Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F₁ mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F₁ mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F₁ fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F₁ mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.     

Chronic alcohol consumption potentiates the development of diabetes through pancreatic β-cell dysfunction

Chronic ethanol consumption is well established as a major risk factor for type-2 diabetes(T2D), which is evidenced by impaired glucose metabolism and insulin resistance. However, the relationships between alcoholconsumption and the development of T2 D remain controversial. In particular, the direct effects of ethanol consumption on proliferation of pancreatic β-cell and the exact mechanisms associated with ethanolmediated β-cell dysfunction and apoptosis remain elusive. Although alcoholism and alcohol consumption are prevalent and represent crucial public health problems worldwide, many people believe that low-tomoderate ethanol consumption may protect against T2 D and cardiovascular diseases. However, the J- or U-shaped curves obtained from cross-sectional and large prospective studies have not fully explained the relationship between alcohol consumption and T2 D. This review provides evidence for the harmful effects of chronic ethanol consumption on the progressive development of T2 D, particularly with respect to pancreatic β-cell mass and function in association with insulin synthesis and secretion. This review also discusses a conceptual framework for how ethanolproduced peroxynitrite contributes to pancreatic β-cell dysfunction and metabolic syndrome.     

Epigenetics: The missing link to understanding β-cell dysfunction in the pathogenesis of type 2 diabetes

Type 2 diabetes (T2D) is a growing health problem worldwide. While peripheral insulin resistance is common during obesity and aging in both animals and people, progression to T2D is largely due to insulin secretory dysfunction and significant apoptosis of functional β-cells, leading to an inability to compensate for insulin resistance. It is recognized that environmental factors and nutrition play an important role in the pathogenesis of diabetes. However, our knowledge surrounding molecular mechanisms by which these factors trigger β-cell dysfunction and diabetes is still limited. Recent discoveries raise the possibility that epigenetic changes in response to environmental stimuli may play an important role in the development of diabetes. In this paper, we review emerging knowledge regarding epigenetic mechanisms that may be involved in β-cell dysfunction and pathogenesis of diabetes, including the role of nutrition, oxidative stress and inflammation. We will mainly focus on the role of DNA methylation and histone modifications but will also briefly review data on miRNA effects on the pancreatic islets. Further studies aimed at better understanding how epigenetic regulation of gene expression controls β-cell function may reveal potential therapeutic targets for prevention and treatment of diabetes.     

Pancreatic cancer-derived exosomal microRNA-19a induces β-cell dysfunction by targeting ADCY1 and EPAC2

New-onset diabetes mellitus has a rough correlation with pancreatic cancer (PaC), but the underlying mechanism remains unclear. This study aimed to explore the exosomal microRNAs and their potential role in PaC-induced β-cell dysfunction. The pancreatic β cells were treated with isolated exosomes from PaC cell lines, SW1990 and BxPC-3, before measuring the glucose-stimulated insulin secretion (GSIS), validating that SW1990 and BxPC-3 might disrupt GSIS of both β cell line MIN6 and primary mouse pancreatic islets. The difference in expression profiles between exosomes and exosome-free medium of PaC cell lines was further defined, revealing that miR-19a secreted by PaC cells might be an important signaling molecule in this process. Furthermore, adenylyl cyclase 1 (Adcy1) and exchange protein directly activated by cAMP 2 (Epac2) were verified as the direct targets of exogenous miR-19a, which was involved in insulin secretion. These results indicated that exosomes might be an important mediator in the pathogenesis of PaC-DM, and miR-19a might be the effector molecule. The findings shed light on the pathogenesis of PaC-DM.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.     

CD147 confers temozolomide resistance of glioma cells via the regulation of β-TrCP/Nrf2 pathway

Background: Drug resistance is one of the biggest challenges in cancer therapy. temozolomide (TMZ) represents the most important chemotherapeutic option for glioma treatment. However, the therapeutic efficacy of TMZ remains very limited due to its frequent resistance in glioma, and the underlying mechanisms were not fully addressed. Herein, we demonstrate that the elevated expression of CD147 contributes to TMZ resistance in glioma cells, potentially through the post-translational regulation of Nrf2 expression.Methods: Cell-based assays of CD147 triggered drug resistance were performed through Edu-incorporation assay, CCK8 assay, TUNEL staining assay and flow cytometric assay. Luciferase reporter assay, protein stability related assays, co-immunoprecipitation assay were used to determine CD147 induction of Nrf2 expression through β-TrCP dependent ubiquitin system. Finally, the effect of the CD147/Nrf2 signaling on glioma progression and TMZ resistance were evaluated by functional experiments and clinical samples.Results: Based on the analysis of clinical glioma tissues, CD147 is highly expressed in glioma tissues and positively associated with tumor malignancy. Suppression of CD147 expression increased the inhibitory effect of TMZ on cell survival in both U251 and T98G cells, whereas the gain of CD147 function blocked TMZ-induced ROS production and cell death. Mechanistic study indicates that CD147 inhibited GSK3β/β-TrCP-dependent Nrf2 degradation by promoting Akt activation, and subsequently increased Nrf2-mediated anti-oxidant gene expressions. Supporting the biological significance, the reciprocal relationship between CD147 and Nrf2 was observed in glioma tissues, and associated with patient outcome.Conclusions: Our data provide the first evidence that glioma resistance to TMZ is potentially due to the activation of CD147/Nrf2 axis. CD147 promotes Nrf2 stability through the suppression of GSK3β/β-TrCP dependent Nrf2 protein degradation, which results in the ablation of TMZ induced ROS production. As such, we point out that targeting CD147/Nrf2 axis may provide a new strategy for the treatment of TMZ resistant gliomas.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule,H2-Q10

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD⁺, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD⁺. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.     

Differential regulation of embryonic and adult β cell replication

Diabetes results from an inadequate functional β cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with β cell failure (Type 2 diabetes). Strategies to enhance β cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic β cell mass expansion differ from those regulating replication ofβ cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal β cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult β cell replication. We have already shown that CTGF is required for embryonicβ cell proliferation and is sufficient to induce replication of embryonic β cells. Here we examine whether adult β cell replication and expansion of β cell mass can be enhanced by increased CTGF expression in mature β cells.     

PINK1 deficiency in β-cells increases basal insulin secretion and improves glucose tolerance in mice

The Parkinson''s disease (PD) gene, PARK6, encodes the PTEN-induced putative kinase 1 (PINK1) mitochondrial kinase, which provides protection against oxidative stress-induced apoptosis. Given the link between glucose metabolism, mitochondrial function and insulin secretion in β-cells, and the reported association of PD with type 2 diabetes, we investigated the response of PINK1-deficient β-cells to glucose stimuli to determine whether loss of PINK1 affected their function. We find that loss of PINK1 significantly impairs the ability of mouse pancreatic β-cells (MIN6 cells) and primary intact islets to take up glucose. This was accompanied by higher basal levels of intracellular calcium leading to increased basal levels of insulin secretion under low glucose conditions. Finally, we investigated the effect of PINK1 deficiency in vivo and find that PINK1 knockout mice have improved glucose tolerance. For the first time, these combined results demonstrate that loss of PINK1 function appears to disrupt glucose-sensing leading to enhanced insulin release, which is uncoupled from glucose uptake, and suggest a key role for PINK1 in β-cell function.     

β-Cells retain a pool of insulin-containing secretory vesicles regulated by adherens junctions and the cadherin-binding protein p120 catenin

The β-cells of the islets of Langerhans are the sole producers of insulin in the human body. In response to rising glucose levels, insulin-containing vesicles inside β-cells fuse with the plasma membrane and release their cargo. However, the mechanisms regulating this process are only partly understood. Previous evidence indicated reductions in α-catenin elevate insulin release, while reductions in β-catenin decrease insulin release. α- and β-catenin contribute to cellular regulation in a range of ways but one is as members of the adherens junction complex. Therefore, we investigated the effects of adherens junctions on insulin release. We show in INS-1E β-cells knockdown of either E- or N-cadherin had only small effects on insulin secretion, but simultaneous knockdown of both cadherins resulted in a significant increase in basal insulin release to the same level as glucose-stimulated release. This double knockdown also significantly attenuated levels of p120 catenin, a cadherin-binding partner involved in regulating cadherin turnover. Conversely, reducing p120 catenin levels with siRNA destabilized both E- and N-cadherin, and this was also associated with an increase in levels of insulin secreted from INS-1E cells. Furthermore, there were also changes in these cells consistent with higher insulin release, namely reductions in levels of F-actin and increased intracellular free Ca²⁺ levels in response to KCl-induced membrane depolarization. Taken together, these data provide evidence that adherens junctions play important roles in retaining a pool of insulin secretory vesicles within the cell and establish a role for p120 catenin in regulating this process.     

Decreased Expression of Cilia Genes in Pancreatic Islets as a Risk Factor for Type 2 Diabetes in Mice and Humans

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Ellagic acid regulates hyperglycemic state through modulation of pancreatic IL-6 and TNF- α immunoexpression

BackgroundType 2 diabetes (T2DM) is a chronic metabolic disorder. Although therapeutic pharmaceutical agents continue to advance, herbal medicines are potential complementary treatments for the promotion of glucose homeostasis, with minimal adverse effects. Conventionally, ellagic acid (EA) has been utilized for the therapy of a range of pathologies owing to its anti-inflammatory and anti-diabetic actions.ObjectiveThe aim of this study is to determine the activity of EA on serum α-amylase and lipase titers, and on pancreatic tumor necrosis factor-α (TNF-α), proliferating cell nuclear antigen (PCNA) and interleukin-6 (IL-6) concentrations using the streptozocin-induced T2DM rodent model.MethodsEA extract synthesized from fresh strawberry fruit was employed for therapy. 50 adults male Wistar rats were randomized into either control, EA, diabetic, co-treated or post- treated cohorts.ResultsEA diminished fasting blood glucose levels, altered lipase, amylase, IL-6, PCNA and TNF- α expression and enhanced islet cell renewal, insulin, and immunoreactivities.ConclusionInflammatory indicators are elevated in the presence of T2DM. Extract of EA has overall tissue reparative and safeguarding properties, as indicated by the augmented β- cell population and enhanced glucose homeostasis. Thus, EA may be an innovative treatment approach for the maintenance of normoglycemia in individuals with T2DM.     

Hepatic and pancreatic effects of polyenoylphosphatidylcholine in rats with alloxan-induced diabetes

Polyenoylphosphatidylcholine (PPC: 100 or 300 mg kg^?1 b.w., by gastric intubation for 30 days) produced a clearcut protection of the liver of rats treated with alloxan (150 mg kg^?1 b.w., i.p.). The liver of rats treated with alloxan was characterized by hydropic dystrophy and lymphocytic infiltrations. Treatment with alloxan increased serum γ-GT and ALAT activities. The liver structure of rats treated with PPC did not differ from the liver of control animals. PPC normalized the biochemical abnormalities caused by the diabetes. The number of pancreatic islets and β/α; cell ratio decreased in the diabetic rats. A number of β-cells in this group did not contain granules. PPC prevented the decrease in the number of islets and the β/α; cell ratio in the pancreas of the diabetic rats. The intensity of staining of β-cell granules in the pancreas of PPC-treated rats had a position intermediate between the control and diabetic groups. Alloxan increased the blood glucose content where treatment with PPC decreased this. The results suggest that PPC acts as a cytoprotector in the liver and pancreas of rats with experimental diabetes induced by alloxan.     

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3^H]-thymidine incorporation, protein abundance, and mRNA expression.     

Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects β-cells from virally mediated lysis

Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated β-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in β-cells functions to limit viral replication and subsequent β-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide–dependent manner.     

Characterization and structural analyses of a novel glycosyltransferase acting on the β-1,2-glucosidic linkages

The IALB_1185 protein, which is encoded in the gene cluster for endo-β-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are β-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward β-1,2-glucooligosaccharides, and that the kinetic parameters for β-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the β-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites −1 and +1, whereas in the E343Q mutant–sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites −1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of β-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined β-1,2-glucooligosaccharide:d-glucoside β-d-glucosyltransferase as a systematic name and β-1,2-glucosyltransferase as an accepted name.     

The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.