Inactivation of the dual Bmp/Wnt inhibitor Sostdc1 enhances pancreatic islet function

Current endeavors in the type 2 diabetes (T2D) field include gaining a better understanding of extracellular signaling pathways that regulate pancreatic islet function. Recent data suggest that both Bmp and Wnt pathways are operative in pancreatic islets and play a positive role in insulin secretion and glucose homeostasis. Our laboratory found the dual Bmp and Wnt antagonist Sostdc1 to be upregulated in a mouse model of islet dysmorphogenesis and nonimmune-mediated lean diabetes. Because Bmp signaling has been proposed to enhance β-cell function, we evaluated the role of Sostdc1 in adult islet function using animals in which Sostdc1 was globally deleted. While Sostdc1-null animals exhibited no pancreas development phenotype, a subset of mutants exhibited enhanced insulin secretion and improved glucose homeostasis compared with control animals after 12-wk exposure to high-fat diet. Loss of Sostdc1 in the setting of metabolic stress results in altered expression of Bmp-responsive genes in islets but did not affect expression of Wnt target genes, suggesting that Sostdc1 primarily regulates the Bmp pathway in the murine pancreas. Furthermore, our data indicate that removal of Sostdc1 enhances the downregulation of the closely related Bmp inhibitors Ctgf and Gremlin in islets after 8-wk exposure to high-fat diet. These data imply that Sostdc1 regulates expression of these inhibitors and provide a means by which Sostdc1-null animals show enhanced insulin secretion and glucose homeostasis. Our studies provide insights into Bmp pathway regulation in the endocrine pancreas and reveal new avenues for improving β-cell function under metabolic stress.     

The what, where, when and how of Wnt/β-catenin signaling in pancreas development

The Wnt/β-catenin signaling pathway, conserved across the animal kingdom, is critical for the development of numerous tissues. Several recent studies have focused on the roles that this pathway plays at different stages of pancreatic organogenesis, including specification, proliferation, differentiation and function. Whereas, during early endoderm development, inhibition of the pathway is required for pancreatic specification, subsequent growth and differentiation of the fetal organ depends on the pathway being active. This appears especially true for exocrine acinar cells, the specification and differentiation of which also depend on β-catenin function. Whether the pathway plays an important role in development or function of endocrine islet cells, including insulin-producing β-cells, remains controversial. This question is particularly important in light of recent studies that implicate a downstream component of the pathway, TCF7L2, in human β-cell function. This review will cover recent work on Wnt/β-catenin signaling in pancreas development, emphasizing those points of controversy that most urgently require further investigation.     

The what,where, when and how of Wnt/β-catenin signaling in pancreas development

The Wnt/β-catenin signaling pathway, conserved across the animal kingdom, is critical for the development of numerous tissues. Several recent studies have focused on the roles that this pathway plays at different stages of pancreatic organogenesis, including specification, proliferation, differentiation and function. Whereas, during early endoderm development, inhibition of the pathway is required for pancreatic specification, subsequent growth and differentiation of the fetal organ depends on the pathway being active. This appears especially true for exocrine acinar cells, the specification and differentiation of which also depend on β-catenin function. Whether the pathway plays an important role in development or function of endocrine islet cells, including insulin-producing β-cells, remains controversial. This question is particularly important in light of recent studies that implicate a downstream component of the pathway, TCF7L2, in human β-cell function. This review will cover recent work on Wnt/β-catenin signaling in pancreas development, emphasizing those points of controversy that most urgently require further investigation.     

Glucagon-like peptide-1 activation of TCF7L2-dependent Wnt signaling enhances pancreatic beta cell proliferation

The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3beta but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3beta. Inhibition of Wnt signaling by small interfering RNAs to beta-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes.     

TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta

The proglucagon gene (glu) encodes glucagon, expressed in pancreatic islets, and the insulinotropic hormone GLP-1, expressed in the intestines. These two hormones exert critical and opposite effects on blood glucose homeostasis. An intriguing question that remains to be answered is whether and how glu gene expression is regulated in a cell type-specific manner. We reported previously that the glu gene promoter in gut endocrine cell lines was stimulated by beta-catenin, the major effector of the Wnt signaling pathway, whereas glu mRNA expression and GLP-1 synthesis were activated via inhibition of glycogen synthase kinase-3beta, the major negative modulator of the Wnt pathway (Ni, Z., Anini, Y., Fang, X., Mills, G. B., Brubaker, P. L., & Jin, T. (2003) J. Biol. Chem. 278, 1380-1387). We now show that beta-catenin and the glycogen synthase kinase-3beta inhibitor lithium do not activate glu mRNA or glu promoter expression in pancreatic cell lines. In the intestinal GLUTag cell line, but not in the pancreatic InR1-G9 cell line, the glu promoter G2 enhancer-element was activated by lithium treatment via a TCF-binding motif. TCF-4 is abundantly expressed in the gut but not in pancreatic islets. Furthermore, both TCF-4 and beta-catenin bind to the glu gene promoter, as detected by chromatin immunoprecipitation. Finally, stable introduction of dominant-negative TCF-4 into the GLUTag cell line repressed basal glu mRNA expression and abolished the effect of lithium on glu mRNA expression and GLP-1 synthesis. We have therefore identified a unique mechanism that regulates glu expression in gut endocrine cells only. Tissue-specific expression of TCF factors thus may play a role in the diversity of the Wnt pathway.     

染色质免疫沉淀技术分析HePG2细胞TCF7L2蛋白与IDE基因转录启动子的结合

TCF7L2是一种重要的转录因子,通过Wnt信号途径,调节葡萄糖代谢.胰岛素降解酶(IDE)是细胞水平催化胰岛素降解的最关键的酶,与2型糖尿病(T2DM)高血糖、胰岛素抵抗、高胰岛素血症密切相关.为了检测HePG2细胞内转录因子TCF7L2与IDE基因启动子区的结合情况,采用染色质免疫沉淀技术结合PCR技术检测IDE基因启动子序列.结果表明,在特异性TCF7L2抗体免疫沉淀的DNA片段中扩增出IDE基因启动子序列,因此证实在HePG2细胞内,TCF7L2蛋白可与IDE基因转录启动子的特异区域结合,进而可能参与IDE基因的表达调控.     

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors’ islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

A study of circadian regulation of lipid metabolism in human pancreatic islets reveals that Type 2 Diabetes leads to global and temporal alterations of phospholipid and sphingolipid metabolism in islets, resulting in decreased membrane fluidity and insulin secretion defects.     

Insulin alters the expression of components of the Wnt signaling pathway including TCF-4 in the intestinal cells

Background

Epidemiological and experimental evidence that support the correlation between Type 2 diabetes mellitus (T2D) and increased risks of colorectal cancer formation have led us to hypothesize the existence of molecular crosstalk between insulin and canonical Wnt signaling pathways. Insulin was shown to stimulate Wnt target gene expression, utilizing the effector of the Wnt signaling pathway. Whether insulin affects expression of components of Wnt pathway has not been extensively examined.

Methods

cDNA microarray was utilized to assess the effect of insulin on gene expression profile in the rat intestinal non-cancer IEC-6 cell line, followed by real-time RT-PCR, Western blotting and reporter gene analyses in intestinal cancer and non-cancer cells.

Results

Insulin was shown to alter the expression of a dozen of Wnt pathway related genes including TCF-4 (= TCF7L2) and frizzled- (Fzd-4). The stimulatory effect of insulin on TCF-4 expression was then confirmed by real-time RT-PCR, Western blotting and luciferase reporter analyses, while the activation on Fzd-4 was confirmed by real-time PCR.

General significance

Our observations suggest that insulin may crosstalk with the Wnt signaling pathway in a multi-level fashion, involving insulin regulation of the expression of Wnt target genes, a Wnt receptor, as well as mediators of the Wnt signaling pathway.     

Wnt3‐frizzled 1 chimera as a model to study canonical Wnt signaling

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low‐density lipoprotein receptor‐related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF‐luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF‐luciferase activation. DKK‐1 protein blocks upregulation of the TCF‐luciferase reporter by the Wnt3–Fz1 chimera, suggesting involvement of LRP in Wnt3–Fz1 signaling. Expression of a Wnt3–Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3–Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt–Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases. J. Cell. Biochem. 109: 876–884, 2010. © 2009 Wiley‐Liss, Inc.