Metabolomic analysis of pancreatic beta cells following exposure to high glucose

Background

Chronic exposure to hyperglycaemic conditions has been shown to have detrimental effects on beta cell function. The resulting glucotoxicity is a contributing factor to the development of type 2 diabetes. The objective of this study was to combine a metabolomics approach with functional assays to gain insight into the mechanism by which glucotoxicity exerts its effects.

Methods

The BRIN-BD11 and INS-1E beta cell lines were cultured in 25 mM glucose for 20 h to mimic glucotoxic effects. PDK-2 protein expression, intracellular glutathione levels and the change in mitochondrial membrane potential and intracellular calcium following glucose stimulation were determined. Metabolomic analysis of beta cell metabolite extracts was performed using GC–MS, ¹H NMR and ¹³C NMR.

Results

Conditions to mimic glucotoxicity were established and resulted in no loss of cellular viability in either cell line while causing a decrease in insulin secretion. Metabolomic analysis of beta cells following exposure to high glucose revealed a change in amino acids, an increase in glucose and a decrease in phospho-choline, n−3 and n−6 PUFAs during glucose stimulated insulin secretion relative to cells cultured under control conditions. However, no changes in calcium handling or mitochondrial membrane potential were evident.

Conclusions

Results indicate that a decrease in TCA cycle metabolism in combination with an alteration in fatty acid composition and phosphocholine levels may play a role in glucotoxicity induced impairment of glucose stimulated insulin secretion.

General significance

Alterations in certain metabolic pathways play a role in glucotoxicity in the pancreatic beta cell.     

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small “scars” on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of “provisional clot”-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.     

Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60)

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 μM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 μM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.     

Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine,Pyrimidine, and Cytokinin Ribosides

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.Nucleoside hydrolases or nucleoside N-ribohydrolases (NRHs; EC 3.2.2.-) are glycosidases that catalyze the cleavage of the N-glycosidic bond in nucleosides to enable the recycling of the nucleobases and Rib (Fig. 1A). The process by which nucleosides and nucleobases are recycled is also known as salvaging and is a way of conserving energy, which would otherwise be needed for the de novo synthesis of purine- and pyrimidine-containing compounds. During the salvage, bases and nucleosides can be converted into nucleoside monophosphates by the action of phosphoribosyltransferases and nucleoside kinases, respectively, and further phosphorylated into nucleoside diphosphates and triphosphates (Moffatt et al., 2002; Zrenner et al., 2006; Fig. 1B). Uridine kinase and uracil phosphoribosyl transferase are key enzymes in the pyrimidine-salvaging pathway in plants (Mainguet et al., 2009; Chen and Thelen, 2011). Adenine phosphoribosyltransferase and adenosine kinase (ADK) are important in purine salvaging (Moffatt and Somerville, 1988; Moffatt et al., 2002), and their mutants cause reductions in fertility or sterility, changes in transmethylation, and the formation of abnormal cell walls. In addition, both enzymes were also reported to play roles in cytokinin metabolism (Moffatt et al., 1991, 2000; von Schwartzenberg et al., 1998; Schoor et al., 2011). Cytokinins (N⁶-substituted adenine derivatives) are plant hormones that regulate cell division and numerous developmental events (Mok and Mok, 2001; Sakakibara, 2006). Cytokinin ribosides are considered to be transport forms and have little or no activity.Open in a separate windowFigure 1.A, Scheme of the reactions catalyzed by plant NRHs when using purine (inosine), pyrimidine (uridine), and cytokinin (iPR) ribosides as the substrates. B, Simplified schematic overview of cytokinin, purine, and pyrimidine metabolism in plants. The diagram is adapted from the work of Stasolla et al. (2003) and Zrenner et al. (2006) with modifications. The metabolic components shown are as follows: 1, cytokinin nucleotide phosphoribohydrolase; 2, adenine phosphoribosyltransferase; 3, adenosine kinase; 4, 5′-nucleotidase; 5, adenosine phosphorylase; 6, purine/pyrimidine nucleoside ribohydrolase; 7, cytokinin oxidase/dehydrogenase; 8, AMP deaminase; 9, hypoxanthine phosphoribosyltransferase; 10, inosine kinase; 11, inosine-guanosine phosphorylase; 12, IMP dehydrogenase; 13, xanthine dehydrogenase; 14, 5′-nucleotidase; 15, GMP synthase; 16, hypoxanthine-guanine phosphoribosyltransferase; 17, guanosine deaminase; 18, guanine deaminase; 19, guanosine kinase; 20, uracil phosphoribosyltransferase; 21, uridine cytidine kinase; 22, pyrimidine 5′-nucleotidase; 23, cytidine deaminase; 24, adenosine/adenine deaminase. CK, Cytokinin; CKR, cytokinin riboside; CKRMP, cytokinin riboside monophosphate.NRHs are metalloproteins first identified and characterized in parasitic protozoa such as Trypanosoma, Crithidia, and Leishmania species that rely on the import and salvage of nucleotide derivatives. They have since been characterized in other organisms such as bacteria, yeast, and insects (Versées and Steyaert, 2003) but never in mammals (Parkin et al., 1991). They have been divided into four classes based on their substrate specificity: nonspecific NRHs, which hydrolyze inosine and uridine (IU-NRHs; Parkin et al., 1991; Shi et al., 1999); purine-specific inosine/adenosine/guanosine NRHs (Parkin, 1996); the 6-oxopurine-specific guanosine/inosine NRHs (Estupiñán and Schramm, 1994); and the pyrimidine nucleoside-specific cytidine/uridine NRHs (CU-NRHs; Giabbai and Degano, 2004). All NRHs exhibit a stringent specificity for the Rib moiety and differ in their preferences regarding the nature of the nucleobase. Crystal structures are available for empty NRH or in complex with inhibitors from Crithidia fasciculata (CfNRH; Degano et al., 1998), Leishmania major (LmNRH; Shi et al., 1999), and Trypanosoma vivax (TvNRH; Versées et al., 2001, 2002). The structures of two CU-NRHs from Escherichia coli, namely YeiK (Iovane et al., 2008) and YbeK (rihA; Muzzolini et al., 2006; Garau et al., 2010), are also available. NRHs are believed to catalyze N-glycosidic bond cleavage by a direct displacement mechanism. An Asp from a conserved motif acts as a general base and abstracts a proton from a catalytic water molecule, which then attacks the C1′ atom of the Rib moiety of the nucleoside. Kinetic isotope-effect studies on CfNRH (Horenstein et al., 1991) showed that the substrate’s hydrolysis proceeds via an oxocarbenium ion-like transition state and is preceded by protonation at the N7 atom of the purine ring, which lowers the electron density on the purine ring and destabilizes the N-glycosidic bond. A conserved active-site His is a likely candidate for this role in IU-NRHs and CU-NRHs. In the transition state, the C1′-N9 glycosidic bond is almost 2 Å long, with the C1′ atom being sp² hybridized while the C3′ atom adopts an exo-conformation, and the whole ribosyl moiety carries a substantial positive charge (Horenstein et al., 1991).Several NRH enzymes have been identified in plants, including a uridine-specific NRH from mung bean (Phaseolus radiatus; Achar and Vaidyanathan, 1967), an inosine-specific NRH (EC 3.2.2.2) and a guanosine-inosine-specific NRH, both from yellow lupine (Lupinus luteus; Guranowski, 1982; Szuwart et al., 2006), and an adenosine-specific NRH (EC 3.2.2.7) from coffee (Coffea arabica), barley (Hordeum vulgare), and wheat (Triticum aestivum; Guranowski and Schneider, 1977; Chen and Kristopeit, 1981; Campos et al., 2005). However, their amino acid sequences have not been reported so far. A detailed study of the NRH gene family from Arabidopsis (Arabidopsis thaliana) has recently been reported (Jung et al., 2009, 2011). The AtNRH1 enzyme exhibits highest hydrolase activity toward uridine and xanthosine. It can also hydrolyze the cytokinin riboside N⁶-(2-isopentenyl)adenosine (iPR), which suggests that it may also play a role in cytokinin homeostasis. However, Riegler et al. (2011) analyzed the phenotypes of homozygous nrh1 and nrh2 single mutants along with the homozygous double mutants and concluded that AtNRHs are probably unimportant in cytokinin metabolism.Here, we identify and characterize plant IU-NRHs from two different model organisms, Physcomitrella patens and maize (Zea mays), combining structural, enzymatic, and in planta functional approaches. The moss P. patens was chosen to represent the bryophytes, which can be regarded as being evolutionarily basal terrestrial plants, and is suitable for use in developmental and metabolic studies (Cove et al., 2006; von Schwartzenberg, 2009), while maize is an important model system for cereal crops. We report the crystal structures of NRH enzymes from the two plant species, PpNRH1 and ZmNRH3. Based on these structures, we performed site-directed mutagenesis experiments and kinetic analyses of point mutants of PpNRH1 in order to identify key residues involved in nucleobase interactions and catalysis. To analyze the physiological role of the PpNRHs, single knockout mutants were generated. NRH deficiency caused significant changes in the levels of purine, pyrimidine, and cytokinin metabolites relative to those seen in the wild type, illustrating the importance of these enzymes in nucleoside and cytokinin metabolism.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.