Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes

Salt-inducible kinases (SIKs), members of the 5′-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.     

CREB研究进展

CREB是cAMP应答元件结合蛋白(cAMPresponseelementbindingprotein)的简称,它于80年代后期被发现。CREB由341个氨基酸残基组成,分子量43000。分子结构分两个区域,N端区域与调节转录的功能有关,C端区域是与启动子结合的部位。CREB是CREBATF家族中的一个成员,它包括8种分子亚型,其中CREBα和CREBΔα最为重要。CREB是一种细胞核内调控因子,它通过自身磷酸化实现调节转录的功能。CREB与学习记忆分子神经机制的关系特别受到注意。CREB能促进果蝇和小鼠等动物长时程记忆的形成。开展对CREB在人类大脑记忆活动中功能的研究,是分子神经生物学家感兴趣的课题。     

CREB信号通路在神经系统中的调控及功能

cAMP应答元件结合蛋白(cAMP response element binding protein,CREB)在神经元生成、突触可塑性及学习记忆等方面都具有重要的调节作用,这使得与CREB信号通路相关的分子成为较受关注的神经系统疾病干预的药物靶点.本文概述了CREB的基本构成、相关信号通路、其目的基因表达调控及其在阿尔茨海默病(Alzheimer’s disease,AD)中的作用.     

CREB与其相关的非编码RNA在肿瘤发生发展中的作用

cAMP反应元件结合蛋白（cAMP responsive element binding protein, CREB）是亮氨酸拉链家族转录因子。新近研究发现,其在肿瘤组织中的表达显著高于癌旁,被认为是体内的原癌基因之一。非编码RNA（non-coding RNA, ncRNA）是生物体内不能翻译成蛋白质的RNA,主要包括微小RNA（microRNA, miRNA）和长链非编码RNA（long non-coding RNA, lncRNA）等,其异常表达与肿瘤的发生发展密切相关,是目前肿瘤研究的热点。研究表明,CREB与ncRNA之间存在互动效应,并且二者之间的相互作用影响肿瘤的发生发展,然而miRNA和lncRNA的作用机制却不相同。肿瘤细胞内高表达的CREB在影响下游靶基因表达时能够正调控miRNA,而对lncRNA则有促进和抑制两方面的作用。反之,肿瘤细胞中一些低表达的miRNA能促进CREB的表达;有趣的是,高表达的lncRNA能够促进CREB的表达和诱导其活性增强。在影响下游靶基因表达时miRNA仅仅发挥抑制作用,而lncRNA则分别具有促进和抑制作用。本文结合我们的系列报道和最新的研究结果,对ncRNA与CREB的互动效应及其与肿瘤的发生发展之间的关系作一综述。     

Involvement of the cAMP response element binding protein, CREB, and cyclin D1 in LPA-induced proliferation of P19 embryonic carcinoma cells

Lysophosphatidic acid (LPA) is a lipid growth factor that induces proliferation of fibroblasts by activating the cAMP response element binding protein (CREB). Here, we further investigated whether LPA induces proliferation of P19 cells, a line of pluripotent embryonic carcinoma cells. 5′-Bromo-2-deoxyuridine incorporation and cell viability assays showed that LPA stimulated proliferation of P19 cells. Immunoblot experiments with P19 cells revealed that the mitogen activated protein kinases, including p-ERK, p38, pAKT, glycogen synthase kinase 3β, and CREB were phosphorylated by treatment with 10 μM LPA. LPA-induced phosphorylation of CREB was efficiently blocked by U0126 and H89, inhibitors of the MAP kinases ERK1/2 and mitogen- and stress-activated protein kinase 1, respectively. Involvement of cyclin D1 in LPA-induced P19 cell proliferation was verified by immunoblot analysis in combination with pharmacological inhibitor treatment. Furthermore, LPA up-regulated CRE-harboring cyclin D1 promoter activity, suggesting that CREB and cyclin D1 play significant roles in LPA-induced proliferation of P19 embryonic carcinoma cells.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation

Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.     

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H⁺-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca²⁺ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.     

Pathogenesis of Selective Insulin Resistance in Isolated Hepatocytes

The development of insulin resistance (IR) in the liver is a key pathophysiologic event in the development of type 2 diabetes. Although insulin loses its ability to suppress glucose production, it largely retains its capacity to drive lipogenesis. This selective IR results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. The delineation of two branched pathways of insulin receptor (InsR) signaling to glucose versus triglyceride production, one through FoxO and the other through SREBP-1c, provides a mechanism to account for this pathophysiological abnormality. We tested the complementary hypothesis that selective IR arises due to different intrinsic sensitivities of glucose production versus de novo lipogenesis to insulin as a result of cell-autonomous down-regulation of InsR number in response to chronic hyperinsulinemia. We demonstrate in mouse primary hepatocytes that chronic hyperinsulinemia abrogates insulin''s inhibition of glucose production, but not its stimulation of de novo lipogenesis. Using a competitive inhibitor of InsR, we show that there is a 4-fold difference between levels of InsR inhibition required to cause resistance of glucose production versus lipogenesis to the actions of insulin. Our data support a parsimonious model in which differential InsR activation underlies the selective IR of glucose production relative to lipogenesis, but both processes require signaling through Akt1/2.     

LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation

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