Inositol 1,4,5-trisphosphate and the endoplasmic reticulum Ca2+ cycle of a rat insulinoma cell line

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.     

Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca²⁺ to glucose and depolarization but to a lesser degree suggesting that altered Ca²⁺ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

556例多发伤患者的急诊救治临床分析

目的探讨多发伤患者的救治策略。方法回顾分析我科2000年1月至2008年5月急诊抢救的556例多发伤患者的临床资料。结果 16例患者经抢救无效死亡,死亡率2.88%;其余患者均经紧急抢救及行必要实验室检查,病情稳定,好转率达97.12%。平均抢救时间为(1.37±1.05)h。结论强化多发伤的急诊科早期救治,树立创伤急救"黄金1 h"观念,是提高多发伤患者生存率及降低死亡率的关键。     

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.     

The a2b adenosine receptor modulates glucose homeostasis and obesity

Background

High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples.

Methodology/Principal Findings

Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression.

Conclusions/Significance

Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.     

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.     

Sporogenesis in Eusporangiate Ferns: II. Polyplastidic Meiosis in Ophioglossum (Ophioglossales)

Ophioglossum petiolatum . Unlike Angiopteris (Marattiales), which is monoplastidic, Ophioglossum undergoes polyplastidic meiosis like members of the fern-seed plant clade. The meiotic spindle is distinctly multipolar in origin and is consolidated into a bipolar spindle that is variously twisted and curved to accommodate the large number of chromosomes. Although a phragmoplast forms after first meiosis, no wall is deposited. Instead, an organelle band consisting of intermingled plastids and mitochondria is formed in the equatorial region between the dyad domains. Following second meiosis, a complex of phragmoplasts forms among sister and non-sister nuclei. Cell plates are deposited first between sister nuclei and then in the region of the organelle band resulting in a tetrad of spores each with a equal allotment of organelles. Received 30 January 2001/ Accepted in revised form 24 April 2001