Increase In Cell Mass Research Articles

Involvement of sulfoquinovosyl diacylglycerol in DNA synthesis in Synechocystis sp. PCC 6803

BackgroundSulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria and their postulated progeny, plastids, in plants. A cyanobacterium, Synechocystis sp. PCC 6803, requires SQDG for growth: its mutant (SD1) with the sqdB gene for SQDG synthesis disrupted can grow with external supplementation of SQDG. However, upon removal of SQDG from the medium, its growth is retarded, with a decrease in the cellular content of SQDG throughout cell division, and finally ceases. Concomitantly with the decrease in SQDG, the maximal activity of photosynthesis at high-light intensity is repressed by 40%.FindingsWe investigated effects of SQDG-defect on physiological aspects in Synechocystis with the use of SD1. SD1 cells defective in SQDG exhibited normal photosynthesis at low-light intensity as on culturing. Meanwhile, SD1 cells defective in SQDG were impaired in light-activated heterotrophic growth as well as in photoautotrophic growth. Flow cytometric analysis of the photoautotrophically growing cells gave similar cell size histograms for the wild type and SD1 supplemented with SQDG. However, the profile of SD1 defective in SQDG changed such that large part of the cell population was increased in size. Of particular interest was the microscopic observation that the mitotic index, i.e., population of dumbbell-like cells with a septum, increased from 14 to 29% in the SD1 culture without SQDG. Flow cytometric analysis also showed that the enlarged cells of SD1 defective in SQDG contained high levels of Chl, however, the DNA content was low.ConclusionsOur experiments strongly support the idea that photosynthesis is not the limiting factor for the growth of SD1 defective in SQDG, and that SQDG is responsible for some physiologically fundamental process common to both photoautotrophic and light-activated heterotrophic growth. Our findings suggest that the SQDG-defect allows construction of the photosynthetic machinery at an elevated level for an increase in cell mass, but represses DNA synthesis. SQDG may be essential for normal replication of chromosomal DNA for completion of the cell cycle.

Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered Escherichia coli

In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce byproduct formation and increase succinate accumulation,pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates,such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus,the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l−1 with a yield of1.02 g g−1 total sugars during anaerobic fermentation.

Bioconversion of lignin model compounds with oleaginous Rhodococci

Although economically efficient biomass conversion depends on the utilization of the complete cell wall (biorefinery concept), including polysaccharides and lignin, current biofuels research concentrate mostly on cellulose conversion, while lignin is viewed as a side-product that is used primarily as a thermal resource. Microbiological conversion of lignin is almost exclusive to fungi, usually resulting in increased cell mass and lignolytic enzymes. Some bacteria can also degrade lignin-related compounds using the β-ketoadipate pathway; for example, Rhodococcus opacus DSM 1069 can degrade coniferyl alcohol and grow on it as sole carbon source. Moreover, this strain belongs to the actinomycetes group that is also known for oleaginous species with lipid accumulation over 20%. Present work shows that R. opacus DSM 1069 and PD630 strains under nitrogen limiting conditions can convert lignin model compounds into triacylglycerols, also known as neutral lipids. 4-Hydroxybenzoic and vanillic acid lignin model compounds were used as sole carbon sources, and after brief adaptation periods, the cells not only began growing but accumulated lipids to the level of oleaginicity. These lipids were extracted for transesterification and analysis of fatty acid methyl esters showed good composition for biodiesel applications with no aromatics. Furthermore, the two strains showed distinct substrate metabolism and product profiles.

Improved Outcome of Islet Transplantation in Partially Pancreatectomized Diabetic Mice by Inhibition of Dipeptidyl Peptidase-4 With Sitagliptin

Glucagon-like peptide-1 (GLP-1) is known to promote beta cell proliferation, and dipeptidyl peptidase-4 (DPP-4) inhibitor increases GLP-1 levels by preventing its degradation. This study was designed to evaluate the effects of sitagliptin (sita), a DPP-4 inhibitor, on the outcome of islet transplantation (ITx) in diabetic mice after partial pancreatectomy (Px). A diabetic mouse model was prepared by performing 70% Px in C57BL/6 mice. The diabetic mice were treated with sita, subjected to ITx, or both treated with sita and subjected to ITx. After 12 days of sita treatment, the pancreatic remnants and transplanted islets were histologically examined. Dipeptidyl peptidase-4 inhibitor increased the concentration of plasma active GLP-1 regardless of ITx and improved glycemic control in the ITx group. The beta cell mass of the pancreatic remnants increased in the ITx group, and mice that received combined treatment with ITx and sita showed a greater increase in the beta cell mass. Dipeptidyl peptidase-4 inhibitor seems to induce proliferation and inhibit apoptosis of beta cells in pancreatic remnants. The DPP-4 inhibitor favorably affects ITx in partially pancreatectomized diabetic mice by increasing the beta cell mass through cell proliferation and inhibition of beta cell apoptosis.

Polycythemia From Mast Cell Activation Syndrome: Lessons Learned

A middle-aged woman presented with fatigue and mild increases in hematocrit and red cell mass. Polycythemia vera was diagnosed. She underwent therapeutic phlebotomy but clinically worsened. On reevaluation, other problems were noted including episodic malaise, nausea, rash and vasomotor issues. The JAK2V617F mutation was absent; paraneoplastic erythrocytosis was investigated. Serum tryptase and urinary N-methylhistamine were normal, but urinary prostaglandin D2 was elevated. Skin and marrow biopsies showed no mast cell abnormalities. Extensive other evaluation was negative. Gastrointestinal tract biopsies were histologically normal but revealed increased, aberrant mast cells on immunohistochemistry; the KITD816V mutation was absent. Mast cell activation syndrome, recently identified as a clonal disorder involving assorted KIT mutations, was diagnosed. Imatinib 200mg/d rapidly effected complete, sustained response. Diagnosis of mast cell activation syndrome is hindered by multiple factors, but existing therapies for mast cell disease are usually achieve significant benefit, highlighting the importance of early diagnosis. Multiple important aspects of clinical reasoning are illustrated by the case.

A Peritoneal Dialysis Regimen Low in Glucose and Glucose Degradation Products Results in Increased Cancer Antigen 125 and Peritoneal Activation

Glucose and glucose degradation products (GDPs) in peritoneal dialysis fluids (PDFs) are both thought to mediate progressive peritoneal worsening. In a multicenter, prospective, randomized crossover study, incident continuous ambulatory peritoneal dialysis patients were treated either with conventional lactate-buffered PDF (sPD regimen) or with a regimen low in glucose and GDPs: Nutrineal×1, Extraneal×1, and Physioneal×2 (NEPP regimen; all solutions: Baxter Healthcare, Utrecht, The Netherlands). After 6 months, patients were switched to the alternative regimen for another 6 months. After 6 weeks of run-in, before the switch, and at the end of the study, 4-hour peritoneal equilibration tests were performed, and overnight effluents were analyzed for cells and biomarkers. Differences between the regimens were assessed by multivariate analysis corrected for time and regimen sequence. The 45 patients who completed the study were equally distributed over both groups. During NEPP treatment, D(4)/D(0) glucose was lower (p < 0.01) and D/P creatinine was higher (p = 0.04). In NEPP overnight effluent, mesothelial cells (p < 0.0001), cancer antigen 125 (p < 0.0001), hyaluronan (p < 0.0001), leukocytes (p < 0.001), interleukins 6 (p = 0.001) and 8 (p = 0.0001), and vascular endothelial growth factor (VEGF, p < 0.0001) were increased by a factor of 2-3 compared with levels in sPD effluent. The NEPP regimen was associated with higher transport parameters, but that association disappeared after the addition of VEGF to the model. The association between NEPP and higher effluent levels of VEGF could not be attributed to glucose and GDP loads. Study results indicate preservation of the mesothelium and increased peritoneal activation during NEPP treatment. Whether the increase in VEGF reflects an increase in mesothelial cell mass or whether it points to another, undesirable mechanism cannot be determined from the present study. Longitudinal studies are needed to finally evaluate the usefulness of the NEPP regimen for further clinical use.

Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation

Epidermal growth factor receptor (EGFR) signalling is essential for the proper fetal development of pancreatic islets and in the postnatal formation of an adequate beta cell mass. In this study we investigated the role of EGFR signalling in the physiological states of beta cell mass expansion in adults during metabolic syndrome and pregnancy, as well as in regeneration after pancreatic duct ligation. Heterozygous Pdx1-EGFR-dominant-negative (E1-DN) mice, which have a kinase-negative EGFR under the Pdx1 promoter, and wild-type mice were both subjected to a high-fat diet, pregnancy and pancreatic duct ligation. The beta cell mass of wild-type mice fed the high-fat diet increased by 70% and the mice remained normoglycaemic; the E1-DN mice became diabetic and failed to show any compensatory beta cell mass expansion. Similarly, pregnant wild-type mice had four times more proliferating beta cells and a 75% increase in beta cell mass at mid-gestation, in contrast to the pregnant E1-DN mice, which did not show any significant beta cell compensation and were hyperglycaemic in an intraperitoneal glucose tolerance test. However, after pancreatic duct ligation, both the wild-type and E1-DN mice showed similar expression of Ngn3 (also known as Neurog3) and beta cell proliferation increased to a similar level in the ligated part of pancreas. EGFR signalling is essential in beta cell mass expansion during a high-fat diet and pregnancy where replication is the primary mechanism for compensatory beta cell mass expansion. In contrast, EGFR signalling appears not to be crucial to increased beta cell proliferation after pancreatic duct ligation.

R-spondin1 deficiency in mice improves glycaemic control in association with increased beta cell mass

Roof plate-specific spondin (R-spondin1; RSPO1) is a modulator of canonical Wg (wingless) plus Int1 (chromosomal integration site of mouse mammary tumour virus on mouse chromosome 15) (cWNT) signalling that induces cWNT target genes. We have demonstrated that Rspo1 is expressed in murine beta cells, and that it stimulates proliferation and insulin secretion, and inhibits cytokine-induced apoptosis, in mouse insulinoma (MIN6) and beta cells. We thus investigated the role of RSPO1 in beta cells in vivo using Rspo1 ( -/- ) mice. The effects of Rspo1 deficiency were assessed by determination of cWNT signalling, glucose tolerance and beta cell mass. Rspo1 ( -/- ) mice demonstrated an 82% reduction in RSPO1 transcripts and a 61% reduction in the signal detected by an RSPO1 antibody, as well as a 47% decrease in islet cWNT signalling. Despite no differences in body and pancreatic weights or in fasting glycaemia and insulinaemia compared with Rspo1 (+/+) mice, Rspo1 ( -/- ) animals had improved glycaemic control after oral glucose challenge (p < 0.05), with no difference in insulin sensitivity, but an enhanced insulin response over 30 min (p < 0.05); glucagon responses were normal. Rspo1 deficiency also resulted in a twofold increase in beta cell mass (p < 0.05) in association with 2- and 12-fold increases in the number of beta cells positive for antigen identified by monoclonal antibody Ki67 (Ki67) (p < 0.01) and insulin-positive ductal cells (p < 0.05), respectively. No change in the number of TUNEL-positive beta cells was detected. Islets isolated from Rspo1 ( -/- ) animals displayed no differences in glucose-induced insulin secretion or in glucose suppression of glucagon. The present study reveals an unexpected role for RSPO1 as a regulator of both beta cell proliferation and neogenesis in vivo, and reinforces the importance of cWNT signalling for the maintenance of normal pancreatic beta cell behaviour.

Improvement of Ethanol Production Using Saccharomyces cerevisiae by Enhancement of Biomass and Nutrient Supplementation

Optimization of ethanol production through addition of substratum and protein-lipid additives was studied. Oilseed meal extract was used as protein lipid supplement, while rice husk was used as substratum. The effect of oil seed meal extract and rice husk was observed at varying concentration of medium sugar from 8% to 20%. Of the three oil seed meal extracts used, viz. groundnut, safflower, and sunflower, safflower was found to be most efficient. The use of oilseed meal extract at 4% was found to enhance ethanol production by almost 50% and enhanced sugar tolerance from 8% to 16%. A further increase of almost 48% ethanol was observed on addition of 2 g of rice husk per 100 ml of medium. An increase in cell mass with better sugar attenuation was observed. Further optimization was sought through use of sugarcane juice as the sugar source. While 8.9% ethanol yield with 75% sugar attenuation was observed at 20% sucrose concentration, it was found to increase to 12% (v/v) with almost complete utilization of medium sugar when sugarcane juice was used. Cell weight was also observed to increase by 26%.

Upregulation of alpha cell glucagon-like peptide 1 (GLP-1) in Psammomys obesus—an adaptive response to hyperglycaemia?

The hormone glucagon-like peptide 1 (GLP-1) is released in response to a meal from the intestinal L-cells, where it is processed from proglucagon by the proconvertase (PC)1/3. In contrast, in the adult islets proglucagon is processed to glucagon by the PC2 enzyme. The aim of the study was to evaluate if, during the development of diabetes, alpha cells produce GLP-1 that, in turn, might trigger beta cell growth. Beta cell mass, GLP-1 and insulin levels were measured in the gerbil Psammomys obesus (P. obesus), a rodent model of nutritionally induced diabetes. Furthermore, the presence of biologically active forms of GLP-1 and PC1/3 in alpha cells was demonstrated by immunofluorescence, and the release of GLP-1 from isolated P. obesus, mouse and human islets was investigated. During the development of diabetes in P. obesus, a significant increase in GLP-1 was detected in the portal vein (9.8 ± 1.5 vs 4.3 ± 0.7 pmol/l, p < 0.05), and in pancreas extracts (11.4 ± 2.2 vs 5.1 ± 1.3 pmol/g tissue, p < 0.05). Freshly isolated islets from hyperglycaemic animals released more GLP-1 following 24 h culture than islets from control animals (28.2 ± 4.4 pmol/l vs 5.8 ± 2.4, p < 0.01). GLP-1 release was increased from healthy P. obesus islets following culture in high glucose for 6 days (91 ± 9.1 pmol/l vs 28.8 ± 6.6, p < 0.01). High levels of GLP-1 were also found to be released from human islets. PC1/3 colocalised weakly with alpha cells. GLP-1 release from alpha cells is upregulated in P. obesus during the development of diabetes. A similar response is seen in islets exposed to high glucose, which supports the hypothesis that GLP-1 released from alpha cells promotes an increase in beta cell mass and function during metabolic challenge such as diabetes.

Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered Escherichia coli

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. L-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and L-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with L-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and L-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 gl(-1) glucose and 7.5 gl(-1) L-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 gl(-1), 1,350 mgl(-1), 32 mg g cells(-1), and 40 mgl(-1)h(-1), respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.

Enhanced Production of Glutamate Decarboxylase by Batch, Fed-Batch, and Repeated Batch Cultivations of Escherichia coli

In order to obtain high yields of glutamate decarboxylase (GAD) to satisfy the growing market for gamma-aminobutyric acid (GABA), production of GAD by Escherichia coli was examined in batch, fed-batch, and repeated batch cultures. In fed-batch mode, the yield of dry cell mass was enhanced, which is beneficial for GAD production, and corresponded to 155.57% improvement of GAD production as compared to batch cultivation. However, extending the culture time to more than 52.5 h resulted in loss of cell mass and decreased GAD activity. Repeated batch culture demonstrated increases in average cell mass and GAD productivity (around 0.27 g L-1 h-1 and 1.8 U mL-1 h-1) in five cycles. An overall faster fermentation with a steady increase in yield (50.6% to 80.6%) occurred in the first three cycles by repeatedly replacing a portion of culture with fresh medium. Nevertheless, long-term operation (after the fifth batch) is not beneficial for cell growth or GAD production. Growth-associated characteristics of GAD production (GAD yield increased or decreased as the amount of cell mass altered) were observed in different cultivation modes of E. coli. The importance of this work was to first investigate various cultivation modes for GAD yield enhancement, which will serve as important research references for industrial purposes and fill in the research gap in comprehensively elucidating the performance of different feeding strategies with respect to GAD yield, productivity, by-product accumulation, process stability, and incubation cycle.

Prevalence of Venous Thromboembolism In Patients with Secondary Polycythemia

AbstractAbstract 3164 Background:Polycythemia vera is associated with an increased risk for venous thromboembolism (VTE). Although phlebotomy is employed as an adjunct to treatment with hydroxyurea and/or aspirin for VTE risk reduction, emerging data suggest that hematocrit is less of a determinant of VTE risk than leukocyte count and JAK2 V617F gene mutation allele burden. The role of secondary polycythemia as a risk factor for VTE is unknown, but phlebotomy for thrombosis risk reduction is frequently practiced. Based on the polycythemia vera model, however, we hypothesize that secondary polycythemia does not increase VTE risk. The purpose of this study was to determine the prevalence of VTE in patients with secondary polycythemia and to investigate the factors associated with VTE in this population. Methods:We performed a case control study that included patients admitted to Dartmouth-Hitchcock Medical Center with a diagnosis of chronic obstructive pulmonary disease (COPD) and a hematocrit greater than or equal to 50% from August 2004 to July 2009. The controls were matched for age and sex and carried a diagnosis of chronic obstructive pulmonary disease (COPD) without evidence of secondary polycythemia. Data were collected on body mass index (BMI), VTE history, comorbid conditions, thrombophilia and smoking history. Clinical characteristics of patients with and without secondary polycythemia were analyzed using chi square and t-test to evaluate for significant differences in the two populations. Results:Eighty-six patients with secondary polycythemia and 86 controls were included in the study. The mean hematocrit was 53.5% in the case group and 43.7% in the control group (p=<0.005). Among cases, a history of VTE was documented in 17/86 (19.8%), 10 of which (58.8%) were judged to be idiopathic. In the control group, VTE was documented in 12/86 (14%), 4 of which (33.3%) were judged to be idiopathic. There was no statistically significant difference in the number of total (OR=1.52, p=0.42) or idiopathic (OR=2.7; p=0.16) VTE between cases and controls, respectively. There were no statistically significant differences noted in age, sex, body mass index, presence of diabetes mellitus, smoking history or the presence of malignancy in the two groups. Patients with VTE in both groups had higher BMI, however, compared to patients without VTE. Conclusions:We did not observe an increased prevalence of VTE in patients with secondary polycythemia compared to age- and sex-matched controls. Our findings suggest that the high prevalence of VTE observed in patients with secondary polycythemia is more likely related to known risk factors such as obesity rather than hyperviscosity due to increased cell mass. The role of phlebotomy for VTE risk reduction in patients with secondary polycythemia is therefore questionable. Disclosures:No relevant conflicts of interest to declare.

GLP-1: What is known, new and controversial in 2010?

Over the past 2 years, more than 1300 manuscripts have been published on glucagon-like peptide-1 (GLP-1) and yet, what do we know about it for sure? The European Club for the Study of GLP-1 (EuCSGLP-1) has debated the latest controversies concerning GLP-1, including both fundamental and clinical aspects, and concluded that the control of glucose metabolism by GLP-1 requires paracrine activation of the enteric nervous system to regulate numerous physiological functions. This involves—but is not limited to—the endocrine pancreas, liver, cardiovascular system, gastric-emptying and the brain. For this reason, the role of GLP-1 as an endocrine hormone has come under question. As systemic concentration of the peptide was not thought to be relevant to its physiological action, it was proposed that dipeptidyl peptidase-4 (DPP4) inhibitors would involve the control of enteric, rather than circulating, DPP4 activity to effectively regulate glycaemia. In any case, the concomitant insulinotropic and glucagonostatic roles of GLP-1 were believed to be of equal importance to glucose control. Another important question was related to the role of GLP-1 on beta cell apoptosis, regeneration and differentiation in type 2 diabetic patients. Although evidence in vitro showed that GLP-1 controls these functions, such effects remain elusive in humans in vivo. Nevertheless, the consensus was that GLP-1 could control glucose responsiveness, one of the first impaired physiological functions at the onset of diabetes. The therapeutic efficiency of GLP-1 would be related to the initial restoration of glucose competence, while an increase of beta cell mass has not yet been demonstrated. From a clinical and fundamental point of view, it was concluded that, at the onset of diabetes, an initial triggering of GLP-1 secretion—by metformin coupled with a DPP4 inhibitor—would help to activate the gut–peripheral axis and, hence, restore adequate regulation of glycaemia. GLP-1 analogues would certainly be helpful in association with long-acting insulin (albeit off-label) in patients with impaired beta cells and GLP-1 secretory potential. However, a reliable and routine feasible test to systematically assess dynamic insulin secretion is essential. More important, factors that influence therapeutic response, such as compliance and lifestyle, as well as pharmacokinetics and dosing, disease duration, age, gender, ethnicity, patients’ clinical characteristics, autonomic nervous system integrity and genotype characteristics also need to be considered. A few innovative perspectives have been debated, such as the recently discovered cardiovascular protective effects of the native GLP-1 peptide and its degradation product GLP-1 9-36, as well as the neuroprotection offered by GLP-1. Although still considered speculative, these perspectives remain hopeful and promising.

The DPP-4 inhibitor vildagliptin increases pancreatic beta cell mass in neonatal rats

The present study addressed the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor vildagliptin ((1-[[(3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine), LAF237) on pancreatic beta cell mass in neonatal rats. Newborn rats were treated orally with vildagliptin (60 mg/kg) or vehicle once daily for 19 days starting from postnatal day 2. Pancreatic immunohistochemistry and morphometric analysis were performed to evaluate changes in beta cell mass, cell apoptosis (Apoptag stain) and replication (5′-Bromo-2′-deoxyuridine (BrdU)-incorporation) on days 7, 21, and 33. On day 7, an eight-fold increase in BrdU-positive pancreatic beta cells and a 71% decrease in Apoptag-positive cells were observed. On day 21, vildagliptin produced a two-fold increase in pancreatic beta cell mass compared to placebo (0.06 ± 0.01 mg vs 0.11 ± 0.02 mg, P < 0.05). Beta cell mass remained elevated (90%, 0.09 ± 0.02 mg vs 0.16 ± 0.03 mg, P < 0.05) on day 33, twelve days after discontinuing vildagliptin treatment. These data show that the DPP-4 inhibitor vildagliptin increased pancreatic beta cell mass through enhanced beta cell replication and reduced apoptosis. The increased beta cell mass was sustained for 12 days after vildagliptin washout. This study demonstrates that DPP-4 inhibitors can elicit beneficial effects on beta cell turnover that could help to prevent or retard the progression of type 2 diabetes.

MRNA expression analysis of cell cycle genes in islets of pregnant mice

Aims/hypothesisPregnancy requires an increase in the functional beta cell mass to match metabolic needs for insulin. To understand this adaptation at the molecular level, we undertook a time course analysis of mRNA expression in mice.MethodsTotal RNA extracted from C57Bl6/J mouse islets every 3 days during pregnancy was hybridised on commercially available expression arrays. Gene network analysis was performed and changes in functional clusters over time visualised. The function of putative novel cell cycle genes was assessed via silencing in replicating mouse insulinoma 6 (MIN6) cells.ResultsGene network analysis identified a large gene cluster associated with cell cycle control (67 genes, all upregulated by ≥1.5-fold, p < 0.001). The number of upregulated cell cycle genes and the mRNA expression levels of individual genes peaked at pregnancy day (P)9.5. Filtering of poorly annotated genes with enhanced expression in islets at P9.5, and in MIN6 cells and thymus resulted in further studies with G7e (also known as D17H6S56E-5) and Fignl1. Gene knock-down experiments in MIN6 cells suggested that these genes are indeed involved in adequate cell cycle accomplishment.Conclusions/interpretationA sharp peak of cell cycle-related mRNA expression in islets occurs around P9.5, after which beta cell replication is increased. As illustrated by the identification of G7e and Fignl1 in islets of pregnant mice, further study of this distinct transcriptional peak should help to unravel the complex process of beta cell replication.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-010-1912-8) contains supplementary material, which is available to authorised users.

Sterol Regulatory Element-binding Protein (SREBP)-1-mediated Lipogenesis Is Involved in Cell Senescence

Increased cell mass is one of the characteristics of senescent cells, but this event has not been clearly defined. When subcellular organellar mass was estimated with organelle-specific fluorescence dyes, we observed that most membranous organelles progressively increase in mass during cell senescence. This increase was accompanied by an increase in membrane lipids and augmented expression of lipogenic enzymes, such as fatty acid synthase (FAS), ATP citrate lyase, and acetyl-CoA carboxylase. The mature form of sterol regulatory element-binding protein (SREBP)-1 was also elevated. Increased expression of these lipogenic effectors was further observed in the liver tissues of aging Fischer 344 rats. Ectopic expression of mature form of SREBP-1 in both Chang cells and primary young human diploid fibroblasts was enough to induce senescence. Blocking lipogenesis with FAS inhibitors (cerulenin and C75) and via siRNA-mediated silencing of SREBP-1 and ATP citrate lyase significantly attenuated H(2)O(2)-induced senescence. Finally, old human diploid fibroblasts were effectively reversed to young-like cells by challenging with FAS inhibitors. Our results suggest that enhanced lipogenesis is not only a common event, but also critically involved in senescence via SREBP-1 induction, thereby contributing to the increase in organelle mass (as a part of cell mass), a novel indicator of senescence.

Hypoxia Increases the Expression of Stem-Cell Markers and Promotes Clonogenicity in Glioblastoma Neurospheres

Hypoxia promotes the expansion of non-neoplastic stem and precursor cell populations in the normal brain, and is common in malignant brain tumors. We examined the effects of hypoxia on stem-like cells in glioblastoma (GBM). When GBM-derived neurosphere cultures are grown in 1% oxygen, hypoxia-inducible factor 1alpha (HIF1alpha) protein levels increase dramatically, and mRNA encoding other hypoxic response genes, such as those encoding hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor, are induced over 10-fold. Hypoxia increases the stem-like side population over fivefold, and the percentage of cells expressing CD133 threefold or more. Notch pathway ligands and targets are also induced. The rise in the stem-like fraction in GBM following hypoxia is paralleled by a twofold increase in clonogenicity. We believe HIF1alpha plays a causal role in these changes, as when oxygen-stable HIF1alpha is expressed in normoxic glioma cells CD133 is induced. We used digoxin, which has been shown to lower HIF protein levels in vitro and in vivo, to inhibit the hypoxic response. Digoxin suppressed HIF1alpha protein expression, HIF1alpha downstream targets, and slowed tumor growth in vivo. In addition, pretreatment with digoxin reduced GBM flank xenograft engraftment of hypoxic GBM cells, and daily intraperitoneal injections of digoxin were able to significantly inhibit the growth of established subcutaneous glioblastoma xenografts, and suppressed expression of vascular endothelial growth factor.

Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass.

Prolonged exposure of pancreatic beta cells to excessive levels of glucose and fatty acids, referred to as glucolipotoxicity, is postulated to contribute to impaired glucose homeostasis in patients with type 2 diabetes. However, the relative contribution of defective beta cell function vs diminished beta cell mass under glucolipotoxic conditions in vivo remains a subject of debate. We therefore sought to determine whether glucolipotoxicity in rats is due to impaired beta cell function and/or reduced beta cell mass, and whether older animals are more susceptible to glucolipotoxic condition. Wistar rats (2 and 6months old) received a 72h infusion of glucose + intravenous fat emulsion or saline control. In vivo insulin secretion and sensitivity were assessed by hyperglycaemic clamps. Ex vivo insulin secretion, insulin biosynthesis and gene expression were measured in isolated islets. Beta cell mass and proliferation were examined by immunohistochemistry. A 72h infusion of glucose + intravenous fat emulsion in 2-month-old Wistar rats did not affect insulin sensitivity, insulin secretion or beta cell mass. In 6-month-old rats by contrast it led to insulin resistance and reduced insulin secretion in vivo, despite an increase in beta cell mass and proliferation. This was associated with: (1) diminished glucose-stimulated second-phase insulin secretion and proinsulin biosynthesis; (2) lower insulin content; and (3) reduced expression of beta cell genes in isolated islets. In this in vivo model, glucolipotoxicity is characterised by an age-dependent impairment of glucose-regulated beta cell function despite a marked increase in beta cell mass.

Inactivation and Mineralization of Aerosol Deposited Model Pathogenic Microorganisms over TiO2and Pt/TiO2

Air disinfection from bacteria and viruses by means of photocatalytic oxidation is investigated with microorganisms loaded over photocatalysts' films from aerosols. Deposition method and equipment have been developed to load Mycobacterium smegmatis , Bacillus thuringiensis , vaccinia virus, and influenza A (H3N2) virus on slides with undoped TiO(2) and platinized sulfated TiO(2) (Pt/TiO(2)). Inactivation dynamics was measured under UVA irradiation and in the dark. About 90% inactivation is reached in 30 min irradiation on TiO(2) and from 90 to 99.8% on Pt/TiO(2). The first-order inactivation rate coefficient ranged from 0.18 to 0.03 min(-1), over Pt/TiO(2) being higher than on TiO(2) for all microorganisms except Bacillus thuringiensis. The photocatalytic mineralization of Bacillus thuringiensis was performed on TiO(2) and Pt/TiO(2) with different photocatalyst and microorganism loadings. Completeness of mineralization depended on the TiO(2) to bacteria mass ratio. The rate of the photocatalytic carbon dioxide production grows with both the cell mass increase and the photocatalyst mass increase. Pt/TiO(2) showed increased rate of mineralization as well as of the inactivation likely due to a better charge carrier separation in the doped semiconductor photocatalyst. The results demonstrate that photocatalytic filters with deposited TiO(2) or Pt/TiO(2) are able to inactivate aerosol microorganisms and completely decompose them into inorganic products and Pt/TiO(2) provides higher disinfection and mineralization rates.