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Mitochondrial dynamics and bioenergetics are central to glucose-stimulated insulin secretion by pancreatic beta cells. Previously, we demonstrated that a disturbance in glucose-invoked fission impairs insulin secretion by compromising glucose catabolism. Here, we investigated whether the overexpression of mitochondrial fission regulator Drp1 in MIN6 cells can improve or rescue insulin secretion. Although Drp1 overexpression slightly improves the triggering mechanism of insulin secretion of the Drp1-knockdown cells and has no adverse effects on mitochondrial metabolism in wildtype MIN6 cells, the constitutive presence of Drp1 unexpectedly impairs insulin content, which leads to a reduction in the absolute values of secreted insulin. Coherent with previous studies in Drp1-overexpressing muscle cells, we found that the upregulation of ER stress-related genes (BiP, Chop, and Hsp60) possibly impacts insulin production in MIN6 cells. Collectively, we confirm the important role of Drp1 for the energy-coupling of insulin secretion but unravel off-targets effects by Drp1 overexpression on insulin content that warrant caution when manipulating Drp1 in disease therapy. [ABSTRACT FROM AUTHOR]

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]

The inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP 7), is thought to negatively regulate the critical insulin signaling protein Akt/PKB. Knockdown of the IP 7 -generating inositol hexakisphosphate kinase 1 (IP6K1) results in a concomitant increase in signaling through Akt/PKB in most cell types so far examined. Total in vivo knockout of IP6K1 is associated with a phenotype resistant to high-fat diet, due to enhanced Akt/PKB signaling in classic insulin regulated tissues, counteracting insulin resistance. In contrast, we have shown an important positive role for IP6K1 in insulin exocytosis in the pancreatic β-cell. These cells also possess functional insulin receptors and the feedback loop following insulin secretion is a key aspect of their normal function. Thus we examined the effect of silencing IP6K1 on the activation of Akt/PKB in β-cells. Silencing reduced the glucose-stimulated increase in Akt/PKB phosphorylation on T308 and S473. These effects were reproduced with the selective pan-IP6K inhibitor TNP. The likely explanation for IP 7 reduction decreasing rather than increasing Akt/PKB phosphorylation is that IP 7 is responsible for generating the insulin signal, which is the main source of Akt/PKB activation. In agreement, insulin receptor activation was compromised in TNP treated cells. To test whether the mechanism of IP 7 inhibition of Akt/PKB still exists in β-cells, we treated them at basal glucose with an insulin concentration equivalent to that reached during glucose stimulation. TNP potentiated the Akt/PKB phosphorylation of T308 induced by exogenous insulin. Thus, the IP 7 regulation of β-cell Akt/PKB is determined by two opposing forces, direct inhibition of Akt/PKB versus indirect stimulation via secreted insulin. The latter mechanism is dominant, masking the inhibitory effect. Consequently, pharmacological strategies to knock down IP6K activity might not have the same positive output in the β-cell as in other insulin regulated tissues. Unlabelled Image • Silencing IP6K1 inhibits glucose-induced Akt/PKB activation in pancreatic β-cells. • A pan IP6K inhibitor, TNP, also reduces glucose-stimulated Akt/PKB phosphorylation. • IP6K1 inhibition impairs Akt/PKB by curtailing insulin secretion and its autocrine feedback. • There are both positive and negative actions of IP 7 on Akt/PKB. • The dominant positive action of IP 7 on Akt/PKB is due to unique insulin autocrine feedback. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP 7 , is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP 7 regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP 7 . Although the inhibitors have diverse targets, they all perturbed IP 7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP 7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K m for ATP makes IP 7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP 7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP 7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP 7 ’s role in cellular signal transduction is likely to have been underestimated. [ABSTRACT FROM AUTHOR]

A picornavirus (Ljungan virus) has been associated with diabetes in its wild rodent reservoir and in diabetes-prone biobreeding (DP-BB) rats. We attempted to alter the development of diabetes in DP-BB rats using two anti-picornavirus compounds (pleconaril and APO-N039), singly or in combination. Antiviral therapy was initiated 2 weeks before expected onset of diabetes. Pleconaril or APO-N039 alone did not affect the debut of diabetes. However, animals receiving a combination of both compounds were protected for at least the entire period of treatment (4 weeks after expected time of diabetes onset). Immunohistochemistry demonstrated that the presence and distribution of virus antigen in the pancreatic islets coincided with the clinical status of the animal. Data indicate that a treatable picornavirus can be involved in the cellular assault resulting in diabetes and in these cases the disease mechanism appears to involve a virus present in the pancreatic beta cell mass itself. [ABSTRACT FROM AUTHOR]

Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM-derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro . Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation. [ABSTRACT FROM AUTHOR]

The voltage-gated Ca (Ca) channel acts as a key player in β cell physiology and pathophysiology. β cell Ca channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca concentration and thereby Ca-triggered β cell apoptosis. The present study was aimed at revealing the subtypes of Ca1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of β cell Ca1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic β cells from Ca1 channel knockout and corresponding control mice. We now show that functional Ca1.3 channels are expressed in a subgroup of islet β cells from Ca1.2 knockout mice (Ca1.2). T1D serum enhanced whole-cell Ca currents in islet β cells from Ca1.3 knockout mice (Ca1.3). T1D serum increased the open probability and number of functional unitary Ca1 channels in Ca1.2 and Ca1.3 β cells. These data demonstrate that T1D serum hyperactivates both Ca1.2 and Ca1.3 channels by increasing their conductivity and number. These findings suggest Ca1.2 and Ca1.3 channels as potential targets for anti-diabetes therapy. [ABSTRACT FROM AUTHOR]

Defects in pancreatic β-cell function and survival are key components in type 2 diabetes (T2D). An age-dependent deterioration in β-cell function has also been observed, but little is known about the molecular mechanisms behind this phenomenon. Our previous studies indicate that the regulation of cytoplasmic free Ca 2+ concentration ([Ca 2+ ] i ) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca 2+ ] i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca 2+ via voltage-dependent Ca 2+ channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5) P 3 )-mediated liberation of Ca 2+ from intracellular stores. Our previous work has indicated a direct relationship between disruption of Ins(1,4,5) P 3 -mediated Ca 2+ regulation and loss of β-cell function, including disturbed [Ca 2+ ] i dynamics and compromised insulin secretion. To investigate these processes in aging we used three mouse models, a premature aging mitochondrial mutator mouse, a mature aging phenotype (C57BL/6) and an aging-resistant phenotype (129). Our data suggest that age-dependent impairment in mitochondrial function leads to modest changes in [Ca 2+ ] i dynamics in mouse β-cells, particularly in the pattern of [Ca 2+ ] i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5) P 3 -mediated Ca 2+ mobilization from intracellular stores and decreased β-cell Ca 2+ influx over the plasma membrane. Our findings underscore an important concept, namely that even relatively small, time-dependent changes in β-cell signal-transduction result in compromised insulin release and in a diabetic phenotype. [ABSTRACT FROM AUTHOR]

The function and survival of pancreatic β cells critically rely on complex electrical signaling systems composed of a series of ionic events, namely fluxes of K, Na, Ca and Cl across the β cell membranes. These electrical signaling systems not only sense events occurring in the extracellular space and intracellular milieu of pancreatic islet cells, but also control different β cell activities, most notably glucose-stimulated insulin secretion. Three major ion fluxes including K efflux through ATP-sensitive K (K) channels, the voltage-gated Ca (Ca) channel-mediated Ca influx and K efflux through voltage-gated K (K) channels operate in the β cell. These ion fluxes set the resting membrane potential and the shape, rate and pattern of firing of action potentials under different metabolic conditions. The K channel-mediated K efflux determines the resting membrane potential and keeps the excitability of the β cell at low levels. Ca influx through Ca1 channels, a major type of β cell Ca channels, causes the upstroke or depolarization phase of the action potential and regulates a wide range of β cell functions including the most elementary β cell function, insulin secretion. K efflux mediated by K2.1 delayed rectifier K channels, a predominant form of β cell K channels, brings about the downstroke or repolarization phase of the action potential, which acts as a brake for insulin secretion owing to shutting down the Ca channel-mediated Ca entry. These three ion channel-mediated ion fluxes are the most important ionic events in β cell signaling. This review concisely discusses various ionic mechanisms in β cell signaling and highlights K channel-, Ca1 channel- and K2.1 channel-mediated ion fluxes. [ABSTRACT FROM AUTHOR]