC
February 2007

Intensive therapy with ceftobiprole medocaril of experimental foreign-body infection by methicillin-resistant Staphylococcus aureus.

Antimicrob Agents Chemother 2005 Sep;49(9):3789-93

Service of Infectious Diseases, University Hospitals of Geneva, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.

The therapeutic activity of ceftobiprole medocaril, the water-soluble prodrug of ceftobiprole, was compared to that of vancomycin in a rat tissue cage model of chronic methicillin-resistant Staphylococcus aureus (MRSA) foreign-body infection. The MICs and MBCs of ceftobiprole and vancomycin in Mueller-Hinton broth for strain MRGR3 were 1 and 4 and 1 and 2 microg/ml, respectively. In vitro elimination rates of strain MRGR3 of 4 and 8 microg/ml of ceftobiprole or vancomycin were equivalent. After 2 weeks of infection, mean +/- standard error of the mean viable counts of strain MRGR3 were 6.83 +/- 0.11 log CFU/ml of tissue cage fluid (n = 87). High-dose regimens of ceftobiprole medocaril (equivalent to 150 mg/kg of ceftobiprole) or 50 mg/kg vancomycin produced nearly identical average peak and trough levels of ceftobiprole and vancomycin in tissue cage fluid, which exceeded the MBC of either antibiotic towards strain MRGR3 for > or =75% of each dosing interval. After 7 days of therapy with ceftobiprole medocaril or vancomycin, average counts of MRGR3 decreased significantly (P < 0.02) by 0.68 +/- 0.28 (n = 29) and 0.88 +/- 0.22 (n = 28) log CFU/ml of tissue cage fluid, respectively, compared with cages of untreated animals, but were not significantly different from each other. No resistant mutants were detected on ceftobiprole-supplemented agar following therapy with this cephalosporin. The in vivo activity of ceftobiprole medocaril against chronic MRSA foreign-body infections was equivalent to that of vancomycin and did not lead to the emergence of resistant subpopulations.
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http://dx.doi.org/10.1128/AAC.49.9.3789-3793.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1195398PMC
September 2005

Beta-cell secretory products activate alpha-cell ATP-dependent potassium channels to inhibit glucagon release.

Diabetes 2005 Jun;54(6):1808-15

Department of Cell PhysiologyMetabolism, University Medical Centre, 1211 Geneva 4, Switzerland.

Glucagon, secreted from islet alpha-cells, mobilizes liver glucose. During hyperglycemia, glucagon secretion is inhibited by paracrine factors from other islet cells, but in type 1 and type 2 diabetic patients, this suppression is lost. We investigated the effects of beta-cell secretory products zinc and insulin on isolated rat alpha-cells, intact islets, and perfused pancreata. Islet glucagon secretion was markedly zinc sensitive (IC(50) = 2.7 micromol/l) more than insulin release (IC(50) = 10.7 micromol/l). Glucose, the mitochondrial substrate pyruvate, and the ATP-sensitive K(+) channel (K(ATP) channel) inhibitor tolbutamide stimulated isolated alpha-cell electrical activity and glucagon secretion. Zinc opened K(ATP) channels and inhibited both electrical activity and pyruvate (but not arginine)-stimulated glucagon secretion in alpha-cells. Insulin transiently increased K(ATP) channel activity, inhibited electrical activity and glucagon secretion in alpha-cells, and inhibited pancreatic glucagon output. Insulin receptor and K(ATP) channel subunit transcripts were more abundant in alpha- than beta-cells. Transcript for the glucagon-like peptide 1 (GLP-1) receptor was not detected in alpha-cells nor did GLP-1 stimulate alpha-cell glucagon release. beta-Cell secretory products zinc and insulin therefore inhibit glucagon secretion most probably by direct activation of K(ATP) channels, thereby masking an alpha-cell metabolism secretion coupling pathway similar to beta-cells.
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http://dx.doi.org/10.2337/diabetes.54.6.1808DOI Listing
June 2005

Loss of connexin36 channels alters beta-cell coupling, islet synchronization of glucose-induced Ca2+ and insulin oscillations, and basal insulin release.

Diabetes 2005 Jun;54(6):1798-807

Department of Cell Physiology and Metabolism, University of Geneva, C.M.U., 1 rue Michel Servet, 1211 Geneva 4, Switzerland.

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36(-/-)) featured beta-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36(-/-) mice did not display the regular oscillations of intracellular calcium concentrations ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.
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http://dx.doi.org/10.2337/diabetes.54.6.1798DOI Listing
June 2005

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors.

J Clin Invest 2004 Feb;113(4):635-45

Institute of Pharmacology and Toxicology, Lausanne, Switzerland.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.
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http://dx.doi.org/10.1172/JCI20518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC338268PMC
February 2004

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice.

EMBO J 2003 Aug;22(15):3844-54

Institut für Pharmakologie und Toxikologie, TU München, Biedersteiner Strasse 29, D-80802 München, Germany.

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.
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http://dx.doi.org/10.1093/emboj/cdg389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC169062PMC
August 2003

Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.

Nat Cell Biol 2003 Apr;5(4):330-5

Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, 1211 Geneva 4, Switzerland.

Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from beta-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from alpha-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet alpha- and beta-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although alpha-cells, like beta-cells, possess the inherent capacity to respond to nutrients, secretion from alpha-cells is normally suppressed by the simultaneous activation of beta-cells. Zinc released from beta-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.
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http://dx.doi.org/10.1038/ncb951DOI Listing
April 2003

Discrimination between signaling pathways in regulation of specific gene expression by insulin and growth hormone in hepatocytes.

Endocrinology 2002 Oct;143(10):3766-72

Division of Clinical Biochemistry and Experimental Diabetes Research, University of Geneva School of Medicine, Switzerland.

Insulin and GH can activate common signaling elements in many tissues and cell lines. We investigated the possibility of overlap in signaling pathways activated by insulin and GH in a key target cell, the hepatocyte. In primary cultures of rat hepatocytes, GH caused a dose- and time-dependent increase in tyrosine phosphorylation of signal transducer and activator of transcription 5. This was accompanied by the induction of the mRNA encoding suppressor of cytokine signaling 2. Neither of these effects took place in companion hepatocytes challenged with insulin. By contrast, insulin caused a rapid and sustained phosphorylation of protein kinase B, accompanied by a massive induction of the mRNA encoding glucokinase. GH had no detectable effect on phosphorylation of protein kinase B or level of glucokinase mRNA. Insulin also elicited brief hyperphosphorylation of ERK1 and 2, an effect not seen in GH-stimulated hepatocytes. Thus, there was a clear demarcation of signaling events triggered in hepatocytes by insulin and GH, and this was accompanied by hormone-specific responses with respect to the induction of gene expression. Additionally, the current results show that signal transducer and activator of transcription 5 activation is neither necessary nor sufficient for the insulin-dependent induction of hepatic glucokinase.
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http://dx.doi.org/10.1210/en.2002-220304DOI Listing
October 2002

Implication of glutamate in the kinetics of insulin secretion in rat and mouse perfused pancreas.

Diabetes 2002 Feb;51 Suppl 1:S99-102

Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, Geneva, Switzerland.

It is intriguing that the kinetics of glucose-stimulated insulin secretion from the in situ perfused pancreas differ between the rat and the mouse. Here we confirm that insulin release in the rat is clearly biphasic, whereas in the mouse glucose essentially elicits a transient monophasic insulin release. Glucose-derived glutamate has been suggested to participate in the full development of the secretory response. The present report shows that the expression of glutamate dehydrogenase is lower in mouse than in rat or human islets, paralleling the insulin secretion profile. Addition of glutamic acid dimethyl ester mainly enhances insulin release at an intermediate glucose concentration in the rat pancreas. In the mouse preparation, glutamic acid dimethyl ester induces a sustained secretory response, both at 7.0 and 16.7 mmol/l glucose. These results are compatible with a role for glucose-derived glutamate principally in the sustained phase of nutrient-stimulated insulin secretion.
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http://dx.doi.org/10.2337/diabetes.51.2007.s99DOI Listing
February 2002