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Abstract: Over the last two decades we have focused on beta cell signal transduction, bringing many new insights, especially in the context of insulin signal transduction, the role of inositol polyphosphates and the regulation of cytoplasmic free Ca2+ concentration. However, there has been a growing awareness that the beta cell, which is mandatory for insulin secretion, has a unique context within the micro-organ of the pancreatic Islet of Langerhans. In this environment the beta cell both mediates and receives paracrine regulation, critical for the control of blood glucose homeostasis. Failure of an appropriate beta cell function leads to the development of diabetes mellitus. In our quest to understand the molecular events maintaining beta cell function we have faced two key challenges. Firstly, whilst there are many similarities between signal transduction in pancreatic islets between the much used rodent models and humans there are some notable differences. Critical distinctions between rodent and primate can be made in the structure of the islet, including the arrangement of the islet cells, the innervation pattern and the microcirculation. This means that important signaling interactions between islets cells, mediated through for example insulin, glucagon, GABA, glutamate and ATP, will have a unique human framework. The second challenge was to be able to take the discoveries we have made using in vitro systems and examine them in an in vivo context. Advances in in vivo imaging achieved by utilizing the anterior chamber of the eye as a transplantation site for pancreatic islets make it possible for non-invasive, longitudinal studies at single cell resolution in real time of islet cell physiology and pathology. Thus it is becoming possible to study the insulin secreting pancreatic beta cell within the framework of the unique micro-organ, the Islet of Langerhans, for the first time in a physiological context, i.e. when being innervated and connected to the blood supply. [Copyright &y& Elsevier]

Abstract: Pancreatic β-cells release insulin in appropriate amounts in order to keep blood glucose levels within physiological limits. Failure to do so leads to the most common metabolic disorder in man, diabetes mellitus. The glucose-stimulus/insulin-secretion coupling represents a sophisticated interplay between glucose and a variety of modulatory factors. These factors are provided by the blood supply (such as nutrients, vitamins, incretins etc.), the nerval innervations, cell-cell contacts as well as by paracrine and autocrine feedback loops within the pancreatic islet of Langerhans. However, the underlying mechanisms of their action remain poorly understood. In the present mini-review we discuss novel aspects of selective insulin signaling in the β-cell and novel insights into the role of higher inositol phosphates in insulin secretion. Finally we present a newly developed experimental platform that allows non-invasive and longitudinal in vivo imaging of pancreatic islet/β-cell biology at single-cell resolution. [Copyright &y& Elsevier]

The appropriate function of insulin-producing pancreatic β-cells is crucial for the regulation of glucose homeostasis, and its impairment leads to diabetes mellitus, the most common metabolic disorder in man. In addition to glucose, the major nutrient factor, inputs from the nervous system, humoral components, and cell-cell communication within the islet of Langerhans act together to guarantee the release of appropriate amounts of insulin in response to changes in blood glucose levels. Data obtained within the past decade in several laboratories have revitalized controversy over the autocrine feedback action of secreted insulin on β-cell function. Although insulin historically has been suggested to exert a negative effect on β-cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion, proliferation, and β-cell survival. Current insights on the role of insulin on pancreatic β-cell function are discussed. [ABSTRACT FROM AUTHOR]

Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the β-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (α and ∈) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9712-9716). Using confocal microscopy, we have now established that in response to glucose, PKC-∈ but not PKC-α associates with insulin granules and that green fluorescent protein-tagged PKC-∈ changes its distribution within the cell periphery upon stimulation of β-cells with glucose. Definite evidence of PKC-∈ requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wildtype PKC-∈ led to a significant increase in insulin exocytosis. These results suggest that association of PKC-∈ with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose. [ABSTRACT FROM AUTHOR]

Pancreatic β-cell function is essential for the regulation of glucose homeostasis and its impairment leads to diabetes mellitus. Besides glucose, the major nutrient factor, inputs from neural and humoral components and intraislet cell–cell communication act together to guarantee an appropriate pancreatic β-cell function. Data obtained over the last 5 years in several laboratories have revitalized a controversial concept, namely the autocrine feedback action of secreted insulin on β-cell function. While, historically, insulin was suggested to exert a negative effect on β-cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion and β-cell survival. [Copyright &y& Elsevier]

Pancreatic β-cell function is essential for the regulation of glucose homeostasis in humans, and its impairment leads to the development of type 2 diabetes. Inputs from glucose and cell surface receptors act together to initiate the β-cell stimulus-response coupling that ultimately leads to the release of insulin. Phosphorylated inositol compounds have recently emerged as key players at all levels of the stimulus-secretion coupling process. In this current review, we seek to highlight recent advances in β-cell phosphoinositide research by dividing our examination into two sections. The first involves the events that lead to insulin secretion. This includes both new roles for inositol polyphosphates, particularly inositol hexakisphosphate, and both conventional and 3-phosphorylated inositol lipids. In the second section, we deal with the more novel concept of the autocrine role of insulin. Here, released insulin initiates signal transduction cascades, principally through the activity of phosphatidylinositol 3-kinase. This new round of signal transduction has been established to activate key β-cell genes, particularly the insulin gene itself. More controversially, this insulin feedback has also been suggested to either terminate or enhance insulin secretion events. [ABSTRACT FROM AUTHOR]

The authors focus on a study published in journal "Nature Medicine" related to noninvasively control of gene expression by the application of radiogenetics and magnetic field in mice. Topics discussed include use of nanoparticle to control transgene expression and glucose homeostasis in a diabetic mouse, activation of tethered TRPV1Ca2+ channel by iron nanoparticles through radio waves or magnetic field and conversion of oscillatory magnetic field in oscillatory pattern.

The article reports on the link of Sirt1, an enzyme having the capacity to remove acetyl groups from specific nuclear proteins, to the regulation of aging. The enzyme is able to control glucose metabolism by serving as a sensor of the metabolic status in the hepatocytes. It regulates the activity of a variety of transcription factors and transcriptional coregulators. Sirt1 modulates aging and may consequently serve as a key decoder that translates caloric restriction to increase lifespan.

Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6)P5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PTEN can degrade both this lipid and Ins(1,3,4,5)P4. Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5)P3, a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5)P3. We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5)P3 in vitro. These data illustrate the importance of MINPP1′s confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1′s ER seclusion. [ABSTRACT FROM AUTHOR]

Insulin resistance and β-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and β-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca2+]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants. [ABSTRACT FROM AUTHOR]

Abstract: Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. It induces the endocytosis of plasma membrane Na,K-ATPase molecules, and this results in a reduced capacity of the cells to transport sodium. Dopamine induces the phosphorylation of Ser-18 in the α1-subunit of Na,K-ATPase. Fluorescence resonance energy transfer analysis of cells expressing YFP-α1 and β1-CFP reveals that treatment of the cells with dopamine increases energy transfer between CFP and YFP. This is consistent with a protein conformational change that results in the N-terminal end of α1 moving closer to the internal face of the plasma membrane. [Copyright &y& Elsevier]

The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells. [ABSTRACT FROM AUTHOR]

The dynamics of cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic β cells is central to our understanding of β‐cell physiology and pathology. In this context, there are numerous in vitro studies available but existing in vivo data are scarce. We now critically evaluate the anterior chamber of the eye as an in vivo, non‐invasive, imaging site for measuring [Ca2+]i dynamics longitudinally in three dimensions and at single‐cell resolution. By applying a fluorescently labeled glucose analogue 2‐(N‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)Amino)‐2‐Deoxyglucose in vivo, we followed how glucose almost simultaneously distributes to all cells within the islet volume, resulting in [Ca2+]i changes. We found that almost all β cells in healthy mice responded to a glucose challenge, while in hyperinsulinemic, hyperglycemic mice about 80% of the β cells could not be further stimulated from fasting basal conditions. This finding indicates that our imaging modality can resolve functional heterogeneity within the β‐cell population in terms of glucose responsiveness. Importantly, we demonstrate that glucose homeostasis is markedly affected using isoflurane compared to hypnorm/midazolam anesthetics, which has major implications for [Ca2+]i measurements. In summary, this setup offers a powerful tool to further investigate in vivo pancreatic β‐cell [Ca2+]i response patterns at single‐cell resolution in health and disease. [ABSTRACT FROM AUTHOR]

Although convincing in genetic models, the relevance of β-cell insulin resistance in diet-induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes-prone, male, C57B1/6J mice being fed different combinations of Western-style diet, we show that β-cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased β-cell workload. Within 8 wk of being fed a high-fat, high-sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin-1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional β-cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional β-cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet-monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional β-cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation. [ABSTRACT FROM AUTHOR]

Pancreatic islets are micro-organs composed of a mixture of endocrine and non-endocrine cells, where the former secrete hormones and peptides necessary for metabolic homeostasis. Through vasculature and innervation the cells within the islets are in communication with the rest of the body, while they interact with each other through juxtacrine, paracrine and autocrine signals, resulting in fine-tuned sensing and response to stimuli. In this context, cellular protrusion in islet cells, such as primary cilia and filopodia, have gained attention as potential signaling hubs. During the last decade, several pieces of evidence have shown how the primary cilium is required for islet vascularization, function and homeostasis. These findings have been possible thanks to the development of ciliary/basal body specific knockout models and technological advances in microscopy, which allow longitudinal monitoring of engrafted islets transplanted in the anterior chamber of the eye in living animals. Using this technique in combination with optogenetics, new potential paracrine interactions have been suggested. For example, reshaping and active movement of filopodia-like protrusions of δ-cells were visualized in vivo , suggesting a continuous cell remodeling to increase intercellular contacts. In this review, we discuss these recent discoveries regarding primary cilia and filopodia and their role in islet homeostasis and intercellular islet communication. [ABSTRACT FROM AUTHOR]

A hallmark of type 2 diabetes mellitus (T2DM) is the development of pancreatic β cell failure, which results in insulinopenia and hyperglycemia. We show that the adipokine adipsin has a beneficial role in maintaining β cell function. Animals genetically lacking adipsin have glucose intolerance due to insulinopenia; isolated islets from these mice have reduced glucose-stimulated insulin secretion. Replenishment of adipsin to diabetic mice treated hyperglycemia by boosting insulin secretion. We identify C3a, a peptide generated by adipsin, as a potent insulin secretagogue and show that the C3a receptor is required for these beneficial effects of adipsin. C3a acts on islets by augmenting ATP levels, respiration, and cytosolic free Ca2+. Finally, we demonstrate that T2DM patients with β cell failure are deficient in adipsin. These findings indicate that the adipsin/C3a pathway connects adipocyte function to β cell physiology, and manipulation of this molecular switch may serve as a therapy in T2DM. [ABSTRACT FROM AUTHOR]

The islets of Langerhans constitute the endocrine part of the pancreas and are responsible for maintenance of blood glucose homeostasis. They are deeply embedded in the exocrine pancreas, limiting their accessibility for functional studies. Understanding regulation of function and survival and assessing the clinical outcomes of individual treatment strategies for diabetes requires a monitoring system that continuously reports on the endocrine pancreas. We describe the application of a natural body window that successfully reports on the properties of in situ pancreatic islets. As proof of principle, we transplanted "reporter islets" into the anterior chamber of the eye of leptin-deficient mice. These islets displayed obesity-induced growth and vascularization patterns that were reversed by leptin treatment. Hence, reporter islets serve as optically accessible indicators of islet function in the pancreas, and also reflect the efficacy of specific treatment regimens aimed at regulating islet plasticity in vivo. [ABSTRACT FROM AUTHOR]

Peptide hormones are powerful regulators of various biological processes. To guarantee continuous availability and function, peptide hormone secretion must be tightly coupled to its biosynthesis. A simple but efficient way to provide such regulation is through an autocrine feedback mechanism in which the secreted hormone is "sensed" by its respective receptor and initiates synthesis at the level of transcription and/or translation. Such a secretion-biosynthesis coupling has been demonstrated for insulin; however, because of insulin's unique role as the sole blood glucose-decreasing peptide hormone, this coupling is considered an exception rather than a more generally used mechanism. Here we provide evidence of a secretion-biosynthesis coupling for glucagon, one of several peptide hormones that increase blood glucose levels. We show that glucagon, secreted by the pancreatic a cell, up-regulates the expression of its own gene by signaling through the glucagon receptor, PKC, and PKA, supporting the more general applicability of an autocrine feedback mechanism in regulation of peptide hormone synthesis. [ABSTRACT FROM AUTHOR]

Phosphatidylinositide 3-kinases (PI3Ks) play central roles in insulin signal transduction. While the contribution of class Ia PI3K members has been extensively studied, the role of class II members remains poorly understood. The diverse actions of class II PI3K-C2α have been attributed to its lipid product PI(3)P. By applying pharmacological inhibitors, transient overexpression and small-interfering RNA-based knockdown of PI3K and PKB/Akt isoforms, together with PI-lipid profiling and live-cell confocal and total internal reflection fluorescence microscopy, we now demonstrate that in response to insulin, PI3K-C2α generates PI(3,4)P2, which allows the selective activation of PKBα/Akt1. Knockdown of PI3K-C2α expression and subsequent reduction of PKBα/Akt1 activity in the pancreatic β-cell impaired glucose-stimulated insulin release, at least in part, due to reduced glucokinase expression and increased AS160 activity. Hence, our results identify signal transduction via PI3K-C2α as a novel pathway whereby insulin activates PKB/Akt and thus discloses PI3K-C2α as a potential drugable target in type 2 diabetes. The high degree of codistribution of PI3K-C2α and PKBα/Akt1 with insulin receptor B type, but not A type, in the same plasma membrane microdomains lends further support to the concept that selectivity in insulin signaling is achieved by the spatial segregation of signaling events. [ABSTRACT FROM AUTHOR]

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Insulin exerts pleiotropic effects at the cellular level. Signaling via the two isoforms of the insulin receptor (IR) may explain the activation of different signaling cascades, while it remains to be explored how selectivity is achieved when utilizing the same IR isoform. We now demonstrate that insulin-stimulated transcription of c-fos and glucokinase genes is activated simultaneously in the insulin-producing β-cell via IR-B localized in different cellular compartments. Insulin activates the glucokinase gene from plasma membrane-standing IR-B, while c-fos gene activation is dependent on clathrin-mediated IR-B-endocytosis and signaling from early endosomes. Moreover, glucokinase gene up-regulation requires the integrity of the juxtamembrane IR-B NPEY-motif and signaling via PI3K-C2α-like/PDK1/PKB, while c-fos gene activation requires the intact C-terminal YTHM-motif and signaling via PI3K Ia/Shc/MEK1/ERK. By using IR-B as an example it is thus possible to demonstrate how spatial segregation allows simultaneous and selective signaling via the same receptor isoform in the same cell. [ABSTRACT FROM AUTHOR]

In pancreatic β-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11 -) and B-type (Ex11 +), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform-specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12-amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion. [ABSTRACT FROM AUTHOR]

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-1ike proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate β-cell apoptosis was demonstrated by inhibiting caspase-3-1ike proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose- and cytokine-induced apoptosis in the β-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 ± 23 min (n = 9) and 257 ± 59 min (n = 4; mean ± SE) after activation of apoptosis with staurosporine (6 µmol/l), showing that this method worked in insulinproducing cells. [ABSTRACT FROM AUTHOR]

Oxidative stress is induced in pancreatic β-cells under diabetic conditions and causes β-cell dysfunction. Antioxidant treatment of diabetic animals leads to recovery of insulin biosynthesis and increases the expression of its controlling transcription factor, pancreatic duodenal homeobox-1 (PDX-1), in pancreatic β-cells. Here, we show that PDX-1 is translocated from the nuclei to the cytoplasm of pancreatic β-cells in response to oxidative stress. When oxidative stress was charged upon β-cellderived HIT-T15 cells, both endogenous PDX-1 and exogenously introduced green fluorescent proteintagged PDX-1 moved from the nuclei to the cytoplasm. The addition of a dominant negative form of c-Jun NH[sub 2]-terminal kinase (JNK) inhibited oxidative stressinduced PDX-1 translocation, suggesting an essential role of JNK in mediating this phenomenon. Whereas the nuclear localization signal (NLS) in PDX-1 was not affected by oxidative stress, leptomycin B, a specific inhibitor of the classical leucine-rich nuclear export signal (NES), inhibited nucleo-cytoplasmic translocation of PDX-1 induced by oxidative stress. Moreover, we identified an NES at position 82-94 of the mouse PDX-1 protein. Thus, our present results revealed a novel mechanism that negatively regulates PDX-1 function. The identification of the NES, which overrides the function of the NLS in an oxidative stress-responsive, JNK-dependent manner, supports the complicated regulation of PDX-1 function in vivo and may further the understanding of β-cell pathophysiology in diabetes. [ABSTRACT FROM AUTHOR]

Multiple inositol polyphosphate phosphatase (MIPP) is an enzyme that, in vitro, has the interesting property of degrading higher inositol polyphosphates to the Ca[sup 2+] second messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P[sub 3]), independently of inositol lipid breakdown. We hypothesized that a truncated cytosolic form of the largely endoplasmic reticulum-confined MIPP (cyt-MIPP) could represent an important new tool in the investigation of Ins(1,4,5) P[sub 3]dependent intracellular Ca[sup 2+] homeostasis. To optimize our ability to judge the impact of cyt-MIPP on intracellular Ca[sup 2+] concentration ([Ca[sup 2+]][sub i]) we chose a poorly responsive β-cell line (HIT M222.2) with an abnormally low [Ca[sup 2+]][sub i]. Our results show for the first time in an intact mammalian cell that cyt-MIPP expression leads to a significant enhancement of Ins(1,4,5)P[sub 3] concentration. This is achieved without a significant interference from other cyt-MIPP-derived inositol phosphates. Furthermore, the low basal [Ca[sup 2+]][sub i] of these cells was raised to normal levels (35 to 115 nM) when they expressed cyt-MIPP. Noteworthy is that the normal feeble glucose-induced Ca[sup 2+] response of HIT M2.2.2 cells was enhanced dramatically by mechanisms related to this increase in basal [Ca[sup 2+]][sub i]. These data support the use of cyt-MIPP as an important tool in investigating Ins(1,4,5)P[sub 3]dependent Ca[sup 2+] homeostasis and suggest a close link between Ins(1,4,5)P[sub 3] concentration and basal [Ca[sup 2+]][sub i], the latter being an important modulator of Ca[sup 2+] signaling in the pancreatic β-cell. [ABSTRACT FROM AUTHOR]

Examines the role of inositol (Ins) hexakisphosphate in improving dynamin-medicated endocytosis in the pancreatic cell. Molecular mechanism regulating the membrane homeostasis; Metabolic control of the Ins; Formation of the phosphatidylinositol biphosphate following activation of protein kinase C.

Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon stimulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell. [ABSTRACT FROM AUTHOR]

Loss of pancreatic β-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of β-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional β-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of β-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents. [ABSTRACT FROM AUTHOR]

Presents a study on the effect of short-term glucose stimulation on insulin gene expression. Methodology used in the study; Hypothesis about the existence of a short-term transcriptional control; Expression of insulin promoter-driven green fluorescent protein expression in single insulin-producing cells; Suggestions on short-term signaling.

Modification of gene expression in pancreatic islets can be a powerful strategy for understanding the pathology of diabetes and developing novel therapeutic strategies against it. However, amenability of the isolated islets to genetic manipulation has been limited to only a subset of cells at the periphery due to poor penetration of transduction particles. To address this issue, we developed a standardized islet model, produced by optimized dissociation and controlled scaffold-free reaggregation of primary human islet cells. This process allowed for an ideal experimental window for accessing and manipulating the pancreatic endocrine cells at their single cell state, while enabling production of uniform islet microtissues displaying long-term (>28 days) and robust function. We used an adenovirus that allows tracking of transduced total cells, endocrine cells and beta cells by labeling them with three specific fluorescent reporters expressed from a single back-bone. To define the optimal transduction conditions, we introduced the virus at various titers during three different production stages; after islet dispersion, during and post reaggregation. We quantified transduction efficiency and viral penetration via 3D confocal microscopy followed by assessment of insulin secretory function, insulin content, and cell viability of transduced islet microtissues. Highly efficient (>75%) and uniform transduction was achieved when the virus was added after cell dispersion and during reaggregation. Approximately 80-95% of transduced cells were endocrine cells, of which 50-63% corresponded to β-cells. Although highly transduced islet microtissues displayed decreased chronic (35-50%), basal (55-62%) and stimulated (65-75%) insulin secretion, a significant fold induction of insulin secretion and unaltered insulin/ATP content was observed. Here we present efficient genetic manipulation of functional reaggregated islets by viral transduction as a novel tool for diabetes research. Disclosure: B. Yesildag: None. J. Mir-Coll: Employee; Self; InSphero. Employee; Spouse/Partner; Roche Pharma. A. Neelakandhan: None. F. Forschler: Employee; Self; InSphero. A. Biernath: None. I.B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. P. Berggren: None. T. Moede: None. C. Ammala: None. [ABSTRACT FROM AUTHOR]