Synaptotagmins (SYT) are calcium-binding proteins that participate in regulated exocytosis. Although SYTI to IX isoforms are expressed in insulin-producing cell lines, hitherto only SYTIX has been associated with native beta-cell insulin granules and implicated in exocytosis. SYTVII was also proposed to regulate insulin exocytosis, but its subcellular location and number of alternative splice variants produced remain controversial. Only transcripts of SYTVII alpha, beta, and a novel splice variant delta are expressed in beta-cells and INS-1E cells. Western blotting revealed that INS-1E cells predominantly produced SYTVII alpha and low levels of SYTVII beta, whereas SYTVII delta was undetectable. The protein colocalized with insulin granules but not with synaptic-like microvesicles. Overexpression of SYTVII alpha resulted in decreased insulin granule content with a concomitant translocation of the variant to the plasma membrane, while SYTVII beta retained largely a granular pattern. Overexpressed SYTVII delta exhibited a distribution different to that of insulin granules and inhibited exocytosis when assessed by whole cell patch clamp capacitance recording. Silencing of SYTVII alpha by targeted RNA interference suppressed secretion, while repression of beta slightly increased release. Our results demonstrate that SYTVII is expressed on insulin granules and that only SYTVII alpha is implicated in exocytosis under physiological conditions.

In pancreatic β-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mm glucose, 0.4 mm palmitate, 0.4 mm oleate and transiently to H2O2. Culture at physiological 5.6 mm glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in β-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, whereas the glutamate carrier GC1 and the aspartate/glutamate carrier AGC1 were down-regulated by palmitate and oleate, respectively. High glucose decreased mRNA levels of key transcription factors (HNF4A, IPF1, PPARA and TFAM) and energy-sensor SIRT1. High glucose also reduced expression of 11 mtDNA-encoded respiratory chain subunits. Interestingly, transcript levels of the carriers for aspartate/glutamate AGC2, malate DIC and malate/oxaloacetate/aspartate UCP2 were increased by high glucose, a profile suggesting important mitochondrial anaplerotic/cataplerotic activities and NADPH-generating shuttles. Chronic exposure to high glucose impaired glucose-stimulated insulin secretion, decreased insulin content, promoted caspase-3 cleavage and cell death, revealing glucotoxicity. Overall, expression profile of mitochondrion-associated genes was selectively modified by glucose, delineating a glucotoxic-specific signature.

Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Mitochondria play a central role in coupling glucose metabolism to insulin secretion. However, little is known on mitochondrial responses to specific stresses; i.e. low versus high glucose, saturated versus unsaturated fatty acids, or oxidative stress. INS-1E cells were exposed for 3 days to 5.6 mM glucose, 25 mM glucose, 0.4 mM palmitate, and 0.4 mM oleate. Culture at standard 11.1 mM glucose served as no-stress control and transient oxidative stress (200 µM H2O2 for 10 min at day 0) served as positive stressful condition. Mito-array analyzed transcripts of 60 mitochondrion-associated genes with special focus on members of the Slc25 family. Transcripts of interest were evaluated at the protein level by immunoblotting. Bioinformatics analyzed the expression profiles to delineate comprehensive networks. Chronic exposure to the different metabolic stresses impaired glucose-stimulated insulin secretion; revealing glucotoxicity and lipo-dysfunction. Both saturated and unsaturated fatty acids increased expression of the carnitine/acylcarnitine carrier CAC, whereas the citrate carrier CIC and energy sensor SIRT1 were specifically upregulated by palmitate and oleate, respectively. High glucose upregulated CIC, the dicarboxylate carrier DIC and glutamate carrier GC1. Conversely, it reduced expression of energy sensors (AMPK, SIRT1, SIRT4), metabolic genes, transcription factor PDX1, and anti-apoptotic Bcl2. This was associated with caspase-3 cleavage and cell death. Expression levels of GC1 and SIRT4 exhibited positive and negative glucose dose-response, respectively. Expression profiles of energy sensors and mitochondrial carriers were selectively modified by the different conditions, exhibiting stress-specific signatures.

The paired/homeodomain transcription factor Pax4 is essential for islet b-cell generation during pancreas development and their survival in adulthood. High Pax4 expression was reported in human insulinomas indicating that deregulation of the gene may be associated with tumorigenesis. We report that rat insulinoma INS-1E cells express 25-fold higher Pax4 mRNA levels than rat islets. In contrast to primary b-cells, activin A but not betacellulin or glucose induced Pax4 mRNA levels indicating dissociation of Pax4 expression from insulinoma cell proliferation. Short hairpin RNA adenoviral constructs targeted to the paired domain or homeodomain (viPax4PD and viPax4HD) were generated. Pax4 mRNA levels were lowered by 73 and 50% in cells expressing either viPax4PD or viPax4HD. Transcript levels of the Pax4 target gene bcl-xl were reduced by 53 and 47%, whereas Pax6 and Pdx1 mRNA levels were unchanged. viPax4PD-infected cells displayed a twofold increase in spontaneous apoptosis and were more susceptible to cytokine-induced cell death. In contrast, proliferation was unaltered. RNA interference-mediated repression of insulin had no adverse effects on either Pax4 or Pdx1 expression as well as on cell replication or apoptosis. These results indicate that Pax4 is redundant for proliferation of insulinoma cells, whereas it is essential for survival through upregulation of the antiapoptotic gene bcl-xl.

Unseparated splenocytes (SPCs) or purified SPC subsets from diabetes-prone BB (BBdp) or diabetic BB (BBd) rats were activated in vitro with either phorbol myristate acetate (PMA) and ionomycin (I) or concanavalin A (ConA). Such activated SPCs were then injected intravenously into 30-day-old BBdp rats, and their capacity to induce adoptive transfer (AT) of diabetes was studied. The proliferative response in vitro of BBd unseparated SPCs or purified W3/13+ SPCs (i.e., T lymphocytes + large granular lymphocytes) to PMA + I far exceeded that of ConA, resulting in mean stimulation indices of 68 and 112 (PMA + I) and 1.9 and 30 (ConA). The incidence of AT was similar when equal numbers of unseparated SPCs from the same BBd donor were injected after activation by either PMA + I + interleukin 2 (PII) or ConA (57 vs. 50%, respectively); however, injection of PII-activated and macrophage-depleted W3/13+ SPCs from BBd animals resulted in a significantly higher incidence of AT (90%, P less than 0.05). As few as 0.5 x 10(6) PII-activated W3/13+ SPCs were sufficient to induce AT. Sixteen percent of recipients developed diabetes after injection of activated W3/13+ cells from 40-day-old BBdp donors. To determine which W3/13+ cells might mediate such transfer, purified and PII-preactivated CD4 T lymphocytes from BBd rats were injected, and they succeeded in AT in 44% of the recipients. Preactivated BBd B lymphocytes were unable to induce AT. Although a possible role for large granular lymphocytes cannot be excluded, the results demonstrate that in the BB rat, the beta-cell destruction can be induced by CD4 T lymphocytes.

Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-cells and human islets were exposed for 3 days to glucose (5.5–25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E β-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the β-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.

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In order to investigate frequency and extent of liver damage in children with inflamatory bowel disease(IB0) we followed 34 patients with ulcerative colitis(UC) and 12 with Crohn's disease(CD). Ihe children were followed prospectively from onset of disease whereby clinical snd biochemical activity was compared to liver function tests(LFT). The liver damage was assessed according to LFT and biopsy was performed in a l l cases with LFT elevated above 3 times normal levels.In g the liver damage was not related to duration and/or extent: .~a l l a b r i g a . H o s p i t a l I n f a n t i l "Val1 d*Aebr6n", Barcelona, Spain LFT 0 mild severe C r y p t o s p o r i d i a s i s u s u a l l y i s diagnosed i n immunodeficient p a t i e n t s . We i n v e s t i g a t e d t h e incidence i n childhood d i a r r h o e a . METHODS: s t o o l c o l o r a t i o n a l c o h o l -a c i d r e s i s t e n t technique. We s t u d i e d 20 primary and secondary immunodeficient p a t i e n t s with d i a r r h o e a (group A ); 43 c a s e s a t t e n d i n g a day c a r e c e n t e r during a g a s t r o e n t e r i t i s outbreak (group 9) and 40 c a s e s with a c u t e d i a r r h o e a (group C ) .RESULTS: f e c e s were p o s i t i v e f o r cryptos.woridium i n : qroup A: 2 c h i l d r e n ; t h e p a r a s i t e was a l s o found i n j e j u n a l mucosa; only 1 responded t o Spyramicine. Gr0u.p B: 17 c a s e s ; 2 had low IqA l e v e l s ; t h e d i a r r h o e a l a s t e d from 1 week t o 2 months and cured without t r e a t m e n t . Group C: 9 c h i l d r e n ; a l l were immunoloqically normal and cured without t r e a t m e n t .CONCLUSIONS: because of t h i s h i g h i n c i d e n c e , cryptosporidium should b e i n v e s t i g a t e d i n childhood d i a r r h o e a , even i n c h i l d r e n w i t h a normal immunological s t a t u s .The signs of damage were permanent in only I case. N o relation was found to treatment and relapses. N o correlation was found between LFT and other laboratory tests. Biopsy in the 7 cases with suspected severe damage showed cirrhosis(l), cholangitis(2), chronic aggressive hepatitis(l1, steatosis(1) and minor changes(2 cases).In g 4 of the 12 patients had signs of mild liver damage, which could not be related to activity or duration of the disease or treatment. It i s concluded that there seems to be a high risk of liver damage in childhood IBO and that severe forms seem to occur a t the time of onset or early in the course of the disease. y p t o s p o r l ium was known a s a cause of s e v e r e d i a r r h e a i n immunod e f i c i e n t p a t i e n t s . Recently i t appeared t o be a l s o involved i n normal population. A 12 month p r o s p e c t i v e study was performed i n Tours, a town w i t h a mainly r u r a l population, i n c h i l d r e n h o s p i t al i z e d w i t h d i a r r h e a . 200 s t o o l s from 190 c h i l d r e n were evaluated f o r t h e presence of Cryptosporidium by a sugar f l o t a t i o n method.4 c h i l d r e n had p o s i t i v e s t o o l s . ...