A common transcriptional mechanism involving R-loop and RNA abasic site regulates an enhancer RNA of APOE.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.

RNA abasic sites in yeast and human cells.

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.

Increased frequency of ß cells with abnormal NKX6.1 expression in type 2 diabetes but not in subjects with higher risk for type 2 diabetes.

BACKGROUND: NKX6.1 is a transcription factor for insulin, as well as a marker for ß cell maturity. Abnormal NKX6.1 expression in ß cells, such as translocation from the nucleus to cytoplasm or lost expression, has been shown as a marker for ß cell dedifferentiation. METHODS: We obtained pancreatic sections from organ donors and immunofluorescence staining with NKX6.1 and insulin was performed to characterize NKX6.1 expression in subjects with or without type 2 diabetes mellitus (T2DM). RESULTS: Our results showed that cells with insulin expression but no nucleic NKX6.1 expression (NKX6.1Nuc-Ins+), and cells with cytoplasmic NKX6.1 expression but no insulin expression (NKX6.1cytIns-) were significantly increased in T2DM subjects and positively correlated with glycated hemoglobin (HbA1c), indicating the elevated ß cell dedifferentiation with NKX6.1 inactivation in T2DM. To investigate whether ß cell dedifferentiation has initiated in subjects with higher risks for T2DM, we next analyzed the association between ß-cell dedifferentiation level in ND subjects with different ages, body mass index, and HbA1c. The results showed the absolute number and percentage of dedifferentiated ß cells with NKX6.1 inactivation did not significantly change in subjects with advanced aging, obesity, or modest hyperglycemia, indicating that the ß cell dedifferentiation might mainly occur after T2DM was diagnosed. CONCLUSION: Our results suggested that NKX6.1 expression in ß cells was changed in type 2 diabetic subjects, evidenced by significantly increased NKX6.1Nuc-Ins+ and NKX6.1cytIns- cells. This abnormality did not occur more frequently in subjects with a higher risk for T2DM, suggesting that ß cell dedifferentiation might be secondary to the pathological changes in T2DM.

Hypoxia-inducible factor-1α mediates the expression of mature ß cell-disallowed genes in hypoxia-induced ß cell dedifferentiation.

Hypoxia affects the function of pancreatic ß cells, and the molecular mechanism underlying hypoxia-related ß cell dysfunction in human type 2 diabetes mellitus (T2DM) remains to be elucidated. In this study, by comparing the gene expression profiles of islets from nondiabetic and T2D subjects using gene chip array, we aimed to elucidate that hypoxia signaling pathways are activated in human T2DM islets. CoCl2 treatment, which was employed to mimic hypoxic stimulation in human islets, decreased insulin secretion, insulin content, and the functional gene expression of human islets. In parallel, the expression of mature ß cell-disallowed genes was upregulated by CoCl2, including progenitor cell marker NGN3, ß cell differentiation marker ALDH1A3, and genes that are typically inhibited in mature ß cells, namely, GLUT1 and LDHA, indicating that CoCl2-mimicked hypoxia induced ß cell dedifferentiation of human islets. This finding in human islets was confirmed in mouse ß cell line NIT-1. By using Dimethyloxalylglycine (DMOG) to activate hypoxia-inducible factor-1α (HIF-1α) or siRNAs to knockdown HIF-1α, we found that HIF-1α was a key regulator of hypoxia-induced dedifferentiation of ß cells by upregulating mature ß cell-disallowed genes. Our findings suggested that HIF-1α activation might be an important contributor to ß cell dedifferentiation in human T2DM islets, and HIF-1α-targeted therapies may have the potential to reverse ß cell dedifferentiation of human T2DM islets.

Opposing effects of IL-1ß/COX-2/PGE2 pathway loop on islets in type 2 diabetes mellitus.

The cyclooxygenase2 (COX-2) enzyme catalyzes the first step of prostanoid biosynthesis, and is known for its crucial role in the pathogenesis of several inflammatory diseases including type 2 diabetes mellitus (T2DM). Although a variety of studies revealed that COX-2 played a role in the IL-1ß induced ß cell dysfunction, the molecular mechanism remains unclear. Here, using a cDNA microarray and in silico analysis, we demonstrated that inflammatory responses were upregulated in human T2DM islets compared with non-diabetic (ND) islets. COX-2 expression was significantly enhanced in human T2DM islets, correlated with the high inflammation level. PGE2, the catalytic product of COX-2, downregulated the functional gene expression of PDX1, NKX6.1, and MAFA and blunted the glucose induced insulin secretion of human islets. Conversely, inhibition of COX-2 activity by a pharmaceutical inhibitor prevented the ß-cell dysfunction induced by IL-1ß. COX-2 inhibitor also abrogated the IL-1ß autostimulation in ß cells, which further resulted in reduced COX-2 expression in ß cells. Together, our results revealed that COX-2/PGE2 signaling was involved in the regulation of IL-1ß autostimulation, thus forming an IL-1ß/COX-2/PGE2 pathway loop, which may result in the high inflammation level in human T2DM islets and the inflammatory impairment of ß cells. Breaking this IL-1ß/COX-2/PGE2 pathway loop provides a potential therapeutic strategy to improve ß cell function in the treatment of T2DM patients.

Ethylenecarbodiimide-fixed splenocytes carrying whole islet antigens decrease the incidence of diabetes in NOD mice via down-regulation of effector memory T cells and autoantibodies.

Type 1 diabetes mellitus (T1DM) is a syndrome of loss of glucose homeostasis caused by the loss of ß cell chronic autoimmunity against islet cells. Islet-specific epitopes coupled antigen presenting cells by Ethylenecarbodiimide (ECDI) is a promising strategy to induce antigen-specific tolerance. However, single epitope induced tolerance is insufficient to prevent the onset of T1DM. The aim of this study is to evaluate the efficacy of whole islet antigens in preventing the onset and progression of T1DM and identify the underlying immune mechanism in NOD mice. In this study, the whole islet antigens, derived from islet lysate isolated from BALB/c mice, were coupled to splenocytes of BALB/c mice by ECDI fixation (SP-Islet lysate), and then intravenously administrated to NOD mice. The results showed that, compared with control group, SP-Islet lysate group significantly decreased T1DM incidence and improved the survival of NOD mice. SP-Islet lysate treated mice had reduced insulitis score and autoantibody levels, and improved glucose tolerance and insulin/glucagon production. Furthermore, the effector memory T cells (TEMs) were downregulated and regulatory T cells (Tregs) were upregulated by the SP-Islet lysate treatment, with reduced populations of Th1&Th17 cells. In conclusion, ECDI-fixed splenocytes carrying whole islet antigens effectively prevented the onset of T1DM in NOD mice, via suppressing the production of autoantibodies and inducing anergy of autoreactive T cells.

HIF-1α/FOXO1 axis regulated autophagy is protective for ß cell survival under hypoxia in human islets.

ß cells suffer from hypoxia due to the rapid metabolic rate to supply insulin production. Mechanistic study of ß cell survival under hypoxia may shed light on the ß cell mass loss in type 2 diabetes mellitus (T2DM). Here, we found that the expressions of LC3 and p62/SQSTM1, two key autophagy regulators, were significantly higher in ß cells than that in non-ß endocrine cells in both non-diabetic and T2DM human pancreases, and the autophagy process was accelerated upon Cobalt Chloride (CoCl2) treatment in ex vivo cultured primary human islets. Meanwhile, CoCl2 induced the upregulation of FOXO1 in human islets, where HIF-1α played a key role. CoCl2 treatment caused the increase of ß cell apoptosis, yet inhibiting autophagy by Chloroquine or by FOXO1 knockdown further aggravated apoptosis, suggesting that FOXO1-regulated autophagy is protective for ß cell survival under hypoxia. Immunofluorescence staining showed that LC3 and p62/SQSTM1 expressions were significantly decreased in T2DM patients and negatively correlated with HbA1c, indicating that the autophagy capacity of ß cells is impaired along with the progression of the disease. Our study revealed that HIF-1α/FOXO1 regulated autophagy benefits ß cell survival under hypoxia and autophagy dysregulation may account for ß cell mass loss in T2DM. BRIEF SUMMARY: Our study revealed that HIF-1α/FOXO1 regulated autophagy benefits ß cell survival under hypoxia and autophagy dysregulation may account for ß cell mass loss in T2DM.

Dynamic Change of ß to α Ratio in Islets of Chinese People With Prediabetes and Type 2 Diabetes Mellitus.

OBJECTIVES: The present study aimed to investigate the dynamic change of α cells and ß cells, and their ratios in prediabetes and type 2 diabetes in the Chinese population. METHODS: Pancreata from 27 nondiabetic (ND), 8 prediabetic (PreD), and 19 type 2 diabetic (T2D) organ donors were subjected to immunofluorescence staining with insulin and glucagon. RESULTS: The ß to α ratio in islets (ß/α) in PreD was significantly higher than that in ND, resulting from an increase of ß cells and a decrease of α cells per islet, but that in T2D was significantly lower than that in ND, resulting from a decrease of ß cells and an increase of α cells per islet. The ß-cell percentage and ß/α ratio positively correlated and α-cell percentage negatively correlated with HbA1c (glycated hemoglobin) in ND and PreD, but these correlations disappeared when T2D subjects were included. CONCLUSIONS: The islet ß to α ratio increased in PreD individuals because of a relative α-cell loss and ß-cell compensation and decreased after T2D onset because of both ß-cell loss and α-cell reexpansion.

Adipose Tissue-Derived Stem Cells from Type 2 Diabetics Reveal Conservative Alterations in Multidimensional Characteristics.

BACKGROUND AND OBJECTIVES: Adipose tissue-derived mesenchymal stem cells (ASCs) are recognized as an advantaged source for the prevention and treatment of diverse diseases including type 2 diabetes mellitus (T2DM). However, alterations in characteristics of ASCs from the aforementioned T2DM patients are still obscure, which also hinder the rigorous and systematic illumination of progression and pathogenesis. METHODS AND RESULTS: In this study, we originally isolated peripancreatic adipose tissue-derived mesenchymal stem cells from both human type 2 diabetic and non-diabetic donors (T2DM-ASCs, ND-ASCs) with the parental consent, respectively. We noticed that T2DM-ASCs exhibited indistinguishable immunophenotype, cell vitality, chondrogenic differentiation and stemness as ND-ASCs. Simultaneously, there's merely alterations in migration and immunoregulatory capacities in T2DM-ASCs. However, differing from ND-ASCs, T2DM-ASCs exhibited deficiency in adipogenic and osteogenic differentiation, and in particular, the delayed cell cycle and different cytokine expression spectrum. CONCLUSIONS: The conservative alterations of T2DM-ASCs in multifaceted characteristics indicated the possibility of autologous application of ASCs for cell-based T2DM treatment in the future.

Mesenchymal stem cells ameliorate ß cell dysfunction of human type 2 diabetic islets by reversing ß cell dedifferentiation.

BACKGROUND: A physiological hallmark of patients with type 2 diabetes mellitus (T2DM) is ß cell dysfunction. Despite adequate treatment, it is an irreversible process that follows disease progression. Therefore, the development of novel therapies that restore ß cell function is of utmost importance. METHODS: This study aims to unveil the mechanistic action of mesenchymal stem cells (MSCs) by investigating its impact on isolated human T2DM islets ex vivo and in vivo. FINDINGS: We propose that MSCs can attenuate ß cell dysfunction by reversing ß cell dedifferentiation in an IL-1Ra-mediated manner. In response to the elevated expression of proinflammatory cytokines in human T2DM islet cells, we observed that MSCs was activated to secret IL-1R antagonist (IL-1Ra) which acted on the inflammed islets and reversed ß cell dedifferentiation, suggesting a crosstalk between MSCs and human T2DM islets. The co-transplantation of MSCs with human T2DM islets in diabetic SCID mice and intravenous infusion of MSCs in db/db mice revealed the reversal of ß cell dedifferentiation and improved glycaemic control in the latter. INTERPRETATION: This evidence highlights the potential of MSCs in future cell-based therapies regarding the amelioration of ß cell dysfunction.

The dual DPP4 inhibitor and GPR119 agonist HBK001 regulates glycemic control and beta cell function ex and in vivo.

Glucagon like peptide-1 (GLP-1) plays a vital role in glucose homeostasis and sustaining ß-cell function. Currently there are two major methods to enhance endogenous GLP-1 activity; inhibiting dipeptidyl peptidase-4 (DPP4) or activating G protein-coupled receptor 119 (GPR119). Here we describe and validate a novel dual-target compound, HBK001, which can both inhibit DPP4 and activate GPR119 ex and in vivo. We show that HBK001 can promote glucose-stimulated insulin secretion in mouse and human primary islets. A single administration of HBK001 in ICR mice can increase plasma incretins levels much more efficiently than linagliptin, a classic DPP4 inhibitor. Long-term treatment of HBK001 in KKAy mice can ameliorate hyperglycemia as well as improve glucose tolerance, while linagliptin fails to achieve such glucose-lowing effects despite inhibiting 95% of serum DPP4 activity. Moreover, HBK001 can increase first-phase insulin secretion in KKAy mice, suggesting a direct effect on islet ß-cells via GPR119 activation. Furthermore, HBK001 can improve islet morphology, increase ß-cell proliferation and up-regulate genes involved in improved ß-cell function. Thus, we have identified, designed and synthesized a novel dual-target compound, HBK001, which represents a promising therapeutic candidate for type 2 diabetes, especially for patients who are insensitive to current DPP4 inhibitors.

Angiopoietin-3 inhibits pulmonary metastasis by inhibiting tumor angiogenesis.

Angiopoietins (Ang-1, Ang-2, and Ang-3) are the ligands of Tie-2 receptor tyrosine kinase. The essential roles of Ang-1 and Tie-2 in embryonic angiogenesis have been established, and studies have demonstrated the involvement of Ang-1 and Ang-2 in tumor angiogenesis. However, the role of Ang-3 in tumor angiogenesis and metastasis and the mechanism underlying its function are totally unknown. We have shown recently that Ang-3 is tethered on cell surface via heparan sulfate proteoglycans. In our current study, we have demonstrated that overexpression of Ang-3 inhibits pulmonary metastasis of Lewis lung carcinoma and TA3 mammary carcinoma (TA3) cells by inhibiting tumor angiogenesis and promoting apoptosis of the tumor cells. In addition, we have demonstrated that the binding of Ang-3 to the cell surface is required for the effective inhibition of Ang-3 on tumor metastasis and that Ang-3 inhibits endothelial cell proliferation and survival and blocks Ang-1- and vascular endothelial growth factor-induced activation of extracellular signal-regulated kinase 1/2 and Akt kinases, which likely underlie the Ang-3-mediated inhibition on tumor angiogenesis and metastasis.

Angiopoietin-3 is tethered on the cell surface via heparan sulfate proteoglycans.

Angiopoietins are a family of factors that play important roles in angiogenesis, and their receptor, Tie-2 receptor tyrosine kinase, is expressed primarily by endothelial cells. Three angiopoietins have been identified so far, angiopoietin-1 (Ang-1), angiopietin-2 (Ang-2), and angiopoietin-3 (Ang-3). It has been established that Ang-1 and Tie-2 play essential roles in embryonic angiogenesis. We have demonstrated recently that, unlike Ang-2, Ang-1 binds to the extracellular matrix, which regulates the availability and activity of Ang-1 (Xu, Y., and Yu, Q. (2001) J. Biol. Chem. 276, 34990-34998). However, the role and biochemical characteristics of Ang-3 are unknown. In our current study, we demonstrated that, unlike Ang-1 and Ang-2, Ang-3 is tethered on cell surface via heparan sulfate proteoglycans (HSPGs), especially perlecan. The cell surface-bound Ang-3 is capable of binding to its receptor, Tie-2; suggesting HSPGs concentrate Ang-3 on the cell surface and present Ang-3 to its receptor to elicit specific local reaction. Mutagenesis experiment revealed that the coiled-coil domain of Ang-3 is responsible for its binding to the cell surface. In addition, we demonstrated that the cell surface-bound Ang-3 but not soluble Ang-3 induces retraction and loss of integrity of endothelial monolayer, indicating the binding of Ang-3 to the cell surface via HSPGs is required for this bioactivity of Ang-3.