Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

Down-regulation of proliferation does not affect the secretory function of transformed ß-cell lines regardless of their anatomical configuration.

AIMS AND OBJECTIVES: Proliferation in transformed ß-cell lines is high compared to primary islet cells and is accompanied by reduced insulin content and release. Our aim was to determine whether experimental reduction of proliferation restores the cells to a more authentic ß-cell phenotype in terms of secretory function and to investigate the potential beneficial effect of their configuration as islet-like structures. RESULTS: Mitosis inhibitor mitomycin c treatment neither altered the rate of proliferation nor improved the secretory responses of MIN6 monolayer cells. The proliferative rate of MIN6 cells was not affected by pseudoislet formation, but in contrast to monolayer cells, pseudoislets responded to 20 mM glucose with a 2.6-fold increase in insulin secretion. MMC reduced proliferation in MIN6 pseudoislets, but did not further improve their secretory responsiveness. Withdrawal of doxycycline resulted in complete growth-arrest in R7T1 cells, but monolayer and pseudoislet R7T1 cells were unresponsive to glucose and remained so upon growth-arrest although insulin content was increased in growth-arrested pseudoislets. METHODS: MIN6 monolayer and pseudoislet cells were treated with MMC whereas growth-arrest was induced in R7T1 monolayer and pseudoislet cells by withdrawal of doxycycline. Proliferation rates were determined by immunocytochemical measurements of BrdU incorporation and insulin secretion was assessed by radioimmunoassay. CONCLUSIONS: Secretory function of transformed ß-cells is not influenced by experimental reduction of proliferation, but can be modulated by enhanced cell-cell contact within islet-like structures. These results have implications for future studies of islet cell redifferentiation and for the generation of islet-like material for transplantation therapy in Type 1 diabetes.

Pseudoislets as primary islet replacements for research: report on a symposium at King's College London, London UK.

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying ß-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 ß-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed 'pseudoislets' largely recapitulates the function of primary islet ß-cells. The Diabetes Research Group at King's College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on "Pseudoislets as primary islet replacements for research", which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or ß-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

Analysis of lipid export in hydrocarbonoclastic bacteria of the genus Alcanivorax: identification of lipid export-negative mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9.

Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source. In this study, we developed a screening procedure to isolate lipid export-negative transposon-induced mutants of bacteria of the genus Alcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposon-induced mutants of A. jadensis and seven of A. borkumensis impaired in lipid secretion were isolated. All isolated mutants were still capable of synthesizing and accumulating these lipids intracellularly and exhibited no growth defect. In the A. jadensis mutants, the transposon insertions were mapped in genes annotated as encoding a putative DNA repair system specific for alkylated DNA (Aj17), a magnesium transporter (Aj7), and a transposase (Aj5). In the A. borkumensis mutants, the insertions were mapped in genes encoding different proteins involved in various transport processes, like genes encoding (i) a heavy metal resistance (CZCA2) in mutant ABO_6/39, (ii) a multidrug efflux (MATE efflux) protein in mutant ABO_25/21, (iii) an alginate lyase (AlgL) in mutants ABO_10/30 and ABO_19/48, (iv) a sodium-dicarboxylate symporter family protein (GltP) in mutant ABO_27/29, (v) an alginate transporter (AlgE) in mutant ABO_26/1, or (vi) a two-component system protein in mutant ABO_27/56. Site-directed MATE, algE, and algL gene disruption mutants, which were constructed in addition, were also unable to export neutral lipids and confirmed the phenotype of the transposon-induced mutants. The putative localization of the different gene products and their possible roles in lipid excretion are discussed. Beside this, the composition of the intra- and extracellular lipids in the wild types and mutants were analyzed in detail.

Impaired islet turnover in human donor pancreata with aging.

OBJECTIVE: The prevalence of type 2 diabetes mellitus escalates with aging although beta-cell mass, a primary parameter of beta-cell function, is subject to compensatory regulation. So far it is unclear whether the proliferative capacity of pancreatic islets is restricted by senescence. MATERIALS AND METHODS: Human pancreatic tissue from n=20 non-diabetic organ donors with a mean age of 50.2+/-3.5 years (range 7-66 years) and mean body mass index of 25.7+/-0.9 kg/m(2) (17.2-33.1 kg/m(2)) was morphometrically analyzed to determine beta-cell volume, beta-cell replication, beta-cell apoptosis, islet neogenesis, and pancreatic duodenal homeobox-1 (PDX-1) expression. RESULTS: Relative beta-cell volume in human pancreata (mean 2.3+/-0.2%) remains constant with aging (r=0.26, P=ns). Beta-cell replication (r=0.71, P=0.0004) decreases age-dependently, while beta-cell apoptosis does not change significantly (r=0.42, P=0.08). Concomitantly, PDX-1 expression is downregulated with age in human pancreatic tissue (r=0.65, P=0.002). The rate of islet neogenesis is not affected by aging (r=0.13, P=ns). CONCLUSIONS: In non-diabetic humans, aging is linked with impaired islet turnover possibly due to reduced PDX-1 expression. As beta-cell replication is considered to be the main mechanism responsible for beta-cell regeneration, these changes restrict the flexibility of the aging human pancreas to adapt to changing demands for insulin secretion and increase the risk for the development of diabetes mellitus in older subjects.

Proliferation of colo-357 pancreatic carcinoma cells and survival of patients with pancreatic carcinoma are not altered by insulin glargine.

OBJECTIVE: It was reported that the long-acting insulin analogue glargine induces cell proliferation in a human osteosarcoma cell line and therefore might induce or accelerate tumor growth. Induction of cell proliferation would be particularly relevant for insulin treatment of subjects with diabetes and the potential of bearing tumor cells (e.g., a history of a malignant disease). RESEARCH DESIGN AND METHODS: Proliferation, apoptosis, and the expression levels of insulin receptor, IGF-I receptor, and insulin receptor substrate (IRS) 2 were analyzed in human pancreatic cancer cells (Colo-357) after incubation (72 h) with insulin glargine or regular human insulin at 0-100 nmol/l. A total of 125 subjects, after partial or total pancreatectomy due to pancreatic carcinoma, were analyzed over a median follow-up period of 22 months. RESULTS: There was no significant difference between glargine and regular human insulin with respect to regulation of proliferation and apoptosis of Colo-357 cells. The expression levels of insulin receptor, IGF-I receptor, and IRS2 as a downstream molecule of both receptor signaling pathways were not altered at any concentration tested. The insulin receptor was downregulated to a similar degree by glargine and regular human insulin at high insulin concentrations (P < 0.0001 for glargine, P = 0.002 for regular human insulin). The median survival time after pancreatic surgery was 15 months. Survival analysis showed that the time-dependent proportion of patients who survived was identical in patients receiving insulin glargine versus insulin treatment without glargine and control subjects without diabetes after surgery (P = 0.4, three-sample comparison). CONCLUSIONS: Regular human insulin and insulin glargine may be used to treat diabetes in patients with pancreatic cancer.