Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.

AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: The global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

Normal values for pancreatic stone protein in different age groups.

BACKGROUND: Pancreatic stone protein (PSP) has been identified as a promising sepsis marker in adults, children and neonates. However, data on population-based reference values are lacking. This study aimed to establish age-specific reference values for PSP. METHODS: PSP was determined using a specific ELISA. PSP serum concentrations were determined in 372 healthy subjects including 217 neonates, 94 infants and children up to 16 years, and 61 adults. The adjacent categories method was used to determine which age categories had significantly different PSP concentrations. RESULTS: PSP circulating levels were not gender-dependent and ranged from 1.0 to 99.4 ng/ml with a median of 9.2 ng/ml. PSP increased significantly between the age categories, from a median of 2.6 ng/ml in very preterm newborns, to 6.3 ng/ml in term newborns, to 16.1 ng/ml in older children (p < 0.001). PSP levels were higher on postnatal day three compared to levels measured immediately post delivery (p < 0.001). Paired umbilical artery and umbilical vein samples were strongly correlated (p < 0.001). Simultaneously obtained capillary heel-prick versus venous samples showed a good level of agreement for PSP (Rho 0.89, bias 19 %). CONCLUSIONS: This study provides age-specific normal values that may be used to define cut-offs for future trials on PSP. We demonstrate an age-dependent increase of PSP from birth to childhood.

Pancreatic adenocarcinoma: the impact of preneoplastic lesion pattern on survival.

Pancreatic adenocarcinoma is associated with a very poor prognosis, characterized with a 5-year survival rate of only 5%. Surgery is the only curative treatment for selected patients. Nevertheless, recurrence is very frequent. Identifying prognostic factors is thus warranted. Like numerous other tumors, adenocarcinomas are preceded by preneoplastic lesions. The role and the impact of these lesions remain unclear. This study aimed to assess the impact of the preneoplastic lesion pattern and histo-morphological features, on survival after pancreatic resection. Thirty-five patients who underwent pancreatic resection for pancreatic adenocarcinoma were identified from a prospective database of a single center, between 2003 and 2008. We considered demographics, tumor characteristics and type of treatment. The major outcome was survival. Analyzes were separated into two groups, according to the preneoplastic lesions: Pancreatic intraepithelial neoplasia (PanIN)-related carcinomas and intracanalar papillary mucinous neoplasia (IPMN)-related carcinomas. The former were more frequent, accounting for 63% (22/35). Moreover, they displayed more aggressive features, with a higher tumor stage (p = 0.01) and higher rate of positive lymph nodes (p = 0.019). Lymphatic (p = 0.009) and perinervous (p = 0.019) invasions were also more frequent. Survival was negatively influenced by PanIN preneoplastic lesions (p = 0.015), T3-4 tumor stage (p = 0.038), positive lymph nodes (p = 0.044), lymphatic (p = 0.019) and vascular (p = 0.029) invasions. Pancreatic adenocarcinoma displays different behavior according to its preneoplastic lesion. Indeed, PanIN-related adenocarcinoma showed more aggressive features and lower survival rate. Preneoplastic lesions may represent predictive factors for survival. Their role and predictive value should be investigated more thoroughly.

Computed tomography-guided percutaneous biopsy of pancreatic masses using pneumodissection

Objective To describe the technique of computed tomography-guided percutaneous biopsy of pancreatic tumors with pneumodissection. Materials and Methods In the period from June 2011 to May 2012, seven computed tomography-guided percutaneous biopsies of pancreatic tumors utilizing pneumodissection were performed in the authors' institution. All the procedures were performed with an automatic biopsy gun and coaxial system with Tru-core needles. The biopsy specimens were histologically assessed. Results In all the cases the pancreatic mass could not be directly approached by computed tomography without passing through major organs and structures. The injection of air allowed the displacement of adjacent structures and creation of a safe coaxial needle pathway toward the lesion. Biopsy was successfully performed in all the cases, yielding appropriate specimens for pathological analysis. Conclusion Pneumodissection is a safe, inexpensive and technically easy approach to perform percutaneous biopsy in selected cases where direct access to the pancreatic tumor is not feasible.

Sorcin links pancreatic β cell lipotoxicity to ER Ca2+ stores.

NlmCategory="UNASSIGNED">Preserving β cell function during the development of obesity and insulin resistance would limit the worldwide epidemic of type 2 diabetes (T2DM). Endoplasmic reticulum (ER) calcium (Ca(2+)) depletion induced by saturated free fatty acids and cytokines causes β cell ER stress and apoptosis, but the molecular mechanisms behind these phenomena are still poorly understood. Here, we demonstrate that palmitate-induced sorcin (SRI) down-regulation, and subsequent increases in glucose-6-phosphatase catalytic subunit-2 (G6PC2) levels contribute to lipotoxicity. SRI is a calcium sensor protein involved in maintaining ER Ca(2+) by inhibiting ryanodine receptor activity and playing a role in terminating Ca(2+)-induced Ca(2+) release. G6PC2, a GWAS gene associated with fasting blood glucose, is a negative regulator of glucose-stimulated insulin secretion (GSIS). High fat feeding in mice and chronic exposure of human islets to palmitate decreases endogenous SRI expression while levels of G6PC2 mRNA increase. Sorcin null mice are glucose intolerant, with markedly impaired GSIS and increased expression of G6pc2. Under high fat diet, mice overexpressing SRI in the β cell display improved glucose tolerance, fasting blood glucose and GSIS, whereas G6PC2 levels are decreased and cytosolic and ER Ca(2+) are increased in transgenic islets. SRI may thus provide a target for intervention in T2DM.

Positron emission tomography in the diagnosis and staging of pancreatic and neuroendocrine tumors

Tutkimuksen tausta ja tavoitteet: Viimeaikaisesta perinteisten kuvantamismenetelmien kehityksestä huolimatta sekä haima- että neuroendokriinisten (NE) kasvaimien diagnostiikka on haastavaa. Uudentyyppinen kuvantamismenetelmä, fuusio positroniemissiotomografia-tietokonetomografia (PET/TT), on lupaava näiden kasvainten erotusdiagnostiikassa ja levinneisyyden arvioinnissa. Huolimatta alustavista lupaavista tutkimustuloksista, PET/TT:n rooli on toistaiseksi vielä epäselvä sekä haima- että NE-kasvaimissa eikä se näin ollen ole vakiintunut kliiniseen hoitokäytäntöön. Väitöskirjatyön tavoitteena oli selvittää PET/TT -menetelmän käyttökelpoisuutta haima- ja NE-kasvaimien diagnostiikassa. Kahden ensimmäisen osatyön prospektiivisessa tutkimuksessa potilaat, joilla epäiltiin haimakasvainta, kuvannettiin PET/TT:llä käyttäen merkkiaineena fluorideoxyglukoosia (¹⁸F-FDG) kasvaimen aineenvaihdunnan arvioimiseksi ja kasvaimen verenvirtausta arviointiin käyttäen merkkiaineena radiovettä (¹⁵O-H₂O). Kolmen muun osatyön tavoitteena oli selvittää dihydroxyfenylalaniini (18F-DOPA) PET -menetelmää erilaisten NE-kasvaimien diagnostiikassa ja levinneisyyden arvioinnissa. Tulokset: Haimakasvaimien ensivaiheen diagnostiikassa ¹⁸F-FDG-PET/TT:llä oli korkeampi diagnostinen tarkkuus verrattuna titokoneleike- (TT) ja magneettikuvantamiseen (MK) (89% vs. 76% ja 79%). Etenkin pahanlaatuiseksi epäillyn sappitiehytahtauman erotusdiagnostiikassa ¹⁸F-FDG-PET/TT:n positiivinen ennustearvo (92%) oli korkea. Haimasyövän levinneisyyden arvioinnissa ¹⁸F-FDG-PET/TT:n herkkyys oli huono (30%) paikallisen taudin osoittamisessa. Sen sijaan etäpesäkkeiden osoittamisessa ¹⁸F-FDG-PET/TT oli merkittävästi herkempi menetelmä verrattuna TT ja magneettikuvantamiseen (88% vs. 38%). Verrattaessa erilaisten haimakasvaimien ja normaalin haimakudoksen aineenvaihduntaa ja verenvirtausta, aineenvaihdunta/verenvirtaus suhde oli merkittävästi korkeampi pahanlaatuisissa haimakasvaimissa (P<0.05). Lisäksi kasvaimen korkea aineenvaihdunta/verenvirtaus suhde viittasi huonompaan taudin ennusteeseen. ¹⁸F-DOPA-PET löysi seitsemän kahdeksasta insulinoomasta ja oli positiivinen myös kahdella potilaalla, joilla todettiin haiman saarakesoluhyperplasia. Perustuen alustaviin tuloksiin, rutiinikäytössä oleva karbidopa esilääke ennen ¹⁸F-DOPA-PET kuvantamista peitti insulinooma löydöksen kahdella potilaalla kolmesta. NE-kasvaiminen diagnostiikassa 82 potilaan aineisto osoitti ¹⁸F-DOPA PET kuvantamisen tarkkuudeksi 90%. Etenkin feokromosytoomien ensivaiheen diagnostiikassa ja NE-kasvaimen uusiutumaa epäiltäessä menetelmän tarkkuus oli korkea. Kokonaisuudessaan 59%:lla aineiston potilaista ¹⁸F-DOPA-PET kuvantamisella oli vaikutusta kliinisiin hoitoratkaisuihin. Johtopäätökset: PET/TT käyttäen merkkiaineena ¹⁸F-FDG:tä ja radiovettä osoittautui käyttökelpoiseksi menetelmäksi haimakasvaimien erotusdiagnostiikassa. Lisäksi ¹⁸F-FDG-PET/TT oli hyödyllinen haimasyövän etäpesäkkeiden arvioinnissa. Tutkimus osoitti myös ¹⁸F-DOPA-PET kuvantamisen olevan luotettava menetelmä insulinoomien ja muiden vatsan alueen NE-kasvaimien ensivaiheen diagnostiikassa sekä levinneisyyden arvioinnissa, etenkin muiden kuvantamislöydösten ollessa ristiriitaisia. PET kuvantamisella oli merkittävä vaikutus potilaiden kliiniseen hoitokäytäntöön sekä haima- että NE-kasvaimissa.

Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of a2b1 Integrin and E-Cadherin Function

In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLex) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLex and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLex and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Low levels of sex hormone-binding globulin and hyperproinsulinemia as markers of increased pancreatic ß-cell demand in men

Low levels of sex hormone-binding globulin (SHBG) are considered to be an indirect index of hyperinsulinemia, predicting the later onset of diabetes mellitus type 2. In the insulin resistance state and in the presence of an increased pancreatic ß-cell demand (e.g. obesity) both absolute and relative increases in proinsulin secretion occur. In the present study we investigated the correlation between SHBG and pancreatic ß-cell secretion in men with different body compositions. Eighteen young men (30.0 ± 2.4 years) with normal glucose tolerance and body mass indexes (BMI) ranging from 22.6 to 43.2 kg/m2 were submitted to an oral glucose tolerance test (75 g) and baseline and 120-min blood samples were used to determine insulin, proinsulin and C-peptide by specific immunoassays. Baseline SHBG values were significantly correlated with baseline insulin (r = -0.58, P<0.05), proinsulin (r = -0.47, P<0.05), C-peptide (r = -0.55, P<0.05) and also with proinsulin at 120 min after glucose load (r = -0.58, P<0.05). Stepwise regression analysis revealed that proinsulin values at 120 min were the strongest predictor of SHBG (r = -0.58, P<0.05). When subjects were divided into obese (BMI >28 kg/m2, N = 8) and nonobese (BMI £25 kg/m2, N = 10) groups, significantly lower levels of SHBG were found in the obese subjects. The obese group had significantly higher baseline proinsulin, C-peptide and 120-min proinsulin and insulin levels. For the first time using a specific assay for insulin determination, a strong inverse correlation between insulinemia and SHBG levels was confirmed. The finding of a strong negative correlation between SHBG levels and pancreatic ß-cell secretion, mainly for the 120-min post-glucose load proinsulin levels, reinforces the concept that low SHBG levels are a suitable marker of increased pancreatic ß-cell demand.

Bradykinin enhances membrane electrical activity of pancreatic beta cells in the presence of low glucose concentrations

In most of cells bradykinin (BK) induces intracellular calcium mobilization. In pancreatic beta cells intracellular calcium is a major signal for insulin secretion. In these cells, glucose metabolism yields intracellular ATP which blocks membrane potassium channels. The membrane depolarizes, voltage-dependent Ca2+ channels are activated and the intracellular calcium load allows insulin secretion. Repolarization occurs due to activation of the Ca2+-dependent K+ channel. The insulin secretion depends on the integrity of this oscillatory process (bursts). Therefore, we decided to determine whether BK (100 nM) induces bursts in the presence of a non-stimulatory glucose concentration (5.6 mM). During continuous membrane voltage recording, our results showed that bursts were obtained with 11 mM glucose, blocked with 5.6 mM glucose and recovered with 5.6 mM glucose plus 100 nM BK. Thus, the stimulatory process obtained in the presence of BK and of a non-stimulatory concentration of glucose in the present study suggests that BK may facilitate the action of glucose on beta cell secretion.

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42%) and NO synthase (NOS) activity (50%) in diabetic but not in control samples. When NO production was blocked by N G-monomethyl-L-arginine (L-NMMA) (600 µM), SOD activity increased (15.21 ± 1.23 vs 24.40 ± 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 ± 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 ± 0.02 vs 0.20 ± 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 ± 0.05); spermine nonoate (100 µM) abolished the decrease in lipoperoxide level (0.70 ± 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat.

Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.