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Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors. Diabetes 61:1100-1111, 2012, Pancreatic β-cells have a highly developed endoplasmic reticulum (ER) that reflects their physiological function as insulin-secreting cells. There is ample evidence indicating that alterations in ER homeostasis in β-cells affect [...]

OBJECTIVE--Sarco-endoplasmic reticulum [Ca.sup.2+]-ATPase 2b (SERCA2b) and SERCA3 pump [Ca.sup.2+] in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.ER]) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS--β-Cell [[[Ca.sup.2+]].sub.ER] of [SERCA3.sup.+/+] and [SERCA3.sup.-/-] mice was monitored with an adenovirus encoding the low [Ca.sup.2+]-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]) and [[[Ca.sup.2+]].sub.ER] were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS--Glucose elicited synchronized [[[Ca.sup.2+]].sub.ER] and [[[Ca.sup.2+]].sub.c] oscillations. [[[Ca.sup.2+]].sub.ER] oscillations were smaller in [SERCA3.sup.-/-] than in [SERCA3.sup.+/+] β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [[[Ca.sup.2+]].sub.ER] rise in [SERCA3.sup.+/+] and [SERCA.sup.3-/-] β-cells. In a [Ca.sup.2+]-free medium, glucose moderately raised [[Ca.sup.2+]].sub.ER] from a highly buffered cytosolic [Ca.sup.2+] pool. Increasing [[[Ca.sup.2+]].sub.c] with high [K] elicited a [[[Ca.sup.2+]].sub.ER] rise that was larger but more transient in [SERCA3.sup.+/+] than [SERCA3.sup.-/-] β-cells because of the activation of a [Ca.sup.2+] release from the ER in [SERCA3.sup.+/+] β-cells. Glucose-induced insulin release was larger in [SERCA3.sup.-/-] than [SERCA3.sup.+/+] islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS--[[[Ca.sup.2+]].sub.c] and [[[Ca.sup.2+]].sub.ER] oscillate in phase in response to glucose. Upon [[[Ca.sup.2+]].sub.c] increase, [Ca.sup.2+] is taken up by SERCA2b and SERCA3. Strong [Ca.sup.2+] influx triggers a [Ca.sup.2+] release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs [Ca.sup.2+] uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress. Diabetes 60:2533-2545, 2011, Pancreatic β-cells stimulated by glucose display oscillations of the free cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]) resulting from intermittent [Ca.sup.2+] influx (1,2). Their endoplasmic reticulum (ER) takes up cytosolic [Ca.sup.2+] by two [...]

Task division in multicellular organisms ensures that differentiated cell types produce cell-specific proteins that fulfill tasks for the whole organism. In some cases, the encoded mRNA species is so abundant that it represents a sizeable fraction of total mRNA in the cell. In this study, we have used a probe- and primer-free technique to quantify such abundant mRNA species in order to assess regulatory effects of in vitro and in vivo conditions. As a first example, we were able to quantify the regulation of proinsulin mRNA abundance in β-cells by food intake or by the glucose concentration in tissue culture. The second example of application of this technique is the effect of corticosteroids on growth hormone mRNA in enriched somatrotrophs. It is anticipated that other examples exist in which measurement of very abundant mRNAs in dedicated cells will help to understand biological processes, monitor disease states, or assist biotechnological manufacturing procedures., Now that increasing numbers of genomes have been sequenced, a major goal in biology is to understand how specialized cells use parts of the genome to produce the proteins required [...]

Type 1 and type 2 diabetes are characterized by progressive β-cell failure. Apoptosis is probably the main form of β-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced β-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1β, nuclear factor (NF)-κB, and Fas. We review herein the similarities and differences between the mechanisms of β-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ IL-1β and/or TNF-α plus IFN-γ induce β-cell apoptosis via the activation of β-cell gene networks under the control of the transcription factors NF-κB and STAT-1. NF-κB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticnium (ER) calcium. The execution of β-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes β-cell dysfunction and may induce β-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially 'glucose hypersensitization' and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1β, NF-κB, or inducible nitric oxide synthase in rat or human β-cells in vitro or in vivo in Psammomys obesus. FFAs may cause β-cell apoptosis via ER stress, which is NF-κB and NO independent. Thus, cytokines and nutrients trigger β-cell death by fundamentally different mechanisms, namely an NF-κB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-κB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of β-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent β-cell death in type I and type 2 diabetes., Clinical definitions of disease often obscure different mechanistic subtypes. This is particularly relevant for complex diseases such as diabetes, where combinations of multiple genes and environmental factors eventually lead to [...]

Two sarcoendoplasmic reticulum [Ca.sup.2+]-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to β-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in β-cell cytosolic-free [Ca.sup.2+] concentration ([[Ca.sup.2+]].sub.c) regulation, insulin secretion, and glucose homeostasis. Basal [[[Ca.sup.2+]].sub.c] was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [[[Ca.sup.2+]].sub.2+] that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. [Ca.sup.2+] mobilization by acetylcholine was normal in SERCA3-deficient β-cells. In contrast, [[[Ca.sup.2+]].sub.c] oscillations resulting from intermittent glucose-stimulated [Ca.sup.2+] influx and [[[Ca.sup.2+]].sub.c] transients induced by pulses of high [K.sup.+] were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases [Ca.sup.2+] that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [[[Ca.sup.2+]].sub.c] regulation in β-cells. SERCA3 becomes operative when [[Ca.sup.2+]].sub.c] rises and is required for normal [[[Ca.sup.2+]].sub.c] oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice., The sarcoendoplasmic reticulum [Ca.sup.2+]-ATPase (SERCA) transfers [Ca.sup.2+] from the cytosol to the lumen of the reticulum. Three SERCA genes (SERCA1-3) have been identified, all of which can generate several alternatively [...]

When fed a high-energy (HE) diet, diabetes-prone (DP) Psammomys obesus develop type 2 diabetes with altered glucose-stimulated insulin secretion (GSIS). β-Cell stimulus-secretion coupling was investigated in islets isolated from DP P. obesus fed a low-energy (LE) diet (DP-LE) and after 5 days on a HE diet (DP-HE). DP-LE islets cultured overnight in 5 mmol/l glucose displayed glucose dose-dependent increases in NAD(P)H, mitochondrial membrane potential, ATP/ (ATP + ADP) ratio, cytosolic calcium concentration ([[[Ca.sup.2+]].sub.c]), and insulin secretion. In comparison, DP-HE islets cultured overnight in 10 mmol/l glucose were 80% degranulated and displayed an increased sensitivity to glucose at the level of glucose metabolism, [[[Ca.sup.2+]].sub.c], and insulin secretion. These changes in DP-HE islets were only marginally reversed after culture in 5 mmol/l glucose and were not reproduced in DP-LE islets cultured overnight in 10 mmol/l glucose, except for the 75% degranulation. Diabetes-resistant P. obesus remain normoglycemic on HE diet. Their β-cell stimulus-secretion coupling was similar to that of DP-LE islets, irrespective of the type of diet. Thus, islets from diabetic P. obesus display an increased sensitivity to glucose at the level of glucose metabolism and a profound β-cell degranulation, both of which may affect their in vivo GSIS., Stimulation of insulin secretion by glucose requires oxidative metabolism of the sugar in β-cells, with acceleration of ATP production and increase of the ATP/ADP ratio (1-4). The subsequent closure of [...]

That oscillations of the cytoplasmic free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in β-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence [...]

The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free [Ca.sup.2+] ([[[Ca.sup.2+]].sub.c]) in β-cells play a [...]

Rapid and sustained stimulation of β-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each β-cell rather than heterogeneity in [...]

Hypertrophy is one mechanism of pancreatic β-cell growth and is seen as an important compensatory response to insulin resistance. We hypothesized that the induction of protective genes contributes to the [...]

An increase in cytoplasmic [Ca.sup.2+] in Β-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free [Ca.sup.2+]. ([[[Ca.sup.2+]].sub.i]) directly regulate secretion remains disputed. This question [...]

Recently, we showed that Β-cell dysfunction in hyperglycemic 90%-pancreatectomized rats is associated with downregulation of key islet genes and transcription factors concomitant with marked upregulation of several suppressed genes, including [...]

Type 2 diabetes is characterized by a reduction in both insulin sensitivity and glucose-stimulated insulin secretion (GSIS). Although insulin resistance usually precedes Β-cell dysfunction, the role of the latter in [...]

Hypertrophy of [Beta]-cells develops as a compensatory mechanism for meeting an increased insulin demand. We have previously shown that [Beta]-cell hypertrophy in hyperglycemic 90% pancreatectomised (Px) rats is associated with [...]

Non-diabetic insulin resistant subjects have increased insulin secretion in response to glucose; however, little is known about the adaptive process. In the present study, changes in [Beta]-cell mass and secretory [...]

Inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) isoforms 1,2, and 3 mediate cellular [Ca.sup.2+] regulation in response to IP3. IP3R mRNA and protein expression were characterized in isolated rat islets using RT-PCR [...]

Excessive activation of the endoplasmic reticulum (ER) stress pathway by chronic hyperglycemia may contribute to [beta]-cell apoptosis in type 2 diabetes. To test whether glucose induces ER stress in [beta]-cells, XBP1 mRNA unconventional splicing and GADD153 mRNA and protein levels were measured in rat islets precultured for 1 week in 10 mmol/l glucose (G10) and further cultured for 18 h in G2, G5, G10, or G30. As expected, 1 [micro]mol/l thapsigargin, an inhibitor of ER [Ca.sup.2+]-ATPases that triggers ER stress in various cell types, increased islet XBP1 mRNA splicing and GADD153 expression (3- and 10-fold, respectively) in G10. In contrast, glucose exerted opposite effects on these two ER stress markers: a two- and threefold increase in XBP1 mRNA splicing after culture in G10 and G30 versus G2-G5 and an ~40 and ~90% reduction in GADD153 expression after culture in G5 and G10-G30 versus G2. After an overnight culture in G5 and further culture in G30, the increase in XBP1 mRNA splicing and decrease in GADD153 mRNA levels occurred within 2 h of stimulation. In conclusion, in contrast with thapsigargin, glucose activates only part of the ER stress pathway while reducing expression of the pro-apoptotic factor GADD153. These rapid glucose effects may play a physiological role in nutrient-induced maintenance of the [beta]-cell phenotype., From the Unit of Endocrinology and Metabolism, Faculty of Medicine, Universite Catholique de Louvain, Brussels, Belgium. Address correspondence to Jean-Christophe Jonas. E-mail: jonas@ [...]

Glucose-induced insulin secretion from hyperglycemic 90% pancreatectomized rats is markedly impaired, possibly because of loss of β cell differentiation. Association of these changes with β cell hypertrophy, increased mRNA levels of the transcription factor c-Myc, and their complete normalization by phlorizin treatment suggested a link between chronic hyperglycemia, increased c-Myc expression, and altered β cell function. In this study, we tested the effect of hyperglycemia on rat pancreatic islet c-Myc expression both in vivoand in vitro. Elevation of plasma glucose for 1–4 days (glucose infusion/clamp) was followed by parallel increases in islet mRNA levels (relative to TATA-binding protein) of c-Myc and two of its target genes, ornithine decarboxylase and lactate dehydrogenase A. Similar changes were observed in vitroupon stimulation of cultured islets or purified β cells with 20 and 30 mmol·liter−1glucose for 18 h. These effects of high glucose were reproduced by high potassium-induced depolarization or dibutyryl-cAMP and were inhibited by agents decreasing cytosolic Ca2+or cAMP concentrations. In conclusion, the expression of the early response gene c-Myc in rat pancreatic β cells is stimulated by high glucose in a Ca2+-dependent manner and by cAMP. c-Myc could therefore participate to the regulation of β cell growth, apoptosis, and differentiation under physiological or pathophysiological conditions.

Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets.

Differentiated pancreatic β cells are unique in their ability to secrete insulin in response to a rise in plasma glucose. We have proposed that the unique constellation of genes they express may be lost in diabetes due to the deleterious effect of chronic hyperglycemia. To test this hypothesis, Sprague-Dawley rats were submitted to a 85–95% pancreatectomy or sham pancreatectomy. One week later, the animals developed mild to severe chronic hyperglycemia that was stable for the next 3 weeks, without significant alteration of plasma nonesterified fatty acid levels. Expression of many genes important for glucose-induced insulin release decreased progressively with increasing hyperglycemia, in parallel with a reduction of several islet transcription factors involved in β cell development and differentiation. In contrast, genes barely expressed in sham islets (lactate dehydrogenase A and hexokinase I) were markedly increased, in parallel with an increase in the transcription factor c-Myc, a potent stimulator of cell growth. These abnormalities were accompanied by β cell hypertrophy. Changes in gene expression were fully developed 2 weeks after pancreatectomy. Correction of blood glucose by phlorizin for the next 2 weeks normalized islet gene expression and β cell volume without affecting plasma nonesterified fatty acid levels, strongly suggesting that hyperglycemia triggers these abnormalities. In conclusion, chronic hyperglycemia leads to β cell hypertrophy and loss of β cell differentiation that is correlated with changes in c-Myc and other key transcription factors. A similar change in β cell differentiation could contribute to the profound derangement of insulin secretion in human diabetes.

1The cytoplasmic Ca2+concentration ([Ca2+]i) was measured in single cells and cell clusters of different sizes prepared from mouse pancreatic islets.2During stimulation with 15 mM glucose, 20 % of isolated cells were inert, whereas 80 % showed [Ca2+]ioscillations of variable amplitude, duration and frequency. Spectral analysis identified a major frequency of 0.14 min−1and a less prominent one of 0.27 min−1.3In contrast, practically all clusters (2–50 cells) responded to glucose, and no inert cells were identified within the clusters. As compared to single cells, mean [Ca2+]iwas more elevated, [Ca2+]ioscillations were more regular and their major frequency was slightly higher (but reached a plateau at ≈0.25 min−1). In some cells and clusters, faster oscillations occurred on top of the slow ones, between them or randomly.4Image analysis revealed that the regular [Ca2+]ioscillations were well synchronized between all cells of the clusters. Even when the Ca2+response was irregular, slow and fast [Ca2+]ioscillations induced by glucose were also synchronous in all cells.5In contrast, [Ca2+]ioscillations resulting from mobilization of intracellular Ca2+by acetylcholine were restricted to certain cells only and were not synchronized.6Heptanol and 18α‐glycyrrhetinic acid, two agents widely used to block gap junctions, altered glucose‐induced Ca2+oscillations, but control experiments showed that they also exerted effects other than a selective uncoupling of the cells.7The results support theoretical models predicting an increased regularity of glucose‐dependent oscillatory events in clusters as compared to isolated islet cells, but contradict the proposal that the frequency of the oscillations increases with the number of coupled cells. Islet cell clusters function better as electrical than biochemical syncytia. This may explain the co‐ordination of [Ca2+]ioscillations driven by depolarization‐dependent Ca2+influx during glucose stimulation.

The potential roles of protein tyrosine kinases (TKs) and of phosphotyrosine phosphatases (PTPs) in pancreatic islet function are not known. In this study, we investigated whether vanadate, a potent PTP inhibitor, affects phosphoinositide (PI) metabolism by a TK-dependent pathway in isolated mouse islets. To avoid the confounding effects of changes in Ca2+ influx, all experiments were performed in the absence of Ca2+. In the presence of 15 mM glucose, vanadate, acetylcholine (ACh) or [Arg]vasopressin (AVP) strongly stimulated InsP production. Vanadate also increased PtdInsP levels in membranes. The TK inhibitor genistein (not its inactive analogues genistin and daidzein) significantly reduced vanadate effects, but was without effect in the absence of stimulation or in the presence of ACh or AVP. Islet proteins resolved by SDS/PAGE were analysed by immunoblotting with anti-phosphotyrosine antibody. Under control conditions, several phosphotyrosyl-proteins (PYPs) were present. Vanadate increased phosphotyrosine residues on several PYPs, notably two proteins of 145 and 85 kDa. This effect was prevented by genistein. p145 and p85 could correspond to phospholipase Cγ (PLCγ) and the regulatory subunit of PtdIns-3-kinase (PtdIns-3K) respectively. Both proteins are expressed in islets, as revealed by immunoblots with specific antibodies. Tungstate, another PTP inhibitor, reproduced vanadate effects, but inhibition of PtdIns-3K by wortmannin failed to affect vanadate-increased PtdInsP levels. Incubation of the islets in the presence of 10% (v/v) fetal calf serum instead of BSA increased InsP production and this effect was prevented by genistein. These results suggest that inhibition of PTP increases InsP production in mouse islets by a TK-dependent pathway. They also provide evidence for a potential role of TK and PTP in pancreatic B-cell function.

Pharmacological inhibition of AQP1 prevents H2O2transport and hypertrophy of cardiac myocytes.