Search

Endogenous expression of tailored nanoparticles in cells followed by application of low-frequency radio waves or a magnetic field can be used to noninvasively modulate gene expression. This approach successfully induces [...]

In type 1 diabetes (TID), there is a specific destruction of the insulin secreting pancreatic [beta] cell. Although the exact molecular mechanisms underlying [beta] cell destruction are not known, sera from TID patients have been shown to promote [Ca.sup.2+]-induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]]i) and [beta] cell death. The apoCIII-induced increase in [[Ca.sup.2+]]i reflects an activation of the voltage-gated L-type [Ca.sup.2+] channel. Both the effects of T1D sera and apoCIII on the [beta] cell are abolished in the presence of antibody against apoCIII. Increased serum levels of apoCIII can thus account for the increase in [beta] cell [[Ca.sup.2+]]i and thereby [beta] cell apoptosis associated with TID.

Pancreatic [beta]-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 [beta]-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 [beta]-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 [beta]-cell genes, particularly the insulin gene itself. More controversially, this insulin feedback has also been suggested to either terminate or enhance insulin secretion events. inositol polyphosphates; inositol lipids; insulin receptor; insulin secretion; pancreatic [beta]-cell

Interaction of syntaxin 1 with the [[Alpha].sub.1D] subunit of the voltage-gated L type [Ca.sup.2+] channel was investigated in the pancreatic [Beta] cell. Coexpression of the enhanced green fluorescent protein-linked [[Alpha].sub.1D] subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the [[Alpha].sub.1D] subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the [[Alpha].sub.1D] subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type [Ca.sup.2+] channel activity because there was a faster [Ca.sup.2+] current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with [K.sup.+] or exposure to high [Ca.sup.2+]. Exchanging [Ca.sup.2+] for [Ba.sup.2+] abolished the effect of antisyntaxin 1 on both [Ca.sup.2+] channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on [Ca.sup.2+]-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the [[Alpha].sub.1D] subunit of the L type [Ca.sup.2+] channel and the exocytotic machinery in the pancreatic [Beta] cell.

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]

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released [Ca.sup.2+] from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger [Ca.sup.2+] spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release [Ca.sup.2+] from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released [Ca.sup.2+] from ER, giving rise to a slow and small increase in [[[Ca.sup.2+]].sub.i] in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered [Ca.sup.2+] release by 4-CEP, giving rise to [Ca.sup.2+] spikes. In glucose-stimulated beta cells forskolin induced [Ca.sup.2+] spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting [Beta]TC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor ([RY.sub.2]). We conclude that in situ activation of [RY.sub.2] in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Author(s): Ingo B Leibiger [1]; Per-Olof Berggren [1] A growing body of evidence suggests that restricted caloric intake extends the lifespan of a number of organisms including yeast, worms, flies [...]