Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

Synchronous plasma membrane electrochemical potential oscillations during yeast colony development and aging

Microorganisms that survive in natural environments form organized multicellular communities, biofilms and colonies with specific properties. During stress and nutrient limitation, slow growing and senescent cells in such communities retain vital processes by maintaining plasma membrane integrity and retaining the ability to generate transmembrane electrochemical gradients. We report the use of a Saccharomyces cerevisiae colonial model to show that population growth in a multicellular community depends on nutrient diffusion and that resting cells start to accumulate from the beginning of the second acidic phase of colony development. Despite differentiation of colony members, synchronous transmembrane potential oscillation was detected in the organized colony. The electrochemical membrane potential periodically oscillated at frequencies between those for circadian to infradian rhythms during colony aging and transiently decreased at time points previously linked with rebuilding of yeast metabolism. Despite extensive decreases in the intracellular ATP concentration and in the amount and activity of the plasma membrane proton pump during nutrient limited growth and colony aging, the transmembrane electrochemical potential appeared to be maintained above a level critical for population survival.     

2 型糖尿病患者胰高血糖素及胰岛素水平检测及临床意义

目的:探讨2型糖尿病患者经标准馒头餐试验后胰高血糖素、胰岛素水平变化及其临床意义。方法:选取我院2014年3月至2015年3月收治的80例2型糖尿病患者为试验组,均接受标准馒头餐试验,对其餐前及餐后0.5 h、1 h、2 h胰高血糖素及胰岛素水平进行检测,并与同期80例血糖无异常的健康对照组作比较。结果:健康对照组餐前及餐后胰高血糖素水平无明显变化(P0.05),而试验组餐前胰高血糖素水平明显高于健康对照组(P0.05),且餐后明显升高,在餐后1 h达到峰值,餐后0.5 h、1h、2 h均明显高于健康对照组(P0.01)。健康对照组餐后胰岛素水平明显升高,在餐后1 h达到峰值后开始降低,较餐前略高,但差异平无统计学意义(P0.05);试验组餐前胰岛素水平明显低于健康对照组(P0.05),且餐后水平升高幅度缓慢,餐后0.5 h、1 h、2h均明显低于健康对照组(P0.05);实验组胰岛素抵抗指数(HOMA-IR)明显高于健康对照组,经Pearson相关分析,试验组餐后0.5 h、1 h、2 h胰高血糖素水平与HOMA-IR均呈明显的正相关(r=0.273、0.335、0.368,P0.05)。结论:2型糖尿病患者血糖高表达与胰高血糖素、胰岛素水平密切相关,胰高血糖素可拮抗胰岛素,及时检测二者的水平对临床准确评估病情具有重要的意义。     

微生态制剂改善非酒精性脂肪性肝病患者胰岛素抵抗的临床研究

目的:观察美常安对非酒精性脂肪性肝病(NAFLD)患者临床症状及胰岛素抵抗(IR)的作用。方法:选取2009年4月至2013年3月我院收治的88例NAFLD患者,随机分为治疗组和对照组。对照组给予基础治疗,治疗组在对照组的基础上,加用美常安胶囊进行治疗。比较两组疗效及治疗前后两组血清谷丙转氨酶(ALT)、甘油三酯(TG)及稳态胰岛素抵抗指数(HOMA-IR)水平,并分析血浆D乳酸、TNF-α、内毒素与HOMA-IR的相关性,观察两组不良反应情况。结果:治疗组治疗有效率为52.3%,明显高于对照组的29.5%(P0.05)。治疗后治疗组ALT、TG、HOMA-IR下降幅度,均明显优于对照组(P0.05)。治疗后治疗组患者血浆D乳酸、TNF-α、内毒素水平明显低于对照组(P0.05),且治疗组患者HOMA-IR与患者血浆D乳酸、血清TNF-α、内毒素水平呈正相关(r=0.352,0.44,0.48;均P0.05)。不良反应发生率低,且两组不良反应发生率无显著差异(P0.05)。结论:美常安治疗NAFLD疗效显著,可降低ALT、TG水平,降低肠道通透性,改善肠源性内毒素血症,改善IR,且安全性较高。     

Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes

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Highlights

• •Insulin Affects the Phosphorylation of G2L1, MARK2, CLIP2, EB1, AGAP3, and CKAP5.
• •Insulin Increases CLASP2 +TIP Density and Decreases CLASP2 +TIP Velocity.
• •Insulin Stimulates CLASP2 and G2L1 Trailing Along Microtubules.
• •Insulin Stimulates α-Tubulin Acetylation at Lysine 40 and Microtubule Stabilization.