Lessons from in vitro perifusion of pancreatic islets isolated from 80 human pancreases.

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve Δ-AUCa)], and the correlation among them was determined. SI positively correlated with Δ-AUCa (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = −0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01)

Chronic administration of green tea extract to TRAMP mice induces the collapse of Golgi apparatus in prostate secretory cells and results in alterations of protein post-translational processing.

Considering its long latency, prostate cancer (PCa) represents an ideal target for chemoprevention strategies. Green tea extract (GTE) has been proved to be one of the most promising natural substances capable of inhibiting PCa progression in animal models (transgenic adenocarcinoma of mouse prostate), as well as in humans. However, the cellular targets of the GTE action are mostly unknown. The main objective of this work was to investigate whether the endoplasmic reticulum (ER) and the Golgi apparatus (GA), known to be actively involved in sensing stress stimuli and initiating and propagating cell death signalling, may represent the subcellular targets of GTE action. To this end, 42 TRAMP mice were divided into four experimental groups: groups II and IV, received GTE in tap water (0.3 g/100 ml solution) starting at 8 weeks of age and up to the time of sacrifice. Groups I and III were respective age-matched water-fed controls. The animals were sacrificed after 4 weeks (groups I and II) or 40 weeks of treatment (groups II and IV). We also treated TRAMP-C2 cells with GTE (20 µg/ml for 7 days) to check the expression profile of clusterin (CLU), a protein involved in prostate tumourigenesis, extensively processed through ER-GA before being secreted through the plasma membrane. In vivo we found that chronic administration of GTE in TRAMP mice results in collapse of ER and GA in prostate epithelial cells. Consistently, in vitro we found that the mature, fully processed form of CLU, sCLU, is strongly reduced by GTE treatment in TRAMP-C2 cells. Taking into account the sCLU biogenesis dependence on the ER-GA integrity and the proposed anti-apoptotic role of sCLU, the possibility for GTE to counteract PCa progression by interfering with sCLU biogenesis is suggested

Combined Small Interfering RNA Therapy and In Vivo Magnetic Resonance Imaging in Islet Transplantation

OBJECTIVE Recent advances in human islet transplantation are hampered by significant graft loss shortly after transplantation and inability to follow islet fate directly. Both issues were addressed by utilizing a dual-purpose therapy/imaging small interfering RNA (siRNA)-nanoparticle probe targeting apoptotic-related gene caspase-3. We expect that treatment with the probe would result in significantly better survival of transplanted islets, which could be monitored by in vivo magnetic resonance imaging (MRI). RESEARCH DESIGN AND METHODS We synthesized a probe consisting of therapeutic (siRNA to human caspase-3) and imaging (magnetic iron oxide nanoparticles, MN) moieties. In vitro testing of the probe included serum starvation of the islets followed by treatment with the probe. Caspase-3 gene silencing and protein expression were determined by RT-PCR and Western blot, respectively. In vivo studies included serial MRI of NOD-SCID mice transplanted with MN-small interfering (si)Caspase-3–labeled human islets under the left kidney capsule and MN-treated islets under the right kidney capsule. RESULTS Treatment with MN-siCaspase-3 probe resulted in decrease of mRNA and protein expression in serum-starved islets compared with controls. In vivo MRI showed that there were significant differences in the relative volume change between MN-siCaspase-3–treated grafts and MN-labeled grafts. Histology revealed decreased caspase-3 expression and cell apoptosis in MN-siCaspase-3–treated grafts compared with the control side. CONCLUSIONS Our data show the feasibility of combining siRNA therapy and in vivo monitoring of transplanted islets in mice. We observed a protective effect of MN-siCaspase-3 in treated islets both in vitro and in vivo. This study could potentially aid in increasing the success of clinical islet transplantation

Long-term beneficial effect of islet transplantation on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients

OBJECTIVE: Our aim was to evaluate the long-term effects of transplanted islets on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients. RESEARCH DESIGN AND METHODS: A total of 34 type 1 diabetic kidney-transplanted patients underwent islet transplantation and were divided into two groups: successful islet-kidney transplantation (SI-K; 21 patients, fasting C-peptide serum concentration &gt;0.5 ng/ml for &gt;1 year) and unsuccessful islet-kidney transplantation (UI-K; 13 patients, fasting C-peptide serum concentration &lt;0.5 ng/ml). Patients cumulative survival, cardiovascular death rate, and atherosclerosis progression were compared in the two groups. Skin biopsies, endothelial dependent dilation (EDD), nitric oxide (NO) levels, and atherothrombotic risk factors [von Willebrand factor (vWF) and D-dimer fragment (DDF)] were studied cross-sectionally. RESULTS: The SI-K group showed a significant better patient survival rate (SI-K 100, 100, and 90% vs. UI-K 84, 74, and 51% at 1, 4, and 7 years, respectively, P = 0.04), lower cardiovascular death rate (SI-K 1/21 vs. UI-K 4/13, chi(2) = 3.9, P = 0.04), and lower intima-media thickness progression than the UI-K group (SI-K group: delta1-3 years -13 +/- 30 micro m vs. UI-K group: delta1-3 years 245 +/- 20 micro m, P = 0.03) with decreased signs of endothelial injuring at skin biopsy. Furthermore, the SI-K group showed a higher EDD than the UI-K group (EDD: SI-K 7.8 +/- 4.5% vs. UI-K 0.5 +/- 2.7%, P = 0.02), higher basal NO (SI-K 42.9 +/- 6.5 vs. UI-K 20.2 +/- 6.8 micro mol/l, P = 0.02), and lower levels of vWF (SI-K 138.6 +/- 15.3 vs. UI-K 180.6 +/- 7.0%, P = 0.02) and DDF (SI-K 0.61 +/- 0.22 vs. UI-K 3.07 +/- 0.68 micro g/ml, P &lt; 0.01). C-peptide-to-creatinine ratio correlated positively with EDD and NO and negatively with vWF and DDF. CONCLUSIONS: Successful islet transplantation improves survival, cardiovascular, and endothelial function in type 1 diabetic kidney-transplanted patient

Energy Expenditure Evaluation in Humans and Non-Human Primates by SenseWear Armband : Validation of Energy Expenditure Evaluation by SenseWear Armband by Direct Comparison with Indirect Calorimetry

Introduction:The purpose of this study was to compare and validate the use of SenseWear Armband (SWA) placed on the arm (SWA ARM) and on the back (SWA BACK) in healthy humans during resting and a cycle-ergometer exercise and to evaluate the SWA to estimate Resting Energy Expenditure (REE) and Total Energy Expenditure (TEE) in healthy baboons.Methods:We studied 26 (15F/11M) human subjects wearing SWA in two different anatomical sites (arm and back) during resting and a cycle-ergometer test and directly compared these results with indirect calorimetry evaluation (IC), performed at the same time. We then inserted the SWA in a metabolic jacket for baboons and evaluated the TEE and REE in free living condition for 6 days in 21 (8F/13M) non-human primates.Results:In humans we found a good correlation between SWA place on the ARM and on the BACK with IC during the resting experiment (1.1\ub10.3 SWAs, 1\ub10.2 IC kcal/min) and a slight underestimation in the SWAs data compared with IC during the cycle-ergometer exercise (5\ub11.9 SWA ARM, 4.5\ub11.5 SWA BACK and 5.4\ub12.1 IC kcal/min). In the non-human primate (baboons) experiment SWA estimated a TEE of 0.54\ub10.009 kcal/min during free living and a REE of 0.82\ub10.06 kcal/min.Conclusion:SWA, an extremely simple and inexpensive apparatus, provides quite accurate measurements of energy expenditure in humans and in baboons. Energy expenditure data obtained with SWA are highly correlated with the data obtained with "gold standard", IC, in humans. \ua9 2013 Casiraghi et al

Exenatide regulates pancreatic islet integrity and insulin sensitivity in the nonhuman primate baboon Papio hamadryas.

The glucagon-like peptide-1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, β cell and α cell function and relative volumes, and islet cell apoptosis and replication in nondiabetic nonhuman primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an l-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated, and the remnant pancreas (head-body) was harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by about 2-fold. β, α, and δ cell relative volumes in exenatide-treated baboons were significantly increased compared with saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on β, α, and δ cells and produces a robust increase in insulin sensitivity in nonhuman primates

RNAi Screen of DAF-16/FOXO Target Genes in C. elegans Links Pathogenesis and Dauer Formation

The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production

Angiopoietin 2 Alters Pancreatic Vascularization in Diabetic Conditions

Islet vascularization, by controlling beta-cell mass expansion in response to increased insulin demand, is implicated in the progression to glucose intolerance and type 2 diabetes. We investigated how hyperglycaemia impairs expansion and differentiation of the growing pancreas. We have grafted xenogenic (avian) embryonic pancreas in severe combined immuno-deficient (SCID) mouse and analyzed endocrine and endothelial development in hyperglycaemic compared to normoglycaemic conditions. 14 dpi chicken pancreases were grafted under the kidney capsule of normoglycaemic or hyperglycaemic, streptozotocin-induced, SCID mice and analyzed two weeks later. Vascularization was analyzed both quantitatively and qualitatively using either in situ hybridization with both mouse- and chick-specific RNA probes for VEGFR2 or immunohistochemistry with an antibody to nestin, a marker of endothelial cells that is specific for murine cells. To inhibit angiopoietin 2 (Ang2), SCID mice were treated with 4 mg/kg IP L1-10 twice/week. In normoglycaemic condition, chicken-derived endocrine and exocrine cells developed well and intragraft vessels were lined with mouse endothelial cells. When pancreases were grafted in hyperglycaemic mice, growth and differentiation of the graft were altered and we observed endothelial discontinuities, large blood-filled spaces. Vessel density was decreased. These major vascular anomalies were associated with strong over-expression of chick-Ang2. To explore the possibility that Ang2 over-expression could be a key step in vascular disorganization induced by hyperglycaemia, we treated mice with L1-10, an Ang-2 specific inhibitor. Inhibition of Ang2 improved vascularization and beta-cell density. this work highligghted an important role of Ang2 in pancreatic vascular defects induced by hyperglycemia