ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells.

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

ER stress increases store-operated Ca2+ entry (SOCE) and augments basal insulin secretion in pancreatic beta cells.

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress-inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion-activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

The effect of macromolecular crowding on single-round transcription by Escherichia coli RNA polymerase.

Previous works have reported significant effects of macromolecular crowding on the structure and behavior of biomolecules. The crowded intracellular environment, in contrast to in vitro buffer solutions, likely imparts similar effects on biomolecules. The enzyme serving as the gatekeeper for the genome, RNA polymerase (RNAP), is among the most regulated enzymes. Although it was previously demonstrated that macromolecular crowding affects association of RNAP to DNA, not much is known about how crowding acts on late initiation and promoter clearance steps, which are considered to be the rate-determining steps for many promoters. Here, we demonstrate that macromolecular crowding enhances the rate of late initiation and promoter clearance using in vitro quenching-based single-molecule kinetics assays. Moreover, the enhancement's dependence on crowder size notably deviates from predictions by the scaled-particle theory, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions could be affected by crowded conditions in the cellular milieu.

Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells.

The free calcium (Ca2+) levels in pancreatic beta cell organelles have been the subject of many recent investigations. Under pathophysiological conditions, disturbances in these pools have been linked to altered intracellular communication and cellular dysfunction. To facilitate studies of subcellular Ca2+ signaling in beta cells and, particularly, signaling between the endoplasmic reticulum (ER) and mitochondria, we designed a novel dual Ca2+ sensor which we termed DS-1. DS-1 encodes two stoichiometrically fluorescent proteins within a single plasmid, G-CEPIA-er, targeted to the ER and R-CEPIA3-mt, targeted to mitochondria. Our goal was to simultaneously measure the ER and mitochondrial Ca2+ in cells in real time. The Kds of G-CEPIA-er and R-CEPIA3-mt for Ca2+ are 672 and 3.7 µM, respectively. Confocal imaging of insulin-secreting INS-1 832/13 expressing DS-1 confirmed that the green and red fluorophores correctly colocalized with organelle-specific fluorescent markers as predicted. Further, we tested whether DS-1 exhibited the functional properties expected by challenging an INS-1 cell to glucose concentrations or drugs having well-documented effects on the ER and mitochondrial Ca2+ handling. The data obtained were consistent with those seen using other single organelle targeted probes. These results taken together suggest that DS-1 is a promising new approach for investigating Ca2+ signaling within multiple organelles of the cell.

The Endoplasmic Reticulum and Calcium Homeostasis in Pancreatic Beta Cells.

The endoplasmic reticulum (ER) mediates the first steps of protein assembly within the secretory pathway and is the site where protein folding and quality control are initiated. The storage and release of Ca2+ are critical physiological functions of the ER. Disrupted ER homeostasis activates the unfolded protein response (UPR), a pathway which attempts to restore cellular equilibrium in the face of ER stress. Unremitting ER stress, and insufficient compensation for it results in beta-cell apoptosis, a process that has been linked to both type 1 diabetes (T1D) and type 2 diabetes (T2D). Both types are characterized by progressive beta-cell failure and a loss of beta-cell mass, although the underlying causes are different. The reduction of mass occurs secondary to apoptosis in the case of T2D, while beta cells undergo autoimmune destruction in T1D. In this review, we examine recent findings that link the UPR pathway and ER Ca2+ to beta cell dysfunction. We also discuss how UPR activation in beta cells favors cell survival versus apoptosis and death, and how ER protein chaperones are involved in regulating ER Ca2+ levels. Abbreviations: BiP, Binding immunoglobulin Protein ER; endoplasmic reticulum; ERAD, ER-associated protein degradation; IFN, interferon; IL, interleukin; JNK, c-Jun N-terminal kinase; KHE, proton-K+ exchanger; MODY, maturity-onset diabetes of young; PERK, PRKR-like ER kinase; SERCA, Sarco/Endoplasmic Reticulum Ca2+-ATPases; T1D, type 1 diabetes; T2D, type 2 diabetes; TNF, tumor necrosis factor; UPR, unfolded protein response; WRS, Wolcott-Rallison syndrome.