The main beta cell mission consists in its ability to secrete insulin for maintenance of organismal glucose homeostasis. This hallmark requires expression of a set of genes (allowed genes) and the concomitant repression of others (disallowed genes)[1, 2]. Indeed, betacells have a dedicated metabolism comprised of a tightly regulated chain of elements from glucose sensors to a specialized exocytosis machinery. Glucose metabolism in betacells linked to insulin secretion has several exclusive features. First, glucose transport is not limiting for its metabolism, and it results in a rapid equilibrium between intracellular and extracellular glucose at all glycemic levels. The second particular trait is that glycolysis is initiated by glucokinase having a high Km for glucose associated to an elevated Vmax, and being devoid of feedback control. As betacells express low levels of plasma membrane monocarboxylate transporters, little or no monocarboxylates, including lactate, enter the cells. Further, the low lactate dehydrogenase (LDH) activity of the betacells accounts for the fact that a major fraction of the glucose entering the betacells is oxidized by the mitochondria[4, 5]. These distinctive beta cell properties must be maintained to achieve efficient oxidative phosphorylation, lipid metabolism and signaling, thus making optimal glucose sensing possible. It is thought that beta cell dysfunction in type 2 diabetes could result from a reduced expression of key genes, triggering the loss of beta cell identity and hence promoting dedifferentiation[4, 6, 7]. Importantly, grounds have been gleaned in favor of beta cell dedifferentiation in human type 2 diabetes. Indeed, dedifferentiated cells account for almost 40% of the betacells in type 2 diabetes patients compared to 8% in controls. Thus, it appears that the maintenance of beta cell identity throughout life is crucial for glucose homeostasis and organismal physiology.
The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic betacells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E betacells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3- dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized betacells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes.
Quantification of mature miRNA levels
Expression of mature miRNAs was quantified with the miRCURY LNA TM Universal RT microRNA PCR kit (Exiqon). Briefly, 200 ng of exosomal or cellular RNAs were used for reverse transcription (RT) in a final volume of 20 μl. Each RT reaction was diluted 10 times in RNase- free water and 8 μl of the cDNA template was combined to the ExiLENT SYBRgreen master mix. qPCR reactions were carried out in triplicates using the CFX Real-Time PCR Detection System (BioRad). miRNA expression in exosomes was normalized to the amount of RNA and expressed as Relative Fluorescence Units (RFU) or com- pared to control condition whenever possible. The UniSp6 RNA spike-in control (Exiqon) was used as additional in- ternal reference. For this purpose, 0.15 fmol of UniSp6 (corresponding to about 10 8 copies) was added to the RT reaction and was measured by qPCR using specific primers. miRNA expression in beta-cells was normal- ized to U6 content.
Plant foods contain flavonoids, polyphenolic compounds with protective effects in several chronic diseases. One of the most widely distributed flavonoids in the human diet, quer- cetin (3,3′,4′,5,7-pentahydroxyflavone) (Hertog et al., 1993), exhibits numerous beneficial effects in diabetes. The long- term consumption of quercetin appears to control blood glucose levels in vivo in animals with streptozotocin-induced diabetes (Coskun et al., 2005; Ramachandra et al., 2005; Adewole et al., 2006; Kim et al., 2011). Quercetin has also been shown to normalize glucose tolerance, reduce oxidative stress markers, regenerate pancreatic islets and preserve the integrity of pancreatic betacells (Vessal et al., 2003; Mahesh and Menon, 2004; Coskun et al., 2005; Kim et al., 2011). Recently, quercetin was also shown to prevent the inhibition of insulin secretion induced by IL-1b in betacells of the rat insulinoma cell line RINm5F (Cho et al., 2012).
2.4. Western blot assay
Total or immunoprecipitated protein extracts were obtained from cells after exposure to treatment and/or transfection with speciﬁc siRNA [5,7] . Proteins were fractioned in a 10% SDS–PAGE and transferred to a nitrocellulose membrane. Immunoblot analy- sis was performed with anti-IKK a , IKK-b, IKK c , P-I j B a (Santa Cruz, Biotechnology, Santa Cruz – CA, USA), TRAF6, TAK1, total JNK, P-JNK, anti-polyubiquitin and anti- a -tubulin (Cell Signaling, Beverly, MA) antibodies, followed by incubation with a secondary horseradish peroxidase-labeled anti-IgG (Santa Cruz Biotechnology or Cell Signaling). The quantiﬁcation of the speciﬁc bands was done
3.9 Effect of electric field on the electrophysiological behavior of murine islet cellsCells from 100 mouse islets were seeded into 1-well MEAs. The bottom half (last 4 rows) of the MEA was designated as the control group and the top half (first 4 rows) was desig- nated as the treated group. A 1 V electric potential was applied to the first 4 rows of electrodes for 3 min, generating an electric field in only the top half of MEAs. The MEAs were then cultured for 3 days as usual to ensure good cell adher- ence and gap-junction formation between β-cells. 14 The elec- trical activity of cells was evaluated by recording their response to glucose and various drugs and subsequently ana- lyzed offline. Normal glucose-induced electrical activity of β-cells is characterized by a low-frequency signal, termed a slow potential, that arises from β-cells linked to one another by gap junctions, just as islets are in situ. 14 We investigated the response of electrophoresed cells to increasing concentra- tions of glucose. We observed that slow potentials were very rare or just absent (0.018 ± .005 Hz) during low glucose (3 mM) incubation and arose to 0.360 ± .015 Hz during high glucose (15 mM) incubation (Fig. 5). This was not signifi- cantly different to control cells (0.016 ± .005 Hz and 0.369 ± .015 Hz, respectively) and in accordance with what is already published. 14,39,41 Therefore, the cells fully retain their dis- criminatory response to glucose. These glucose-induced sig- nals were suppressed by the addition of adrenaline (5 μM, Fig. 5, G15 + Adr) or nifedipine (25 μM, not shown), two inhibitors of β-cell electrical activity. 14 This further confirms that electrophoresis does not affect the ability of the biosen- sor to detect glucose variations, hyperglycemic hormones or molecules targeting Ca 2+ ion channels. The addition of the
Of special interest to type 1 and 2 diabetes pathogenesis is the constitutive profile of the β cell proteasomes and their regulation. Type 1 diabetes (T1D) is an autoimmune disease, in which tolerance to β cells is broken, with proinsulin serving as a major autoantigen. T1D is his- tologically characterized by pancreatic islet inflammation with increased levels of cytokines i.e. IL-1β, INF-γ/β and TNF-α, in the islet microenvironment [ 30 ]. Type 2 diabetes (T2D) arises when insulin secretion fails to meet demands mainly due to impaired insulin sensitivity, with β-cell oxidative and endoplasmic reticulum stress, lipotoxicity and glucotoxicity as conse- quences causing progressive loss of β cell functional mass [ 31 ]. All these cellular stresses induce an inflammatory response or are exacerbated by or associated with low-grade systemic inflam- mation via production of interleukin 1β (IL-1β) and IL-6 and recruitment and activation of innate immune cells [ 32 , 33 ]. As i- and int-proteasomes can modify e.g. signal transduction and MHC I peptide presentation, their constitutive and/or induced expression in β cells by inflammatory cytokines is of high interest and therapeutic potential.
In contrast to mammals, the adult zebrafish has the remarkable capacity to rapidly and spontaneously regenerate its betacells following their selective destruction . Our findings showing that ductal cells, such as CACs, behave as pancreatic progenitors/stem cells in normal – non-diabetic – adult animals and during regeneration strongly suggest that they could also constitute a source of regenerated betacells in diabetic mammalian models. In mouse, adult murine CACs display endocrine and exocrine progenitor potential in vitro with self-renewing ability , but evidences of their potential in vivo as progenitors of endocrine cells are missing. Indeed, CRE-based cell tracing of Hes1+ terminal duct cells/CACs in adult mice failed to show any islet progenitor capacity while these cells seemed to contribute to the ductal tree . One explanation for this difference would be that while zebrafish CACs present pancreatic progenitor activity, mammalian CACs have retained a very limited capacity in vivo which could not be evidenced in the mouse model of beta cell regeneration used. Another explanation would be that CACs form a heterogeneous cell population. This latter hypothesis could be verified by the analysis of the expression of different ductal markers identified in our transcriptome that could help determine the existence of different ductal cell subpopulations.
E hb = D hb 关5共R hb /R DA 兲 12 − 6共R hb /R DA 兲 10 兴cos 4 共 DHA 兲, 共7兲 where D hb has the physical meaning as the depth of the H-bond energy and R hb as the zero force distance for the H bond if cos共 DHA 兲 keeps constant, and those two are param- eters from Dreiding model for the H bond. The parameter R DA is the distance between the donor 共N here兲 and the ac- ceptor 共O here兲, and DHA is the angle for ⬔NHO as shown in Fig. 1共a兲 . The reason to choose the Dreiding force field to describe the force-extension behavior of hydrogen bonds is that it provides an explicit expression of the hydrogen bond energy, including angular terms. By using this expression, we are able to obtain an explicit expression of the elastic stiffness of the H bond. Since the length of the covalent bond N-H is a 1 in the Dreiding force field, by taking the average distance between two neighboring beta strands as a constant, the geometric variants in Eq. 共 7 兲 can be expressed as
associated with beta numeration. It is well known that they induce an aperiodic multiple tiling of their representation space, and there are several topological, combinatorial, and arithmetical conditions that imply the tiling property. In the irreducible unit context, having an aperiodic tiling is equivalent to having a periodic one [IR06]. The situation is different when we switch to the reducible and non-unit cases. In order to have a periodic tiling, a certain algebraic hypothesis (QM), first introduced in [ST09] for substitutions, must hold, and our attention is naturally restricted to a certain stripe space when dealing with the non-unit case.
Figure 3 Flow cytometry data for IMR-90 cells transfected with EGFP using the leading polymers. Transfection efficacy expressed as percent green fluorescent protein- positive live cells (A) following a single dose and (B) after three doses of B4s4e7 (68%). graphs show the mean ± standard error of the mean, n$5. (C) Fluorescence microscopy images of untreated (left) and transfected (right) IMR-90 fibroblasts, showing good viability in both cases.
Notes: acrylate monomers (Bx), amino-alcohol monomers (sy), and end-capping groups (ez). The number “x” following the acrylate monomers “B” refers to the number of carbons between acrylate groups in the monomer, the number “y” following the amino-alcohol monomers “s” refers to the number of carbons between the amine group and the alcohol group in the sidechain, and the number “z” following the end-capping groups “e” refers to an arbitrary number used to designate a particular end-capping group. Abbreviation: EGFP, enhanced green fluorescent protein.
Transient transfection and luciferase assay
Vectors encoding human C/EBPa or b (SC303472 and SC319561; Origene, Burlington, ON) and vectors encoding C/ EBPb-2 and C/EBPb-3 (No. 15738 and No. 15737; Addgene, Cambridge, MA) were obtained commercially. The cDNA encoding the human galectin-7 (provided by Dr. T. Magnaldo) was cloned in the sra eukaryotic expression vector (kind gift of Dr. Franc¸ois Denis) using SpeI and BamHI restriction sites. Cells were transfected using the Lipofectamine 2000 reagent (Life Technol- ogies) according to the manufacturer’s protocol. For reporter assays, NF-kB or a C/EBP luciferase reporter vectors (Cat. No. 219078 and 240112 respectively; Stratagene, Santa Clara, CA) were used. The (empty) pCMV5 vector was used as a control. Transfection efficiency was measured using the pCMV/b-gal reporter vector (Promega, Madison, WI). Luciferase activity was measured using the Luciferase Assay System protocol (Promega) and a luminometer (Lumat LB 9507, Berthold). b-galactosidase activity was detected using a colorimetric enzyme assay using the Luminescent b-Galactosidase Detection Kit II according to the manufacturer’s instructions (Clontech Laboratories, Mountain View, CA). Luciferase expression levels were normalized to the levels of b-galactosidase expression.
In ionizing radiation metrology, the energy spectra of beta decays are often needed, especially when measurements are carried out using Liquid Scintillation Counting, since the mod- eling of the light emission used in the triple to double coinci- dence ratio (TDCR) method, used to establish a relationship between the detection eﬃciency and the experimental TDCR ratio, requires the knowledge of the shape of the beta spec- tra for beta-decaying nuclides (Broda et al., 2007 ). Due to the threshold eﬀect, the reliability of the TDCR-Cerenkov tech- nique is even more sensitive to the accuracy of the shape of beta spectra (Bobin et al., 2010 ).
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