Methylglyoxal accumulation de-regulates HoxA5 expression,thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-?B-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-?B-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-?B-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-?B-p65, thereby impairing the angiogenic ability of endothelial cells.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Activation and mitochondrial translocation of protein kinase Cdelta are necessary for insulin stimulation of pyruvate dehydrogenase complex activity in muscle and liver cells

In L6 skeletal muscle cells and immortalized hepatocytes, insulin induced a 2-fold increase in the activity of the pyruvate dehydrogenase (PDH) complex. This effect was almost completely blocked by the protein kinase C (PKC) delta inhibitor Rottlerin and by PKCdelta antisense oligonucleotides. At variance, overexpression of wild-type PKCdelta or of an active PKCdelta mutant induced PDH complex activity in both L6 and liver cells. Insulin stimulation of the activity of the PDH complex was accompanied by a 2.5-fold increase in PDH phosphatases 1 and 2 (PDP1/2) activity with no change in the activity of PDH kinase. PKCdelta antisense blocked insulin activation of PDP1/2, the same as with PDH. In insulin-exposed cells, PDP1/2 activation was paralleled by activation and mitochondrial translocation of PKCdelta, as revealed by cell subfractionation and confocal microscopy studies. The mitochondrial translocation of PKCdelta, like its activation, was prevented by Rottlerin. In extracts from insulin-stimulated cells, PKCdelta co-precipitated with PDP1/2. PKCdelta also bound to PDP1/2 in overlay blots, suggesting that direct PKCdelta-PDP interaction may occur in vivo as well. In intact cells, insulin exposure determined PDP1/2 phosphorylation, which was specifically prevented by PKCdelta antisense. PKCdelta also phosphorylated PDP in vitro, followed by PDP1/2 activation. Thus, in muscle and liver cells, insulin causes activation and mitochondrial translocation of PKCdelta, accompanied by PDP phosphorylation and activation. These events are necessary for insulin activation of the PDH complex in these cells.     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Vestibular mechanisms involved in idiopathic scoliosis

Patients affected by idiopathic scoliosis (IS) show not only a spinal deformity, but also postural and oculomotor deficits suggesting that such syndrome can be related to a vestibular disfunction. It appears, however, that, in children, a slight unbalance in the activity of vestibular complex of both sides escapes the neuronal mechanisms responsible for vestibular compensation and leads to the spinal curvature which characterises IS. Such process could be reinforced by a disrupted integration of vestibular and visual signals at cortical level, leading to an altered perception of the vertical and to abnormal motor commands. In addition to the classical ascending and descending pathways arising from the vestibular nuclei, which utilize glutamate or GABA as neurotransmitters, labyrinthine afferents may also affect spinal, cerebellar and cerebrocortical structures, through the noradrenergic and serotoninergic systems, which originate from the locus coeruleus and the raphe nuclei, respectively. Due to the role of these neuromodulators in brain plasticity, a disruption in the activity of monoaminergic neurons could favour the development of postural and oculomotor deficits. An impaired release of monoamine at cerebrocortical level could also explain the cognitive deficits which may occur in IS patients.